Novel lactic acid bacteria

Abstract

The present invention relates to Streptococcus thermophilus strains that are lactose-positive and sucrose-negative. These strains carry one or more mutations in one or more genes of sucrose regulators, and optionally carry one or more further mutations that affect glucose transporters, such as those affecting glcU and / or expression thereof. The strains of the invention can further carry mutations in genes encoding a mannose-glucose specific PTS protein and a glucokinase, a mannose-glucose specific PTS protein and a catabolic metabolite control protein A (CcpA), and / or a mannose-glucose specific PTS protein, a glucokinase and a CcpA. When the strains of the invention ferment milk, these strains can achieve this without or with the need to add reduced amounts of sucrose, optionally providing low lactose fermented milk and / or fermented milk that is not subjected to post-acidification when stored at fermentation temperatures. The invention also relates to a composition comprising at least one lactose-positive sucrose-negative Streptococcus thermophilus strain of the invention, and the use of such a strain or composition for the manufacture of a fermented milk product.

Description

Technical Field

[0001] The present invention relates to lactose-positive sucrose-negative Streptococcus thermophilus strains. These strains carry one or more mutations in one or more genes of sucrose regulon, and optionally carry one or more further mutations affecting glucose transporter (non-PTS glucose permease), for example affecting glcU and / or its expression. These strains of the present invention can further carry mutations in genes encoding mannose-glucose specific PTS protein and glucokinase, mannose-glucose specific PTS protein and catabolite control protein A (CcpA), and / or mannose-glucose specific PTS protein, glucokinase and CcpA.

[0002] When the strains of the invention ferment milk containing any sucrose, the strains can do so without consuming that sucrose, thereby reducing the need for added sucrose, or where a reduced amount of sucrose needs to be added, while still obtaining the desired sweetness level from the sucrose. Optionally, the strains of the invention provide low lactose fermented milk and / or fermented milk that does not undergo post-acidification when stored at fermentation temperatures. The invention also relates to a composition comprising at least one lactose-positive, sucrose-negative thermophilic Streptococcus strain of the invention, and the use of such strains or compositions for the manufacture of fermented milk products. Background Art

[0003] The food industry uses bacteria to improve the taste and texture of food products or feed products. In the dairy industry, lactic acid bacteria are often used, for example, to cause acidification of milk (by lactose fermentation) and to change the texture of the products into which they are incorporated. For example, lactic acid bacteria belonging to the species Streptococcus thermophilus (S. thermophilus) are widely used alone or in combination with other bacteria in the manufacture of fresh fermented milk products such as cheese or yogurt.

[0004] One of the difficulties in using lactic acid bacteria in dairy technology is post-acidification, i.e. the production of lactic acid by lactic acid bacteria after the target pH (the pH required for the technology) has been achieved [termination of fermentation]. Therefore, the phenomenon of post-acidification is not only a problem for dairy product manufacturers (who want to have a flexible manufacturing process without the need for a rapid cooling step immediately after the target pH has been achieved) but also for consumers (the production of lactic acid bacteria causes an increase in acidity and a decrease in the quality of the fermented product).

[0005] Another difficulty in the dairy industry is that any added or already present sucrose may be consumed by the culture during fermentation. In order to obtain the desired sweet taste of the fermented product, it is necessary to add sucrose to the milk before, during or at the end of fermentation. Another difficulty in the dairy industry is that any added sucrose may be consumed by the culture during fermentation. Therefore, in order to achieve the desired sweet taste, it may be necessary to add excess sucrose to the milk. In addition, it is conceivable to reduce the amount of added sucrose even more by releasing part or all of the glucose produced by the hydrolysis of lactose by the culture.

[0006] Furthermore, there is a trend among dairy consumers to use fermented products with reduced or lower lactose content (lactose intolerance).

[0007]

[0006] Therefore, there is a need for improved methods of producing fermented milk products that are satisfactory to both manufacturers and consumers, do not undergo post-acidification, require less added sucrose, and have a reduced lactose content. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 : with scrA 829 FS and pscrB 829 DNA sequence alignment of the chromosomal locus of mutant strain DGCC8368 with that of ST1. The dots in the DGCC8368 sequence indicate identical nucleotides in the two sequences. The nucleotides in bold in the DGCC8368 sequence indicate and name the differences between the two sequences. Difference pscrB 829 Corresponds to the replacement of a T nucleotide with a G nucleotide at position 36 of the sequence; difference scrA 829 FS This corresponds to the insertion of an A nucleotide at sequence position 384. The start codon is italicized and underlined, the open reading frame is italicized, and the putative -10 frame is bolded and underlined.

[0009] Figure 2 : DNA sequence alignment of the promoter region of the glcU gene of strain ST1 with the promoter region of various sucrose-negative, glucose-positive mutants described in Example 5. The dots in the mutant sequences indicate the same nucleotides in all sequences. The nucleotides in bold in the mutant sequences are named and indicate sequence differences or black triangles indicate insertions of IS1191. Mutation pglcU SNP_C Corresponds to the replacement of a T nucleotide with a C nucleotide at position 96 of the sequence; difference pglcU IN_T and pglcU IN_C Corresponds to the insertion of a T or C nucleotide at position 102 of the sequence. ISThis corresponds to the insertion of IS1191 between positions 111 and 112 of the sequence (or between positions 110 and 111 of the sequences of, for example, ST1 and ST1-ABU4). The start codon is indicated in italics and underlined, while the open reading frame is indicated in italics.

[0010] Figure 3 : For the wild-type (parent) strain (black), their respective scrA 829 FS -pscrB 829 Calculation of μ for the mutant (white) and its glucose-positive derivative (grey) carrying the mutation within pglcU 葡萄糖 / μmax 乳糖 Comparison of ratios.

[0011] Figures 4 to 8 :Indicates using ST1( Figure 4 )、ST1-GM( Figure 5 )、ST1-GM-AB( Figure 6 )、ST1-GM-ABU1( Figure 7 )、ST1-CM-ABU1( Figure 8 ) Graph of the pH evolution over time (A) and graph of the velocity as a function of pH (B) of fermented plain milk (1) and milk supplemented with 7% (w / v) sucrose (2). DETAILED DESCRIPTION

[0012] The starter culture of Streptococcus thermophilus used in the art will generally consume a certain amount of any sucrose added to the milk for sweetening purposes during yogurt fermentation; the consequences are as follows:

[0013] a) More sucrose needs to be added than is (theoretically) required to achieve a certain sweetness level because the strain is using the sucrose (rather than using lactose).

[0014] b) In sweet milk, the function of the controlled pH phenotype produced by fermentation of thermophilic Streptococcus with mutations in manM (and / or manL and / or manN) and glcK and / or ccpA (as described in the examples of WO 2019197051) will be reduced or lost.

[0015] c) In sweet milk, glucose release produced by fermentation of thermophilic Streptococcus having only a mutation in glcK and / or a mutation in ccpA or a mutation in glcK and / or ccpA and a mutation in manM (and / or manL and / or manN) (as described in the examples of WO 2019122365 and WO 2019197051) may be reduced or absent.

[0016] d) In sweet milk, the ability of Streptococcus thermophilus having mutations in glcK, ccpA, glck / ccpA, glcK / manM (manL / manN), ccpA / manM (manL / manN) and glcK / ccpA / manM (manL / manN) to overconsume lactose by fermentation (as described in the examples of WO2019122365 and WO 2019197051) may be reduced or absent.

[0017] The present invention has solved each of these problems in the art and provides solutions regarding specific mutations that modify sugar metabolism, which can be used to design improved Streptococcus thermophilus strains that can be used to obtain low-lactose fermented milk products and / or can be used to produce fermented milk containing sucrose that does not undergo post-acidification even when stored at fermentation temperatures and does not face the above-mentioned difficulties.

[0018] Thus, in a first aspect, the present invention relates to a lactose-positive, sucrose-negative Streptococcus thermophilus strain carrying one or more mutations selected from the group consisting of:

[0019] 1) one or more mutations in one or more genes of a sucrose regulator; the mutation significantly reduces the ability of the strain to grow on sucrose and / or transport sucrose; and the mutation is

[0020] i) one or more genes of the sucrose regulator, such as the scrA gene (encoding the sucrose-specific EIIABC of the phosphoenolpyruvate-sugar phosphotransferase system (PTS) 蔗糖 component) and / or scrB gene (encoding sucrose-6-phosphate hydrolase) and / or scrR gene (encoding a putative sucrose operon regulator), and

[0021] ii) optionally further mutations in the promoter region of the sucrose regulon regulating the expression of the scrA, scrB and / or scrR genes, e.g. mutations that protect the strain from reversion to a sucrose negative phenotype; and / or

[0022] 2) optionally one or more further mutations affecting a glucose transporter, such as glcU and / or its expression, which mutations restore or improve glucose consumption in said strain.

[0023] It should be understood that in some embodiments, the lactose-positive, sucrose-negative thermophilic Streptococcus strain according to the present invention has a mandatory mutation in one or more of the scrA gene and / or the scrB gene and / or the scrR gene. In some embodiments, the lactose-positive, sucrose-negative thermophilic Streptococcus strain according to the present invention has a) a further mutation in the promoter region regulating the expression of the scrA, scrB and / or scrR genes, such as a mutation that protects the strain from reversing from a sucrose-negative phenotype; or

[0024] b) one or more further mutations affecting a glucose transporter, such as glcU and / or its expression, which mutations restore or improve glucose consumption by the strain when the strain is grown on glucose as the sole carbohydrate source; or both a) and b).

[0025] In a second aspect, the present invention relates to a composition comprising at least one, in particular one, lactose-positive, sucrose-negative Streptococcus thermophilus strain according to the present invention, in particular in combination with another lactic acid bacterium, in particular in combination with one or more strains selected from the group consisting of: a strain of the genus Lactobacillus, such as a strain of Lactobacillus delbrueckii subsp bulgaricus; a strain of the genus Lactococcus, such as a strain of Lactococcus lactis; or a strain of the genus Bifidobacterium.

[0026] In a third aspect, the present invention relates to a method for producing a fermented milk product, in particular fermented milk, comprising inoculating a milk substrate with a lactose-positive, sucrose-negative Streptococcus thermophilus strain according to the invention or with a composition according to the invention, and fermenting said inoculated milk to obtain a fermented milk product.

[0027] In another aspect, the present invention relates to the use of a lactose-positive, sucrose-negative Streptococcus thermophilus strain according to the present invention or the above-mentioned composition according to the present invention for the manufacture of a fermented milk product.

[0028] In another aspect, the present invention relates to a fermented milk product comprising at least one, in particular one, lactose-positive, sucrose-negative Streptococcus thermophilus strain according to the present invention or a lactose-positive, sucrose-negative Streptococcus thermophilus strain obtained by the above-mentioned method according to the present invention.

[0029] In another aspect, the present invention relates to a Streptococcus thermophilus strain, such as a lactose-positive, sucrose-negative Streptococcus thermophilus strain selected from the group consisting of:

[0030] - strains deposited on January 18, 2022 in the name of DuPont Nutrition Biosciences ApS under the Budapest Treaty under the number DSM 34132 at the Leibniz-Institut DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH;

[0031] - strain deposited on January 18, 2022 in the name of DuPont Nutrition Biosciences under the number DSM34134 at the Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures in accordance with the Budapest Treaty;

[0032] - strain deposited on January 18, 2022 in the name of DuPont Nutrition Biosciences under the number DSM34138 at the Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures in accordance with the Budapest Treaty;

[0033] - strain deposited on January 18, 2022 in the name of DuPont Nutrition Biosciences under the number DSM34139 at the Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures in accordance with the Budapest Treaty;

[0034] - strains deposited on January 18, 2022 in the name of DuPont Nutrition Biosciences under the number DSM34140 at the Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures in accordance with the Budapest Treaty;

[0035] - strain deposited on January 18, 2022 in the name of DuPont Nutrition Biosciences under the number DSM34146 at the Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures in accordance with the Budapest Treaty;

[0036] - strain deposited on January 18, 2022 in the name of DuPont Nutrition Biosciences under the number DSM34147 at the Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures in accordance with the Budapest Treaty;

[0037] -The strain deposited on February 16, 2022 at the Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures under the number DSM34172 in the name of DuPont Nutrition Biosciences under the Budapest Treaty.

[0038] In another aspect, the present invention relates to a lactose-positive, sucrose-negative Streptococcus thermophilus strain selected from the group consisting of:

[0039] A strain corresponding to the Streptococcus thermophilus strain deposited at the Leibniz Institute DSMZ under the Budapest Treaty on January 14, 2014 under the number DSM28255 in the name of Danisco Deutschland GmbH [referred to herein as ST4 or DSM28255 strain], wherein the sequence of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, resulting in a frameshift such that a truncated EIIABC is translated 蔗糖 (scrA 829 FS );

[0040] a strain corresponding to the Streptococcus thermophilus strain deposited at the Leibniz Institute DSMZ under the Budapest Treaty on January 14, 2014 under the number DSM28255 in the name of Danisco Deutschland GmbH [referred to herein as ST4 or DSM28255 strain], wherein the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto (pscrB 829 );

[0041] a strain corresponding to the Streptococcus thermophilus strain deposited on January 14, 2014 at the Leibniz Institute DSMZ under the Budapest Treaty under the number DSM28255 in the name of Danisco Deutschland GmbH [referred to herein as ST4 or DSM28255 strain], wherein the locus (pglcU) as the promoter region upstream of the glcU gene is replaced by the corresponding sequence as shown in any one of SEQ ID NOs: 217 to 220 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pglcU SNP_C ,pglcU IN_T ,pglcU IN_C ,pglcU IS );

[0042] a strain corresponding to the Streptococcus thermophilus strain deposited at the Leibniz Institute DSMZ under the Budapest Treaty on January 14, 2014 under the number DSM28255 in the name of Danisco Deutschland GmbH [referred to herein as ST4 or DSM28255 strain], wherein the sequence of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, resulting in a frameshift, such that a truncated EIIABC is translated 蔗糖 (scrA 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pscrB 829 );

[0043] a strain corresponding to the Streptococcus thermophilus strain deposited at the Leibniz Institute DSMZ under the Budapest Treaty on January 14, 2014 under the number DSM28255 in the name of Danisco Deutschland GmbH [referred to herein as ST4 or DSM28255 strain], wherein the sequence of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, resulting in a frameshift, such that a truncated EIIABC is translated 蔗糖 (scrA 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pscrB 829 ), and the gene locus (pglcU) as the promoter region upstream of the glcU gene is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217-220 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pglcU SNP_C ,pglcU IN_T ,pglcU IN_C ,pglcU IS );

[0044] a strain corresponding to the Streptococcus thermophilus strain deposited at the Leibniz Institute DSMZ under the Budapest Treaty on January 14, 2014 under the number DSM28255 in the name of Danisco Deutschland GmbH [referred to herein as ST4 or DSM28255 strain], wherein the sequence of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, resulting in a frameshift, such that a truncated EIIABC is translated 蔗糖 (scrA 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pscrB 829 ), and the gene locus (pglcU) as the promoter region upstream of the glcU gene is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217-220 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pglcU SNP_C ,pglcU IN_T ,pglcU IN_C ,pglcU IS ), and optionally wherein the glcK gene encoding glucokinase comprises a sequence as defined by SEQ ID NO: 25 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, wherein the amino acid at position 275 is lysine (glcK 378 ), and optionally further comprising generating an encoding of the EIIC Man A mutation of the open reading frame shift of the gene for the protein, resulting in a stop codon at position 180 of SEQ ID NO: 222, for example, by inserting a nucleotide C at any of positions 438, 439 or 440 of SEQ ID NO: 223 in the manM gene (manM 12997 ); and optionally further comprises the ccpA sequence of SEQ ID NO:71 or a ccpA variant sequence having at least 95% identity with SEQ ID NO:71.

[0045] a strain of Streptococcus thermophilus, such as the strain deposited on 14 January 2014 at the Leibniz Institute DSMZ under the Budapest Treaty in the name of Danisco Deutschland GmbH under the number DSM 28255 [referred to herein as strain ST4 or DSM 28255], which strain comprises one or more of the following characteristics:

[0046] 1) The sequence of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, resulting in a frame shift, so that a truncated EIIABC is translated 蔗糖 (scrA 829 FS ),

[0047] 2) The sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pscrB 829 ),

[0048] 3) the locus (pglcU) as the promoter region upstream of the glcU gene is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217-220 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pglcU SNP_C ,pglcU IN_T ,pglcU IN_C ,pglcU IS ),

[0049] 4) The glcK gene encoding glucokinase comprises a sequence as defined in SEQ ID NO: 25 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, wherein the amino acid at position 275 is lysine (glcK 378 ),

[0050] 5) Generate the EIIC code Man A mutation of the open reading frame shift of the gene for the protein, resulting in a stop codon at position 180 of SEQ ID NO: 222, for example, by inserting a nucleotide C at any of positions 438, 439 or 440 of SEQ ID NO: 223 in the manM gene (manM 12997 );

[0051] 6) a ccpA sequence having the ccpA sequence of SEQ ID NO:71 or a ccpA variant sequence having at least about 90%, such as at least 95%, identity to SEQ ID NO:71.

[0052] In another aspect, the present invention relates to a lactose-positive, sucrose-negative Streptococcus thermophilus strain selected from the group consisting of:

[0053] - A strain corresponding to the Streptococcus thermophilus strain deposited at the Leibniz Institute DSMZ under the number DSM34132 on January 18, 2022 in the name of DuPont Nutrition Biosciences under the Budapest Treaty [referred to herein as ST2 or DSM34132 strain], wherein the sequence of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, resulting in a frameshift, such that a truncated EIIABC is translated 蔗糖 (scrA 829 FS );

[0054] - A strain corresponding to the Streptococcus thermophilus strain deposited at the Leibniz Institute DSMZ under the number DSM34132 on January 18, 2022 in the name of DuPont Nutrition Biosciences under the Budapest Treaty [referred to herein as ST2 or DSM34132 strain], wherein the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto (pscrB 829 );

[0055] - A strain corresponding to the Streptococcus thermophilus strain deposited at the Leibniz Institute DSMZ under the number DSM34132 on January 18, 2022 in the name of DuPont Nutrition Biosciences under the Budapest Treaty [referred to herein as ST2 or DSM34132 strain], wherein the locus (pglcU) as the promoter region upstream of the glcU gene is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217-220 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pglcU SNP_C ,pglcU IN_T ,pglcU IN_C ,pglcU IS );

[0056] - A strain corresponding to the Streptococcus thermophilus strain deposited at the Leibniz Institute DSMZ under the number DSM34132 on January 18, 2022 in the name of DuPont Nutrition Biosciences under the Budapest Treaty [referred to herein as ST2 or DSM34132 strain], wherein the sequence of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, resulting in a frameshift, such that a truncated EIIABC is translated 蔗糖 (scrA 829FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pscrB 829 );

[0057] - A strain corresponding to the Streptococcus thermophilus strain deposited at the Leibniz Institute DSMZ under the number DSM34132 on January 18, 2022 in the name of DuPont Nutrition Biosciences under the Budapest Treaty [referred to herein as ST2 or DSM34132 strain], wherein the sequence of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, resulting in a frameshift, such that a truncated EIIABC is translated 蔗糖 (scrA 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pscrB 829 ), and the gene locus (pglcU) as the promoter region upstream of the glcU gene is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217-220 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pglcU SNP_C ,pglcU IN_T ,pglcU IN_C ,pglcU IS );

[0058] - A strain corresponding to the Streptococcus thermophilus strain deposited at the Leibniz Institute DSMZ under the number DSM34132 on January 18, 2022 in the name of DuPont Nutrition Biosciences under the Budapest Treaty [referred to herein as ST2 or DSM34132 strain], wherein the sequence of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, resulting in a frameshift, such that a truncated EIIABC is translated 蔗糖 (scrA 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pscrB 829), and the gene locus (pglcU) as the promoter region upstream of the glcU gene is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217-220 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pglcU SNP_C ,pglcU IN_T ,pglcU IN_C ,pglcU IS ), and optionally wherein the glcK gene encoding glucokinase contains a sequence as defined in SEQ ID NO: 25, wherein the amino acid at position 275 is lysine (glcK 378 ), and optionally further comprising generating an encoding of the EIIC Man A mutation of the open reading frame shift of the gene for the protein, resulting in a stop codon at position 180 of SEQ ID NO: 222, for example, by inserting a nucleotide C at any of positions 438, 439 or 440 of SEQ ID NO: 223 in the manM gene (manM 12997 ); and optionally further comprises the ccpA sequence of SEQ ID NO:71 or a ccpA variant sequence having at least about 90%, such as at least 95%, identity to SEQ ID NO:71.

[0059] - a strain of Streptococcus thermophilus, such as the strain deposited on January 18, 2022 at the Leibniz Institute DSMZ under the number DSM34132 in the name of DuPont Nutrition Biosciences under the Budapest Treaty [referred to herein as ST2 or DSM34132 strain], which strain comprises one or more of the following characteristics:

[0060] 1) The sequence of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, resulting in a frame shift, so that a truncated EIIABC is translated 蔗糖 (scrA 829 FS ),

[0061] 2) The sequence upstream of the start codon of the scrB gene was replaced by the corresponding sequence shown in SEQ ID NO: 213 (pscrB 829 ),

[0062] 3) the locus (pglcU) as the promoter region upstream of the glcU gene is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217-220 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pglcU SNP_C ,pglcU IN_T ,pglcU IN_C ,pglcU IS ),

[0063] 4) The glcK gene encoding glucokinase comprises a sequence as defined in SEQ ID NO: 25 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, wherein the amino acid at position 275 is lysine (glcK 378 ),

[0064] 5) Generate the EIIC code Man A mutation of the open reading frame shift of the gene for the protein, resulting in a stop codon at position 180 of SEQ ID NO: 222, for example, by inserting a nucleotide C at any of positions 438, 439 or 440 of SEQ ID NO: 223 in the manM gene (manM 12997 );

[0065] 6) a ccpA sequence having the ccpA sequence of SEQ ID NO:71 or a ccpA variant sequence having at least about 90%, such as at least 95%, identity to SEQ ID NO:71.

[0066] In another aspect, the present invention relates to a lactose-positive, sucrose-negative Streptococcus thermophilus strain selected from the group consisting of:

[0067] - A strain corresponding to the Streptococcus thermophilus strain deposited with the DSMZ [German Collection of Microorganisms and Cell Cultures, Inhoffenstrasse 7B, D-38124 Braunschweig-Germany] on September 29, 2020 under the deposit number DSM33651 (hereinafter referred to as ST3 or DSM33651), wherein the sequence of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, resulting in a frameshift, so that a truncated EIIABC is translated 蔗糖(scrA 829 FS );

[0068] - A strain corresponding to the Streptococcus thermophilus strain deposited with the DSMZ [German Collection of Microorganisms and Cell Cultures, Inhofenstrasse 7B, Braunschweig, Germany, D-38124] on September 29, 2020 under the accession number DSM33651 (referred to herein as ST3 or DSM33651), wherein the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto (pscrB 829 );

[0069] - A strain corresponding to the Streptococcus thermophilus strain deposited on September 29, 2020 with the accession number DSM33651 at DSMZ [German Collection of Microorganisms and Cell Cultures, Inhofenstrasse 7B, Braunschweig, Germany, D-38124] (referred to herein as ST3 or DSM33651), wherein the locus (pglcU) as the promoter region upstream of the glcU gene is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217-220 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, or the promoter comprises the sequence of SEQ ID NO: 221 (pglcU SNP_C ,pglcU IN_T ,pglcU IN_C ,pglcU IS );

[0070] - A strain corresponding to the Streptococcus thermophilus strain deposited with the DSMZ [German Collection of Microorganisms and Cell Cultures, Inhofenstrasse 7B, D-38124 Braunschweig, Germany] on September 29, 2020 under the accession number DSM33651 (referred to herein as ST3 or DSM33651), wherein the sequence of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, resulting in a frameshift, such that a truncated EIIABC is translated 蔗糖 (scrA 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pscrB 829 );

[0071] - A strain corresponding to the Streptococcus thermophilus strain deposited with the DSMZ [German Collection of Microorganisms and Cell Cultures, Inhofenstrasse 7B, D-38124 Braunschweig, Germany] on September 29, 2020 under the accession number DSM33651 (referred to herein as ST3 or DSM33651), wherein the sequence of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, resulting in a frameshift, such that a truncated EIIABC is translated 蔗糖 (scrA 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pscrB 829 ), and the gene locus (pglcU) as the promoter region upstream of the glcU gene is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217-220 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pglcU SNP_C ,pglcU IN_T ,pglcU IN_C ,pglcU IS );

[0072] - A strain corresponding to the Streptococcus thermophilus strain deposited with the DSMZ [German Collection of Microorganisms and Cell Cultures, Inhofenstrasse 7B, D-38124 Braunschweig, Germany] on September 29, 2020 under the accession number DSM33651 (referred to herein as ST3 or DSM33651), wherein the sequence of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, resulting in a frameshift, such that a truncated EIIABC is translated 蔗糖 (scrA 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pscrB 829 ), and the locus (pglcU) as the promoter region upstream of the glcU gene is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217-220, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence (pglcU) having at least about 90%, such as at least about 95% identity thereto.SNP_C ,pglcU IN_T ,pglcU IN_C ,pglcU IS ), and optionally wherein the glcK gene encoding glucokinase comprises a sequence as defined by SEQ ID NO: 25 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, wherein the amino acid at position 275 is lysine (glcK 378 ), and optionally further comprising generating an encoding of the EIIC Man A mutation of the open reading frame shift of the gene for the protein, resulting in a stop codon at position 180 of SEQ ID NO: 222, for example, by inserting a nucleotide C at any of positions 438, 439 or 440 of SEQ ID NO: 223 in the manM gene (manM 12997 ); and optionally further comprises the ccpA sequence of SEQ ID NO:71 or a ccpA variant sequence having at least about 90%, such as at least 95%, identity to SEQ ID NO:71.

[0073] - A strain of Streptococcus thermophilus deposited on September 29, 2020 with the accession number DSM33651 at DSMZ [German Collection of Microorganisms and Cell Cultures, Inhofenstrasse 7B, D-38124 Braunschweig, Germany] (hereinafter referred to as ST3 or DSM33651), comprising one or more of the following characteristics:

[0074] 1) The sequence of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, resulting in a frame shift, so that a truncated EIIABC is translated 蔗糖 (scrA 829 FS ),

[0075] 2) The sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pscrB 829 ),

[0076] 3) the locus (pglcU) as the promoter region upstream of the glcU gene is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217-220 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pglcU SNP_C ,pglcUIN_T ,pglcU IN_C ,pglcU IS ),

[0077] 4) The glcK gene encoding glucokinase comprises a sequence as defined in SEQ ID NO: 25 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, wherein the amino acid at position 275 is lysine (glcK 378 ),

[0078] 5) Generate the EIIC code Man A mutation of the open reading frame shift of the gene for the protein, resulting in a stop codon at position 180 of SEQ ID NO: 222, for example, by inserting a nucleotide C at any of positions 438, 439 or 440 of SEQ ID NO: 223 in the manM gene (manM 12997 );

[0079] 6) a ccpA sequence having the ccpA sequence of SEQ ID NO:71 or a ccpA variant sequence having at least about 90%, such as at least 95%, identity to SEQ ID NO:71.

[0080] In another aspect, the present invention relates to a lactose-positive, sucrose-negative Streptococcus thermophilus strain selected from the group consisting of:

[0081] - A strain corresponding to the Streptococcus thermophilus strain deposited with the DSMZ [German Collection of Microorganisms and Cell Cultures, Inhofenstrasse 7B, D-38124 Braunschweig, Germany] on September 29, 2020 under the accession number DSM34172 (referred to herein as ST1 or DSM34172), wherein the sequence of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, resulting in a frameshift, such that a truncated EIIABC is translated 蔗糖 (scrA 829 FS );

[0082] - A strain corresponding to the Streptococcus thermophilus strain deposited with the DSMZ [German Collection of Microorganisms and Cell Cultures, Inhofenstrasse 7B, Braunschweig, Germany, D-38124] on September 29, 2020 under the accession number DSM34172 (referred to herein as ST1 or DSM34172), wherein the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto (pscrB 829 );

[0083] - A strain corresponding to the Streptococcus thermophilus strain deposited on September 29, 2020 with the accession number DSM34172 at DSMZ [German Collection of Microorganisms and Cell Cultures, Inhofenstrasse 7B, Braunschweig, Germany, D-38124] (referred to herein as ST1 or DSM34172), wherein the locus (pglcU) as the promoter region upstream of the glcU gene is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217-220 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, or the promoter comprises the sequence of SEQ ID NO: 221 (pglcU SNP_C ,pglcU IN_T ,pglcU IN_C ,pglcU IS );

[0084] - A strain corresponding to the Streptococcus thermophilus strain deposited with the DSMZ [German Collection of Microorganisms and Cell Cultures, Inhofenstrasse 7B, D-38124 Braunschweig, Germany] on September 29, 2020 under the accession number DSM34172 (referred to herein as ST1 or DSM34172), wherein the sequence of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, resulting in a frameshift, such that a truncated EIIABC is translated 蔗糖 (scrA 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pscrB 829 );

[0085] - A strain corresponding to the Streptococcus thermophilus strain deposited with the DSMZ [German Collection of Microorganisms and Cell Cultures, Inhofenstrasse 7B, D-38124 Braunschweig, Germany] on September 29, 2020 under the accession number DSM34172 (referred to herein as ST1 or DSM34172), wherein the sequence of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, resulting in a frameshift, such that a truncated EIIABC is translated 蔗糖 (scrA 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pscrB 829), and the gene locus (pglcU) as the promoter region upstream of the glcU gene is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217-220 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pglcU SNP_C ,pglcU IN_T ,pglcU IN_C ,pglcU IS );

[0086] - A strain corresponding to the Streptococcus thermophilus strain deposited with the DSMZ [German Collection of Microorganisms and Cell Cultures, Inhofenstrasse 7B, D-38124 Braunschweig, Germany] on September 29, 2020 under the accession number DSM34172 (referred to herein as ST1 or DSM34172), wherein the sequence of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, resulting in a frameshift, such that a truncated EIIABC is translated 蔗糖 (scrA 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pscrB 829 ), and the locus (pglcU) as the promoter region upstream of the glcU gene is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217-220, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence (pglcU) having at least about 90%, such as at least about 95% identity thereto. SNP_C ,pglcU IN_T ,pglcU IN_C ,pglcU IS ), and optionally wherein the glcK gene encoding glucokinase comprises a sequence as defined by SEQ ID NO: 25 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, wherein the amino acid at position 275 is lysine (glcK 378 ), and optionally further comprising generating an encoding of the EIIC Man A mutation of the open reading frame shift of the gene for the protein, resulting in a stop codon at position 180 of SEQ ID NO: 222, for example, by inserting a nucleotide C at any of positions 438, 439 or 440 of SEQ ID NO: 223 in the manM gene (manM12997 ); and optionally further comprises the ccpA sequence of SEQ ID NO:71 or a ccpA variant sequence having at least about 90%, such as at least 95%, identity to SEQ ID NO:71.

[0087] - A strain corresponding to the Streptococcus thermophilus strain deposited with the DSMZ [German Collection of Microorganisms and Cell Cultures, Inhofenstrasse 7B, D-38124 Braunschweig, Germany] under the accession number DSM34172 on September 29, 2020 (referred to herein as ST1 or DSM34172), which strain comprises one or more of the following characteristics:

[0088] 1) The sequence of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, resulting in a frame shift, so that a truncated EIIABC is translated 蔗糖 (scrA 829 FS ),

[0089] 2) The sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pscrB 829 ),

[0090] 3) the locus (pglcU) as the promoter region upstream of the glcU gene is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217-220 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pglcU SNP_C ,pglcU IN_T ,pglcU IN_C ,pglcU IS ),

[0091] 4) The glcK gene encoding glucokinase comprises a sequence as defined in SEQ ID NO: 25 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, wherein the amino acid at position 275 is lysine (glcK 378 ),

[0092] 5) Generate the EIIC code ManA mutation of the open reading frame shift of the gene for the protein, resulting in a stop codon at position 180 of SEQ ID NO: 222, for example, by inserting a nucleotide C at any of positions 438, 439 or 440 of SEQ ID NO: 223 in the manM gene (manM 12997 );

[0093] 6) a ccpA sequence having the ccpA sequence of SEQ ID NO:71 or a ccpA variant sequence having at least about 90%, such as at least 95%, identity to SEQ ID NO:71.

[0094] In another aspect, the present invention relates to a lactose-positive, sucrose-negative Streptococcus thermophilus strain selected from the group consisting of:

[0095] - a strain of a Streptococcus thermophilus strain, wherein the sequence of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, resulting in a frameshift, such that a truncated EIIABC is translated 蔗糖 (scrA 829 FS );

[0096] - a strain of Streptococcus thermophilus, wherein the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto (pscrB 829 );

[0097] - A strain of Streptococcus thermophilus, wherein the locus (pglcU) as the promoter region upstream of the glcU gene is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217-220 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, or the promoter comprises the sequence of SEQ ID NO: 221 (pglcU SNP_C ,pglcU IN_T ,pglcU IN_C ,pglcU IS );

[0098] - a strain of Streptococcus thermophilus, wherein the sequence of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, resulting in a frameshift, such that a truncated EIIABC is translated 蔗糖 (scrA 829 FS), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pscrB 829 );

[0099] - a strain of Streptococcus thermophilus, wherein the sequence of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, resulting in a frameshift, such that a truncated EIIABC is translated 蔗糖 (scrA 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pscrB 829 ), and the gene locus (pglcU) as the promoter region upstream of the glcU gene is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217-220 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pglcU SNP_C ,pglcU IN_T ,pglcU IN_C ,pglcU IS );

[0100] - a strain of Streptococcus thermophilus, wherein the sequence of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, resulting in a frameshift, such that a truncated EIIABC is translated 蔗糖 (scrA 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pscrB 829 ), and the gene locus (pglcU) as the promoter region upstream of the glcU gene is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217-220 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pglcU SNP_C ,pglcU IN_T ,pglcUIN_C ,pglcU IS ), and optionally wherein the glcK gene encoding glucokinase comprises a sequence as defined by SEQ ID NO: 25 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, wherein the amino acid at position 275 is lysine (glcK 378 ), and optionally further comprising generating an encoding of the EIIC Man A mutation of the open reading frame shift of the gene for the protein, resulting in a stop codon at position 180 of SEQ ID NO: 222, for example, by inserting a nucleotide C at any of positions 438, 439 or 440 of SEQ ID NO: 223 in the manM gene (manM 12997 ); and optionally further comprises the ccpA sequence of SEQ ID NO:71 or a ccpA variant sequence having at least about 90%, such as at least 95%, identity to SEQ ID NO:71.

[0101] - A strain of Streptococcus thermophilus comprising one or more of the following characteristics:

[0102] 1) The sequence of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, resulting in a frame shift, so that a truncated EIIABC is translated 蔗糖 (scrA 829 FS ),

[0103] 2) The sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pscrB 829 ),

[0104] 3) the locus (pglcU) as the promoter region upstream of the glcU gene is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217-220 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pglcU SNP_C ,pglcU IN_T ,pglcU IN_C ,pglcU IS ),

[0105] 4) The glcK gene encoding glucokinase comprises a sequence as defined in SEQ ID NO: 25 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, wherein the amino acid at position 275 is lysine (glcK 378 ),

[0106] 5) Generate the EIIC code Man A mutation of the open reading frame shift of the gene for the protein, resulting in a stop codon at position 180 of SEQ ID NO: 222, for example, by inserting a nucleotide C at any of positions 438, 439 or 440 of SEQ ID NO: 223 in the manM gene (manM 12997 );

[0107] 6) a ccpA sequence having the ccpA sequence of SEQ ID NO:71 or a ccpA variant sequence having at least about 90%, such as at least 95%, identity to SEQ ID NO:71.

[0108] In another aspect, the present invention relates to a method for selecting a lactose-positive sucrose-negative Streptococcus thermophilus strain, the method comprising the following steps:

[0109] 1) providing a strain of thermophilic Streptococcus, comprising

[0110] a) one or more mutations in one or more genes of a sucrose regulator; the mutation significantly reduces the ability of the strain to grow on sucrose and / or transport sucrose; and the mutation comprises at least

[0111] i) a mutation in one or more of the scrA gene and / or the scrB gene and / or the scrR gene, and

[0112] ii) optionally further mutations in the promoter region regulating the expression of the scrA, scrB and / or scrR genes, For example, mutations that protect strains from reversion to a sucrose-negative phenotype ;

[0113] 2) screening a strain of Streptococcus thermophilus from step 1) having a significantly reduced ability to grow on sucrose and to transport sucrose; and

[0114] 3) optionally before, simultaneously with or after any of steps 1 and 2, screening for S. thermophilus strains having restored or improved glucose consumption, optionally from strains having one or more further mutations affecting a glucose transporter, e.g., affecting glcU and / or its expression.

[0115] In a specific embodiment, a "variant sequence" of a strain as defined herein has at least 90% or at least 95% sequence identity with the specific SEQ ID NO mentioned or with the genomic sequence of the parent strain from which the variant sequence is obtained (including or excluding the specific mutations or insertions of the strain), in particular at least 90%, at least 91%, at least 95%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.92%, at least 99.94%, at least 99.96%, at least 99.98% or at least 99.99% identity with the specific SEQ ID NO mentioned or with the genomic sequence of the parent strain from which the variant is obtained. Identity is described by comparing the two sequences over their entire length (global alignment) and can be calculated using any program based on the Needleman-Wunsch algorithm.

[0116] In dairy applications, it is expected that the thermophilic Streptococcus strains of the present invention do not consume sucrose, i.e., have a sucrose negative phenotype. Therefore, as used herein, when referring to the phenotype of a strain within the scope of the present invention, "sucrose negative" means a strain that has very limited or no ability to grow on a medium containing sucrose as the sole carbohydrate source. This is measured by Test A described herein. In the context of the present invention, a thermophilic Streptococcus strain that has very limited or no ability to grow on a medium containing sucrose as the sole carbohydrate source refers to a strain that, when tested with Test A described herein, has a ratio of colonies (cfu) formed on sucrose (sucrose positive colonies) to colonies (cfu) formed on lactose of less than 10. -2 , for example, less than 10 -3 , 10 -4 , 10 -5 , 10 -6 or 10 -7 As used herein, a mutation that causes a strain to have "a significantly reduced ability to grow on and transport sucrose" is a mutation in the strain that induces or helps induce the strain to acquire a "sucrose negative phenotype."

[0117] If the ratio of colonies (cfu) formed by a strain of S. thermophilus on sucrose (sucrose positive colonies) to colonies (cfu) formed on lactose is close to 1 or greater than 10 when tested using Test A described herein, -2 When the thermophilic Streptococcus strain is considered to be "sucrose positive".

[0118] Alternatively, enzymatic approaches can be considered to obtain the ability of the strain to grow on sucrose. For example, by measuring the ability of resting cells of scrA mutants to transport sucrose; or measuring the ability of intracellular extracts of scrB mutants to hydrolyze phosphorylated sucrose (sucrose-6-phosphate).

[0119] The inventors of the present invention have shown that by mutating selected genes in the S. thermophilus sucrose regulon, optionally in combination with one or more mutations affecting a glucose transporter, such as glcU and / or its expression, the S. thermophilus strain will display a "sucrose negative phenotype" and optionally a "glucose positive phenotype" as defined above.

[0120] In some embodiments, the present invention relates to a lactose-positive, sucrose-negative Streptococcus thermophilus strain, which further carries mutations in genes selected from the group consisting of: 1) at least one gene encoding a mannose-glucose-specific PTS protein and a glcK gene, 2) at least one gene encoding a mannose-glucose-specific PTS protein and a ccpA gene, and 3) a gene encoding a mannose-glucose-specific PTS protein, a glcK gene, and a ccpA gene, as described in the examples of WO2019122365 and WO 2019197051 patents.

[0121] In some embodiments, the lactose-positive sucrose-negative thermophilic Streptococcus strain carries a mutation in a gene encoding a mannose-glucose-specific PTS protein and a glcK gene. In an embodiment, the gene encoding the mannose-glucose-specific PTS protein is selected from the group consisting of: manL gene, manM gene, manN gene and manO gene. In an embodiment, the gene encoding the mannose-glucose-specific PTS protein is selected from the group consisting of: manL gene, manM gene and manN gene. In an embodiment, the lactose-positive sucrose-negative thermophilic Streptococcus strain carries a mutation in a gene selected from the group consisting of manL gene, manM gene and manN gene. In an embodiment, the lactose-positive sucrose-negative thermophilic Streptococcus strain carries a mutation in 2 genes selected from the group consisting of manL gene, manM gene and manN gene. In an embodiment, the lactose-positive sucrose-negative thermophilic Streptococcus strain carries a mutation in manL gene, manM gene and manN gene. In some embodiments, the lactose-positive, sucrose-negative S. thermophilus strain carries a mutation in only one gene encoding a mannose-glucose-specific PTS protein, the manM gene, as described in the examples of WO 2019122365 and WO 2019197051.

[0122] In another aspect, the lactose-positive sucrose-negative Streptococcus thermophilus strain according to the present invention releases glucose during milk fermentation. As described herein, the lactose-positive sucrose-negative Streptococcus thermophilus strain of the present invention can be further characterized by its ability to release glucose when used for fermenting milk. In this article, when the strain of the present invention is used for fermenting milk, this ability is defined by the concentration of glucose released into the milk. In an embodiment, fermentation conditions are described in Example 6, and the concentration of glucose released is determined. Therefore, in some embodiments, the lactose-positive sucrose-negative Streptococcus thermophilus strain according to the present invention releases the glucose concentration measured as described in Example 6, and the glucose concentration is at least 50mM, such as at least 60, 70, 80 or 90mM, or compared with the glucose concentration released by the corresponding parent strain without the mutation described in the present invention, the glucose concentration released by the strain increases by at least 150% or at least 200%, such as 300%, 400%, 500%, when both strains are measured as described in Example 6. In some embodiments, the mutation affecting the glucose transporter affects glcU, such as a mutation in the promoter region of glcU, which provides for such increased release at a glucose concentration determined as described in Example 6 of at least 50 mM, such as at least 60, 70, 80, or 90 mM. In some embodiments, the mutation in the promoter region of glcU is as described herein.

[0123] In some embodiments, the present invention relates to a lactose-positive, sucrose-negative Streptococcus thermophilus strain, which carries mutations in 2 or 3 genes selected from the group consisting of: 1) a gene encoding a mannose-glucose-specific PTS protein and a glcK gene, 2) a gene encoding a mannose-glucose-specific PTS protein and a ccpA gene, and 3) a gene encoding a mannose-glucose-specific PTS protein, a glcK gene, and a ccpA gene (as described in the examples of WO 2019122365 and WO2019197051).

[0124] In some embodiments, the lactose-positive, sucrose-negative Streptococcus thermophilus strain of the present invention does not carry any mutation in a gene selected from the group consisting of a gene encoding a mannose-glucose-specific PTS protein, a glcK gene, and a ccpA gene.

[0125] For the avoidance of doubt, the species Streptococcus thermophilus is understood to be a strain of Streptococcus salivarius subsp. thermophilus.

[0126] The expression "lactose positive" means that the Streptococcus thermophilus strain is able to grow on lactose as the sole carbohydrate source, in particular on M17 medium supplemented with 3% lactose as described in Test A. In one embodiment, the "lactose positive" phenotype is determined by inoculating an overnight culture of the Streptococcus thermophilus strain to be tested into M17 broth containing 3% lactose at a rate of 1% and incubating at 37° C. for 20 hours or alternatively for 24 hours, and wherein a pH of 5.5 or less at the end of the incubation is indicative of a lactose positive phenotype.

[0127] The expression "galactose negative" means that the thermophilus strain cannot grow on galactose as the sole carbohydrate source, in particular on M17 medium supplemented with 2% lactose. In a specific embodiment, the "galactose negative" phenotype is determined by the following method: an overnight culture of the thermophilus strain to be tested is inoculated at a rate of 1% into M17 broth containing 2% galactose and incubated at 37°C for 20 hours, and wherein a pH of 6 or higher at the end of the incubation indicates a galactose negative phenotype.

[0128] The expression "derivative" (e.g., DSM28255 derivative) about the parent strain refers to a strain obtained from the original strain (e.g., from the DSM28255 strain) by replacing one of its genes (e.g., scrA, glcK, manM ...) with another allele of the same gene (especially an allele carrying some specific mutations), or by replacing a gene with a mutant gene. In an embodiment, a derivative is obtained by replacing a gene and its promoter of the original strain with another allele of the gene and its promoter. In an embodiment, a derivative is obtained by replacing all or part of the coding sequence of a gene of the original strain with another allele of the gene or part thereof.

[0129] For embodiments requiring any of glcK sequencing, transferring the glcK allele of a suitable strain into the genome of other S. thermophilus strains, screening and selecting for glucose excretion properties of S. thermophilus strains, identifying relevant mutations in the glcK gene, identifying and using further glcK mutations (e.g., a non-conservative amino acid difference G144S), identifying and using specific mutations in the glcK gene (e.g., a non-conservative amino acid difference E275K in the GlcK sequence), reference is made to the co-pending international patent application with publication number WO2019197051A1.

[0130] A derivative of the DSM28255 strain was designed, in which the glcK gene encodes a glucokinase in which the glutamic acid (E) at position 275 is replaced by the amino acid lysine (K). This derivative (DGCC12534) was deposited in the DSMZ on August 15, 2017 under the deposit number DSM32587. The sequence of its GlcK protein is defined in SEQ ID NO: 22.

[0131] Milk acidification performance

[0132] As described in Example 5, the acidification properties of thermophilic Streptococcus strains can be evaluated by recording the change in pH over time during milk fermentation. The pH was monitored for 24 hours using the CINAC system (Alliance Instruments, France; pH electrode was Mettler 405DPAS SC (Toledo, Spain)). The pH was measured and recorded every 5 or 25 minutes. The slope between pH 6.0 and pH 5.5 (UpH / min) was calculated [Slope pH6-5.5] using CINAC v2.07 software.

[0133] Therefore, the present invention relates to a sucrose negative strain of Streptococcus thermophilus. This phenotype of the Streptococcus thermophilus strain is achieved by one or more of the following mutations:

[0134] 1) one or more mutations in one or more genes of a sucrose regulator; the mutation significantly reduces the ability of the strain to grow on sucrose and / or transport sucrose; and the mutation is in one or more genes of a sucrose regulator, such as scrA and / or scrB genes and / or scrR genes, optionally further mutations in promoter regions that regulate expression of scrA, scrB and / or scrR genes; and

[0135] 2) optionally one or more further mutations affecting a glucose transporter, such as glcU and / or its expression, which mutations restore or improve glucose consumption in said strain.

[0136] Sucrose phosphoenolpyruvate-sugar phosphotransferase system (PTS) and sucrose regulon

[0137] The term "sucrose phosphoenolpyruvate-sugar phosphotransferase system (PTS)" refers to the sucrose phosphoenolpyruvate-sugar phosphotransferase system (PTS) in its usual sense used by bacteria for sugar uptake, where the energy source is from phosphoenolpyruvate (PEP). It consists of two nonspecific energy coupling components involved in all PTSs, namely enzyme I (EI) and heat stable protein (HPr), and a sucrose-specific multidomain permease EIIABC encoded by scrA.蔗糖 (the enzyme internalizes sucrose into sucrose-6-phosphate) composition.Three other genes related to the scrA (same locus) inheritance on the sucrose regulon; i.e. scrB (encoding sucrose-6-phosphate hydrolase), scrR (encoding GalR-LacI type transcriptional regulator) and scrK (encoding fructokinase). They are located on the operons of 2 bidirectional transcriptions (scrAK on one side, scrBR on the other side), and the center is the operon operator gene. Therefore, the term "sucrose regulon" as used herein refers to the scrA gene and the gene related to the scrA inheritance, i.e. scrB, scrR and scrK.

[0138] In some embodiments, the sucrose negative phenotype is conferred by a mutation in the scrA gene. The present inventors have used a single mutation (scrA) in three different genetic backgrounds. 829 ), which is a single base pair (bp) insertion at position 133 that causes a frameshift in the open reading frame (ORF), resulting in a 45-aa truncation of the 539-aa native protein. 蔗糖 Protein, which is actually a gene disruption, inactivates the truncated protein product. Therefore, it can be expected that most frameshifts (insertions or deletions (indels), except for 3bp and multiples of 3bp) in genes will have the same gene disruption effect, inactivating the protein product. SNPs (single nucleotide polymorphisms), especially those that produce stop codons or affect amino acids involved in protein catalytic sites, are expected to have similar effects that affect enzyme active sites and inhibit activity.

[0139] In some embodiments, the sucrose negative phenotype is conferred by a mutation in the scrB gene. In some embodiments, the sucrose negative phenotype is conferred by a mutation in the scrR gene. In some embodiments, one or more genes of the sucrose regulon are related to one or more mutations in one or more genes in genes scrA, scrB, and scrR, destroying the function of one or more genes in these genes.

[0140] Mutations in the promoter region that regulate expression of the scrA, scrB and / or scrR genes.

[0141] The pscrB region is the operator region that drives bidirectional transcriptional gene expression of the sucrose regulon. This promoter region drives transcription of scrA and scrK on one side and scrB and scrR on the other side. We have characterized a single mutation, termed "pscrB 829". This particular mutation was transferred into multiple strains with the same phenotypic outcome, promoting the strains to become sucrose negative. The mutation was a SNP located in a region that could be the scrB promoter region (in a region that could contain the -10 and -35 sequences of the promoter), or specifically in the scrAB promoter region, resulting in a T to G transition at position -38 of the scrB promoter.

[0142] In some embodiments, the sucrose negative phenotype is conferred by a mutation in the pscrB region.

[0143] Mutations affecting glucose transporters, such as glcU, and / or their expression.

[0144] The gene glcU encodes a non-PTS transporter of glucose, also called a "non-PTS glucose permease" or simply a "glucose transporter". It has its own promoter region (pglcU). Some mutations in pglcU allow the expression of proteins with scrA 829 and / or pscrB 829 The mutant strain recovers a growth rate similar to that of the parent strain on glucose. The inventors of the present invention have characterized 4 different mutations. One is SNP, 2 of which are insertions of different nucleotides at the same position, and the 3rd is the insertion of IS. All these mutations allow the growth on glucose to be fully restored. The inventors of the present invention have characterized mutations in 3 independent genetic backgrounds. All three strains have fully recovered the ability to grow on glucose. Therefore, this mutation of glucose transporter can restore or improve the glucose consumption of strains. As used herein, "recovery or improvement of glucose consumption" related to the mutation affecting glucose transporter by strain refers to the following strain: after this mutation, the strain recovers from, for example, a mutation in a sucrose regulon to a growth rate similar to or higher than that of a parent strain without such mutation in a sucrose regulon, or alternatively, the mutation of the glucose transporter affecting the strain only improves the ability of the strain to grow on glucose. In certain embodiments, this term "recovery or improvement of glucose consumption" refers to that such a mutation will confer a glucose-positive phenotype on the strain and is considered to be glucose-positive as defined herein.

[0145] The present inventors have carried out reverse genetics research to a pglcU mutant. The pglcU mutation in one of the mutants reverts to the original wild type (WT, parent) sequence. This reversal causes a slow-growing bacterial strain on glucose, proving that the pglcU mutation itself is the reason for the glucose-positive phenotype of the triple mutant (scrA, pscrB, pglcU). In addition, by genetic engineering, the pglcU mutation is introduced in the bacterial strain that grows slowly on sucrose negative and glucose to make the bacterial strain glucose-positive.

[0146] In a specific embodiment, the mutation affecting the glucose transporter is identified as pglcU IN_T A mutation in which insertion of a T nucleotide at the position corresponding to position -75 of the glcU promoter in the poly-T restored / improved glucose uptake and consumption.

[0147] In another specific embodiment, the mutation affecting the glucose transporter is identified as pglcU IN_C A mutation in which insertion of a C nucleotide at the position corresponding to position -75 of the glcU promoter in the poly-T restored / improved glucose uptake and consumption.

[0148] In another specific embodiment, the mutation affecting the glucose transporter is identified as pglcU SNP_C Mutations wherein a mutation of the T to C nucleotide at the position corresponding to position -81 of the glcU promoter in the poly-T restored / improved glucose uptake and consumption.

[0149] In another specific embodiment, the mutation affecting the glucose transporter is identified as pglcU IS A mutation wherein a sequence is inserted at a position between nucleotides 110 and 111 relative to the sequence of strain ST1-ABU4 identified herein or ST1 identified by SEQ ID NO: 216 of the glcU promoter, which insertion restores and / or improves glucose uptake and consumption.

[0150] Expected scrA 829 The mutation may have pleiotropic effects on the expression of other genes involved in carbohydrate utilization. This effect may occur at the transcriptional level (as suggested by the location of the mutation in pglcU and the common knowledge about the regulation of carbohydrate catabolism). 829 The mutation may downregulate glcU expression, and the mutation in pglcU may reverse this downregulation, possibly making the expression constitutive.

[0151] Therefore, in some embodiments, the lactose-positive sucrose-negative thermophilic Streptococcus strain according to the present invention is a glucose-positive or slow-growing strain on glucose. Therefore, glucose-positive strains can be propagated and produced using glucose instead of lactose.

[0152] As used herein, a "glucose positive" strain refers to a strain of S. thermophilus that is able to grow on glucose as a sole carbohydrate source, e.g., wherein the growth rate (μ 葡萄糖 ) (as described in Test B described herein corresponding to the maximum growth rate (μmax) of the same strain on lactose 乳糖) and μmax 乳糖 The ratio (μ 葡萄糖 / μmax 乳糖 Ratio) is equal to or higher than 0.25, for example higher than 0.3, such as 0.35, 0.4, 0.5, 0.6, 0.7, 0.8 or 0.9, indicating that the strain grows well on glucose. Alternatively, when μ is defined and measured according to Test B described herein, 葡萄糖 When the value is equal to or higher than 0.20, such as higher than 0.25, 0.3, 0.4, 0.5, 0.6, 0.7 or 0.8, the S. thermophilus strain is considered "glucose positive".

[0153] As used herein, a strain that "grows slowly on glucose" refers to a strain of Streptococcus thermophilus whose ability to grow on glucose as a sole carbohydrate source is not completely abolished but is significantly slower than that of a glucose-positive strain, e.g., wherein the growth rate (μ 葡萄糖 ) (as described in Test B described herein corresponding to the maximum growth rate (μmax) of the same strain on lactose 乳糖 ) and μmax 乳糖 The ratio (μ 葡萄糖 / μmax 乳糖 Ratio) is lower than 0.25, such as lower than 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18 or 0.17, but still higher than 0.05, indicating that the strain has the ability to grow slowly on glucose. Alternatively, the μ of the thermophilic Streptococcus strain defined and measured according to Test B described herein is 葡萄糖 When the value is above 0.01 but below 0.2, such as below 0.18, 0.17, 0.16, 0.15, 0.14, 0.13 or 0.12, the S. thermophilus strain is considered a "slow-growing on glucose" strain.

[0154] In dairy applications, the strains of the present invention are expected to not consume sucrose, or at least consume significantly lower amounts of sucrose than the parent strain.

[0155] In order to evaluate the sucrose negative phenotype, the strain of the present invention is tested for its ability to grow on a culture medium containing sucrose as the sole carbohydrate source. For example, by kinetic measurement of the OD of the strain culture in a synthetic medium (M17) containing sucrose (30 g / L) as the sole carbohydrate source at an appropriate temperature (37 ° C). Alternatively, enzymatic means can be considered. For example, by measuring the ability of resting cells of scrA mutants to transport sucrose; or measuring the ability of intracellular extracts of scrB mutants to hydrolyze phosphorylated sucrose (sucrose-6-phosphate). In a preferred assay, the sucrose negative phenotype of the strain of the present invention is tested as described in Test A defined herein.

[0156] In some embodiments, the strain according to the present invention comprises a mutation in a gene encoding a mannose-glucose specific PTS protein, which mutation alone or in combination with mutations in other genes can reduce or eliminate the input of glucose into the bacteria from the culture medium. In some embodiments, the mutated glcK gene encodes a glucokinase, and the glucokinase activity of the glucokinase in the strain is significantly reduced but not zero. In some embodiments, the lactose-positive, sucrose-negative thermophilic Streptococcus strain carries a mutation in a gene encoding a mannose-glucose specific PTS protein that reduces or eliminates the input of glucose into the bacteria from the culture medium and carries a mutation in the glcK gene encoding glucokinase, so that the glucokinase activity of the glucokinase in the strain is significantly reduced but not zero. In any of these embodiments, the gene encoding the mannose-glucose specific PTS protein can be selected from the group consisting of: a manL gene, a manM gene, and a manN gene.

[0157] In some embodiments, mutations in genes encoding mannose-glucose specific PTS proteins, alone or in combination with mutations in other genes, can reduce or eliminate glucose import from the culture medium into bacteria. In some embodiments, mutations in the ccpA gene produce lactose-positive, sucrose-negative S. thermophilus strains that exhibit a ratio of at least 4.10 for the β-galactosidase activity of the strain as determined by the β-galactosidase activity assay described herein relative to the glucokinase activity of the strain as determined by the glucokinase activity assay. -6 , at least 5.10 -6 , at least 6.10 -6 , at least 7.10 -6 or at least 8.10 -6In some embodiments, the lactose-positive, sucrose-negative S. thermophilus strain carries a mutation in a gene encoding a mannose-glucose-specific PTS protein that reduces or eliminates the import of glucose into the bacteria from the culture medium and a mutation in the ccpA gene such that the ratio of the β-galactosidase activity of the strain as determined by the β-galactosidase activity assay described herein relative to the glucokinase activity of the strain as determined by the glucokinase activity assay is at least 4.10. -6 .

[0158] In some embodiments, mutations in genes encoding mannose-glucose specific PTS proteins, alone or in combination with mutations in other genes, can reduce or eliminate glucose import from the culture medium into bacteria. In some embodiments, the mutated glcK gene encodes a glucokinase, the glucokinase activity of which in the strain is significantly reduced but not zero in the strain. In some embodiments, the lactose-positive, sucrose-negative thermophilic Streptococcus strain carrying a mutation in the glcK gene also carries a mutation in the ccpA gene.

[0159] The following sections describe mutations of the glcK gene, mutations of genes encoding mannose-glucose-specific PTS proteins (eg, mutations of the manL gene, the manM gene, and the manN gene), and mutations of the ccpA gene, respectively.

[0160] Although these mutations are disclosed herein individually (for clarity), any embodiment of one section may be combined with any embodiment of another section or with any embodiments of two other sections to design a lactose-positive, sucrose-negative S. thermophilus strain as defined herein.

[0161] For the avoidance of doubt, in some embodiments, the present invention relates to a lactose positive sucrose negative Streptococcus thermophilus strain as defined herein, wherein the strain carries:

[0162] 1) a mutation in at least one, in particular one, gene encoding a mannose-glucose-specific PTS protein and a mutation in its glcK gene; or

[0163] 2) a mutation in at least one, in particular one, gene encoding a mannose-glucose-specific PTS protein and a mutation in its ccpA gene; or

[0164] 3) a mutation in at least one, in particular one, gene encoding a mannose-glucose-specific PTS protein, as well as a mutation in its glcK gene and a mutation in its ccpA gene.

[0165] Mutations in the glcK gene

[0166] This section describes mutations in the glcK gene, which can be used in combination with mutations in one or more genes of the sucrose regulon, mutations affecting glucose transporters, and mutations in genes encoding, for example, mannose-glucose-specific PTS proteins, and / or in combination with mutations in a gene encoding a mannose-glucose-specific PTS protein as defined herein and a mutation in the ccpA gene as defined herein, in the context of the lactose-positive, sucrose-negative S. thermophilus strains of the invention.

[0167] In some embodiments, the mutant glcK gene of the strain of the present invention encodes a glucokinase, wherein the glucokinase activity of the glucokinase in the strain is significantly reduced but not zero. In fact, the inventors have demonstrated some mutant alleles of the glcK gene encoding glucokinase (GlcK), wherein the glucokinase activity of the glucokinase is significantly reduced but not zero when the mutant glcK gene is present in a lactose-positive thermophilic Streptococcus strain.

[0168] The expression "glcK gene encoding glucokinase" refers to any DNA sequence encoding glucokinase in a thermophilic Streptococcus strain that catalyzes the conversion of glucose and ATP to glucose-6-phosphate (G6P) and ADP. Non-limiting examples of thermophilic Streptococcus glucokinase sequences are disclosed in SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20.

[0169] In some embodiments of the present invention, the glucokinase activity in the thermophilic Streptococcus strain is significantly reduced but not zero due to the glcK gene mutation of the strain. In other words, the allele of the glcK gene carried by the strain causes the glucokinase activity in the strain to be significantly reduced but not zero.

[0170] The expression "the glucokinase activity in the strain is significantly reduced but not zero" means that the glucokinase activity of the strain meets the following two conditions:

[0171] - the glucokinase activity in the strain is significantly reduced, especially compared to the glucokinase activity in a strain carrying a non-mutated glcK gene; and

[0172] - is not zero, ie the activity is detectable by a glucokinase activity assay as defined herein.

[0173] According to an embodiment of the present invention, the feature "glucokinase activity in the strain is significantly reduced but not zero" can be determined by methods well known in the art. Therefore, methods for measuring glucokinase activity in thermophilic Streptococcus strains are known and include enzyme assays using commercially available reactants. Reference is made herein to paragraph 2.4 of Pool et al. (2006. Metabolic Engineering [Metabolic Engineering] 8 (5); 456-464) (incorporated herein by reference). In a specific embodiment, the glucokinase activity in the thermophilic Streptococcus strain of the present invention is determined by glucokinase activity assay [i.e., the thermophilic Streptococcus strain of the present invention is used to perform glucokinase activity assay].

[0174] Glucokinase activity assay:

[0175] A fresh overnight culture of the thermophilic Streptococcus strain to be assayed in M17 containing 30 g / L lactose was obtained and used to inoculate 10 ml of fresh M17 containing 30 g / L lactose at 1% (vol / vol). At an optical density at 600 nm (OD600) of 0.8 + / - 0.2, cells were collected by centrifugation (6000 g, 10 min, 4°C), washed in 5 ml of cold GLCK buffer (5 mM MgCl2, 10 mM K2HPO4 / KH2PO4 [pH 7.2]), and resuspended in 500 μl of cold GLCK buffer. The EDTA-free protease inhibitor "cOmplete" was added as described by the supplier. TM” (Roche, supplier reference number 04693132001) was added to the GLCK buffer. The cells were disrupted by adding 100 mg of glass beads (150-212 μm, Sigma G1145) to 200 μl of resuspended cells and shaking at 30 cycles / s for 6 min in a MM200 oscillating mill (Retsch, Haan, Germany). Cell debris and glass beads were removed by centrifugation (14000 g, 15 min, 4°C) and the supernatant was transferred to 1.5 mL of clean 4% paraformaldehyde kept on ice. Centrifuge tubes. Total protein content was determined by using the Fluka Protein Quantification Kit-Rapid (Ref. 51254). Glucokinase activity in cell extracts was determined spectrophotometrically by glucose-6-phosphate dehydrogenase (G-6PDH, EC 1.1.1.49):NADPH coupled assay (Porter et al., 1982), essentially as described by Pool et al. (2006). Each sample (5, 10 and 20 μL) was added to an assay buffer (10 mM K2HPO4 / KH2PO4[pH7.2], 5mMMgCl2, 1mMATP, 20mM glucose, 1mMNADP, 1UG-6PDH), and the mixture was placed at 30°C for 5min. The optical density at 340nm was measured for 5 minutes using SynergyHT multi-detection microplate reader (Bio-TEK). One unit of glucokinase corresponds to the amount of enzyme that catalyzes the phosphorylation of 1 micromole of D-glucose to D-glucose 6-phosphate per minute under the assay conditions. The glucokinase activity was calculated as follows:

[0176] Glucokinase activity (U / g total protein extract) = dOD x V / [dt xl xε x Qprot], where:

[0177] -dOD is the change in optical density (OD) at 340nm

[0178] - V is the reaction volume (250 μL in this article)

[0179] dt = measurement time (in minutes)

[0180] l = optical path length (0.73 cm in this article)

[0181] ε = molar attenuation coefficient of NADPH; H + (6220cm2 / μmol in this article)

[0182] Qprot = amount of protein in the cuvette (in grams (g))

[0183] Each sample was measured three times, and the glucokinase specific activity values ​​given herein according to the glucokinase activity assay are the average of three independent experiments.

[0184] In a first specific embodiment of the feature "the glucokinase activity in the strain is significantly reduced but not zero", the glucokinase activity in the thermophilic Streptococcus strain of the present invention is 200 to 1500 U / g total protein extract as determined by a glucokinase activity assay. In a specific embodiment, the glucokinase activity in the thermophilic Streptococcus strain of the present invention is 300 to 1200 U / g total protein extract as determined by a glucokinase activity assay. In a specific embodiment, the glucokinase activity in the thermophilic Streptococcus strain of the present invention is 400 to 1000 U / g total protein extract as determined by a glucokinase activity assay. In a specific embodiment, the glucokinase activity in the thermophilic Streptococcus strain of the present invention is between a minimum value selected from the group consisting of 200, 300 and 400 U / g total protein extract and a maximum value selected from the group consisting of 1000, 1200 and 1500 U / g total protein extract as determined by a glucokinase activity assay. It is worth noting that, as mentioned for the glucokinase activity assay, the glucokinase activity values ​​disclosed herein are the average of three independent experiments (triplicate).

[0185] In a second specific embodiment of the feature "the glucokinase activity in the strain is significantly reduced but not zero", the glucokinase activity in the thermophilic Streptococcus strain of the present invention is 5% to 60% of the glucokinase activity of the DGCC7710 strain deposited in the DSMZ on January 14, 2014 under the deposit number DSM28255. "Glucokinase activity of the DSM28255 strain" refers to the activity of the DSM28255 strain glucokinase (i.e., having SEQ ID NO: 2) determined in the DSM28255 strain by the glucokinase activity assay [i.e., the glucokinase activity assay is performed using the DSM28255 strain]. The percentage value is calculated based on the glucokinase activity in the strain of the present invention and the glucokinase activity of the DSM28255 strain, both glucokinase activities being determined by the glucokinase activity assay. In a specific embodiment, the glucokinase activity in the thermophilic Streptococcus strain of the present invention is 10% to 50% of the glucokinase activity of the DSM28255 strain. In a specific embodiment, the glucokinase activity in the thermophilic streptococcus strain of the present invention is 15% to 40% of the glucokinase activity of the strain DSM28255. In a specific embodiment, the glucokinase activity in the thermophilic streptococcus strain of the present invention is between the minimum percentage of the group consisting of 5%, 10% and 15% of the glucokinase activity of the DSM28255 strain and the maximum percentage of the group consisting of 40%, 50% and 60% of the glucokinase activity of the DSM28255 strain. In a specific embodiment, and regardless of the percentage range, the activity of the glucokinase activity is determined by the glucokinase activity assay described herein. It is worth noting that the percentage values ​​disclosed herein are calculated based on the glucokinase activity values, which are the average values ​​of three independent experiments (triplicates) determined by the glucokinase activity assay.

[0186] In the first and second specific embodiments, the following strains can be used as controls in the glucokinase activity assay:

[0187] - As positive control (ie a Streptococcus thermophilus strain representing a strain carrying a non-mutated glcK gene): strain DGCC7710 deposited at the DSMZ on January 14, 2014 under the accession number DSM28255.

[0188] The feature "glucokinase activity in the strain is significantly reduced but not zero" can also be characterized by the maximum forward velocity of glucokinase (referred to herein as Vmax and defined as the rate at which glucose+ATP is converted to G6P+ADP) or by the reciprocal of the affinity of glucokinase for its substrate, i.e., one or both of glucose and ATP (referred to as Km). In some embodiments, the feature of the strain of the present invention "glucokinase activity in the strain is significantly reduced but not zero" is also characterized by the maximum forward velocity (Vmax) of its glucokinase in the strain.

[0189] Therefore, in combination with the first or second specific embodiment of the feature "glucokinase activity in the strain is significantly reduced but not zero" defined herein, the maximum forward velocity (Vmax) of glucokinase in the lactose-positive, sucrose-negative thermophilic Streptococcus strain of the present invention is significantly reduced but not zero. The feature "glucokinase Vmax in the strain is significantly reduced but not zero" can be defined by one or two of the following parameters:

[0190] - Vmax of 200 to 1500 U / g total protein extract as determined by the glucokinase Vmax assay.

[0191] - Vmax is 5% to 60% of the Vmax of the glucokinase of the DSM 28255 strain deposited at the DSMZ on January 14, 2014 under the accession number DSM 28255, when both are determined by the glucokinase Vmax assay.

[0192] In a specific embodiment, the mutated glcK gene of the lactose-positive, sucrose-negative thermophilic Streptococcus strain of the invention encodes a glucokinase, wherein the glucokinase activity in the strain is significantly reduced but not zero (as defined herein), and wherein the maximum forward velocity (Vmax) of its glucokinase in the strain is significantly reduced but not zero and is defined by one or both of the following parameters:

[0193] - Vmax of 200 to 1500 U / g total protein extract as determined by the glucokinase Vmax assay.

[0194] - Vmax is 5% to 60% of the Vmax of the glucokinase of the DSM 28255 strain deposited at the DSMZ on January 14, 2014 under the accession number DSM 28255, when both are determined by the glucokinase Vmax assay.

[0195] The maximum forward velocity (Vmax) of glucokinase in the thermophilus strain of the present invention is determined by the glucokinase Vmax assay [ie, the glucokinase Vmax assay is performed using the thermophilus strain of the present invention].

[0196] Glucokinase Vmax determination ：

[0197] The maximum forward velocity (Vmax) was determined by using a variety of glucose concentrations (0, 5, 10, 15, 20 mM) for the crude extracts prepared as described in the glucokinase activity assay. Each sample was measured three times, and the Vmax values ​​given according to the glucokinase Vmax assay were the mean values ​​of three independent experiments. The linear regression showing the variation of the reciprocal of the specific velocity with the reciprocal of the glucose concentration gave the reciprocal of the maximum forward velocity at the intersection with the Y axis of the graph.

[0198] In a specific embodiment of the maximum forward velocity of glucokinase in the thermophilic Streptococcus strain of the present invention, Vmax is 200U / g to 1500U / g total protein extract as determined by the glucokinase Vmax assay. In a specific embodiment, Vmax is 300U / g to 1200U / g total protein extract as determined by the glucokinase Vmax assay. In a specific embodiment, Vmax is 400 to 1000U / g total protein extract. In a specific embodiment, the Vmax of glucokinase in the thermophilic Streptococcus strain of the present invention is between a minimum value selected from the group consisting of 200, 300 and 400U / g total protein extract and a maximum value selected from the group consisting of 1000, 1200 and 1500U / g total protein extract as determined by the glucokinase Vmax assay.

[0199] In a specific embodiment of the maximum forward velocity of the glucokinase in the thermophilic Streptococcus strain of the present invention, Vmax is 5% to 60% of the Vmax of the glucokinase of the DSM28255 strain. "Vmax of the glucokinase of the DSM28255 strain" refers to the Vmax of the glucokinase of the DSM28255 strain (i.e., having SEQ ID NO: 2) determined in the DSM28255 strain by the glucokinase Vmax assay [i.e., the glucokinase Vmax assay is performed using the DSM28255 strain]. The percentage value is calculated based on the Vmax of the glucokinase in the strain of the present invention and the Vmax of the DSM28255 strain, both of which are determined by the glucokinase Vmax assay. In a specific embodiment, the glucokinase Vmax in the thermophilic Streptococcus strain of the present invention is 10% to 50% of the Vmax of the glucokinase of the DSM28255 strain when both are determined by the glucokinase Vmax assay. In a specific embodiment, the glucokinase Vmax in the thermophilic Streptococcus strain of the present invention is 15% to 40% of the Vmax of the glucokinase of the DSM 28255 strain. In a specific embodiment, the Vmax of the glucokinase in the thermophilic Streptococcus strain of the present invention is between a minimum percentage selected from the group consisting of 5%, 10%, and 15% of the Vmax of the glucokinase activity of the DSM 28255 strain and a maximum percentage selected from the group consisting of 40%, 50%, and 60% of the Vmax of the glucokinase activity of the DSM 28255 strain.

[0200] The lactose-positive, sucrose-negative Streptococcus thermophilus strain of the present invention may carry a mutation in the glcK gene encoding glucokinase, the glucokinase activity of the glucokinase in the strain being significantly reduced but not zero as defined herein, and optionally wherein the maximum forward velocity of the glucokinase in the strain is significantly reduced but not zero as defined herein.

[0201] The "mutation in the glcK gene" in the present invention refers to any nucleotide change in the glcK gene, wherein the change at the nucleotide level significantly reduces the glucokinase activity in the strain carrying the mutated glcK gene (as the only glcK gene) as defined herein, but is not zero, and optionally significantly reduces the maximum forward velocity of glucokinase in the strain as defined herein, but is not zero. In a specific embodiment, the "mutation in the glcK gene" in the present invention refers to any nucleotide change in the open reading frame of the glcK gene, wherein the change at the nucleotide level significantly reduces the glucokinase activity in the strain carrying the mutated glcK gene (as the only glcK gene) as defined herein, but is not zero, and optionally significantly reduces the maximum forward velocity of glucokinase in the strain as defined herein, but is not zero.

[0202] Thus, although two thermophilic Streptococcus strains may differ in the sequence of their respective glcK genes, this does not necessarily mean that one of the two glcK genes is mutated in the sense of the present invention. In fact, the following are not considered mutations in the present invention:

[0203] - changes at the nucleotide level that do not cause any changes at the protein level (silent changes) and do not affect the translation of glcK RNA; and

[0204] - changes at the nucleotide level that do cause changes at the protein level, but wherein this change does not affect the glucokinase activity of the resulting GlcK protein as defined herein and optionally the maximum forward velocity of the resulting GlcK protein. In fact, such changes can be observed at the level of the glcK gene of the thermophilic Streptococcus of the present invention without affecting the scope of protection.

[0205] Non-limiting examples of glcK genes that are not considered to be mutated in the sense of the present invention are:

[0206] - a polynucleotide encoding a glucokinase as defined in SEQ ID NO: 2, in particular a polynucleotide as defined in SEQ ID NO: 1; this GlcK type is one of the strains DGCC7710 deposited at the DSMZ on January 14, 2014 under the deposit number DSM28255;

[0207] - a polynucleotide encoding a glucokinase as defined in SEQ ID NO: 4, in particular a polynucleotide as defined in SEQ ID NO: 3;

[0208] - a polynucleotide encoding a glucokinase as defined in SEQ ID NO: 6, in particular a polynucleotide as defined in SEQ ID NO: 5;

[0209] - a polynucleotide encoding a glucokinase as defined in SEQ ID NO: 8, in particular a polynucleotide as defined in SEQ ID NO: 7;

[0210] - a polynucleotide encoding a glucokinase as defined in SEQ ID NO: 10, in particular a polynucleotide as defined in SEQ ID NO: 9;

[0211] - a polynucleotide encoding a glucokinase as defined in SEQ ID NO: 12, in particular a polynucleotide as defined in SEQ ID NO: 11;

[0212] - a polynucleotide encoding a glucokinase as defined in SEQ ID NO: 14, in particular a polynucleotide as defined in SEQ ID NO: 13;

[0213] - a polynucleotide encoding a glucokinase as defined in SEQ ID NO: 16, in particular a polynucleotide as defined in SEQ ID NO: 15;

[0214] - a polynucleotide encoding a glucokinase as defined in SEQ ID NO: 18, in particular a polynucleotide as defined in SEQ ID NO: 17;

[0215] - a polynucleotide encoding a glucokinase as defined in SEQ ID NO: 20, in particular a polynucleotide as defined in SEQ ID NO: 19.

[0216] In addition, some nucleotide mutations within the glcK gene are considered unsuitable for the purposes of the present invention because these mutations would result in a glucokinase having an activity of zero or below the minimum value defined herein as determined by a glucokinase activity assay. In some embodiments, the thermophilic Streptococcus of the present invention does not carry a mutation selected from the group consisting of mutations causing knockouts of the glcK gene and large deletions within the glcK gene.

[0217] In some embodiments, the lactose-positive, sucrose-negative thermophilic Streptococcus strain of the present invention carries a mutation in the open reading frame of the glcK gene, such that an amino acid in the GlcK protein is substituted, the glucokinase activity of the GlcK protein in the strain carrying the mutated glcK gene is significantly reduced but not zero (as defined herein), and optionally wherein the maximum forward velocity of glucokinase in the strain is significantly reduced but not zero as defined herein. In a specific embodiment, the lactose-positive, sucrose-negative thermophilic Streptococcus strain of the present invention carries a mutation in the glcK gene, such that an amino acid in the GlcK protein is substituted, the glucokinase activity of the GlcK protein in the strain carrying the mutated glcK gene is significantly reduced but not zero (as defined herein), and optionally wherein the maximum forward velocity of glucokinase in the strain is significantly reduced but not zero as defined herein. In a specific embodiment, the thermophilic Streptococcus strain of the invention carries a mutation in the glcK gene such that the length of the GlcK protein is 322 amino acids, and wherein the glucokinase activity in the strain is significantly reduced but not zero as defined herein, and optionally wherein the maximum forward velocity of glucokinase in the strain is significantly reduced but not zero as defined herein.

[0218] As described above, some DNA modifications can be observed at the level of the glcK gene of the thermophilic Streptococcus of the present invention, which modifications do not affect the glucokinase activity of the strain. Based on the glucokinase activity assay defined herein and the control strain defined herein, a person skilled in the art should know how to identify 1) a glcK gene encoding a glucokinase, the glucokinase activity of the glucokinase in the strain carrying this glcK gene is significantly reduced but not zero (as defined herein), and optionally wherein the maximum forward velocity of the glucokinase in the strain carrying this mutated glcK gene is significantly reduced but not zero (as defined herein); 2) a glcK gene with the following modification, which modification has no effect on the glucokinase activity in the strain carrying this modification, or 3) a glcK gene encoding a glucokinase, the glucokinase activity of the glucokinase in the strain carrying this glcK gene is zero (as defined herein).

[0219] The inventors of the present invention have identified two positions within glucokinase whose amino acid properties have been shown to affect the activity of glucokinase such that the glucokinase activity is significantly reduced but not zero as defined herein, and affect the Vmax of glucokinase such that the Vmax is significantly reduced but not zero as defined herein: positions 144 and 275 of glucokinase (i.e., codons 144 and 275 of the glcK gene). It is noteworthy that based on the glucokinase activity assay and glucokinase Vmax assay defined herein and control strains, a person skilled in the art will know how to identify other positions and appropriate amino acids within glucokinase to obtain a significantly reduced but not zero (as defined herein) glucokinase activity and optionally a significantly reduced but not zero maximum forward velocity, and thus the corresponding glcK gene.

[0220] In some embodiments, the amino acid at position 275 of glucokinase (encoded by the glcK gene of the thermophilic Streptococcus strain of the present invention) is not glutamic acid (i.e., is any amino acid other than glutamic acid); therefore, in some embodiments, the codon 275 of the glcK gene carried by the thermophilic Streptococcus strain of the present invention is neither GAA nor GAG. In specific embodiments, the amino acid at position 275 of glucokinase is not an acidic amino acid (i.e., is any amino acid other than an acidic amino acid); therefore, in some embodiments, the codon 275 of the glcK gene carried by the thermophilic Streptococcus strain of the present invention is a codon encoding a non-acidic amino acid. In specific embodiments, the amino acid at position 275 of glucokinase is selected from the group consisting of lysine and any conservative amino acids thereof; therefore, in some embodiments, the codon 275 of the glcK gene carried by the thermophilic Streptococcus strain of the present invention is a codon encoding an amino acid selected from the group consisting of lysine and any conservative amino acids thereof. In a specific embodiment, the amino acid at position 275 of glucokinase is lysine; therefore, in some embodiments, the 275th codon of the glcK gene carried by the thermophilic Streptococcus strain of the present invention is AAA or AAG. In a specific embodiment, nucleotides 823-825 of the glcK gene carried by the thermophilic Streptococcus strain of the present invention are AAA or AAG.

[0221] In a specific embodiment, the sequence of the GlcK protein of the lactose-positive, sucrose-negative thermophilus Streptococcus strain of the invention is selected from the group consisting of:

[0222] a) a sequence as defined in SEQ ID NO: 25, wherein the amino acid at position 275 is any amino acid except glutamic acid, in particular any amino acid except acidic amino acids, in particular lysine; and

[0223] b) a GlcK variant sequence having at least 90% similarity or identity to SEQ ID NO: 25, wherein the amino acid corresponding to position 275 of SEQ ID NO: 25 (or the amino acid at position 275 of glucokinase) is any amino acid except glutamic acid, in particular any amino acid except acidic amino acids, in particular lysine. In a specific embodiment, the length of the GlcK variant sequence is 322 amino acids.

[0224] In another embodiment, the amino acid at position 144 of glucokinase (encoded by the glcK gene of the thermophilic Streptococcus strain of the present invention) is not glycine (i.e., is any amino acid other than glycine); therefore, in some embodiments, the 144th codon of the glcK gene carried by the thermophilic Streptococcus strain of the present invention is not GGT, GGC, GGA or GGG. In a specific embodiment, the amino acid at position 144 of glucokinase is not an aliphatic amino acid (i.e., is any amino acid other than an aliphatic amino acid). In a specific embodiment, the amino acid at position 144 of glucokinase is selected from the group consisting of serine and any conservative amino acids thereof; therefore, in some embodiments, the 144th codon of the glcK gene carried by the thermophilic Streptococcus strain of the present invention is a codon encoding an amino acid selected from the group consisting of serine and any conservative amino acids thereof. In a specific embodiment, the amino acid at position 144 of glucokinase is serine; therefore, in some embodiments, the 144th codon of the glcK gene carried by the thermophilic Streptococcus strain of the present invention is AGT, AGC, TCT, TCC, TCA or TCG. In a specific embodiment, nucleotides 430-432 of the glcK gene carried by the S. thermophilus strain of the present invention are AGT, AGC, TCT, TCC, TCA or TCG.

[0225] In a specific embodiment, the sequence of the GlcK protein of the lactose-positive, sucrose-negative thermophilus Streptococcus strain of the invention is selected from the group consisting of:

[0226] a) a sequence as defined in SEQ ID NO: 46, wherein the amino acid at position 144 is any amino acid except glycine, in particular any amino acid except aliphatic amino acids, in particular serine; and

[0227] b) a GlcK variant sequence having at least 90% similarity or identity to SEQ ID NO: 46, wherein the amino acid corresponding to position 144 of SEQ ID NO: 46 (or the amino acid at position 144 of glucokinase) is any amino acid except glycine, in particular any amino acid except aliphatic amino acids, in particular serine. In a specific embodiment, the length of the GlcK variant sequence is 322 amino acids.

[0228] In order to define a GlcK variant having at least 90% similarity to SEQ ID NO: 25, similarity or identity is herein calculated over the entire length of the two sequences after optimal alignment [i.e., the number of similar or identical amino acid residues in one or more aligned portions of the sequences]; position 275 as defined in SEQ ID NO: 25 is not taken into account when calculating the similarity or identity. In a specific embodiment, the GlcK variant sequence has at least 91, 92, 93, 94, 95, 96, 97, 98 or 99% similarity or identity to SEQ ID NO: 25, wherein the amino acid corresponding to position 275 of SEQ ID NO: 25 (or the amino acid at position 275 of glucokinase) is any amino acid except glutamic acid, in particular any amino acid except acidic amino acids, in particular lysine. In some embodiments, the GlcK variant sequence has at least 95% similarity or identity to SEQ ID NO: 25, wherein the amino acid corresponding to position 275 of SEQ ID NO: 25 (or the amino acid at position 275 of glucokinase) is any amino acid except glutamic acid, in particular any amino acid except acidic amino acids, in particular lysine. In some embodiments, the GlcK variant sequence has at least 97% similarity or identity to SEQ ID NO: 25, wherein the amino acid corresponding to position 275 of SEQ ID NO: 25 (or the amino acid at position 275 of glucokinase) is any amino acid except glutamic acid, in particular any amino acid except acidic amino acids, in particular lysine.

[0229] In a specific embodiment, the GlcK variant sequence differs from SEQ ID NO: 25 by 1 to 30 amino acid substitutions, wherein the amino acid at position 275 of the GlcK variant is any amino acid except glutamic acid, in particular any amino acid except acidic amino acids, in particular lysine (position 275 is not taken into account when calculating the number of one or more substitutions). In a specific embodiment, the GlcK variant sequence differs from SEQ ID NO: 25 by 1 to 20 amino acid substitutions, wherein the amino acid at position 275 of the GlcK variant is any amino acid except glutamic acid, in particular any amino acid except acidic amino acids, in particular lysine. In a specific embodiment, the GlcK variant sequence differs from SEQ ID NO: 25 by 1 to 15 amino acid substitutions, wherein the amino acid at position 275 of the GlcK variant is any amino acid except glutamic acid, in particular any amino acid except acidic amino acids, in particular lysine. In a specific embodiment, the GlcK variant sequence differs from SEQ ID NO: 25 by 1 to 10 amino acid substitutions, wherein the amino acid at position 275 of the GlcK variant is any amino acid except glutamic acid, in particular any amino acid except acidic amino acids, in particular lysine. In a specific embodiment, the GlcK variant sequence differs from SEQ ID NO: 25 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid substitutions, wherein the amino acid at position 275 of the GlcK variant is any amino acid except glutamic acid, in particular any amino acid except acidic amino acids, in particular lysine.

[0230] In order to define a GlcK variant having at least 90% similarity to SEQ ID NO: 46, similarity or identity is herein calculated over the entire length of the two sequences after optimal alignment [i.e., the number of similar or identical amino acid residues in one or more aligned portions of the sequences]; position 144 as defined in SEQ ID NO: 46 is not taken into account when calculating the similarity or identity. In a specific embodiment, the GlcK variant sequence has at least 91, 92, 93, 94, 95, 96, 97, 98 or 99% similarity or identity to SEQ ID NO: 46, wherein the amino acid corresponding to position 144 of SEQ ID NO: 46 (or the amino acid at position 144 of glucokinase) is any amino acid except glycine, in particular any amino acid except aliphatic amino acids, in particular serine. In some embodiments, the GlcK variant sequence has at least 95% similarity or identity to SEQ ID NO: 46, wherein the amino acid corresponding to position 144 of SEQ ID NO: 46 (or the amino acid at position 144 of glucokinase) is any amino acid except glycine, in particular any amino acid except aliphatic amino acids, in particular serine. In some embodiments, the GlcK variant sequence has at least 97% similarity or identity to SEQ ID NO: 46, wherein the amino acid corresponding to position 144 of SEQ ID NO: 46 (or the amino acid at position 144 of glucokinase) is any amino acid except glycine, in particular any amino acid except aliphatic amino acids, in particular serine.

[0231] In a specific embodiment, the GlcK variant sequence differs from SEQ ID NO: 46 by 1 to 30 amino acid substitutions, wherein the amino acid at position 144 of the GlcK variant is any amino acid except glycine, in particular any amino acid except aliphatic amino acids, in particular serine (position 144 is not taken into account when calculating the number of one or more substitutions). In a specific embodiment, the GlcK variant sequence differs from SEQ ID NO: 46 by 1 to 20 amino acid substitutions, wherein the amino acid at position 144 of the GlcK variant is any amino acid except glycine, in particular any amino acid except aliphatic amino acids, in particular serine. In a specific embodiment, the GlcK variant sequence differs from SEQ ID NO: 46 by 1 to 15 amino acid substitutions, wherein the amino acid at position 144 of the GlcK variant is any amino acid except glycine, in particular any amino acid except aliphatic amino acids, in particular serine. In a specific embodiment, the GlcK variant sequence differs from SEQ ID NO: 46 by 1 to 10 amino acid substitutions, wherein the amino acid at position 144 of the GlcK variant is any amino acid except glycine, in particular any amino acid except aliphatic amino acids, in particular serine. In a specific embodiment, the GlcK variant sequence differs from SEQ ID NO: 46 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid substitutions, wherein the amino acid at position 144 of the GlcK variant is any amino acid except glycine, in particular any amino acid except aliphatic amino acids, in particular serine.

[0232] In some embodiments, the sequence of the GlcK protein of the lactose-positive, sucrose-negative thermophilic Streptococcus strain of the present invention is selected from the group consisting of SEQ ID NO: 25, 26, 27, 28, 29, 30, 31, 32, 33 and 34, wherein the amino acid at position 275 of the variant is any amino acid except glutamic acid, in particular any amino acid except acidic amino acids, in particular lysine; therefore, in some embodiments, the glcK gene carried by the thermophilic Streptococcus strain of the present invention encodes a GlcK protein, whose sequence is selected from the group consisting of SEQ ID NO: 25, 26, 27, 28, 29, 30, 31, 32, 33 and 34, wherein the amino acid at position 275 of glucokinase is not glutamic acid, in particular is not an acidic amino acid, in particular is lysine.

[0233] In a specific embodiment, as SEQ ID NO:25 or any GlcK variant sequence as defined herein having at least 90% similarity or identity to SEQ ID NO:25 (particularly SEQ ID NO:26, 27, 28, 29, 30, 31, 32, 33 or 34), the amino acid at position 275 of glucokinase corresponding to SEQ ID NO:25 (or the amino acid at position 275 of glucokinase) is not glutamic acid; therefore, in some embodiments, the codon 275 of the glcK gene carried by the thermophilus Streptococcus strain of the present invention is neither GAA nor GAG; therefore, in some embodiments, the glcK gene carried by the thermophilus Streptococcus strain of the present invention encodes a GlcK protein, the sequence of which is selected from the group consisting of SEQ ID NO:25 and any GlcK variant sequence as defined herein having at least 90% similarity or identity to SEQ ID NO:25 (particularly SEQ ID NO:26, 27, 28, 29, 30, 31, 32, 33 or 34), wherein the amino acid at position 275 of glucokinase corresponding to SEQ ID NO:25 (or the amino acid at position 275 of glucokinase) is not glutamic acid; NO:25 The amino acid at position 275 (or the amino acid at position 275 of glucokinase) is not glutamic acid.

[0234] In a specific embodiment, as SEQ ID NO:25 or any GlcK variant sequence as defined herein having at least 90% similarity or identity to SEQ ID NO:25 (particularly SEQ ID NO:26, 27, 28, 29, 30, 31, 32, 33 or 34), the amino acid corresponding to position 275 of SEQ ID NO:25 (or the amino acid at position 275 of glucokinase) is not an acidic amino acid; therefore, in some embodiments, the 275th codon of the glcK gene carried by the thermophilus Streptococcus strain of the present invention is a codon that does not encode an acidic amino acid; therefore, in some embodiments, the glcK gene carried by the thermophilus Streptococcus strain of the present invention encodes a GlcK protein, whose sequence is selected from the group consisting of SEQ ID NO:25 and any GlcK variant sequence as defined herein having at least 90% similarity or identity to SEQ ID NO:25 (particularly SEQ ID NO:26, 27, 28, 29, 30, 31, 32, 33 or 34), wherein the amino acid corresponding to position 275 of SEQ ID NO:25 (or the amino acid at position 275 of glucokinase) is not an acidic amino acid; NO:25 The amino acid at position 275 (or the amino acid at position 275 of glucokinase) is not an acidic amino acid.

[0235] In a specific embodiment, as SEQ ID NO:25 or any GlcK variant sequence as defined herein having at least 90% similarity or identity to SEQ ID NO:25 (particularly SEQ ID NO:26, 27, 28, 29, 30, 31, 32, 33 or 34), the amino acid corresponding to position 275 of SEQ ID NO:25 in glucokinase (or the amino acid at position 275 of glucokinase) is selected from the group consisting of lysine and any conservative amino acids thereof; therefore, in some embodiments, the 275th codon of the glcK gene carried by the thermophilus Streptococcus strain of the present invention is a codon encoding an amino acid selected from the group consisting of lysine and any conservative amino acids thereof; therefore, in some embodiments, the glcK gene carried by the thermophilus Streptococcus strain of the present invention encodes a GlcK protein, whose sequence is selected from SEQ ID NO:25 and any GlcK variant sequence as defined herein having at least 90% similarity or identity to SEQ ID NO:25 (particularly SEQ ID NO: NO:26, 27, 28, 29, 30, 31, 32, 33 or 34), wherein the amino acid corresponding to position 275 of SEQ ID NO:25 (or the amino acid at position 275 of glucokinase) is lysine and any conservative amino acid thereof.

[0236] In a specific embodiment, as SEQ ID NO:25 or any GlcK variant sequence as defined herein having at least 90% similarity or identity to SEQ ID NO:25 (particularly SEQ ID NO:26, 27, 28, 29, 30, 31, 32, 33 or 34), the amino acid corresponding to position 275 of SEQ ID NO:25 in glucokinase (or the amino acid at position 275 of glucokinase) is lysine; therefore, in some embodiments, the 275th codon of the glcK gene carried by the thermophilic Streptococcus strain of the present invention is a codon encoding lysine, in particular AAA or AAG, respectively; therefore, in a specific embodiment, the sequence of the GlcK protein of the lactose-positive, sucrose-negative thermophilic Streptococcus strain of the present invention is selected from the group consisting of SEQ ID NO:22, 35, 36, 37, 38, 39, 40, 41, 42 and 43; therefore, in some embodiments, the glcK gene carried by the thermophilic Streptococcus strain of the present invention encodes a GlcK protein, the sequence of which is selected from the group consisting of SEQ ID NO: NO:22, 35, 36, 37, 38, 39, 40, 41, 42 and 43; in a specific embodiment, the glcK gene carried by the thermophilic Streptococcus strain of the present invention is defined in SEQ ID NO:21.

[0237] In another embodiment, the sequence of the GlcK protein of the lactose-positive, sucrose-negative thermophilic Streptococcus strain of the present invention is selected from the group consisting of SEQ ID NO:46, 47, 48, 49, 50, 51, 52, 53, 54 and 55, wherein the amino acid at position 144 of the variant is any amino acid except glycine, in particular any amino acid except aliphatic amino acids, in particular serine; therefore, in some embodiments, the glcK gene carried by the thermophilic Streptococcus strain of the present invention encodes a GlcK protein, whose sequence is selected from the group consisting of SEQ ID NO:46, 47, 48, 49, 50, 51, 52, 53, 54 and 55, wherein the amino acid at position 144 of glucokinase is not glycine, in particular is not an aliphatic amino acid, in particular is serine.

[0238] In a specific embodiment, as SEQ ID NO:46 or any GlcK variant sequence defined herein having at least 90% similarity or identity to SEQ ID NO:46 (particularly SEQ ID NO:47, 48, 49, 50, 51, 52, 53, 54 or 55), the amino acid corresponding to position 144 of SEQ ID NO:46 in glucokinase (or the amino acid at position 144 of glucokinase) is not glycine; therefore, in some embodiments, the 144th codon of the glcK gene carried by the thermophilus Streptococcus strain of the present invention is not GGT, GGC, GGA or GGG; therefore, in some embodiments, the glcK gene carried by the thermophilus Streptococcus strain of the present invention encodes a GlcK protein, whose sequence is selected from SEQ ID NO:46 and any GlcK variant sequence defined herein having at least 90% similarity or identity to SEQ ID NO:46 (particularly SEQ ID NO:47, 48, 49, 50, 51, 52, 53, 54 or 55). NO:47, 48, 49, 50, 51, 52, 53, 54 or 55), wherein the amino acid at position 144 of glucokinase corresponding to SEQ ID NO:46 (or the amino acid at position 144 of glucokinase) is not glycine.

[0239] In a specific embodiment, as SEQ ID NO:46 or any GlcK variant sequence as defined herein having at least 90% similarity or identity to SEQ ID NO:46 (particularly SEQ ID NO:47, 48, 49, 50, 51, 52, 53, 54 or 55), the amino acid corresponding to position 144 of SEQ ID NO:46 (or the amino acid at position 144 of glucokinase) is not an aliphatic amino acid; therefore, in some embodiments, the 144th codon of the glcK gene carried by the thermophilus Streptococcus strain of the present invention is a codon that does not encode an aliphatic amino acid; therefore, in some embodiments, the glcK gene carried by the thermophilus Streptococcus strain of the present invention encodes a GlcK protein, whose sequence is selected from the group consisting of SEQ ID NO:46 and any GlcK variant sequence as defined herein having at least 90% similarity or identity to SEQ ID NO:46 (particularly SEQ ID NO:47, 48, 49, 50, 51, 52, 53, 54 or 55), wherein the amino acid corresponding to position 144 of SEQ ID NO:46 in the glucose kinase (or the amino acid at position 144 of the glucose kinase) is not an aliphatic amino acid; The amino acid at position 144 of NO:46 (or the amino acid at position 144 of glucokinase) is not an aliphatic amino acid.

[0240] In a specific embodiment, as SEQ ID NO:46 or any GlcK variant sequence as defined herein having at least 90% similarity or identity to SEQ ID NO:46 (particularly SEQ ID NO:47, 48, 49, 50, 51, 52, 53, 54 or 55), the amino acid corresponding to position 144 of SEQ ID NO:46 in glucokinase (or the amino acid at position 144 of glucokinase) is selected from the group consisting of serine and any conservative amino acids thereof; therefore, in some embodiments, the 144th codon of the glcK gene carried by the thermophilus Streptococcus strain of the present invention is a codon encoding an amino acid selected from the group consisting of serine and any conservative amino acids thereof; therefore, in some embodiments, the glcK gene carried by the thermophilus Streptococcus strain of the present invention encodes a GlcK protein, whose sequence is selected from SEQ ID NO:46 and any GlcK variant sequence as defined herein having at least 90% similarity or identity to SEQ ID NO:46 (particularly SEQ ID NO:47, 48, 49, 50, 51, 52, 53, 54 or 55). NO:47, 48, 49, 50, 51, 52, 53, 54 or 55), wherein the amino acid at position 144 of glucokinase corresponding to SEQ ID NO:46 (or the amino acid at position 144 of glucokinase) is serine and any conserved amino acid thereof.

[0241] In a specific embodiment, as SEQ ID NO:46 or any GlcK variant sequence as defined herein having at least 90% similarity or identity to SEQ ID NO:46 (particularly SEQ ID NO:47, 48, 49, 50, 51, 52, 53, 54 or 55), the amino acid corresponding to position 144 of SEQ ID NO:46 in glucokinase (or the amino acid at position 144 of glucokinase) is serine; therefore, in some embodiments, the 144th codon of the glcK gene carried by the thermophilic Streptococcus strain of the present invention is a codon encoding serine, particularly AAA or AAG; therefore, in a specific embodiment, the sequence of the GlcK protein of the lactose-positive, sucrose-negative thermophilic Streptococcus strain of the present invention is selected from the group consisting of SEQ ID NO:45, 56, 57, 58, 59, 60, 61, 62, 63 and 64; therefore, in some embodiments, the glcK gene carried by the thermophilic Streptococcus strain of the present invention encodes a GlcK protein, the sequence of which is selected from the group consisting of SEQ ID NO: NO:45, 56, 57, 58, 59, 60, 61, 62, 63 and 64; in a specific embodiment, the glcK gene carried by the thermophilic Streptococcus strain of the present invention is defined in SEQ ID NO:44.

[0242] When defining the sequence of the GlcK protein of the lactose-positive, sucrose-negative thermophilic Streptococcus strain of the present invention, according to the teachings of the present application, the glucokinase activity in the strain expressing this GlcK protein is significantly reduced but not zero as defined herein, and optionally the Vmax of glucokinase in this strain is significantly reduced but not zero as defined herein.

[0243] Mutations in genes encoding mannose-glucose specific PTS proteins, particularly manL, manM and manN genes Caused by mutation.

[0244] This section describes mutations in genes encoding mannose-glucose-specific PTS proteins, in particular mutations in the manL, manM and manN genes, which can be used in combination with mutations in the glcK gene as defined herein, or in combination with mutations in the ccpA gene as defined herein, or in combination with both mutations in the glcK gene and mutations in the ccpA gene as defined herein, in the context of the lactose-positive, sucrose-negative Streptococcus thermophilus strains of the present invention.

[0245] In the lactose-positive, sucrose-negative thermophilic Streptococcus strain, any mutation in the gene encoding the mannose-glucose-specific PTS protein is suitable and can be combined with a mutation in the glcK gene as defined herein, or with a mutation in the ccpA gene as defined herein, or with both a mutation in the glcK gene as defined herein and a mutation in the ccpA gene.

[0246] The inventors have demonstrated that mutations in genes encoding mannose-glucose-specific PTS proteins that reduce or eliminate glucose import from the culture medium in lactose-positive sucrose-negative Streptococcus thermophilus strains, particularly mutations in mutated manL genes, mutated manM genes or mutated manN genes, are particularly advantageous in the present invention. In some embodiments, mutations in genes encoding mannose-glucose-specific PTS proteins result in reduced or eliminated glucose import activity of the protein encoded by this gene. In some embodiments, the mutated gene is the manL gene, and mutations in the manL gene result in reduced or eliminated EIIAB Man In some embodiments, the mutated gene is the manM gene, and the mutation of the manM gene causes a decrease or elimination of EIIC Man In some embodiments, the mutated gene is the manN gene, and the mutation of the manN gene causes reduction or elimination of EIID. Man Glucose import activity of proteins.

[0247] In some embodiments, the mutation of the gene encoding the mannose-glucose-specific PTS protein, in particular the mutation of the manL gene, the manM gene or the manN gene, is a mutation that causes gene knockout (ie, complete disruption).

[0248] In some embodiments, the mutation of the gene encoding the mannose-glucose-specific PTS protein, particularly the manL gene, manM gene or manN gene, is a mutation of the promoter of the gene, particularly a mutation of the promoter of the gene that reduces or inhibits gene transcription.

[0249] In some embodiments, a mutation in a gene encoding a mannose-glucose specific PTS protein, in particular a mutation in a manL gene, a manM gene or a manN gene, is a mutation introduced into the coding sequence of the gene, in particular causing a reduction or elimination of the glucose import activity of the protein encoded by the mutated gene, in particular causing EIIAB Man Protein, EIIC Man Protein or EIID Man Mutations that reduce or eliminate the glucose import activity of a protein.

[0250] In some embodiments, the mutation of the gene encoding the mannose-glucose specific PTS protein, in particular the mutation of the manL gene, the manM gene or the manN gene, is a mutation that produces a truncated protein, in particular a truncated EIIAB, in the coding sequence of the gene. Man Protein, truncated EIICMan Protein or truncated EIID Man Proteins, particularly truncated proteins with reduced or abolished glucose import activity (such as truncated EIIAB Man Protein, truncated EIIC Man Protein or truncated EIID Man Protein). Regardless of the position of the truncation, the mutation introduced into the gene is a nucleotide substitution that produces a stop codon, or a deletion, insertion, or deletion / insertion that causes a frameshift of the open reading frame and produces an early stop codon. In some embodiments, the mutation introduced into the gene is a nucleotide substitution that produces a stop codon. In some embodiments, the mutation introduced into the gene is a deletion, insertion, or deletion / insertion that causes a frameshift of the open reading frame and produces an early stop codon.

[0251] Although two thermophilic Streptococcus strains may differ in the sequence of their respective manL, manM or manN genes, this does not necessarily mean that one of these genes is mutated in the sense of the present invention. In fact, the following are not considered mutations of the manL, manM or manN genes in the present invention:

[0252] - changes at the nucleotide level that do not cause any changes at the protein level (silent changes) and do not affect the translation of manL, manM or manN RNA; and

[0253] - In a thermophilic Streptococcus strain with a lactose-positive, sucrose-negative phenotype, a change at the nucleotide level that does cause an alteration at the protein level, but when combined with a mutated glcK gene as defined herein, or when combined with a mutation in the ccpA gene as defined herein, or when combined with both a mutated glcK gene and a mutated ccpA gene, wherein this alteration cannot be achieved.

[0254] The manL, manM and manN genes (encoding EIIAB, respectively) are not considered to be mutated in the sense of the present invention. Man Protein, EIIC Man Proteins and EIID Man Non-limiting examples of proteins are:

[0255] - encoding EIIAB as defined in SEQ ID NO: 78 Man A polynucleotide of a protein, in particular a polynucleotide as defined in SEQ ID NO: 77; this EIIAB Man The type is one of the DSM28255 strains;

[0256] - encoding EIIAB as defined in SEQ ID NO: 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108 and 110 Man Polynucleotides of proteins, in particular polynucleotides as defined in SEQ ID NO: 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107 and 109. EIIAB as defined in SEQ ID NO: 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108 and 110 Man The sequence of the protein is 98.4% to 99.6% identical to SEQ ID NO: 78;

[0257] - encoding EIIC as defined in SEQ ID NO: 130 Man A polynucleotide of a protein, in particular a polynucleotide as defined in SEQ ID NO: 129; Man The type is one of DSM28255;

[0258] - encoding an EIIC as defined in SEQ ID NO: 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154 and 156 Man Polynucleotides of proteins, in particular polynucleotides as defined in SEQ ID NO: 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153 and 155. EIIC as defined in SEQ ID NO: 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154 and 156 Man The sequence of the protein is 98.5% to 99.6% identical to SEQ ID NO: 130;

[0259] -encodes an EIID as defined in SEQ ID NO: 167 Man A polynucleotide of a protein, in particular a polynucleotide as defined in SEQ ID NO: 166; Man The type is one of the DSM28255 strains;

[0260] -encoding an EIID as defined in SEQ ID NO: 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203 and 205 Man Polynucleotides of proteins, in particular polynucleotides as defined in SEQ ID NO: 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202 and 204. EIID as defined in SEQ ID NO: 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203 and 205. Man The sequence of the protein is 97.3% to 99.6% identical to SEQ ID NO:167.

[0261] The present inventors have identified at least one mutation in the manL gene which, when inserted into the manL gene of a starting lactose-positive, sucrose-negative S. thermophilus strain [a mutation in the glcK gene, the ccpA gene or both the glcK and ccpA genes as defined herein], is capable of producing the phenotype of interest in the present invention.

[0262] In some embodiments, mutations in the manL gene cause EIIAB Man The protein is truncated at position 305. In some embodiments, the mutation in the manL gene is a substitution of nucleotide T for nucleotide G at position 916 (resulting in a stop codon at position 306). Man The protein is referred to herein as IIAB Man 305 .

[0263] In some embodiments, the EIIAB truncated at position 305 Man The sequence of the protein is selected from the group consisting of:

[0264] a) a sequence as defined in SEQ ID NO: 112; and

[0265] b) EIIAB having at least about 90%, such as at least 95% similarity or identity to SEQ ID NO: 112 Man Variant sequences, in particular sequences of 305 amino acids in length.

[0266] To define an EIIAB having at least about 90%, such as at least 95%, similarity or identity to SEQ ID NO: 112 Man Variants, herein similarity or identity is calculated over the entire length of the two sequences after optimal alignment [i.e., the number of similar or identical amino acid residues in one or more aligned portions of the sequences]. In a specific embodiment, EIIAB Man The variant sequence has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% similarity or identity to SEQ ID NO:112.

[0267] In certain embodiments, EIIAB Man The variant sequence differs from SEQ ID NO: 112 by 1 to 30 amino acid substitutions. In a specific embodiment, EIIAB Man The variant sequence differs from SEQ ID NO: 112 by 1 to 20 amino acid substitutions. In a specific embodiment, EIIAB Man The variant sequence differs from SEQ ID NO: 112 by 1 to 15 amino acid substitutions. In a specific embodiment, EIIAB Man The variant sequence differs from SEQ ID NO: 112 by 1 to 10 amino acid substitutions. In a specific embodiment, EIIAB Man The variant sequence differs from SEQ ID NO: 112 by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In some embodiments, the EIIAB of the lactose-positive, sucrose-negative thermophilic Streptococcus strain of the invention Man The sequence of the protein is selected from the group consisting of SEQ ID NOs: 112-128.

[0268] In some embodiments, the thermophilic Streptococcus strains of the present invention carry the manL gene encoding EIIAB Man A protein whose sequence is selected from SEQ ID NO: 112 and any EIIAB having at least about 90%, such as at least 95% similarity or identity to SEQ ID NO: 112 as defined herein Man The variant sequences (particularly SEQ ID NOs: 113 to 128) are grouped together. In some embodiments, the manL gene carried by the thermophilic Streptococcus strain of the present invention is as defined in SEQ ID NO:111.

[0269] The inventors have identified at least one mutation in the manM gene which, when inserted into the manM gene of a starting lactose-positive, sucrose-negative S. thermophilus strain [a mutation in the glcK gene, the ccpA gene, or both the glcK and ccpA genes as defined herein], confers certain interesting properties to these strains.

[0270] In some embodiments, mutations in the manM gene cause EIIC Man The protein is truncated at position 208. In some embodiments, the mutation in the manM gene is a substitution of nucleotide T for nucleotide G at position 625 (resulting in a stop codon at position 209). Man The protein is referred to herein as EIIC Man208 .

[0271] In some embodiments, the EIIC is truncated at position 208. Man The sequence of the protein is selected from the group consisting of:

[0272] a) a sequence as defined in SEQ ID NO: 158; and

[0273] b) an EIIC having at least 90% similarity or identity to SEQ ID NO: 158 Man Variant sequences, in particular sequences of 208 amino acids in length.

[0274] To define an EIIC having at least 90% similarity to SEQ ID NO: 158 Man Variants, herein similarity or identity is calculated over the entire length of the two sequences after optimal alignment [i.e., the number of similar or identical amino acid residues in one or more aligned portions of the sequences]. In a specific embodiment, EIIC Man The variant sequence has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% similarity or identity to SEQ ID NO:158.

[0275] In certain embodiments, EIIC Man The variant sequence differs from SEQ ID NO: 158 by 1 to 30 amino acid substitutions. In a specific embodiment, EIIC Man The variant sequence differs from SEQ ID NO: 158 by 1 to 20 amino acid substitutions. In a specific embodiment, EIIC Man The variant sequence differs from SEQ ID NO: 158 by 1 to 15 amino acid substitutions. In a specific embodiment, EIIC ManThe variant sequence differs from SEQ ID NO: 158 by 1 to 10 amino acid substitutions. In certain embodiments, EIIC Man The variant sequence differs from SEQ ID NO: 158 by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In some embodiments, the EIIC of the lactose-positive, sucrose-negative thermophilic Streptococcus strain of the invention is Man The sequence of the protein is selected from the group consisting of SEQ ID NOs: 158-165.

[0276] In some embodiments, the manM gene carried by the thermophilic Streptococcus strain of the present invention encodes EIIC Man A protein whose sequence is selected from SEQ ID NO: 158 and any EIIC having at least about 90%, such as at least 95%, similarity or identity to SEQ ID NO: 158 as defined herein Man The variant sequences (particularly SEQ ID NOs: 159 to 165) are grouped together. In some embodiments, the manM gene carried by the thermophilic Streptococcus strain of the present invention is as defined in SEQ ID NO:157.

[0277] In some embodiments, the mutation in the manM gene is an insertion of a nucleotide C at any of positions 438, 439, or 440 of SEQ ID NO: 129 to produce a frameshift in manM, such that in SEQ ID NO: 130 (manM 12997 ) at position 180. The resulting thermophilic Streptococcus IIC was truncated at position 179. Man Protein is referred to herein as IIC Man179 .

[0278] In some embodiments, the IIC truncated at position 179 Man The sequence of the protein is selected from the group consisting of:

[0279] a) a sequence as defined in SEQ ID NO: 223; and

[0280] b) an IIC having at least 90% similarity or identity to SEQ ID NO: 223 Man Variant sequences, in particular sequences of 179 amino acids in length.

[0281] To define an IIC having at least 90% similarity to SEQ ID NO: 223 ManVariants, herein similarity or identity is calculated over the entire length of the two sequences after optimal alignment [i.e., the number of similar or identical amino acid residues in one or more aligned portions of the sequences]. In a specific embodiment, IIC Man The variant sequence has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% similarity or identity to SEQ ID NO:223.

[0282] In certain embodiments, IIC Man The variant sequence differs from SEQ ID NO: 223 by 1 to 30 amino acid substitutions. In a specific embodiment, IIC Man The variant sequence differs from SEQ ID NO: 223 by 1 to 20 amino acid substitutions. In a specific embodiment, IIC Man The variant sequence differs from SEQ ID NO: 223 by 1 to 15 amino acid substitutions. In a specific embodiment, IIC Man The variant sequence differs from SEQ ID NO: 223 by 1 to 10 amino acid substitutions. In a specific embodiment, IIC Man The variant sequence differs from SEQ ID NO: 223 by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions. In some embodiments, the IIC of the lactose-positive thermophilic Streptococcus strain used in accordance with the present invention is Man The sequence of the protein is selected from the group consisting of SEQ ID NO:223.

[0283] In some embodiments, the thermophilic Streptococcus strain used according to the present invention carries the manM gene encoding IIC Man A protein whose sequence is selected from SEQ ID NO: 223 and any IIC having at least about 90%, such as at least 95%, similarity or identity to SEQ ID NO: 223 as defined herein Man A group of variant sequences.

[0284] The inventors have identified at least one mutation in the manN gene which, when inserted into the manN gene of a starting lactose-positive, sucrose-negative S. thermophilus strain [a mutation in the glcK gene, the ccpA gene, or both the glcK and ccpA genes as defined herein], confers certain interesting properties to these S. thermophilus strains.

[0285] In some embodiments, mutations in the manN gene cause EIID ManThe protein is truncated at position 28. In some embodiments, the mutation in the manN gene is an insertion of a nucleotide A into a stretch of 5 nucleotides A at positions 37-41 (resulting in a fragment with 6 nucleotides A, causing EIID Man The protein has an open reading frame shift and truncation at position 28). This Streptococcus thermophilus EIID is truncated at position 28 Man Protein is referred to herein as IID Man 28 .

[0286] In some embodiments, the EIID is truncated at position 28 Man The sequence of the protein is selected from the group consisting of:

[0287] a) a sequence as defined in SEQ ID NO: 207; and

[0288] b) an EIID having at least 90% similarity or identity to SEQ ID NO: 207 Man Variant sequences, in particular sequences of 28 amino acids in length.

[0289] To define an EIID having at least 90% similarity to SEQ ID NO: 207 Man Variants, herein similarity or identity is calculated over the entire length of the two sequences after optimal alignment [i.e., the number of similar or identical amino acid residues in one or more aligned portions of the sequences]. In a specific embodiment, EIIC Man The variant sequence has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% similarity or identity to SEQ ID NO:207.

[0290] In certain embodiments, the EIID Man The variant sequence differs from SEQ ID NO: 207 by 1 to 10 amino acid substitutions. In a specific embodiment, EIID Man The variant sequence differs from SEQ ID NO: 207 by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In some embodiments, the EIID of the lactose-positive, sucrose-negative thermophilic Streptococcus strain of the invention is Man The sequence of the protein is selected from the group consisting of SEQ ID NOs: 207-211.

[0291] In some embodiments, the manN gene carried by the thermophilic Streptococcus strain of the present invention encodes EIID ManA protein whose sequence is selected from SEQ ID NO: 207 and any EIID having at least 90% similarity or identity to SEQ ID NO: 207 as defined herein Man The variant sequences (particularly SEQ ID NOs: 208 to 211) are described in some embodiments. In some embodiments, the manN gene carried by the thermophilic Streptococcus strain of the present invention is as defined in SEQ ID NO: 206.

[0292] Regardless of the mutation of the glcK gene as defined herein and the mutation of the ccpA gene as defined herein (present alone or in combination in the lactose-positive, sucrose-negative, thermophilic Streptococcus strain of the present invention), at least one gene encoding a mannose-glucose-specific PTS protein is mutated as defined herein. Regardless of which embodiment, the present invention comprises a lactose-positive, sucrose-negative, thermophilic Streptococcus strain carrying a mutation in one, two or three genes selected from the group consisting of the manL gene, the manM gene and the manN gene. In some embodiments, the lactose-positive, sucrose-negative thermophilic Streptococcus strain of the present invention carries a mutation in manL. In some embodiments, the lactose-positive, sucrose-negative thermophilic Streptococcus strain of the present invention carries a mutation in manM. In some embodiments, the lactose-positive, sucrose-negative thermophilic Streptococcus strain of the present invention carries a mutation in manN. In some embodiments, the lactose-positive, sucrose-negative thermophilic Streptococcus strain of the present invention carries a mutation in manL and a mutation in manM. In some embodiments, the lactose-positive, sucrose-negative thermophilic Streptococcus strain of the present invention carries a mutation in manL and a mutation in manN. In some embodiments, the lactose-positive, sucrose-negative S. thermophilus strains of the invention carry a mutation in manM and a mutation in manN. In some embodiments, the lactose-positive, sucrose-negative S. thermophilus strains of the invention carry a mutation in manL, a mutation in manM and a mutation in manN.

[0293] Any method may be used to identify mutations in the gene encoding the mannose-glucose specific PTS protein, in particular the manL gene, the manM gene or the manN gene, in a lactose-positive, sucrose-negative S. thermophilus strain suitable for the present invention.

[0294] For example, in order to identify suitable mutations in the manL gene, manM gene or manN gene, those skilled in the art may operate by the following method:

[0295] a) Provide DSM32587 strain (whose glcK gene is mutated)

[0296] b) mutagenizing the manL, manM or manN gene of the strain in a) by random mutagenesis or targeted mutagenesis to obtain a manL, manM or manN gene having a sequence different from that of the manL, manM or manN gene of DSM32587, thereby obtaining a DSM32587 strain with a man mutation;

[0297] c) Determine whether pH is achieved 终止 Phenotype.

[0298] Alternatively, those skilled in the art may perform the operation by the following method:

[0299] a) providing a strain, such as DSM28255 strain, wherein the ccpA gene of the strain has been replaced with a mutated ccpA gene (ccpA) as defined in SEQ ID NO: 71; Δ1A114-120 ) was replaced, and the strain is referred to herein as DGCC7710-ccpA Δ1A114-120 strain;

[0300] b) mutagenizing the manL, manM or manN gene of the strain in a) by random mutagenesis or targeted mutagenesis to obtain a sequence similar to that of DGCC7710-ccpA Δ1A114-120 The manL, manM or manN gene of the strain has a different sequence, thereby obtaining DGCC7710-ccpA with man mutation Δ1A114-120 strain;

[0301] c) Determine whether pH is achieved 终止 Phenotype.

[0302] After identification, the mutated manL, manM or manN gene according to the present invention can be introduced to replace manL, manM or manN of the lactose-positive sucrose-negative Streptococcus thermophilus strain to obtain the lactose-positive sucrose-negative Streptococcus thermophilus strain of the present invention.

[0303] Mutation of ccpA gene

[0304] This section describes mutations in the ccpA gene, which can be used in combination with mutations in the gene encoding a mannose-glucose-specific PTS protein as defined herein, or in combination with mutations in the gene encoding a mannose-glucose-specific PTS protein as defined herein and a mutation in the glcK gene as defined herein, in the context of the lactose-positive, sucrose-negative Streptococcus thermophilus strains of the present invention.

[0305] In a lactose-positive, sucrose-negative thermophilic Streptococcus strain, any mutation in the ccpA gene is suitable as long as, when combined with a mutated gene encoding a mannose-glucose-specific PTS protein as defined herein, or when combined with both a mutated glcK gene as defined herein and a mutated gene encoding a mannose-glucose-specific PTS protein, a pH of 1.1 to 1.1 is obtained when the strain is used to ferment milk. 终止 Phenotype.

[0306] To determine the ratio of β-galactosidase activity relative to glucokinase activity in the strains of the invention, the β-galactosidase activity assay and the glucokinase activity assay as described herein were used:

[0307] β-Galactosidase activity assay:

[0308] During milk fermentation, lactose contained in milk (as the main carbohydrate source in milk) is input into the thermophilic Streptococcus strain. The intracellular lactose is then cleaved into glucose and galactose by β-galactosidase (so that 1 mol of lactose produces 1 mol of glucose and 1 mol of galactose), which is also called β-galactosidase activity.

[0309] Determination of the β-galactosidase activity in the thermophilus strain of the present invention is performed by a β-galactosidase activity assay [ie, β-galactosidase activity assay is performed using the thermophilus strain of the present invention].

[0310] A fresh overnight culture of the thermophilic Streptococcus strain to be assayed in M17 containing 30 g / L lactose was obtained and used to inoculate 10 ml of fresh M17 containing 30 g / L lactose at 1% (vol / vol). After 3 hours of growth on M17 + 30 g / l lactose at 42°C, cells were collected by centrifugation (6000 g, 10 min, 4°C), washed in 1.5 ml of cold lysis buffer (KPO4 0.1 M), and resuspended in 300 μl of cold lysis buffer. The EDTA-free protease inhibitor "cOmplete" was added as described by the supplier. TM” (Roche, supplier reference 04693132001) was added to the lysis buffer. The cells were disrupted by adding 100 mg of glass beads (150-212 μm, Sigma G1145) to 250 μl of resuspended cells and shaking at 30 cycles / s for 6 min in a MM200 oscillating mill (Retsch, Haan, Germany). Cell debris and glass beads were removed by centrifugation (14000 g, 15 min, 4°C) and the supernatant was transferred to a 1.5 mL clean centrifuge tube kept on ice. The supernatant was quantified by using the FLUKA Protein Quantification Kit (FLUKA Protein Quantification Kit). Kit-Rapid, reference number 51254) to determine the total protein content. The β-galactosidase activity in the cell extract was determined by monitoring the hydrolysis of O-nitro-phenol-β-galactoside (ONPG) to galactose and O-nitro-phenol (ONP) by spectrophotometry. 20 μL of bacterial extract was mixed with 135 μL of reaction buffer (NaPO40.1 M+KCl0.01 M+MgSO40.001 M+ONPG 3mM+β-mercaptoethanol 60mM, pH=6). The production of ONP gives a yellow color in the tube. When this color appears, add 250 μL of stop buffer (Na2CO31 The reaction was blocked by adding 4 μM of β-galactosidase. The optical density at 420 nm was measured using a SynergyHT multi-detection microplate reader (BIO-TEK). One unit of β-galactosidase corresponds to the amount of enzyme that catalyzes the production of 1 micromole of ONP per minute under the assay conditions. The activity of β-galactosidase was calculated as follows:

[0311] β-Galactosidase activity (U / g total protein extract) = dOD x V / [dt xl xε x Qprot], where:

[0312] -dOD is the change in optical density (OD) at 420 nm between the blank sample and the test sample

[0313] - V is the volume of the reaction where the optical density was measured (250 μL in this case)

[0314] -dt = the duration in minutes from the addition of 20 μL of bacterial extract to the addition of 250 μL of stop buffer

[0315] -l = optical path length (0.73 cm in this article)

[0316] -ε = molar attenuation coefficient of ONP (4500 cm in this paper) 2 / μmol)

[0317] -Qprot = amount of protein in the cuvette (in grams (g))

[0318] Each sample was measured at least three times, and the β-galactosidase specific activity values ​​given herein according to the β-galactosidase activity assay are the average of three independent experiments.

[0319] Glucokinase activity assay:

[0320] In order to determine the ratio, the glucokinase activity in the thermophilus strain of the present invention is determined, and the following determination is performed using the thermophilus strain of the present invention.

[0321] A fresh overnight culture of the thermophilic Streptococcus strain to be assayed in M17 containing 30 g / L lactose was obtained and used to inoculate 10 ml of fresh M17 containing 30 g / L lactose at 1% (vol / vol). After growing for 3 hours on M17+30 g / L lactose at 42°C, cells were collected by centrifugation (6000 g, 10 min, 4°C), washed in 1.5 ml of cold GLCK buffer (5 mM MgCl2, 10 mM K2HPO4 / KH2PO4 [pH 7.2]), and resuspended in 300 μl of cold GLCK buffer. The EDTA-free protease inhibitor "cOmplete" was added as described by the supplier. TM” (Roche, supplier reference number 04693132001) was added to the GLCK buffer. The cells were disrupted by adding 100 mg of glass beads (150-212 μm, Sigma G1145) to 250 μl of resuspended cells and shaking at 30 cycles / s for 6 min in a MM200 oscillating mill (Retsch, Haan, Germany). Cell debris and glass beads were removed by centrifugation (14000 g, 15 min, 4°C) and the supernatant was transferred to 1.5 mL of clean 4% paraformaldehyde kept on ice. Centrifuge tubes. Total protein content was determined by using the Fluka Protein Quantification Kit-Rapid (Ref. 51254). Glucokinase activity in cell extracts was determined spectrophotometrically by glucose-6-phosphate dehydrogenase (G-6PDH, EC 1.1.1.49):NADPH coupled assay (Porter et al., 1982), essentially as described by Pool et al. (2006). Each sample (5, 10 and 20 μL) was added to an assay buffer (10 mM K2HPO4 / KH2PO4[pH7.2], 5mMMgCl2, 1mMATP, 20mM glucose, 1mMNADP, 1UG-6PDH), and the mixture was placed at 30°C for 5min. The optical density at 340nm was measured for 5 minutes using SynergyHT multi-detection microplate reader (Bio-TEK). One unit of glucokinase corresponds to the amount of enzyme that catalyzes the phosphorylation of 1 micromole of D-glucose to D-glucose 6-phosphate per minute under the assay conditions. The glucokinase activity was calculated as follows:

[0322] Glucokinase activity (U / g total protein extract) = dOD x V / [dt xl xε x Qprot], where:

[0323] -dOD is the change in optical density (OD) at 340nm

[0324] - V is the reaction volume (250 μL in this article)

[0325] dt = measurement time (in minutes)

[0326] l = optical path length (0.73 cm in this article)

[0327] ε = molar attenuation coefficient of NADPH; H + (6220cm2 / μmol in this article)

[0328] Qprot = amount of protein in the cuvette (in grams (g))

[0329] Each sample was measured three times, and the glucokinase specific activity values ​​given herein according to the glucokinase activity assay are the average of three independent experiments.

[0330] In some embodiments, the ccpA gene mutation is not a mutation that knocks out (ie, completely disrupts) the gene.

[0331] In some embodiments, the ccpA gene mutation is a mutation in the coding sequence of the ccpA gene, in particular in the first 270 nucleotides of the coding sequence of the ccpA gene. In some embodiments, the mutation is a mutation selected from the group consisting of:

[0332] a) a nonsense mutation (i.e., generating a stop codon) between nucleotide 1 and nucleotide 270 of the ccpA gene coding sequence; and

[0333] b) A mutation located in the first quarter of the coding sequence of the ccpA gene (ie, between nucleotide 1 and nucleotide 250) causing an open reading frame shift of the ccpA gene.

[0334] In some embodiments, the mutation causing the open reading frame shift of the ccpA gene is located between the 50th nucleotide and the 200th nucleotide of the ccpA gene coding sequence. In some embodiments, the mutation causing the open reading frame shift of the ccpA gene is located between the 100th nucleotide and the 150th nucleotide of the ccpA gene coding sequence. Regardless of the mutation position causing the frame shift, the mutation is selected from the group consisting of deletion, insertion, or deletion / insertion (all not a multiple of 3).

[0335] Although two thermophilic Streptococcus strains may differ in the sequence of their respective ccpA genes, this does not necessarily mean that one of the two ccpA genes is mutated in the sense of the present invention. In fact, mutations in the ccpA gene that are not considered in the present invention are changes at the nucleotide level that do cause changes at the protein level.

[0336] Non-limiting examples of ccpA genes that are not considered to be mutated in the sense of the present invention are:

[0337] - a polynucleotide as defined in SEQ ID NO: 65 (ccpA type ST1); this ccpA type is one of the DSM28255 strains;

[0338] - a polynucleotide as defined in SEQ ID NO: 66 (ccpA type ST2), which is 99.8% identical to SEQ ID NO: 65;

[0339] - a polynucleotide as defined in SEQ ID NO: 67 (ccpA type ST3), which is 99.8% identical to SEQ ID NO: 65;

[0340] - a polynucleotide as defined in SEQ ID NO: 68 (ccpA type ST4), which is 99.7% identical to SEQ ID NO: 65;

[0341] - a polynucleotide as defined in SEQ ID NO: 69 (ccpA type ST5), which is 99.8% identical to SEQ ID NO: 65; and

[0342] - a polynucleotide as defined in SEQ ID NO: 70 (ccpA type ST6), which polynucleotide is 99.7% identical to SEQ ID NO: 65.

[0343] The present invention may, in some embodiments, relate to a lactose-positive, sucrose-negative Streptococcus thermophilus strain carrying a mutation in the ccpA gene, the mutation being selected from the group consisting of: a nonsense mutation located between the 1st nucleotide and the 270th nucleotide of the coding sequence of the ccpA gene, and a mutation located in the first quarter of the coding sequence of the ccpA gene that causes a frameshift of the open reading frame of the ccpA gene.

[0344] In some embodiments, the mutation of the ccpA gene is a deletion of nucleotide A in a stretch of 7 nucleotide A at positions 114-120 (causing a frame shift of the open reading frame of the ccpA gene). Such a mutated ccpA gene of Streptococcus thermophilus is referred to herein as ccpA Δ1A114-120 .

[0345] In some embodiments, the sequence of the ccpA gene having a stop codon at codon 66 is selected from the group consisting of:

[0346] a) a sequence as defined in SEQ ID NO: 71; and

[0347] b) a ccpA variant sequence having at least 90% identity to SEQ ID NO: 71. The ccpA variant as defined herein carries a mutation as defined above, i.e. selected from the group consisting of: a nonsense mutation located between nucleotide 1 and nucleotide 270 of the coding sequence of the ccpA gene, and a mutation located in the first quarter of the coding sequence of ccpA causing a frameshift of the open reading frame of the ccpA gene.

[0348] To define a ccpA variant that has at least 90% identity to SEQ ID NO:71, identity is calculated herein over the entire length of the two sequences after optimal alignment [i.e., the number of identical nucleotides in one or more aligned portions of the sequences]. In specific embodiments, the ccpA variant sequence has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:71. In specific embodiments, the ccpA variant sequence differs from SEQ ID NO:71 by 1 to 30 nucleotide substitutions. In specific embodiments, the ccpA variant sequence differs from SEQ ID NO:71 by 1 to 20 nucleotide substitutions. In specific embodiments, the ccpA variant sequence differs from SEQ ID NO:71 by 1 to 15 nucleotide substitutions. In specific embodiments, the ccpA variant sequence differs from SEQ ID NO:71 by 1 to 10 nucleotide substitutions. In certain embodiments, the ccpA variant sequence differs from SEQ ID NO: 71 by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide substitutions. In some embodiments, the sequence of the ccpA gene of the lactose-positive, sucrose-negative thermophilic Streptococcus strain of the invention is selected from the group consisting of SEQ ID NO: 71, 72, 73, 74, 75, and 76.

[0349] In this section of the application, the skilled person is guided on how to identify mutations in the ccpA gene other than the specifically disclosed mutations.

[0350] Therefore, those skilled in the art can perform the operation by the following method:

[0351] a) providing a parent strain, such as DSM28255 strain, wherein its manL gene has been replaced with a mutated manL gene (encoding IIAB) as defined in SEQ ID NO: 111 Man 305 protein) replacement, referred to herein as DGCC7710-IIAB Man 305 strain;

[0352] b) mutagenizing the ccpA gene of the strain in a) by random mutagenesis or targeted mutagenesis to obtain a sequence similar to that of DGCC7710-IIAB Man 305 The ccpA gene sequence of the strain is different from ccpA, thereby obtaining the ccpA mutant DGCC7710-IIAB Man 305 strains.

[0353] Alternatively, those skilled in the art may perform the operation by the following method:

[0354] a) providing a parent strain, such as DSM28255 strain, wherein its manM gene has been replaced with a mutated manM gene (encoding EIIC) as defined in SEQ ID NO: 157 Man 208 protein) replacement, referred to herein as DGCC7710-IIC Man 208 strain;

[0355] b) mutagenizing the ccpA gene of the strain in a) by random mutagenesis or targeted mutagenesis to obtain a sequence similar to that of DGCC7710-IIC Man 208 The ccpA gene sequence of the strain is different from that of ccpA, thereby obtaining the ccpA mutant DGCC7710-IIC Man 208 strains.

[0356] Alternatively, those skilled in the art may perform the operation by the following method:

[0357] a) providing a parent strain, such as DSM28255 strain, wherein its manM gene has been replaced with a mutated manM, the mutation producing a gene encoding the EIIC Man A mutation in the open reading frame shift of the gene for the protein results in a stop codon at position 180 of SEQ ID NO: 222, for example, by inserting a nucleotide C at any of positions 438, 439 or 440 of SEQ ID NO: 223 in the manM gene (manM 12997 );

[0358] b) mutagenizing the ccpA gene of the strain in a) by random mutagenesis or targeted mutagenesis to obtain a sequence similar to that of DGCC7710-IIC Man 208 The ccpA gene sequence of the strain is different from that of ccpA, thereby obtaining the ccpA mutant DGCC7710-IIC Man 208 strains.

[0359] Those skilled in the art can also perform the operation by the following method:

[0360] a) providing a parent strain, such as DSM28255 strain, wherein its manN gene has been replaced with a mutated manN gene (encoding IID Man 28protein) replacement, referred to herein as DGCC7710-IID Man 28 strain;

[0361] b) mutagenizing the ccpA gene of the strain in a) by random mutagenesis or targeted mutagenesis to obtain a sequence similar to that of DGCC7710-IID Man 28 The ccpA gene sequence of the strain is different from that of ccpA, thereby obtaining the ccpA mutant DGCC7710-IID Man 28 strains.

[0362] After identification, the mutated ccpA gene identified herein can be introduced to replace the ccpA gene of the lactose-positive, sucrose-negative Streptococcus thermophilus strain, thereby obtaining the lactose-positive, sucrose-negative Streptococcus thermophilus strain of the present invention.

[0363] Further characterization of the strains of the present invention

[0364] The inventors have demonstrated that a strain having a mutation in the gene encoding a mannose-glucose specific PTS protein and a mutation in the glcK gene and / or the ccpA gene, when used to ferment milk (plain milk and milk with added sucrose), is able to:

[0365] 1) obtaining low lactose fermented milk; and / or

[0366] 2) Fermented milk which has not undergone post-acidification can be obtained even when stored at the fermentation temperature.

[0367] In some embodiments, the lactose-positive, sucrose-negative Streptococcus thermophilus strain of the present invention is characterized in that when used to ferment milk, the strain produces low-lactose fermented milk.

[0368] In some embodiments, the lactose-positive, sucrose-negative Streptococcus thermophilus strain of the present invention is characterized in that the strain produces a fermented milk that does not undergo post-acidification when stored at the fermentation temperature.

[0369] In some embodiments, the lactose-positive, sucrose-negative Streptococcus thermophilus strain of the present invention is characterized in that the strain produces a low-lactose fermented milk (when used for fermenting milk) that does not undergo post-acidification when stored at fermentation temperature.

[0370] The expression "low lactose fermented milk" means fermented milk containing a certain amount of residual lactose in the fermented milk, which is less than 60mM, less than 50mM, less than 45mM, less than 40mM, less than 35mM or less than 30mM at the end of fermentation as described in Example 5. It is worth noting that the amount of lactose in the milk measured according to Example 6 before fermentation is about 50g / L.

[0371] The expression "does not undergo post-acidification" means that when the strain of the present invention is inoculated and fermented as described in Example 5, the pH of the milk product decreases to a specific pH value (i.e., pH 50) at which the acidification rate clearly becomes less than 0.1 mU / min. 终止 value), wherein the pH 终止 The value is comprised between 4.4 and 5.3, and optionally the slope between pH 6 and pH 5.5 is at least -0.008 UpH / min.

[0372] As used herein, the term "pH 终止 The term "phenotype" refers to a strain of S. thermophilus which, when used to ferment milk as described in Example 5, will produce a milk product that "has not undergone post-acidification" as defined herein.

[0373] Therefore, the absence of post-acidification is characterized by the fact that the pH value of the fermented milk stops between 4.4 and 5.3. The pH is considered to be terminated (pH) when the acidification rate (ΔpH / Δtime) becomes clearly less than 0.1 mUpH / min (less than 0.0001 UpH / min). 终止 ). "Explicitly become" means that once the pH is obtained 终止 During the remainder of the fermentation described in Example 5 (ie until 24 h at fermentation temperature), the acidification rate remained less than 0.1 mUpH / min.

[0374] In some embodiments, the pH obtained using the strains of the present invention is 终止 Included between 4.7 and 5.2. In some embodiments, the pH obtained using the strains of the present invention is 终止 Included between 4.8 and 5.1. In some embodiments, the pH obtained using the strains of the present invention is 终止 Included between a minimum value selected from the group consisting of 4.4, 4.5, 4.6, 4.7 and 4.8 and a maximum value selected from the group consisting of 5.1, 5.2 and 5.3.

[0375] In some embodiments, the fermented milk that has not undergone post-acidification is further characterized by a slope between pH 6 and pH 5.5. The slope represents the inverse of the speed (speed of acidification). In some embodiments, the slope is at least -0.009 UpH / min. In some embodiments, the slope is at least -0.01 UpH / min.

[0376] Compositions, methods and uses of lactose-positive sucrose-negative thermophilic streptococci strains of the present invention

[0377] The present invention also relates to a bacterial composition comprising or consisting of at least one, in particular one, lactose-positive, sucrose-negative thermophilic Streptococcus strain of the present invention. In a particular embodiment, the bacterial composition is a pure culture, i.e., comprises or consists of a single bacterial strain. In another embodiment, the bacterial composition is a mixed culture, i.e., comprises or consists of one or more lactose-positive, sucrose-negative thermophilic Streptococcus strains of the present invention and at least one other bacterial strain. "At least" (with respect to strains or bacteria) means 1 or more, and in particular 1, 2, 3, 4 or 5 strains.

[0378] Thus, in some embodiments, the bacterial composition of the invention comprises or consists of one or more lactose-positive, sucrose-negative thermophilic Streptococcus strains of the invention and at least one lactic acid bacterium of a species selected from the group consisting of: Lactococcus species, Streptococcus species, Lactobacillus species including Lactobacillus acidophilus, Enterococcus species, Pediococcus species, Leuconostoc species, Bifidobacterium species and Oenococcus species or any combination thereof. Lactococcus species include Lactobacillus acidophilus and Lactococcus lactis, including Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris and Lactococcus lactis subsp. diacetyl lactic acid biovar. Bifidobacterium species include Bifidobacterium animalis, in particular Bifidobacterium animalis subsp lactis. Other lactic acid bacteria species include Leuconostoc species, Streptococcus thermophilus, Lactobacillus delbrueckii subsp bulgaricus and Lactobacillus helveticus.

[0379] In some embodiments, the bacterial composition comprises or consists of one or more lactose-positive sucrose-negative thermophilic Streptococcus strains of the present invention and at least one thermophilic Streptococcus strain different from one or more thermophilic Streptococcus strains of the present invention and / or at least one strain of the genus Lactobacillus and / or any combination thereof. In a specific embodiment, the bacterial composition comprises or consists of one or more thermophilic Streptococcus strains of the present invention, one or more strains of Lactobacillus delbrueckii subsp. bulgaricus and / or one or more strains of Lactobacillus helveticus and / or any combination thereof, and optionally at least one thermophilic Streptococcus strain different from one or more thermophilic Streptococcus strains of the present invention. In a specific embodiment, the bacterial composition comprises or consists of one or more thermophilic Streptococcus strains of the present invention, at least one thermophilic Streptococcus strain different from one or more thermophilic Streptococcus strains of the present invention and a strain of Lactobacillus delbrueckii subsp. bulgaricus. In another specific embodiment, the bacterial composition comprises or consists of one or more thermophilic Streptococcus strains of the present invention and a strain of Lactobacillus delbrueckii subsp. bulgaricus.

[0380] In some embodiments, the bacterial composition comprises or consists of one or more Streptococcus thermophilus strains, Lactococcus lactis subsp. lactis and / or Lactococcus lactis subsp. cremoris of the present invention.

[0381] In a particular embodiment of any bacterial composition in the form of a pure culture or a mixed culture as defined herein, the bacterial composition further comprises at least one probiotic strain, such as Bifidobacterium animalis subsp. lactis, Lactobacillus acidophilus, Lactobacillus paracasei or Lactobacillus casei.

[0382] In a particular embodiment, the bacterial composition as defined above in the form of a pure culture or a mixed culture is in frozen, dried, freeze-dried, liquid or solid form, in the form of pellets or frozen pellets, or in the form of a powder or dry powder. In a particular embodiment, the bacterial composition of the invention is in frozen form or in the form of pellets or frozen pellets, in particular contained in one or more boxes or pouches. In another embodiment, the bacterial composition as defined herein is in the form of a powder, such as a dried or freeze-dried powder, in particular contained in one or more boxes or pouches.

[0383] In a specific embodiment, the bacterial composition of the invention as defined above in the form of a pure culture or a mixed culture and in any form (frozen, dried, freeze-dried, liquid or solid form, in the form of pellets or frozen pellets, or in the form of a powder or dry powder) comprises one or more lactose-positive, sucrose-negative thermophilic Streptococcus strains of the invention in a concentration contained in 10 5Up to 10 12 cfu (colony forming unit) / gram of bacterial composition. In a specific embodiment, the concentration of one or more lactose-positive sucrose-negative thermophilic Streptococcus strains in the bacterial composition of the present invention is 10 7 Up to 10 12 cfu / gram of bacterial composition, and in particular at least 10 7 , at least 10 8 , at least 10 9 , at least 10 10 or at least 10 11 In a specific embodiment, when in the form of a frozen or dried concentrate, the concentration of one or more lactose-positive, sucrose-negative thermophilic Streptococcus strains in the bacterial composition in the form of a pure culture or a mixed culture is 10 8 Up to 10 12 cfu / g frozen concentrate or dried concentrate, and more preferably at least 10 8 , at least 10 9 , at least 10 10 , at least 10 11 or at least 10 12 cfu / g frozen concentrate or dried concentrate.

[0384] The present invention also relates to a method for making a fermented product, the method comprising: a) inoculating a substrate with one or more lactose-positive sucrose-negative thermophilic streptococcus strains of the present invention, and b) fermenting the inoculated substrate to obtain a fermented product. In a specific embodiment, one or more lactose-positive sucrose-negative thermophilic streptococcus strains of the present invention are inoculated in the form of a bacterial composition such as a pure culture or a mixed culture as defined herein. In some embodiments, the substrate added to one or more thermophilic streptococcus strains or bacterial compositions of the present invention is a milk substrate. "Milk substrate" refers to milk of animal and / or plant origin. In a specific embodiment, the milk substrate is of animal origin such as cows, goats, sheep, buffaloes, zebras, horses, donkeys or camels. Milk can be natural state, reconstituted milk, skim milk or milk supplemented with compounds required for bacterial growth or subsequent processing of fermented milk. Therefore, in a specific embodiment, the present invention also provides a method for producing a fermented milk product, the method comprising: a) inoculating a milk substrate with one or more lactose-positive, sucrose-negative Streptococcus thermophilus strains or bacterial compositions of the present invention, and b) fermenting the inoculated milk substrate to obtain a fermented milk product.

[0385] The present invention also relates to the use of one or more lactose-positive, sucrose-negative Streptococcus thermophilus strains of the present invention or the composition of the present invention for the manufacture of a fermented milk product.

[0386] The present invention also relates to a fermented milk product obtained using one or more lactose-positive, sucrose-negative thermophilic Streptococcus strains of the present invention or the bacterial composition of the present invention, in particular obtained by or by the method of the present invention. Therefore, the present invention relates to a fermented milk product comprising one or more lactose-positive, sucrose-negative thermophilic Streptococcus strains of the present invention. In a specific embodiment, the fermented milk food product of the present invention is fresh fermented milk.

[0387] Various features and embodiments of the invention will now be described by way of non-limiting examples.

[0388] Examples

[0389] The assay used in the following examples

[0390] 1. Test A:

[0391] The thermophilic Streptococcus strain to be tested was cultured in 10 mL M17 medium (ThermoScientific TM Oxoid TM , CM0785) at 37°C overnight. Collect cells from each culture by centrifugation (2100g, 10min, 4°C) and resuspend the cell pellet in 10mL of fresh sugar-free M17 medium. One hundred microliters of the cell suspension diluted 10 times in series were inoculated on the surface of an agar plate containing 30g / L lactose or sucrose in M17 medium. Incubate at 37°C under anaerobic conditions (using After incubation for 24 h in a sealed container with a ELISA system (Merck, Darmstadt, Germany), the colonies grown on lactose and on sucrose were counted. The ratio of colonies (cfu) formed on sucrose (sucrose positive colonies) to colonies (cfu) formed on lactose (lactose positive colonies) was calculated.

[0392] 2. Test B:

[0393] The thermophilic Streptococcus strain to be tested was cultured in 10 mL M17 medium (ThermoScientific TM Oxoid TM,CM0785) and cultured overnight at 37°C. Cells were collected from each culture by centrifugation (2100g, 10min, 4°C) and the cell pellet was resuspended in 10mL of fresh sugar-free M17 medium. Two hundred microliters of cell suspension were inoculated into 10mL of M17 medium containing no sugar or 30g / L sugar (lactose or glucose). Two hundred microliters of each medium inoculated under three conditions (sugar-free, lactose or glucose) were distributed in triplicate in 96-well microtiter plates, and 100μL of paraffin oil was added to the top of each well. These microtiter plates were incubated at 37°C. Bacterial growth was monitored at 600nm for 24h (read every 15min) using SpectraMax ID5 (Molecular Devices, San Jose), with the track shaken at high speed for 5 seconds before each reading. In order to evaluate the ability of these strains to grow on glucose as the sole carbohydrate source, their growth rate on glucose (μ 葡萄糖 To ensure appropriate comparisons between strains and to limit the effect of inoculum rate, the strains were grown at a time corresponding to their maximum growth rate on lactose (μmax 乳糖 ) is calculated at the time point. In addition, the value corresponding to μ is determined 葡萄糖 and μmax 乳糖 ratio.

[0394] 3. Test C:

[0395] The thermophilic Streptococcus strain to be tested was placed in 10 mL M17 (ThermoScientific TM Oxoid TM ,CM0785) and cultured overnight at 37°C. Cells were collected from each culture by centrifugation (2100g, 10min, 4°C) and the cell pellet was resuspended in 10mL of fresh sugar-free M17 medium. Two hundred microliters of cell suspension were inoculated with 10mL of M17 medium containing 30g / L lactose or sucrose. After incubation at 37°C for 24 hours, the OD of the culture was measured at 600nm using a Biochrom WPA Biowave DNA spectrophotometer (Biochrom Ltd., Cambridge, UK). 600 , and compare them.

[0396] Numbered embodiments of the present invention:

[0397] 1. A lactose-positive, sucrose-negative Streptococcus thermophilus strain carrying one or more mutations selected from the group consisting of:

[0398] 1) one or more mutations in one or more genes of a sucrose regulator; the mutation significantly reduces the ability of the strain to grow on sucrose and / or transport sucrose; and the mutation at least comprises

[0399] i) a mutation in one or more of the scrA gene and / or the scrB gene and / or the scrR gene, and

[0400] ii) optionally further mutations in the promoter region regulating the expression of the scrA, scrB and / or scrR genes, e.g. mutations that protect the strain from reversion to a sucrose-negative phenotype; and

[0401] 2) optionally one or more further mutations affecting a glucose transporter, such as glcU and / or its expression, which mutations restore or improve glucose consumption by the strain when the strain is grown on glucose as sole carbohydrate source.

[0402] 2. The lactose-positive, sucrose-negative thermophilic Streptococcus strain according to Example 1, which is a glucose-positive strain or a strain that grows slowly on glucose.

[0403] 3. The lactose-positive, sucrose-negative Streptococcus thermophilus strain of embodiment 1 or 2, which has one or more mutations in one or more of the scrA and scrB genes of the sucrose regulon.

[0404] 4. The lactose-positive, sucrose-negative thermophilic Streptococcus strain according to Example 3, wherein the mutation in one or more genes of the sucrose regulon actually destroys the gene function in the scrA gene, for example, causing the mutation to encode EIIABC 蔗糖 Insertion or deletion of the open reading frame of the protein gene results in translation of a truncated EIIABC encoded by the scrA gene. 蔗糖 Protein, for example, by inserting or deleting nucleotides in the open reading frame (ORF) of the scrA gene, such as a mutation by inserting or deleting nucleotides at or around the nucleotide corresponding to position 133 of SEQ ID NO: 212.

[0405] 5. The lactose-positive, sucrose-negative thermophilic Streptococcus strain according to any one of embodiments 1-4, which has a mutation in the promoter region regulating the expression of any one of the scrA, scrB and / or scrR genes.

[0406] 6. The lactose-positive, sucrose-negative thermophilic Streptococcus strain according to embodiment 5, wherein the mutation is a SNP in the promoter region of the scrA and / or scrB gene, such as a SNP in the promoter region of the scrB gene, such as a mutation near the -10 and -35 sequences of the promoter, such as a T to G mutation corresponding to position 38 of SEQ ID NO: 213 upstream of the scrB promoter, such as a mutation comprising the complete sequence identified by SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto (pscrB 829 ).

[0407] 7. A lactose-positive, sucrose-negative thermophilic Streptococcus strain according to any one of embodiments 1-6, which has a mutation affecting a glucose transporter, which restores or improves glucose consumption of the strain when the strain is grown on glucose as the sole carbohydrate source, such as complete restoration of growth of the strain on glucose compared to the lactose-positive and sucrose-positive thermophilic Streptococcus parent strain.

[0408] 8. The lactose-positive, sucrose-negative thermophilic Streptococcus strain according to embodiment 7, wherein the mutation affecting the glucose transporter affects glcU and / or its expression, such as a mutation located in the promoter region of glcU, such as a mutation selected from the following

[0409] a) Insertion of a T nucleotide at position -75 of the glcU promoter identified as SEQ ID NO: 216 in the poly-T region (pglcU IN_T ), for example a mutation containing a T nucleotide at position 102 of SEQ ID NO: 217, for example a mutation comprising the entire sequence identified by SEQ ID NO: 217 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto;

[0410] b) Insertion of a C nucleotide at position -75 of the glcU promoter identified as SEQ ID NO: 216 in the poly-T region (pglcU IN_C ), for example a mutation that will contain a C nucleotide at position 102 of SEQ ID NO: 218, for example a mutation that will comprise the entire sequence identified by SEQ ID NO: 218 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto;

[0411] c) a mutation of a T to C nucleotide at the position corresponding to position -81 of the glcU promoter identified as SEQ ID NO: 216 in the poly-T region (pglcU SNP_C), for example a mutation that would contain a C nucleotide at position 96 of SEQ ID NO: 219, for example a mutation that would comprise the entire sequence identified by SEQ ID NO: 219 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto;

[0412] d) inserting a sequence at a position corresponding to nucleotides 110 and 111 of the glcU promoter identified as SEQ ID NO: 216, for example inserting a sequence identified by nucleotides 1 to 1321 of SEQ ID NO: 221, for example a mutation (pglcU) comprising the complete sequence identified by SEQ ID NO: 220 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, or SEQ ID NO: 221 or a variant sequence having at least about 90%, such as at least about 95% identity thereto IS ).

[0413] 9. The lactose-positive, sucrose-negative thermophilic Streptococcus strain of any one of embodiments 1-8, further comprising one or more mutations selected from the group consisting of:

[0414] a) a mutation in the glcK gene encoding glucokinase, the glucokinase activity of which in the strain is significantly reduced but not zero; and / or

[0415] b) Generate code EIIC Man A mutation of the open reading frame shift of the gene for the protein, resulting in a stop codon at position 180 of SEQ ID NO: 222, for example, by inserting a nucleotide C at any of positions 438, 439 or 440 of SEQ ID NO: 223 in the manM gene (manM 12997 ); and / or a mutation comprising a substitution of nucleotide G to T at position 916 of SEQ ID NO: 79 of the manL gene (manL 13740 ), resulting in a stop codon (IIAB) at position 306 of the protein of SEQ ID: 80 Man ); and / or a mutation comprising a substitution of nucleotide G at position 625 of manM with nucleotide T (manM 16404 ), resulting in a stop codon at position 209; and / or a mutation comprising the insertion of a nucleotide A at a stretch of 5 nucleotides A at positions 37-41, resulting in a frameshift in the manN gene (resulting in manN 13741 mutant allele), making IID Man The protein is truncated at position 28 of SEQ ID NO:167.

[0416] 10. The lactose-positive, sucrose-negative Streptococcus thermophilus strain according to any one of embodiments 1-9, which is a galactose-negative strain, for example by mutations in the glcK gene and / or the ccpA gene, optionally in combination with a mutation in one of the genes encoding a protein of a mannose-glucose-specific PTS.

[0417] 11. The lactose-positive, sucrose-negative thermophilic Streptococcus strain according to Examples 9-10, which has a mutation in the glcK gene encoding glucokinase, wherein the glucokinase activity in the strain is significantly reduced but not zero.

[0418] 12. The lactose-positive, sucrose-negative thermophilic Streptococcus strain of any one of embodiments 9-11, wherein the mutated glcK gene encodes a glucokinase selected from the group consisting of:

[0419] a) a glucokinase having an amino acid at position 275 which is not glutamic acid, in particular not an acidic amino acid, in particular lysine,

[0420] b) The amino acid at position 275 is not glutamic acid, in particular not an acidic amino acid, in particular lysine (glcK 378 ), and a glucokinase with arginine at position 278 and / or serine at position 279; and

[0421] c) a glucokinase having an amino acid at position 144 which is not glycine, in particular not an aliphatic amino acid, in particular serine.

[0422] 13. The lactose-positive, sucrose-negative thermophilic Streptococcus strain of any one of embodiments 9-12, wherein the mutated glcK gene encodes a glucokinase whose sequence is selected from the group consisting of:

[0423] a) a sequence as defined in SEQ ID NO: 25, wherein the amino acid at position 275 is not glutamic acid, in particular is not an acidic amino acid, in particular is lysine; and

[0424] b) a GlcK variant sequence having at least 90% similarity or identity to SEQ ID NO: 25, wherein the amino acid corresponding to position 275 of SEQ ID NO: 25 (or the amino acid at position 275 of the glucokinase) is not glutamic acid, in particular not an acidic amino acid, in particular lysine;

[0425] c) a sequence as defined in SEQ ID NO: 46, wherein the amino acid at position 144 is not glycine, in particular is not an aliphatic amino acid, in particular is serine; and

[0426] d) a GlcK variant sequence having at least 90% similarity or identity to SEQ ID NO: 46, wherein the amino acid at position 144 of the glucose kinase corresponding to SEQ ID NO: 46 (or the amino acid at position 144 of the glucose kinase) is not glycine, in particular is not an aliphatic amino acid, in particular is serine.

[0427] 14. The lactose-positive, sucrose-negative Streptococcus thermophilus strain according to any one of embodiments 1-13, further carrying a mutation in the ccpA gene.

[0428] 15. The lactose-positive, sucrose-negative Streptococcus thermophilus strain according to any one of embodiments 1-14, further carrying a mutation in a gene encoding a mannose-glucose-specific PTS protein, such as a mutation in a gene selected from the group consisting of a manL gene, a manM gene, and a manN gene.

[0429] 16. The lactose-positive, sucrose-negative thermophilic Streptococcus strain according to any one of embodiments 14 or 15, wherein the ccpA gene carries a mutation selected from the group consisting of: a nonsense mutation located between the 1st nucleotide and the 270th nucleotide of the coding sequence of the ccpA gene, and a mutation located in the first quarter of the coding sequence of the ccpA gene that causes a frameshift of the open reading frame of the ccpA gene.

[0430] 17. The lactose-positive, sucrose-negative thermophilic Streptococcus strain of embodiment 16, wherein the sequence of the mutated ccpA gene is selected from the group consisting of:

[0431] a) a sequence as defined in SEQ ID NO: 71; and

[0432] b) a ccpA variant sequence that is at least 90%, such as at least 95%, identical to SEQ ID NO: 71.

[0433] 18. The lactose-positive, sucrose-negative Streptococcus thermophilus strain according to any one of embodiments 1-17, wherein when the strain is capable of fermenting milk as described in Example 5 during a 24-hour fermentation period:

[0434] a) the residual lactose concentration in the fermented milk is less than 60 mM; and / or

[0435] b) The pH of the milk is reduced to the pH at which the acidification rate becomes clearly less than 0.1 mUpH / min (pH 终止 ), wherein the pH 终止 Between 4.4 and 5.3, and optionally between pH 6 and pH 5.5, the slope is at least -0.008 UpH / min.

[0436] 19. The lactose-positive, sucrose-negative thermophilic Streptococcus strain of any one of embodiments 1-18, wherein the strain has the ability to consume lactose in excess compared to a parent strain without the one or more mutations, for example wherein when fermenting sweet milk, the residual amount of lactose as described and determined according to Example 6 is less than 100 mM, for example, less than 90 mM, 80 mM, 70 mM, 60 mM, 50 mM, 40 mM, 30 mM, 20 mM, 10 mM, 8 mM, 6 mM, 4 mM, 2 mM or 1 mM.

[0437] 20. The lactose-positive, sucrose-negative Streptococcus thermophilus strain of any one of embodiments 1-19, wherein the strain has the ability to release increased amounts of glucose compared to a parent strain without the one or more mutations, for example, wherein the glucose release as described and determined according to Example 6 is greater than 10 mM, for example, greater than 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM or 90 mM.

[0438] 21. The lactose-positive, sucrose-negative thermophilic Streptococcus strain of any one of embodiments 1-20, wherein the strain releases a glucose concentration determined as described in Example 6 of at least 50 mM, such as at least 60, 70, 80 or 90 mM, or an increase of at least 150% or at least 200%, such as 300%, 400%, 500% compared to the glucose concentration released by the parent strain, when both strains are determined as described in Example 6.

[0439] 22. A composition comprising at least one, in particular one, lactose-positive, sucrose-negative Streptococcus thermophilus strain according to any one of embodiments 1-21, in particular in combination with another lactic acid bacterium, in particular in combination with one or more strains selected from the group consisting of: a strain of the genus Lactobacillus, such as a strain of Lactobacillus delbrueckii subsp. bulgaricus; a strain of the genus Lactococcus, such as a strain of Lactococcus lactis; or a strain of the genus Bifidobacterium.

[0440] 23. A method for producing a fermented milk product, in particular fermented milk, comprising inoculating a milk substrate with a lactose-positive, sucrose-negative Streptococcus thermophilus strain according to any one of embodiments 1 to 21 or a composition according to embodiment 19, and fermenting the inoculated milk to obtain a fermented milk product.

[0441] 24. Use of the lactose-positive, sucrose-negative Streptococcus thermophilus strain according to any one of embodiments 1 to 21 or the composition according to embodiment 22 for the manufacture of a fermented milk product.

[0442] 25. A fermented milk product comprising at least one, in particular one, lactose-positive, sucrose-negative Streptococcus thermophilus strain according to any one of embodiments 1 to 21 or a lactose-positive, sucrose-negative Streptococcus thermophilus strain obtained by the method according to embodiment 23.

[0443] 26. A thermophilic Streptococcus strain, e.g., a lactose-positive, sucrose-negative thermophilic Streptococcus strain selected from the group consisting of:

[0444] - strain deposited on January 18, 2022 in the name of DuPont Nutrition Biosciences under the number DSM34132 at the Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures in accordance with the Budapest Treaty;

[0445] - strain deposited on January 18, 2022 in the name of DuPont Nutrition Biosciences under the number DSM34134 at the Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures in accordance with the Budapest Treaty;

[0446] - strain deposited on January 18, 2022 in the name of DuPont Nutrition Biosciences under the number DSM34138 at the Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures in accordance with the Budapest Treaty;

[0447] - strain deposited on January 18, 2022 in the name of DuPont Nutrition Biosciences under the number DSM34139 at the Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures in accordance with the Budapest Treaty;

[0448] - strains deposited on January 18, 2022 in the name of DuPont Nutrition Biosciences under the number DSM34140 at the Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures in accordance with the Budapest Treaty;

[0449] - the strain deposited on January 18, 2022 at the Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures under the number DSM34146 in the name of DuPont Nutrition Biosciences under the Budapest Treaty; - the strain deposited on January 18, 2022 at the Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures under the number DSM34147 in the name of DuPont Nutrition Biosciences under the Budapest Treaty; and

[0450] -The strain deposited on February 16, 2022 at the Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures under the number DSM34172 in the name of DuPont Nutrition Biosciences under the Budapest Treaty.

[0451] 27. A lactose-positive, sucrose-negative thermophilic Streptococcus strain selected from the group consisting of:

[0452] a strain corresponding to the Streptococcus thermophilus strain deposited at the Leibniz Institute DSMZ under the Budapest Treaty on January 14, 2014 under the number DSM34172 in the name of Danisco Deutschland GmbH [referred to herein as ST1 or DSM34172 strain], wherein the sequence of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, resulting in a frameshift, such that a truncated EIIABC is translated 蔗糖 (scrA 829 FS );

[0453] a strain corresponding to the Streptococcus thermophilus strain deposited on January 14, 2014 at the Leibniz Institute DSMZ under the Budapest Treaty in the name of Danisco Deutschland GmbH under the number DSM34172 [referred to herein as ST1 or DSM34172 strain], wherein the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto (pscrB 829 );

[0454] a strain corresponding to the Streptococcus thermophilus strain deposited on January 14, 2014 at the Leibniz Institute DSMZ under the Budapest Treaty under the number DSM34172 in the name of Danisco Deutschland GmbH [referred to herein as ST1 or DSM34172 strain], wherein the locus (pglcU) as the promoter region upstream of the glcU gene is replaced by the corresponding sequence as shown in any one of SEQ ID NOs: 217 to 220 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pglcU SNP_C ,pglcU IN_T ,pglcU IN_C ,pglcU IS );

[0455] a strain corresponding to the Streptococcus thermophilus strain deposited at the Leibniz Institute DSMZ under the Budapest Treaty on January 14, 2014 under the number DSM34172 in the name of Danisco Deutschland GmbH [referred to herein as ST1 or DSM34172 strain], wherein the sequence of the open reading frame (ORF) of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, resulting in a frameshift, such that a truncated EIIABC is translated 蔗糖 (scrA 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pscrB 829 );

[0456] a strain corresponding to the Streptococcus thermophilus strain deposited at the Leibniz Institute DSMZ under the Budapest Treaty on January 14, 2014 under the number DSM34172 in the name of Danisco Deutschland GmbH [referred to herein as ST1 or DSM34172 strain], wherein the sequence of the open reading frame (ORF) of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, resulting in a frameshift, such that a truncated EIIABC is translated 蔗糖 (scrA 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pscrB 829 ), and the gene locus (pglcU) as the promoter region upstream of the glcU gene is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217-220 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pglcU SNP_C ,pglcU IN_T ,pglcU IN_C ,pglcU IS );

[0457] a strain corresponding to the Streptococcus thermophilus strain deposited at the Leibniz Institute DSMZ under the Budapest Treaty on January 14, 2014 under the number DSM34172 in the name of Danisco Deutschland GmbH [referred to herein as ST1 or DSM34172 strain], wherein the sequence of the open reading frame (ORF) of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, resulting in a frameshift, such that a truncated EIIABC is translated 蔗糖 (scrA 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pscrB 829 ), and the gene locus (pglcU) as the promoter region upstream of the glcU gene is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217-220 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pglcU SNP_C ,pglcU IN_T ,pglcU IN_C ,pglcU IS ), and optionally wherein the glcK gene encoding glucokinase comprises a sequence as defined by SEQ ID NO: 25 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, wherein the amino acid at position 275 is lysine (glcK 378 ), and optionally further comprising generating an encoding said EIIC Man A mutation of the open reading frame shift of the gene for the protein, resulting in a stop codon at position 180 of SEQ ID NO: 222, for example, by inserting a nucleotide C at any of positions 438, 439 or 440 of SEQ ID NO: 223 in the manM gene (manM 12997 ), and / or a mutation comprising a substitution of nucleotide G to T at position 916 of SEQ ID NO: 79 of the manL gene (manL 13740 ), resulting in a stop codon (IIAB) at position 306 of the protein of SEQ ID: 80 Man ); and / or a mutation comprising a substitution of nucleotide G at position 625 of manM with nucleotide T (manM 16404), resulting in a stop codon at position 209; and / or a mutation comprising the insertion of a nucleotide A at a stretch of 5 nucleotides A at positions 37-41, resulting in a frameshift in the manN gene (resulting in manN 13741 mutant allele), making IID Man The protein is truncated at position 28 of SEQ ID NO: 167; and optionally further comprises the ccpA sequence of SEQ ID NO: 71 or a ccpA variant sequence having at least about 90%, such as at least 95%, identity to SEQ ID NO: 71;

[0458] a strain of Streptococcus thermophilus, such as the strain deposited on 14 January 2014 at the Leibniz Institute DSMZ under the Budapest Treaty in the name of Danisco Deutschland GmbH under the number DSM34172 [referred to herein as strain ST1 or DSM34172], which strain comprises one or more of the following characteristics:

[0459] 1) The sequence of the open reading frame (ORF) of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, resulting in a frame shift, so that a truncated EIIABC is translated 蔗糖 (scrA 829 FS ),

[0460] 2) The sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pscrB 829 ),

[0461] 3) the locus (pglcU) as the promoter region upstream of the glcU gene is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217-220 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pglcU SNP_C ,pglcU IN_T ,pglcU IN_C ,pglcU IS ),

[0462] 4) The glcK gene encoding glucokinase comprises a sequence as defined in SEQ ID NO: 25 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, wherein the amino acid at position 275 is lysine (glcK 378 ),

[0463] 5) With the ability to generate code EIIC Man A mutation of the open reading frame shift of the gene for the protein, resulting in a stop codon at position 180 of SEQ ID NO: 222, for example, by inserting a nucleotide C at any of positions 438, 439 or 440 of SEQ ID NO: 223 in the manM gene (manM 12997 ), and / or a mutation comprising a substitution of nucleotide G to T at position 916 of SEQ ID NO: 79 of the manL gene (manL 13740 ), resulting in a stop codon (IIAB) at position 306 of the protein of SEQ ID: 80 Man ); and / or a mutation comprising a substitution of nucleotide G at position 625 of manM with nucleotide T (manM 16404 ), resulting in a stop codon at position 209; and / or a mutation comprising the insertion of a nucleotide A at a stretch of 5 nucleotides A at positions 37-41, resulting in a frameshift in the manN gene (resulting in manN 13741 mutant allele), making IID Man The protein is truncated at position 28 of SEQ ID NO:167;

[0464] 6) a ccpA sequence having the sequence of SEQ ID NO:71 or a ccpA variant sequence having at least about 90%, such as at least 95%, identity to SEQ ID NO:71;

[0465] a strain corresponding to the Streptococcus thermophilus strain deposited at the Leibniz Institute DSMZ under the Budapest Treaty on January 14, 2014 under the number DSM28255 in the name of Danisco Deutschland GmbH [referred to herein as ST4 or DSM28255 strain], wherein the sequence of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, resulting in a frameshift, such that a truncated EIIABC is translated 蔗糖 (scrA 829 FS );

[0466] a strain corresponding to the Streptococcus thermophilus strain deposited at the Leibniz Institute DSMZ under the Budapest Treaty on January 14, 2014 under the number DSM28255 in the name of Danisco Deutschland GmbH [referred to herein as ST4 or DSM28255 strain], wherein the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto (pscrB829 );

[0467] a strain corresponding to the Streptococcus thermophilus strain deposited on January 14, 2014 at the Leibniz Institute DSMZ under the Budapest Treaty under the number DSM28255 in the name of Danisco Deutschland GmbH [referred to herein as ST4 or DSM28255 strain], wherein the locus (pglcU) as the promoter region upstream of the glcU gene is replaced by the corresponding sequence as shown in any one of SEQ ID NOs: 217 to 220 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pglcU SNP_C ,pglcU IN_T ,pglcU IN_C ,pglcU IS );

[0468] a strain corresponding to the Streptococcus thermophilus strain deposited on January 14, 2014 at the Leibniz Institute DSMZ under the Budapest Treaty under the number DSM28255 in the name of Danisco Deutschland GmbH [referred to herein as ST4 or DSM28255 strain], wherein the sequence of the open reading frame (ORF) of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, resulting in a frameshift, such that a truncated EIIABC is translated 蔗糖 (scrA 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pscrB 829 );

[0469] a strain corresponding to the Streptococcus thermophilus strain deposited on January 14, 2014 at the Leibniz Institute DSMZ under the Budapest Treaty under the number DSM28255 in the name of Danisco Deutschland GmbH [referred to herein as ST4 or DSM28255 strain], wherein the sequence of the open reading frame (ORF) of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, resulting in a frameshift, such that a truncated EIIABC is translated 蔗糖 (scrA 829 FS), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pscrB 829 ), and the gene locus (pglcU) as the promoter region upstream of the glcU gene is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217-220 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pglcU SNP_C ,pglcU IN_T ,pglcU IN_C ,pglcU IS );

[0470] a strain corresponding to the Streptococcus thermophilus strain deposited on January 14, 2014 at the Leibniz Institute DSMZ under the Budapest Treaty under the number DSM28255 in the name of Danisco Deutschland GmbH [referred to herein as ST4 or DSM28255 strain], wherein the sequence of the open reading frame (ORF) of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, resulting in a frameshift, such that a truncated EIIABC is translated 蔗糖 (scrA 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pscrB 829 ), and the gene locus (pglcU) as the promoter region upstream of the glcU gene is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217-220 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pglcU SNP_C ,pglcU IN_T ,pglcU IN_C ,pglcU IS ), and optionally wherein the glcK gene encoding glucokinase comprises a sequence as defined by SEQ ID NO: 25 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, wherein the amino acid at position 275 is lysine (glcK 378 ), and optionally further comprising generating an encoding said EIICMan A mutation of the open reading frame shift of the gene for the protein, resulting in a stop codon at position 180 of SEQ ID NO: 222, for example, by inserting a nucleotide C at any of positions 438, 439 or 440 of SEQ ID NO: 223 in the manM gene (manM 12997 ), and / or a mutation comprising a substitution of nucleotide G to T at position 916 of SEQ ID NO: 79 of the manL gene (manL 13740 ), resulting in a stop codon (IIAB) at position 306 of the protein of SEQ ID: 80 Man ); and / or a mutation comprising a substitution of nucleotide G at position 625 of manM with nucleotide T (manM 16404 ), resulting in a stop codon at position 209; and / or a mutation comprising the insertion of a nucleotide A at a stretch of 5 nucleotides A at positions 37-41, resulting in a frameshift in the manN gene (resulting in manN 13741 mutant allele), making IID Man The protein is truncated at position 28 of SEQ ID NO: 167; and optionally further comprises the ccpA sequence of SEQ ID NO: 71 or a ccpA variant sequence having at least about 90%, such as at least 95%, identity to SEQ ID NO: 71;

[0471] a strain of Streptococcus thermophilus, such as the strain deposited on 14 January 2014 at the Leibniz Institute DSMZ under the Budapest Treaty in the name of Danisco Deutschland GmbH under the number DSM 28255 [referred to herein as strain ST4 or DSM 28255], which strain comprises one or more of the following characteristics:

[0472] 1) The sequence of the open reading frame (ORF) of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, resulting in a frame shift, so that a truncated EIIABC sucrose (scrA) is translated. 829 FS ),

[0473] 2) The sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pscrB 829 ),

[0474] 3) the locus (pglcU) as the promoter region upstream of the glcU gene is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217-220 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pglcU SNP_C ,pglcU IN_T ,pglcU IN_C ,pglcU IS ),

[0475] 4) The glcK gene encoding glucokinase comprises a sequence as defined in SEQ ID NO: 25 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, wherein the amino acid at position 275 is lysine (glcK 378 ),

[0476] 5) With the ability to generate code EIIC Man A mutation of the open reading frame shift of the gene for the protein, resulting in a stop codon at position 180 of SEQ ID NO: 222, for example, by inserting a nucleotide C at any of positions 438, 439 or 440 of SEQ ID NO: 223 in the manM gene (manM 12997 ), and / or a mutation comprising a substitution of nucleotide G to T at position 916 of SEQ ID NO: 79 of the manL gene (manL 13740 ), resulting in a stop codon (IIAB) at position 306 of the protein of SEQ ID: 80 Man ); and / or a mutation comprising a substitution of nucleotide G at position 625 of manM with nucleotide T (manM 16404 ), resulting in a stop codon at position 209; and / or a mutation comprising the insertion of a nucleotide A at a stretch of 5 nucleotides A at positions 37-41, resulting in a frameshift in the manN gene (resulting in manN 13741 mutant allele), making IID Man The protein is truncated at position 28 of SEQ ID NO:167;

[0477] 6) a ccpA sequence having the sequence of SEQ ID NO:71 or a ccpA variant sequence having at least about 90%, such as at least 95%, identity to SEQ ID NO:71;

[0478] - A strain corresponding to the Streptococcus thermophilus strain deposited at the Leibniz Institute DSMZ under the number DSM34132 on January 18, 2022 in the name of DuPont Nutrition Biosciences under the Budapest Treaty [referred to herein as ST2 or DSM34132 strain], wherein the sequence of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, resulting in a frameshift, such that a truncated EIIABC is translated 蔗糖 (scrA 829 FS );

[0479] - A strain corresponding to the Streptococcus thermophilus strain deposited at the Leibniz Institute DSMZ under the number DSM34132 on January 18, 2022 in the name of DuPont Nutrition Biosciences under the Budapest Treaty [referred to herein as ST2 or DSM34132 strain], wherein the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto (pscrB 829 );

[0480] - A strain corresponding to the Streptococcus thermophilus strain deposited at the Leibniz Institute DSMZ under the number DSM34132 on January 18, 2022 in the name of DuPont Nutrition Biosciences under the Budapest Treaty [referred to herein as ST2 or DSM34132 strain], wherein the locus (pglcU) as the promoter region upstream of the glcU gene is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217-220 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pglcU SNP_C ,pglcU IN_T ,pglcU IN_C ,pglcU IS );

[0481] - A strain corresponding to the Streptococcus thermophilus strain deposited at the Leibniz Institute DSMZ under the number DSM34132 on January 18, 2022 in the name of DuPont Nutrition Biosciences under the Budapest Treaty [referred to herein as ST2 or DSM34132 strain], wherein the sequence of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, resulting in a frameshift, such that a truncated EIIABC is translated 蔗糖 (scrA 829FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pscrB 829 );

[0482] - A strain corresponding to the Streptococcus thermophilus strain deposited at the Leibniz Institute DSMZ under the number DSM34132 on January 18, 2022 in the name of DuPont Nutrition Biosciences under the Budapest Treaty [referred to herein as ST2 or DSM34132 strain], wherein the sequence of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, resulting in a frameshift, such that a truncated EIIABC is translated 蔗糖 (scrA 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pscrB 829 ), and the gene locus (pglcU) as the promoter region upstream of the glcU gene is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217-220 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pglcU SNP_C ,pglcU IN_T ,pglcU IN_C ,pglcU IS );

[0483] - A strain corresponding to the Streptococcus thermophilus strain deposited at the Leibniz Institute DSMZ under the number DSM34132 on January 18, 2022 in the name of DuPont Nutrition Biosciences under the Budapest Treaty [referred to herein as ST2 or DSM34132 strain], wherein the sequence of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, resulting in a frameshift, such that a truncated EIIABC is translated 蔗糖 (scrA 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pscrB 829), and the gene locus (pglcU) as the promoter region upstream of the glcU gene is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217-220 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pglcU SNP_C ,pglcU IN_T ,pglcU IN_C ,pglcU IS ), and optionally wherein the glcK gene encoding glucokinase comprises a sequence as defined by SEQ ID NO: 25 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, wherein the amino acid at position 275 is lysine (glcK 378 ), and optionally further comprising generating an encoding said EIIC Man A mutation of the open reading frame shift of the gene for the protein, resulting in a stop codon at position 180 of SEQ ID NO: 222, for example, by inserting a nucleotide C at any of positions 438, 439 or 440 of SEQ ID NO: 223 in the manM gene (manM 12997 ), and / or a mutation comprising a substitution of nucleotide G to T at position 916 of SEQ ID NO: 79 of the manL gene (manL 13740 ), resulting in a stop codon (IIAB) at position 306 of the protein of SEQ ID: 80 Man ); and / or a mutation comprising a substitution of nucleotide G at position 625 of manM with nucleotide T (manM 16404 ), resulting in a stop codon at position 209; and / or a mutation comprising the insertion of a nucleotide A at a stretch of 5 nucleotides A at positions 37-41, resulting in a frameshift in the manN gene (resulting in manN 13741 mutant allele), making IID Man The protein is truncated at position 28 of SEQ ID NO: 167; and optionally further comprises the ccpA sequence of SEQ ID NO: 71 or a ccpA variant sequence having at least about 90%, such as at least 95%, identity to SEQ ID NO: 71;

[0484] - a strain of Streptococcus thermophilus, such as the strain deposited on January 18, 2022 at the Leibniz Institute DSMZ under the number DSM34132 in the name of DuPont Nutrition Biosciences under the Budapest Treaty [referred to herein as ST2 or DSM34132 strain], which strain comprises one or more of the following characteristics:

[0485] 1) The sequence of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, resulting in a frame shift, so that a truncated EIIABC is translated 蔗糖 (scrA 829 FS ),

[0486] 2) The sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pscrB 829 ),

[0487] 3) the locus (pglcU) as the promoter region upstream of the glcU gene is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217-220 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pglcU SNP_C ,pglcU IN_T ,pglcU IN_C ,pglcU IS ),

[0488] 4) The glcK gene encoding glucokinase comprises a sequence as defined in SEQ ID NO: 25 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, wherein the amino acid at position 275 is lysine (glcK 378 ),

[0489] 5) With the ability to generate code EIIC Man A mutation of the open reading frame shift of the gene for the protein, resulting in a stop codon at position 180 of SEQ ID NO: 222, for example, by inserting a nucleotide C at any of positions 438, 439 or 440 of SEQ ID NO: 223 in the manM gene (manM 12997 ), and / or a mutation comprising a substitution of nucleotide G to T at position 916 of SEQ ID NO: 79 of the manL gene (manL 13740 ), resulting in a stop codon (IIAB) at position 306 of the protein of SEQ ID: 80 Man ); and / or a mutation comprising a substitution of nucleotide G at position 625 of manM with nucleotide T (manM 16404), resulting in a stop codon at position 209; and / or a mutation comprising the insertion of a nucleotide A at a stretch of 5 nucleotides A at positions 37-41, resulting in a frameshift in the manN gene (resulting in manN 13741 mutant allele), making IID Man The protein is truncated at position 28 of SEQ ID NO:167;

[0490] 6) a ccpA sequence having the sequence of SEQ ID NO:71 or a ccpA variant sequence having at least about 90%, such as at least 95%, identity to SEQ ID NO:71;

[0491] - A strain corresponding to the Streptococcus thermophilus strain deposited with the DSMZ [German Collection of Microorganisms and Cell Cultures, Inhofenstrasse 7B, D-38124 Braunschweig, Germany] on September 29, 2020 under the accession number DSM33651 (referred to herein as ST3 or DSM33651), wherein the sequence of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, resulting in a frameshift, such that a truncated EIIABC is translated 蔗糖 (scrA 829 FS );

[0492] - A strain corresponding to the Streptococcus thermophilus strain deposited with the DSMZ [German Collection of Microorganisms and Cell Cultures, Inhofenstrasse 7B, Braunschweig, Germany, D-38124] on September 29, 2020 under the accession number DSM33651 (referred to herein as ST3 or DSM33651), wherein the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto (pscrB 829 );

[0493] - A strain corresponding to the Streptococcus thermophilus strain deposited on September 29, 2020 with the accession number DSM33651 at DSMZ [German Collection of Microorganisms and Cell Cultures, Inhofenstrasse 7B, Braunschweig, Germany, D-38124] (referred to herein as ST3 or DSM33651), wherein the locus (pglcU) as the promoter region upstream of the glcU gene is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217-220 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pglcU SNP_C ,pglcUIN_T ,pglcU IN_C ,pglcU IS );

[0494] - A strain corresponding to the Streptococcus thermophilus strain deposited with the DSMZ [German Collection of Microorganisms and Cell Cultures, Inhofenstrasse 7B, D-38124 Braunschweig, Germany] on September 29, 2020 under the accession number DSM33651 (referred to herein as ST3 or DSM33651), wherein the sequence of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, resulting in a frameshift, such that a truncated EIIABC is translated 蔗糖 (scrA 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pscrB 829 );

[0495] - A strain corresponding to the Streptococcus thermophilus strain deposited with the DSMZ [German Collection of Microorganisms and Cell Cultures, Inhofenstrasse 7B, D-38124 Braunschweig, Germany] on September 29, 2020 under the accession number DSM33651 (referred to herein as ST3 or DSM33651), wherein the sequence of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, resulting in a frameshift, such that a truncated EIIABC is translated 蔗糖 (scrA 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pscrB 829 ), and the gene locus (pglcU) as the promoter region upstream of the glcU gene is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217-220 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pglcU SNP_C ,pglcU IN_T ,pglcU IN_C ,pglcU IS );

[0496] - A strain corresponding to the Streptococcus thermophilus strain deposited with the DSMZ [German Collection of Microorganisms and Cell Cultures, Inhofenstrasse 7B, D-38124 Braunschweig, Germany] on September 29, 2020 under the accession number DSM33651 (referred to herein as ST3 or DSM33651), wherein the sequence of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, resulting in a frameshift, such that a truncated EIIABC is translated 蔗糖 (scrA 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pscrB 829 ), and the gene locus (pglcU) as the promoter region upstream of the glcU gene is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217-220 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pglcU SNP_C ,pglcU IN_T ,pglcU IN_C ,pglcU IS ), and optionally wherein the glcK gene encoding glucokinase comprises a sequence as defined by SEQ ID NO: 25 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, wherein the amino acid at position 275 is lysine (glcK 378 ), and optionally further comprising generating an encoding said EIIC Man A mutation of the open reading frame shift of the gene for the protein, resulting in a stop codon at position 180 of SEQ ID NO: 222, for example, by inserting a nucleotide C at any of positions 438, 439 or 440 of SEQ ID NO: 223 in the manM gene (manM 12997 ), and / or a mutation comprising a substitution of nucleotide G to T at position 916 of SEQ ID NO: 79 of the manL gene (manL 13740 ), resulting in a stop codon (IIAB) at position 306 of the protein of SEQ ID: 80 Man ); and / or a mutation comprising a substitution of nucleotide G at position 625 of manM with nucleotide T (manM 16404), resulting in a stop codon at position 209; and / or a mutation comprising the insertion of a nucleotide A at a stretch of 5 nucleotides A at positions 37-41, resulting in a frameshift in the manN gene (resulting in manN 13741 mutant allele), making IID Man The protein is truncated at position 28 of SEQ ID NO: 167; and optionally further comprises the ccpA sequence of SEQ ID NO: 71 or a ccpA variant sequence having at least about 90%, such as at least 95%, identity to SEQ ID NO: 71;

[0497] - A strain of Streptococcus thermophilus deposited on September 29, 2020 with the accession number DSM33651 at DSMZ [German Collection of Microorganisms and Cell Cultures, Inhofenstrasse 7B, D-38124 Braunschweig, Germany] (hereinafter referred to as ST3 or DSM33651), comprising one or more of the following characteristics:

[0498] 1) The sequence of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, resulting in a frame shift, so that a truncated EIIABC is translated 蔗糖 (scrA 829 FS ),

[0499] 2) The sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pscrB 829 ),

[0500] 3) the locus (pglcU) as the promoter region upstream of the glcU gene is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217-220 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pglcU SNP_C ,pglcU IN_T ,pglcU IN_C ,pglcU IS ),

[0501] 4) The glcK gene encoding glucokinase comprises a sequence as defined in SEQ ID NO: 25 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, wherein the amino acid at position 275 is lysine (glcK 378 ),

[0502] 5) With the ability to generate code EIIC Man A mutation of the open reading frame shift of the gene for the protein, resulting in a stop codon at position 180 of SEQ ID NO: 222, for example, by inserting a nucleotide C at any of positions 438, 439 or 440 of SEQ ID NO: 223 in the manM gene (manM 12997 ), and / or a mutation comprising a substitution of nucleotide G to T at position 916 of SEQ ID NO: 79 of the manL gene (manL 13740 ), resulting in a stop codon (IIAB) at position 306 of the protein of SEQ ID: 80 Man ); and / or a mutation comprising a substitution of nucleotide G at position 625 of manM with nucleotide T (manM 16404 ), resulting in a stop codon at position 209; and / or a mutation comprising the insertion of a nucleotide A at a stretch of 5 nucleotides A at positions 37-41, resulting in a frameshift in the manN gene (resulting in manN 13741 mutant allele), making IID Man The protein is truncated at position 28 of SEQ ID NO:167;

[0503] 6) a ccpA sequence having the ccpA sequence of SEQ ID NO:71 or a ccpA variant sequence having at least about 90%, such as at least 95%, identity to SEQ ID NO:71.

[0504] 28. A polynucleotide comprising one or more mutations in one or more genes of a sucrose regulon, the one or more mutations significantly reducing the ability of a strain selected from the group consisting of:

[0505] a) a strain corresponding to the strain of Streptococcus thermophilus deposited on January 14, 2014 at the Leibniz Institute DSMZ under the number DSM 28255 in the name of Danisco Deutschland GmbH under the Budapest Treaty, wherein the polynucleotide replaces the corresponding sequence in DSM 28255;

[0506] b) a strain corresponding to the strain of Streptococcus thermophilus deposited at the Leibniz Institute DSMZ under the number DSM34132 on January 18, 2022 in the name of DuPont Nutrition Biosciences under the Budapest Treaty, wherein the polynucleotide replaces the corresponding sequence in DSM34132;

[0507] c) a strain corresponding to the strain of Streptococcus thermophilus deposited on September 29, 2020 under the accession number DSM33651 in the DSMZ [German Collection of Microorganisms and Cell Cultures, Inhofenstrasse 7B, Braunschweig, Germany, postal code D-38124], wherein the polynucleotide replaces the corresponding sequence in DSM33651;

[0508] d) A strain corresponding to the Streptococcus thermophilus strain deposited at the Leibniz Institute DSMZ under the number DSM34172 on February 16, 2022 in the name of DuPont Nutrition Biosciences under the Budapest Treaty, wherein the polynucleotide replaces the corresponding sequence in DSM34172.

[0509] 29. A polynucleotide comprising one or more mutations affecting a glucose transporter, the one or more mutations restoring or improving glucose consumption in a strain selected from:

[0510] a) a strain corresponding to the strain of Streptococcus thermophilus deposited on January 14, 2014 at the Leibniz Institute DSMZ under the number DSM 28255 in the name of Danisco Deutschland GmbH under the Budapest Treaty, wherein the polynucleotide replaces the corresponding sequence in DSM 28255;

[0511] b) a strain corresponding to the strain of Streptococcus thermophilus deposited at the Leibniz Institute DSMZ under the number DSM34132 on January 18, 2022 in the name of DuPont Nutrition Biosciences under the Budapest Treaty, wherein the polynucleotide replaces the corresponding sequence in DSM34132,

[0512] c) a strain corresponding to the strain of Streptococcus thermophilus deposited on September 29, 2020 under the accession number DSM33651 in the DSMZ [German Collection of Microorganisms and Cell Cultures, Inhofenstrasse 7B, Braunschweig, Germany, postal code D-38124], wherein the polynucleotide replaces the corresponding sequence in DSM33651;

[0513] d) A strain corresponding to the Streptococcus thermophilus strain deposited at the Leibniz Institute DSMZ under the number DSM34172 on February 16, 2022 in the name of DuPont Nutrition Biosciences under the Budapest Treaty, wherein the polynucleotide replaces the corresponding sequence in DSM34172.

[0514] 30. The polynucleotide of any one of embodiments 28 or 29, wherein the one or more mutations are as identified in any one of embodiments 3-8.

[0515] 31. A method for selecting a lactose-positive, sucrose-negative thermophilic Streptococcus strain, the method comprising the steps of:

[0516] 1) providing a strain of thermophilic Streptococcus, comprising

[0517] a) one or more mutations in one or more genes of a sucrose regulator; the mutation significantly reduces the ability of the strain to grow on sucrose and / or transport sucrose; and the mutation comprises at least

[0518] i) a mutation in one or more of the scrA gene and / or the scrB gene and / or the scrR gene, and

[0519] ii) optionally further mutations in the promoter region regulating the expression of the scrA, scrB and / or scrR genes, e.g. mutations that protect the strain from reversion to a sucrose-negative phenotype;

[0520] 2) screening a strain of Streptococcus thermophilus from step 1) having a significantly reduced ability to grow on sucrose and to transport sucrose; and

[0521] 3) optionally before, simultaneously with or after any of steps 1 and 2, screening for S. thermophilus strains having restored or improved glucose consumption, optionally from strains having one or more further mutations affecting a glucose transporter, e.g., affecting glcU and / or its expression.

[0522] 32. The method according to embodiment 31, wherein the strain is defined according to any one of embodiments 1-21.

[0523] Examples

[0524] Example 1: Identification of mutations in the scrA gene and mutations in the promoter of the scrB gene

[0525] S. thermophilus is described as a species that can grow on lactose (lactose positive phenotype) and sucrose (sucrose positive phenotype). A proprietary strain collection was screened for S. thermophilus strains that could not grow on sucrose as the sole carbohydrate source. The S. thermophilus strains to be tested were grown in M17 (ThermoScientific TM Oxoid TM,CM0785) and cultured overnight at 37°C. Cells were collected from each culture by centrifugation (2100g, 10min, 4°C), and the cell pellet was washed twice with tryptone salts (TS; tryptone 1g / L, NaCl 8.5g / L) and then resuspended in 10mL of fresh sugar-free M17 medium. In a 96-well microtiter plate, 200μL of fresh M17 medium containing 30g / L sugar (lactose or sucrose) was inoculated in triplicate with the washed cells at 1% (v / v), and 100μL of paraffin oil was added to the top of each well. These microtiter plates were incubated at 37°C. Bacterial growth was monitored at 620nm for 24h (read every 15min) using SpectraMaxID5 (Molecular Devices, San Jose), and the track was shaken at high speed for 5 seconds before each reading. A lactose-positive strain, DGCC8368, was found to be unable to grow on sucrose as the sole carbohydrate source. The ability to grow on sucrose was defined as the OD of the strain reaching 0.04 after incubation at 37 °C for 24 h. 600 Better than 0.5 capability.

[0526] Several genes of DGCC8368 known to be involved in sucrose catabolism in S. thermophilus (scrA, scrB, scrK, and scrR genes) were sequenced and compared with the corresponding gene sequences of other S. thermophilus strains. First, an insertion of an A nucleotide in the poly-A region was identified at position 133 of the open reading frame (ORF) of scrA of DGCC8368 ( Figure 1 ; SEQ ID NO: 212); the mutation was named scrA 829 FS This insertion results in a frameshift, resulting in the translation of a truncated EIIABC 蔗糖 In addition, a substitution of nucleotide A with nucleotide C was identified at position -38 upstream of the start codon of the scrB gene (position 38 of SEQ ID NO: 213). This mutation was named pscrB 829 , located 2 nucleotides upstream of the putative -10 box ( Figure 1 Neither of these two mutations was found in the scrA gene sequences or scrB promoter sequences of other thermophilic Streptococcus strains in the collection or in public databases.

[0527] Example 2. scrA 829 FS Effects on the growth of Streptococcus thermophilus strains on sucrose as the sole carbohydrate source.

[0528] To evaluate scrA 829 FSThe effect of mutations on the ability of thermophilic S. strains to grow on sucrose, derivatives of thermophilic S. strains ST1 (DSM34172), ST2 (DSM34132) and ST3 (DSM33651) were constructed in which the mutation replaced the native gene allele. To assess the ability of these strains to grow on sucrose as the sole carbohydrate source, these constructs were analyzed using Test A. In Test A, the thermophilic S. strains to be tested were grown in 10 mL M17 medium (Thermo Scientific TM Oxoid TM , CM0785) at 37°C overnight. Collect cells from each culture by centrifugation (2100g, 10min, 4°C) and resuspend the cell pellet in 10mL of fresh sugar-free M17 medium. One hundred microliters of the cell suspension diluted 10 times in series were inoculated on the surface of an agar plate containing 30g / L lactose or sucrose in M17 medium. Incubate at 37°C under anaerobic conditions (using After incubation for 24 hours in a sealed container of the A system (Merck, Darmstadt, Germany), the colonies grown on lactose and on sucrose were counted. The ratio of colonies (cfu) formed on sucrose (sucrose positive colonies) to colonies (cfu) formed on lactose (lactose positive colonies) was calculated. The results are summarized in Table 1.

[0529] scrA 829 FS The mutants (ST1-A, ST2-A, and ST3-A) were barely able to grow on sucrose as the sole carbohydrate source. In test A, the ratio of sucrose-positive colonies to lactose-positive colonies of the mutants was less than 10 -6 , while the ratio of the parent strain was close to 1. Overall, the results suggest that scrA 829 FS The mutation rendered the S. thermophilus strain sucrose negative, meaning that less than 0.01% of the cfu grown on lactose were able to form colonies on sucrose as the sole carbohydrate source.

[0530] Table 1 : Analysis of scrA 829 FS Effects on the ability of Streptococcus thermophilus to grow on sucrose as a sole carbohydrate source.

[0531]

[0532] a , cell counts were established as described in Test A.

[0533] Example 3. Combination of scrA 829FS and pscrB 829 Stabilizes the sucrose-negative phenotype of thermophilic Streptococcus strains.

[0534] In test A, on sucrose-containing agar plates, three scrA 829 FS Low-level bacterium colonies (see Example 2, Table 1) were observed in the culture of mutants (ST1-A, ST2-A and ST3-A), indicating the instability of the negative phenotype of sucrose. When these mutants were cultivated in the culture medium containing sucrose as the sole carbohydrate source, this instability (test C) could be clearly observed. In test C, the thermophilic streptococcus strain to be tested was incubated overnight at 37°C in the 10mLM17 containing 30g / L lactose. Cells were collected from each culture by centrifugation (2100g, 10min, 4°C), and the cell pellet was resuspended in the fresh sugar-free M17 culture medium of 10mL. The M17 culture medium containing 30g / L lactose or sucrose was inoculated with 10mL of cell suspension of two hundred microlitres. After incubation at 37°C for 24 hours, the OD of the culture was measured at 600nm using Biochrom WPA Biowave DNA spectrophotometer (British Bai Xuan Co., Ltd. (Biochrom Ltd.), Cambridge, UK). 600 The results (Table 2) showed that the OD values ​​of the parent strain grown on M17 medium containing sucrose ranged from 2.8 to 6.0. These values ​​were consistent with those of the three scrA 829 FS There were no significant differences in the values ​​measured for the mutants (OD 600 The values ​​ranged from 3.7 to 4.6). However, since strains showing a sucrose-negative phenotype would be expected to show an OD of 600 The value is close to zero; therefore, 829 FS The associated phenotype may be unstable.

[0535] To evaluate the mutation of scrA 829 FS and pscrB 829 The combined effect of the two on the stabilization of the sucrose-negative phenotype, constructed scrA 829 FS A mutant derivative in which pscrB 829 The mutation replaced the native gene allele. Using Test C, the parent strain, scrA 829 FS Single mutants and scrA 829 FS -pscrB 829 The double mutants.829 FS In contrast to the single mutants, scrA 829 FS -pscrB 829 The double mutant maintained a lower OD throughout the 24-h incubation period. 600 value, and OD 600 It never reached a value higher than 0.14. This clearly demonstrated that scrA 829 FS and pscrB 829 Combinations of mutations can stabilize scrA 829 FS The sucrose-negative phenotype conferred by the mutation. It is also noteworthy that in test A, no scrA 829 FS -pscrB 829 The colonies of double mutants with reversion to the sucrose-negative phenotype were below the detection threshold (<0.510 -8 ).

[0536] Table 2 : Analysis of scrA 829 FS and pscrB 829 Effects of combinations of mutations on stabilization of the sucrose-negative phenotype of mutant strains of Streptococcus thermophilus.

[0537]

[0538]

[0539] a , establish the cell count ratio as described in Test A; b , OD600 of strains grown on sucrose for 24 h was measured as described in Test C.

[0540] Example 4. scrA 829 FS Mutations and pscrB 829 Either combination of mutations or none of them impaired the ability of the S. thermophilus strain to grow on glucose; pglcU restored this ability.

[0541] Strains of the species S. thermophilus are described as naturally able to grow on glucose as a sole carbohydrate source. 829 FS Mutations with or without pscrB 829For this control, test B was performed. In test B, the thermophilic Streptococcus strain to be tested was cultured in 10 mL M17 medium (Thermo Scientific TM Oxoid TM , CM0785) was cultured at 37 ° C overnight. Cells were collected from each culture by centrifugation (2100g, 10min, 4 ° C), and the cell pellet was resuspended in 10mL fresh sugar-free M17 medium. Two hundred microliters of the culture medium from the cell suspension were inoculated with 1% (v / v) 10mL of M17 medium without sugar or containing 30g / L sugar (lactose or glucose). Two hundred microliters of each condition (sugar-free, lactose or glucose) of the culture medium were distributed in triplicate in 96-well microtiter plates, and 100 μL of paraffin oil was added to the top of each well. These microtiter plates were incubated at 37 ° C. SpectraMax ID5 (Molecular Devices, San Jose) was used to monitor bacterial growth at 600nm for 24h (read once every 15min), and the track was shaken at high speed for 5 seconds before each reading.

[0542] To evaluate the ability of these strains to grow on glucose as a sole carbohydrate source, their growth rate on glucose (μ 葡萄糖 To ensure appropriate comparisons between strains and to limit the effect of inoculum rate, the strains were grown at a time corresponding to their maximum growth rate on lactose (μmax 乳糖 ) is calculated at the time point. In addition, the value corresponding to μ is determined 葡萄糖 and μmax 乳糖 The data are summarized in Table 3. Figure 3 Provides μ 葡萄糖 / μmax 乳糖 Graphical comparison of the ratios. Typically, for the lactose-positive and glucose-positive S. thermophilus parent strain, the ratio is better than 0.5, indicating that the strain grows well on glucose, even if more slowly than on lactose. Surprisingly, the scrA 829 FS Mutations with or without pscrB 829 The mutant strain was affected in its ability to grow on glucose compared to its parent. 葡萄糖 / μmax 乳糖 The values ​​of the ratios were significantly lower and below 0.25. This suggests that at least scrA 829 FS The mutation had a negative effect on the growth of S. thermophilus on glucose. Growth on glucose was not completely abolished, but was significantly slower; this phenotype was defined as "slow growth on glucose".

[0543] The spontaneous mutants that showed an improved ability to consume glucose after cultivation on glucose as the sole carbohydrate source may have arisen from strains that grow slowly on glucose (possibly explaining their high OD recorded after 24 h of cultivation in M17 glucose medium). 600 , not shown). 829 FS -pscrB 829 Cultures of the strains were plated on M17 agar plates containing 30 g / L glucose as the sole carbohydrate source. Colonies were picked after incubation at 37°C and the ability of the colonies to grow on glucose was investigated as described in Test B. Some selected clones (ST1-ABU1, ST1-ABU4, ST2-ABU1, ST2-ABU2, ST2-ABU3, ST3-ABU1 and ST3-ABU2) showed similar or higher μ values ​​calculated with ST1, ST2 and ST3 parent strains in Test B. 葡萄糖 / μmax 乳糖 Ratio (Table 3). These clones were further investigated for mutations in their genomes. Comparative genomics of the selected clones with their parents allowed the identification of four different mutations, each of which could result in a reversal of the slow growth phenotype on glucose (SEQ ID NOs: 217-220). Each clone was found to have a mutation at a locus corresponding to the promoter region upstream of the glcU gene (pglcU); the glcU gene is predicted to encode a non-PTS transporter of glucose. One mutation was a SNP (pglcU SNP_C ), and two mutations were single nucleotide insertions (same position but different nucleotides) (pglcU IN_T and pglcU IN_C ), and the last mutation is the insertion of IS (pglcU IS )(See Figure 2 ).

[0544] To confirm that the mutation in pglcU was responsible for the reversal of the slow growth phenotype on glucose, we inserted pglcU in the promoter region of glcU SNP_C Mutation ST2-AB was genetically engineered to generate strain ST2-ABU3 ge In test B, ST2-ABU3 ge Compared with ST2-ABU3, the growth rates of the two strains in the medium containing glucose were faster (0.477 and 0.480, respectively) and similar, confirming that pglcU SNP_CThe mutation was responsible for the reversal of the slow growth phenotype on glucose. With the same goal in mind, reverse genetics studies were performed on ST2-ABU3 to replace pglcU with the wild-type allele of pglcU from the parent ST2. SNP_C Mutation. Obtain ST2-ABU wt , and compared with ST2-AB in test B. The growth rates of the two strains in the medium containing glucose were slower (0.161 and 0.081, respectively) and similar, thus confirming again that pglcU SNP_C The mutation is responsible for the reversal of the slow growth phenotype on glucose.

[0545] Table 3 : Analysis of the effect of pglcU mutation on the expression of scrA 829 FS Mutations with or without pscrB 829 Effect of mutants of S. thermophilus on their ability to grow on glucose as a sole carbohydrate source.

[0546]

[0547]

[0548] a , establish the cell count ratio as described in Test A; b , grown on lactose and glucose as described in Test B for 24 h.

[0549] Example 5: In addition to glcK E275K and manM 12997 Combinations of mutations or ccpA 855 and manM 12997 In addition to the combination of mutations, the use of 829 FS and pscrB 829 pH of sweet fresh fermentation products of mutant Streptococcus thermophilus strains (with or without pglcU mutation) 终 end.

[0550] It has been shown that lactose-positive, galactose-negative thermophilic Streptococcus strains carrying mutations in the glcK gene and / or the ccpA gene in combination with a mutation in one of the genes encoding a mannose-glucose-specific PTS protein are of interest for controlling the endpoint pH in fresh fermented products such as yogurt (WO 2019197051). However, for fresh fermented products to which sucrose is added to obtain a sweet product, this property is greatly affected.

[0551] To evaluate the expression of scrA in the S. thermophilus genome 829 FSand pscrB 829 mutations and the effects and benefits of the pglcU mutation on the acidification kinetics of plain and sweetened milks, the following strains were engineered:

[0552] ·ST1-GM-AB, a derivative of ST1-GM (glcK 378 -manM 12997 Double mutant), with scrA 829 FS and pscrB 829 mutation

[0553] ·ST1-GM-ABU1, a derivative of ST1-ABU1 with glcK 378 and manM 12997 mutation

[0554] ST1-CM-ABU1, a derivative of ST1-ABU1 with ccpA 855 and manM 12997 mutation

[0555] With engineered strain and appropriate control strain (ST1 and ST1-GM) fermentation pure milk and sweet milk as follows: with the fresh pre-culture of strain to be tested (in reconstituted milk containing 10% (w / v) skim milk powder, pasteurized at 120 ℃ for 20 minutes) inoculate 100mL pure milk ("Candia Grand Lait" pasteurized at 90 ℃ for 10 minutes, diluted with water at 93% (v / v)) and 100mL sweet milk ("Candia Grand Lait" pasteurized at 90 ℃ for 10min, supplemented with sucrose at 7% (w / v)) at 2% (v / v). The inoculated milk was incubated in a water bath at 43 ℃ for 24h. The acidification characteristics of engineered strains were evaluated by recording pH during milk fermentation. The pH was monitored for 24 hours using a CINAC system (Alliance Instruments, France; pH electrode was a Mettler 405DPAS SC (Toledo, Spain)). The pH was recorded every 5 or 25 minutes.

[0556] The evolution of pH over time Figure 4 The speed between pH 6 and pH 5.5 was calculated as the slope of the linear model derived from the evolution of pH as a function of time for pH values ​​from 6 to 5.5. The slope value is inversely proportional to the speed (Table 4). The evolution of speed as a function of pH is also shown ( Figure 4B1, 4B2, 5B1, 5B2, 6B1, 6B2, 7B1, 7B2, 8B1 and 8B2). The velocity is determined as the instantaneous derivative of the pH evolution as a function of time. pH 终止 The characteristic of the kinetics is that the pH value at which the velocity decrease is undetectable (below 0.1 mU / min) (Table 4). 终止 (TpH 终止 ) corresponding to the time (Table 4).

[0557] The results are shown in Table 4. Under the above conditions, the parent strain (ST1) showed a pH lower than 4.60 in both pure milk and sweet milk. 终止 As expected, using glcK 378 -ManM 12997 pH of pure milk fermented by double mutant (ST1-GM) 终止 In contrast, like the parent strain, the pH in sweet milk 终止 The value is lower than 4.6. In contrast, 829 FS and pscrB 829 The mutant engineered strain ST1-GM-AB showed a high pH above 4.6 in sweet milk 终止 The presence or absence of the pglcU mutation appears to have an effect on the pH of pure and sweet milk. 终止 The pH value has no effect because the pH of ST1-GM-ABU1 终止 The value is still above 4.6. Similarly, except for ccpA 855 and manM 12997 In addition to the mutation (ST1-CM-ABU1), there is also scrA 829 FS ,pscrB 829 The strains with pglcU mutation showed a high pH value above 4.6 in both pure milk and sweet fermented milk. 终止 value.

[0558] Table 4 also shows that when using glcK 378 -ManM 12997 In pure milk fermented with the double mutant, TpH was reached within 450 min. 终止 However, the double mutant still acidified sweet milk after 24 h of incubation; TpH 终止 The value was higher than 1425 minutes. 829 FS and pscrB 829 The TpH of strains with or without pglcU mutation in both pure milk and sweet fermented milk 终止 These data also indicate that the addition of scrA829 FS and pscrB 829 Neither the A mutation nor the pglcU mutation had a negative impact on the acidification kinetics (slope between pH 6 and 5.5), which is still acceptable at the industrial level for the manufacture of fermented dairy products.

[0559] In conclusion, scrA 829 FS and pscrB 829 and the pglcU mutation on glcK 378 -ManM 12997 and ccpA 855 -ManM 12997 The double mutant had no effect on its ability to control the endpoint pH in pure fermented milk. 829 FS and pscrB 829 The mutation allows the extension of this property in milk supplemented with sucrose.

[0560] Table 4 : Analysis of scrA 829 FS ,pscrB 829 and pglcU mutations to glcK 378 、ManM 12997 and ccpA 855 Effects of the mutants on physiological characteristics of fermented and sweetened milk.

[0561]

[0562] a “Candia Grand Lait” was pasteurized at 90°C for 10 minutes and diluted with water at 93% (v / v); b "Candia Grand Lait" pasteurized at 90°C for 10 min, supplemented with sucrose at 7% (w / v); nd, not determined.

[0563] Example 6. In addition to glcK E275K and manM 12997 Mutation or ccpA 855 and manM 12997 In addition to the combination of mutations, the use of 829 FS and pscrB 829 Mutations of S. thermophilus (with or without the pglcU mutation) improved glucose release and lactose consumption.

[0564] In addition to controlling the endpoint pH of fermented milk (see Example 5), lactose-positive, galactose-negative Streptococcus thermophilus strains carrying mutations in the glcK gene and / or the ccpA gene in combination with a mutation in one of the genes encoding the mannose-glucose-specific PTS protein have been shown to be of interest for excessive consumption of lactose and release of glucose (WO 2019197051).

[0565] To evaluate scrA 829 FS and pscrB 829 Mutation and the effect of pglcU mutation on excessive lactose consumption and glucose release, further studied the engineered strains ST1-GM-AB, ST1-GM-ABU1 and ST1-CM-ABU1 described in Example 5. Ferment sweet milk with these strains and appropriate control strains (ST1-GM and ST1), as described in Example 5. Sampling was taken before incubation and after incubation for 24 hours. Biochemical analysis was performed as follows: 5g of each sample was diluted in 25g of 0.025NH2SO4, then centrifuged at 4 ℃ for 10 minutes with 4600rpm. Supernatant was directly filtered into 2ml HPLC vials by 0.2μm nylon filter (Phenomenex, Germany, Aschaffenburg, Germany). The chromatograms were analyzed by high performance liquid chromatography (Agilent 1200 HPLC) equipped with a refractive index detector at 35°C using an Aminex HPX-87H anion exchange column (Bio-Rad Laboratories Inc.) using 12.5 mM H2SO4 as the eluent and 0.6 ml min -1 Glucose and lactose were quantified at a flow rate of 1.5 50 mmol / l. Results were developed using Chemstation reprocessing software (Agilent).

[0566] The results are shown in Table 5. The parent strain (ST1) consumed limited amounts of lactose (less than 50% considering the initial concentration in sweet milk was 145 mM) and no measurable amount of glucose was detected from the hydrolysis of carbohydrates. As expected, and as previously shown (WO 2019197051), the glcK 378 and ManM 12997 The double mutant (ST1-GM) with both mutations was able to consume more lactose (only 44 mM remaining) and release up to 44 mM glucose. 829 FS and pscrB 829The introduction of mutations into the genome of ST1-GM (producing ST1-GM-AB) resulted in a strain that consumed even more lactose (16 mM residual lactose), with the result that more glucose (71 mM) was released. Finally, the addition of mutations in the promoter region of glcU in the ST1-GM-AB genome resulted in a strain (ST1-GM-ABU1) that was able to hydrolyze all the lactose present in milk after 24 hours of fermentation, and more surprisingly (considering that the pglcU mutation improved scrA 829 FS -pscrB 829 mutants grown on glucose) released even more glucose (up to 91 mM). 855 Mutation instead of glcK 378 Similar behavior was observed in the mutated strain (strain ST1-CM-ABU1).

[0567] Finally, scrA 829 FS and pscrB 829 Effects of pglcU mutation on the fermentation of milk by glcK 378 and ManM 12997 ccpA 855 and ManM 12997 The phenotype conferred by the mutations (lactose overconsumption and glucose release) has no negative effects. On the contrary, these mutations allow obtaining strains that hydrolyze even more lactose and release even more glucose.

[0568] Table 5 : Analysis of scrA 829 FS ,pscrB 829 and pglcU mutations to glcK 378 -ManM 12997 and ccpA 855 -ManM 12997 Effects of the mutants on their ability to consume lactose and release glucose during milk fermentation.

[0569]

[0570] a "Candia Grand Lait" was pasteurized at 90°C for 10 minutes and supplemented with sucrose at 7% (w / v).

[0571] Example 7. scrA 829 FS -pscrB 829 The double mutation impairs the transport of sucrose and glucose into the cells, and the pglcU mutation restores the transport of glucose into the double mutant cells.

[0572] To elucidate the mechanism leading to the sucrose-negative and slow-growth phenotype on glucose, ST1 and its scrA 829 FS -pscrB 829 (ST1-AB) and scrA 829 FS -pscrB 829 -pglcU IN_T Experiments were performed to study carbohydrate transport in resting cells (non-growing cells) of the (ST1-ABU1) mutant.

[0573] The strain was pre-cultured in M17 containing 30 g / L lactose at 37°C overnight. The pre-culture was used to inoculate M17 containing 30 g / L lactose, and the final OD 600 The concentration of the cells was 0.05 and incubated at 42°C for 4 hours. The cells were collected by centrifugation at 2450 g for 7 min, washed twice with 4% (w / v) β-glycerophosphate buffer, and then resuspended in 10 mL of β-glycerophosphate buffer. The OD of the final cell suspension was recorded. 600 . The cell suspension was preheated at 42°C, and glucose, lactose or sucrose were added to a final concentration of 3.5 g / L, 7.0 g / L or 7.0 g / L, respectively. After incubation at 42°C for 3 min, the reaction medium was filtered on a 0.2 μm nylon membrane (PHENEXGF / NY), and the carbohydrate dose was measured by high performance liquid chromatography (HPLCAgilent 1100 or 1200). The sample was injected into an ion exchange column and then injected into a guard column (Security GuardCartridges, Carbo-Pb4x3.0mmID, Phenomenex). The ion exchange column was Pb2+ (RezexRPM-monosaccharide Pb+2, 300x 7.8mm, 8μm, Phenomenex). Elution was performed in isocratic mode with ultrapure water, and carbohydrates were detected with a refractometer. Quantification was performed by external calibration. Standard solutions were prepared in 4% (w / v) β-glycerophosphate, ranging from 0 to 7 g / L for each carbohydrate. All experiments were repeated. The sugar consumption rate was calculated in μmol / (u.OD*min.) as follows:

[0574] Carbohydrate uptake activity =[([sugar] T0 x 2 / 1.8x 1000 / MW 糖 )-([sugar] T+3’ x1000 / MW 糖 )] / OD 600 / 3

[0575] Of which: [Sugar] T0: Initial concentration of carbohydrates (in g / L)

[0576] [sugar] T+3’ : Final concentration of carbohydrates measured after 3 minutes of contact (in g / L)

[0577] Volume of cell suspension: 1.8 mL

[0578] Volume of cell suspension and sugar solution: 2 mL

[0579] MW 糖 : Molecular weight of carbohydrate (g / mol)

[0580] OD 600 : Cell concentration at the start of the test

[0581] The results are shown in Table 6. The parent strain ST4 (DSM28255) was able to efficiently transport lactose, sucrose, and glucose, with consumption rates of 0.236, 0.180, and 0.108 μmole / u.OD*min., respectively. 829 FS -pscrB 829 The double mutant (ST4-AB) significantly reduced the rates of sucrose and glucose consumption (0.032 and 0.011 μmole / u.OD*min., respectively), while there was no significant difference in the rate of lactose consumption (0.201 μmole / u.OD*min.). IN_T The introduction of the mutation into the genome of the double mutant (ST4-ABU1) had no significant effect on the transport of lactose into the cell (0.227 μmole / u.OD*min.) and did not allow for an improvement in the transport of sucrose (0.004 μmole / u.OD*min.). IN_T The mutation allowed full restoration of glucose transport into the cell (0.102 μmole / u.OD*min.). Finally, scrA 829 FS -pscrB 829 The double mutation impairs the transport of sucrose and glucose into the cell, and pglcU IN_T The mutation restored glucose transport into cells of the double mutant.

[0582] Table 6 : Transport rates of different carbohydrates by resting cells of the parental ST4 strain and its derivatives ST4-AB and ST4-ABU1.

[0583]

[0584] a RSD, relative standard deviation

[0585] sequence

[0586] SEQ ID NO:2

[0587] MSKKLLGIDLGGTTVKFGILTADGEVQEKWAIETNTFENGSHIVPDIVESLKHRLELYGLTAEDFIGIGMGSPGAVDRENKTVTGAFNLNWAETQEVGSVIEKELGIPFAIDNDANVAALGERWVGAGANNRNVVFITLGTGVGGGVIADGNLIHGVAGAGGEIGHIIVEPDTGFECTCGNKGCLETVASATGIVRVAHHLAEKYEGNSSIKAAVDNGEFVTSKDIIVAATEGDKFADSIVDKVSKYLGLATANISNILNPDSVVIGGGVSAAGEFLRSRVEGYFTRYAFPQVRRTTKVKLAELGNDAGIIGAASLAYSIDK

[0588] SEQ ID NO:22

[0589] MSKKLLGIDLGGTTVKFGILTADGEVQEKWAIETNTFENGSHIVPDIVESLKHRLELYGLTAEDFIGIGMGSPGAVDRENKTVTGAFNLNWAETQEVGSVIEKELGIPFAIDNDANVAALGERWVGAGANNRNVVFITLGTGVGGGVIADGNLIHGVAGAGGEIGHIIVEPDTGFECTCGNKGCLETVASATGIVRVAHHLAEKYEGNSSIKAAVDNGEFVTSKDIIVAATEGDKFADSIVDKVSKYLGLATANISNILNPDSVVIGGGVSAAGKFLRSRVEGYFTRYAFPQVRRTTKVKLAELGNDAGIIGAASLAYSIDK

[0590] SEQ ID NO:25 (the bold underlined X corresponds to the aa at position 275 and can be any naturally occurring amino acid but not Glu)

[0591] MSKKLLGIDLGGTTVKFGILTADGEVQEKWAIETNTFENGSHIVPDIVESLKHRLELYGL

[0592] TAEDFIGIGMGGSPGAVDRENKTTVTGAFNLNWAETQEVGSVIEKELGIPFAIDNDANVAAL

[0593] GERWVGAGNNNRNVVFITLGTGVGGGVIADGNLIHGVAGAGGEIGHIIVEPDTGFECTCG

[0594] NKGCLETVASATGIVRVAHHLAEKYEGNSIKAAVDNGEFVTSKDIIVAATEGDKFADSI

[0595]

[0596] LAELGNDAGIIGASLAYSIDK

[0597] SEQ ID NO:45

[0598] MSKKLLGIDLGGTTVKFGILTADGEVQEKWAIETNTFENGSHIVPDIVESLKHRLELYGLTAEDFIGIGMGSPGAVDRENKTTVTGAFNLNWAETQEVGSVIEKELGIPFAIDNDANVAALGERWVGAGANNRNVVFITLGTGVSGGVIADGNLIHGVAGAG GEIGHIIVEPDTGFECTCGNKGCLETVASATGIVRVAHHLAEKYEGNSIKAAVDNGEFVTSKDIIVAATEGDKFADSIVDKVSKYLGLATANISNILNPDSVVIGGGVSAAGEFLRSRVEGYFTRYAFPQVRRTTKVKLAELGNDAGIIGAASLAYSIDK

[0599] SEQ ID NO:23

[0600] MSKKLLGIDLGGTTTVKFGILTADGEVQEKWAIETNTFENGSHIVPDIVESLKHRLELYGLTAEDFIGIGMGPPGAVDRENKTVTGAFNLNWAETQEVGSVIEKELGIPFAIDNDANVAALGERWVGAGANNRNVVFITLGTGVGGGVIADGNLIHGVAGAGGEIGHIIVEPDTGFECTCGNKGCLETVASATGIVRVAHHLAEKYEGNSSIKAAVDNGEFVTSKDIIVAATEGDKFADSIVDKVSKYLGLATANISNILNPDSVVIGGGVSAAGEFLRSRVEGYFTRYAFPQVRRTTKVKLAELGNDAGIIGAASLAYSIDK

[0601] SEQ ID NO:65

[0602]

[0603] SEQ ID NO:71

[0604]

[0605] SEQ ID NO:78

[0606] MGIGIIIASHGRFAEGIHQSGSMIFGDQEKVQVVTFMPSEGPDDLYAHFNNAIAQFDVDDEILVLADLWSGSPFNQASRIARENPDRKIAIITGLNLPMLIQAYTERMMDANATVEQVAANIIKEAKGGIKALPEELNPAEETTAAPVEAAAPQGAIPEGTVIGDGKLKINLARLDTRLLHGQVVTNWVPYSKADRIIVASDDVAKDELRKELIKQAAPNGIKVNVVPIQKLIDASKDPRFGNTHALVLFETVQDALRAIEGGVPIKELNVGSMAHSTGKTMVNNVLSMDKDDVACFEKLRDLGVEFDVRKVPNDSKKDLFELIKKANVQ

[0607] SEQ ID NO:111

[0608] ATGGGTATCGGTATTATTATTGCCAGCCATGGTAAGGTTCGCTGAAGGAATCCACCAATCAGGCTTCTATGATTTTTGGGGACCAAGAGAAAGTTCAAGTTGTGACTTTCATGCCAAGTGAAGGTC CTGATGATTTGTACGCTCACTTCAACAACGCCATTGCACAATTCGATGTTGATGATGAAATTCTTGTTTTGGCTGACCTTTGGAGTGGTTCACCATTTAACCAAGCTAGTCGAATCGCTAGGGA AAATCCAGATCGCAAGATTGCTATCATCACAGGACTTAACTTGCCAATGCTAATCCAAGCATACACTGAACGTATGATGGATGCTAACGCTACTGTAGAGCAAGTTGCTGCTAATATCATCAAGGAAGCTAAGGGTGGTATCAAGGCACTTCCAGAAGAGCTAAATCCAGCTGAGGAAACAACTGCAGTCCTGTAGAAGCTGCAGCACCTCAAGGAGCTATCCCTGAAGGAACAGTCATCGGAGATG GTAAACTCAAGATTAACTTGGCACGTTTGGACACACGTCTCTTGCATGGTCAAGTAGTAACTAACTGGGTACCTTATTCTAAAGCAGACCGTATTATTGTTGCTTCGGATGACGTTGCCAAAGATGAGCTTCGTAAGGAATTGATCAAACAGGCTGCACCAAACGGTATTAAAGTAAACGTTGTTCCGATTCAAAAATTAATTGATGCTTCTAAAGACCCACGTTTTGGAAATACACATGCGCTTGTCTTGTTCGAAACTGTTCAAGACGCACTTCGTGCTATCGAAGGTGGCGTGCCAATAAAAGAACTTAACGTTGGTTCTATGGCTCACTCAACTGGTAAAACAATGGTTAACAACGTTTTGTCTATGGATAAAGATGATGTTGCTTGTTTTGAAAAATTACGTGACCTTGGCGTTTAATTTGACGTCCGTAAGGTTCCAAACGATTCTAAGAAAGATTTGTTTGAGCTTATCAAGAAAGCTAACGTTCAATAA

[0609] SEQ ID NO:112

[0610] MGIGIIIASHGRFAEGIHQSGSMIFGDQEKVQVVTFMPSEGPDDLYAHFNNAIAQFDVDDEILVLADLWSGSPFNQASRIARENPDRKIAIITGLNLPMLIQAYTERMMDANATVEQVAANIIKEAKGGIKALPEELNPAEETTAAPVEAAAPQGAIPEGTVIGDGKLKINLARLDTRLLHGQVVTNWVPYSKADRIIVASDDVAKDELRKELIKQAAPNGIKVNVVPIQKLIDASKDPRFGNTHALVLFETVQDALRAIEGGVPIKELNVGSMAHSTGKTMVNNVLSMDKDDVACFEKLRDLGV

[0611] SEQ ID NO:130

[0612] MSDMSIISAILVVAVAFLAGLESILDQFQFHQPLVACTLIGAATGNLTAGIMLGGSLQMITLAWANIGAAVAPDVALASVAAAIILVKGGKFTAEGIGVAIAIAILLAVAGLFLTMPVRTASIAFVHAADKAAEHGNIAGVERAYYLALLLQGLRIAVPAALLLAIPAQSVQHALGLMPDWLTHGLVVGGGMVVAVGYAMIINMMATREVWPFFAIGFALAAISQLTLIALSTIGVAIAFIYLNLSKQGGGNGGGNGGGTSSGSGDPIGDILEDY

[0613] SEQ ID NO:157

[0614] ATGTCAGATATGTCAATTATTTCTGCGATTTTGGTCGTAGCTGTTGCCTTCCTTGCTGGTCTTGAAAGTATCCTTGACCAATTCCAATTCCACCAACCACTTGTTGCATGTACCCTCATCGGTGCTGCCACAGGTAACCTCACTGCAGGTATCATGCTTGGTGGTTCTCTTCAAATGATTACCCTTGCTTGGGCAAACATCGGTGCTGCCGTAGCTCCTGACGTTGCCCTTGCATCTGTTGCCGCTGCCATCATTTTGGTTAAAGGTGGTAAATTTACAGCTGAAGGTATCGGTGTTGCGATTGCAATAGCTATCCTGCTTGCAGTTGCAGGTCTCTTCCTAACTATGCCTGTTCGTACAGCATCTATTGCCTTTGTTCATGCTGCAGATAAAGCTGCAGAACACGGAAACATCGCTGGTGTTGAACGTGCATACTACCTCGCTCTCCTTCTTCAAGGTTTGCGTATTGCTGTGCCAGCAGCCCTTCTTCTTGCCATCCCGGCCCAATCTGTTCAACATGCCCTTGGCTTGATGCCTGACTGGCTCACCCATGGTTTGGTTGTCGGTGGTGGTATGGTCGTAGCCGTTGGTTACGCCATGATTATCAATATGATGGCTACTCGTTAAGTTTGGCCATTCTTCGCCATTGGTTTTGCTTTGGCAGCAATTAGCCAATTGACACTTATCGCTCTTAGTACCATTGGTGTTGCCATCGCCTTCATCTACCTCAACCTTTCTAAACAAGGTGGCGGAAATGGTGGCGGAAATGGTGGCGGAACTTCATCTGGTTCAGGCGACCCAATCGGCGATATCTTGGAAGACTACTAG

[0615] SEQ ID NO:158

[0616] MSDMSIISAILVVAVAFLAGLESILDQFQFHQPLVACTLIGAATGNLTAGIMLGGSLQMITLAWANIGAAVAPDVALASVAAAIILVKGGKFTAEGIGVAIAIAILLAVAGLFLTMPVRTASIAFVHAADKAAEHGNIAGVERAYYLALLLQGLRIAVPAALLLAIPAQSVQHALGLMPDWLTHGLVVGGGMVVAVGYAMIINMMATR

[0617] SEQ ID NO:166

[0618] MAEKIQLSQADRKKVWWRSQFLQGAWNYERMQNLGWAYSLIPAIKKLYTNKEDQAAALKRHLEFFNTHPYVAAPIIGVTLALEEEKANGTEIEDAAIQGVKIGMMGPLAGIGDPVFWFTIRPILGALGASLAQAGNIAGPLIFFIGWNLIRMAFLWYTQELGYKAGSEITKDISGGILKDITKGASILGMFILAVLVERWVSVVFTVKLPGKVLPKGAYIEWPKGYVTGDQLKTILGQVNDKLSFDKIQVDTLQKQLDSLIPGLTGLLLTFACMWLLKKKVSPITIIIGLFVVGIVASFF

[0619] GIM

[0620] SEQ ID NO:206

[0621] ATGGCTGAAAAAATTCAATTATCTCAAGCGGATCGTAAAAGGTTTGGTGGCGCTCACAATTCTTGCAAGGTGCATGGAACTATGAACGTATGCAAAACTTGGGTTGGGCTTACTCACTCATTCCTGCTATCAAAACTTTATACTAACAAAGAGGACCAAGCCGCAGCTCTTAAACGTCACTTGGAATTCTTCAACACTCACCCTTACGTAGCTGCTCCTATCAT AGGGGTTACCTTAGCTCTTGAAGAAGAAGAAGCTAATGGTACTGAAATCGAAGATGCGGCTATCCAAGGGGTTAAAATCGGTATGATGGGTCCACTTGCCGGTATCGGTGACCCTGTCTTCTGGTTCACAATTCGTCCAATTCTTGGTGCCCTTGGTGCATCATTGGCACAAGCTGGTAACATTGCTGGTCCACTTATCTTCTTCATTGGTTGGAACCTTATCCGCAT GGCCTTCTTGTGGTACACTCAAGAACTTGGTTACAAAGCAGGTTCAGAAATCACTAAAGACATATCTGGTGGTATCTTGAAAGATATTACTAAAGGGGCATCAATACTTGGTATGTTCATCTTGGCCGTCCTCGTTGAACGTTGGGTATCTGTCGTCTTCACTGTAAAGCTTCCAGGTAAAGTTTTGCCTAAAGGTGCTTATATTGAATGGCCAAAAGGATATGTTACTGGTGACCAACTAAAAACTATCCTTGGTCAAGTCAACGATAAGCTTAGCTTTGATAAGATTCAAGTCGATACCCTACAAAAACAATTGGATTCATTAATTCCAGGTTTGACGGGACTTCTCCTTACTTTTGCATGTATGTGGTTGCTTAAGAAGAAAGTTTCACCAATCACAATCATCATCGGACTCTTTGTAGTTGGTATTGTTGCAAGCTTCTTCGGAATCATGTAA

[0622] SEQ ID NO:207

[0623] MAEKIQLSQADRKKGLVALTILARCMEL

[0624] SEQ ID NO:208

[0625] MAEKIQLSQADRKKGLVALTILARFMEL

[0626] SEQ ID NO:209

[0627] MSEKIQLSQADRKKGLVALTILARFMEL

[0628] SEQ ID NO:210

[0629] MAEKIQLSQADRRKGLVALTILARFMEL

[0630] SEQ ID NO:211

[0631] MSEKIQLSQADHKKGLVALTILARFMEL

[0632] SEQ ID NO:212_partial scrA829

[0633] ATGGATTACAAACAAATTGCAAAAGAAGTCATCGAAGCCCTCGGTGGACGTGAAAATGTTAACAGTGTTGCTCACTGTGCGACACGTCTACGTGTTATGGTTAAAGATGAAAACAAAATCAATAAAGAAAAA A GCTGAGAATATTGAAAAAGTTCAAGG

[0634] SEQ ID NO:213_pscrB829

[0635] ATTTAATAATCTCCTAATTTATTTATTAGTAATATAGGAAACGTTTTACCTAGAAAAATCAACGATTTATAATAAGAAAACAAAAAAATGCTAAACGTTTGACATATGACAGAAAGATGTTAAAATTAATATCGTAAAGAAAAGTGAAACGTTTTCAAAAACAAATTTTGTTAAAGATGTTAAAATTGATATCGTAAAGAAAAGCGAAACGTTTTCAAAAACAAATTTTATTAAGGAGAATTTTGCAA

[0636] SEQ ID NO:214

[0637] ATGGATTACAAACAAATTGCAAAAGAAGTCATCGAAGCCCTCGGTGGACGTGAAAATGTTAACAGTGTTGCTCACTGTGCGACACGTCTACGTGTTATGGTTAAAGATGAAACAAAATCAATAAAGAAAAAGCTGAGAATATTGAAAAAGTTCAAGG

[0638] SEQ ID NO:215

[0639] ATTTAATAATCTCCTAATTTATTTATTAGTAATATAGTAAACGTTTTACCTAGAAAAATCAACGATTTATAATAAGAAAACAAAAAAATGCTAAACGTTTGACATATGACAGAAAGATGTTAAAATTAATATCGTAAAGAAAGTGAAACGTTTTCAAAAACAAATTTTGTTAAAGATGTTAAAATTGATATCGTAAAGAAAGCGAAACGTTTTCAAAAACAAATTTTATTAAGGAGAATTTTGCAA

[0640] SEQ ID NO:216

[0641] CCAAATGGAGAACCGCCTCAAATGATAAATTTATTATTCAACACCAGTTGAGGTAGAGTAGAACCACAGTTATGGTTCTTGTGTATTTTTTTATCTCTTGTTTTTTCCCGAAATAAGAGTAATATAGAGCTATGCTTAATTTTTTAGGTAAAAAAATTATTTTAAAGAGGTAAATATAAAC

[0642] SEQ ID NO:217

[0643] CCAAATGGAGAACCGCCTCAAATGATAAATTTATTATTCAACACCAGTTGAGGTAGAGTAGAACCACAGTTATGGTTCTTGTGTATTTTTTTATCTCTTGTTTTTTTCCCGAAATAAGAGTAATATAGAGCTATGCTTAATTTTTTAGGTAAAAAAATTATTTTAAAGAGGTAAATATAAAC

[0644] SEQ ID NO:218

[0645] CCAAATGGAGAACCGCCTCAAATGATAAATTTATTATTCAACACCAGTTGAGGTAGAGTAGAACCACAGTTATGGTTCTTGTGTATTTTTTTATCTCTTGTTTTTTCCCCGAAATAAGAGTAATATAGAGCTATGCTTAATTTTTTAGGTAAAAAAATTATTTTAAAGAGGTAAATATAAAC

[0646] SEQ ID NO:219

[0647] CCAAATGGAGAACCGCCTCAAATGATAAATTTATTATTCAACACCAGTTGAGGTAGAGTAGAACCACAGTTATGGTTCTTGTGTATTTTTTTATCTCTTGCTTTTTCCCGAAATAAGAGTAATATAGAGCTATGCTTAATTTTTTAGGTAAAAAAATTATTTTAAAGAGGTAAATATAAAC

[0648] SEQ ID NO:220

[0649] CCAAATGGAGAACCGCCTCAAATGATAAATTTATTATTCAACACCAGTTGACGTAGAACC

[0650] ACAGTTATGGTTCTTGTGTATTTTTTTATCTCTTGTTTTTTCCCGAAATAGGTAGTGTAA

[0651] AATAAGTTGTGTAAACACAAAAAGGAATAAATCCGTTATAGTAGAGTTGCAAAACATTAC

[0652] TAGAAAGAGATTTATTCCTATGACTCCGTTTACCACAGAACTACTTAACTTCCTTGCCCA

[0653] AAAGCAAGATATTGATGAATTTTTCCGTACTTCTCTTGAAACAGCTATGAATGATCTGCT

[0654] TCAAGCAGAGTTATCAGCCTTTTTAGGGTATGAACCTTACGATAAATTAGGCTATAATTC

[0655] TGGGAATAGTCGTAACGGAAGCTATGCACGGAAATTCGAAACCAAATATGGGACTGTTCA

[0656] GTTGAGTATTCCTAGAGATCGTAATGGGAACTTTTAGTCCAGCTTTGCTTCCCGCTTATGG

[0657] ACGTCGAGATGACCACTTGGAAGAGATGGTTATCAAACTCTATCAAACCGGTGTAACGAC

[0658] TCGAGAAATTAGTGATATCATCGAGCGGAATGTATGGTCATCACTATAGTCCTGCCACAAT

[0659] TTCTAATATCTCAAAAGCAACTCAGGAGAATGTCGCTACTTTTCATGAGCGAAGCTTAGA

[0660] AGCCAATTACTCTGTTTTATTTCTTGACGGAACCTATCTTCCCTTAAGACGTGGAACCGT

[0661] TAGTAAAGAATGTATTCATATCGCACTTGGCATTACACCAGAAGGACAGAGGCTGTTCTT

[0662] TGGATATGAAATCGCTCCAAATGAAACAAATGCTTCTTGGTCCACCCTGTTAGACAAGCT

[0663] TCAAACCAAGGAATCCAACAGGTTTCTCTTGTAGTGACCGATGGCTTCAAGGGGCTTGA

[0664] AGAGATTATCAATCAGGCTTACCCATTAGCTAAAAC

[0665] AACGTTGCTTAATTCATATTAG

[0666] TCGAAATCTAGCTAGTAAAGTGAAACGAGCAGATAGAGCGGTTATTCTGGAGCAATTTAA

[0667] AACGATTTATCGTGCTGAAAATTTAGAAATGGCAGTGCAAGCTTTAGAGAACTTTATCGC

[0668] CGAATGGAAACCAAAGTATAGGAAAGTCATGGAAAGTCTGGAGAATACGGATAATCTTTT

[0669] AACTTTTTATTCAGTTTCCCTACCAGATTTGGCATAGCATTTATTCGACAAACCTCATTGA

[0670] GTCTCTTAACAAAGAAATCAAACGTCAAACGAAAGAAGGTCCTTTTTCCTAACGAGGA

[0671] GGCTCTGGAACGTTATTTAGTTACCCTGTTTGAAGATTATAATTTCAAGCAAAATCAACG

[0672] CATCCATAAAGGGTTTGGACAATGTACTGACACACTTGAAAGCTTATTTGATTAACATTC

[0673] TTCAACTCTACTTGAGTGTTTACACATAATTATTGACAGTATCCCGAAATAAGAGTAATA

[0674] TAGAGCTATGCTTAATTTTTTAGGTAAAAAATTATTTTAAAGAGGTAAATATAAAC

[0675] SEQ ID NO:221:

[0676] GGTAGTGTAAAATAAGTTGTGTAAACACAAAAAGGAATAAATCCGTTATAGTAGAGTTGC

[0677] AAAACATTACTAGAAAGAGATTTATTCCTATGACTCCGTTTACCACAGAACTACTTAACT

[0678] TCCTTGCCCAAAAGCAAGATATTGATGAATTTTTCCGTACTTCTCTTGAAACAGCTATGA

[0679] ATGATCTGCTTCAAGCAGAGTTATCAGCCTTTTTAGGGTATGAACCTTACGATAAATTAG

[0680] GCTATAATTCTGGGAATAGTCGTAACGGAAGCTATGCACGGAAATTCGAAACCAAATATG

[0681] GGACTGTTCAGTTGAGTATTCCTAGAGATCGTAATGGGAA

[0682] CTTTAGTCCAGCTTGCTTC

[0683] CCGCTTATGGACGTCGAGATGACCACTTGGAAGAGATGGTTATCAAACTCTATCAAACCG

[0684] GTGTAACGACTCGAGAAAATTAGTGATATCATCGAGCGAATGTATGGTCATCACTATAGTC

[0685] CTGCCACAATTTCTAATATCTCAAAAGCAACTCAGGAGAATGTCGCTACTTTTCATGAGC

[0686] GAAGCTTAGAAGCCAATTACTCTGTTTTATTTCTTGACGGAACCTATCTTCCCTTAAGAC

[0687] GTGGAACCGTTAGTAAAGAATGTATTCATATCGCACTTGGCATTACACCAGAAGGACAGA

[0688] AGGCTGTTCTTGGATATGAAATCGCTCCAAATGAAAACAATGCTTCTGGTCCACCCTGT

[0689] TAGACAAGCTTCAAACCAAGGAATCCAACAGGTTTCTCTTGTAGTGACCGATGGCTTCA

[0690] AGGGGCTTGAAGAGATTATCAATCAGGCTTACCCATTAGCTAAACAACAACGTTGCTTAA

[0691] TTCATATTAGTCGAAATCTAGCTAGTAAAGTGAAACGAGCAGATAGAGCGGTTATTCTGG

[0692] AGCAATTTAAAACGATTTATCGTGCTGAAAATTTAGAAATGGCAGTGCAAGCTTTAGAGA

[0693] ACTTTATCGCCGAATGGAAACCAAAGTATAGGAAAGTCATGGAAAGTCTGGAGAATACGG

[0694] ATAATCTTTTAACTTTTTATCAGTTTCCCTACCAGATTTGGCATAGCATTTATTCGACAA

[0695] ACCTCATTGAGTCTCTTAACAAAGAAATCAAACGTCAAACGAAAAAGAAGGTCCTTTTTC

[0696] CTAACGAGGAGGCTCTGGAACGTTATTTAGTTACCCTGTTTGAAGATTATAATTTCAAGC

[0697] AAAATCAACGCATCCATAAAGGGTTTGGACAATGTACTGACACACTTGAAAGCTTATTTG

[0698] ATTAACATTCTTCAACTCTACTTGAGTGTTTACACATAATTATTGACAGTATCCCGAAAT

[0699] A

[0700] SEQ ID NO:222>ST12997_manM_DNA_mutation

[0701] ATGTCAGATATGTCAATTATTTCTGCGATTTTGGTCGTAGCTGTTGCCTTCCTTGCTGGT

[0702] CTTGAAAGTATCCTTGACCAATTCCAATTCCACCAACCACTTGTTGCATGTACCCTCATC

[0703] GGTGCTGCCACAGGTAACCTCACTGCAGGTATCATGCTTGGTGGTTCTCTTCAAATGATT

[0704] ACCCTTGCTTGGGCAAACATCGGTGCTGCCGTAGCTCCTGACGTTGCCCTTGCATCTGTT

[0705] GCCGCTGCCATCATTTTGGTTAAAGGTGGTAAATTTACAGCTGAAGGTATCGGTGTTGCG

[0706] ATTGCAATAGCTATCCTGCTTGCAGTTGCAGGTCTCTTCCTAACTATGCCTGTTCGTACA

[0707] GCATCTATTGCCTTTGTTCATGCTGCAGATAAAGCTGCAGAACACGGAAACATCGCTGGT

[0708] GTTGAACGTGCATACTACCCTCGCTCTCCTTCTTCAAGGTTTGCGTATTGCTGTGCCAGC

[0709] AGCCCTTCTTCTTGCCATCCCGGCCCAATCTGTTCAACATGCCCTTGGCTTGATGCCTGA

[0710] CTGGCTCACCCATGGTTTGGTTGTCGGTGGTGGTATGGTCGTAGCCGTTGGTTACGCCAT

[0711] GATTATCAATATGATGGCTACTCGTGAAGTTTGGCCATTCTTCGCCATTGGTTTTGCTTT

[0712] GGCAGCAATTAGCCAATTGACACTTATCGCTCTTAGTACCATTGGTGTTGCCATCGCCTT

[0713] CATCTACCTCAACCTTTCTAAACAAGGTGGCGGAAATGGTGGCGGAAATGGTGGCGGAAC

[0714] TTCATCTGGTTCAGGCGACCCAATCGGCGATATCTTGGAAGACTACTAG

[0715] SEQ ID NO:223>ST12997_manM_protein mutation

[0716] MSDMSIISAILVVAVAFLAGLESILDQFQFHQPLVACTLIGAATGNLTAGIMLGGSLQMI

[0717] TLAWANIGAAVAPDVALASVAAAIILVKGGKFTAEGIGVAIAIAILLAVAGLFLTMPVRT

[0718] ASIAFVHAADKAAEHGNIAGVERAYYPRSPSSRFAYCCASSPSSCHPGPICSTCPWLDA*

[0719] strain

[0720] The DGCC number is the internal reference of the DuPont Danisco collection; the DSM number is the number assigned by the Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures (Inhoffenstr. 7B, D-38124 Braunschweig) under the Budapest Treaty.

[0721] With respect to the Streptococcus thermophilus strain DGCC7710 deposited on January 14, 2014 with the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures under the Budapest Treaty under the number DSM28255, we hereby confirm that the depositor, Danisco Deutschland GmbH (Busch-Johannsen-Strasse 1, D-25899 Niebüll, Germany), has authorized the applicant (DuPont Nutrition Biosciences ApS) to refer to the deposited biological material in this application. The expressions "DGCC7710 strain" and "DGCC7710 derivative" are used interchangeably with the expressions "DSM28255 strain" and "DSM28255 derivative".

[0722] A derivative of the DGCC7710 strain was designed, in which the glcK gene encodes a glucokinase in which the glutamic acid (E) at position 275 is replaced by the amino acid lysine (K). This derivative (DGCC12534) was deposited in the DSMZ on August 15, 2017 under the deposit number DSM32587.

[0723] -The Streptococcus thermophilus strain deposited on September 29, 2020 with the deposit number DSM33651 in the DSMZ [German Collection of Microorganisms and Cell Cultures, Inhofenstrasse 7B, D-38124 Braunschweig, Germany] (referred to herein as ST3 or DSM33651).

[0724] The following strains were deposited on 18 January 2022 at the Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures in the name of DuPont Nutrition Biosciences under the Budapest Treaty:

[0725] Streptococcus thermophilus strain ST1-ABU1 (deposited as DSM 34134);

[0726] Streptococcus thermophilus strain ST1-ABU4 (deposited as DSM 34140);

[0727] Streptococcus thermophilus strain ST1-GM-ABU1 (deposited as DSM34138);

[0728] Streptococcus thermophilus strain ST1-CM-ABU1 (deposited as DSM34139);

[0729] Streptococcus thermophilus strain ST2 (deposited as DSM 34132);

[0730] Streptococcus thermophilus strain ST2-ABU2 (deposited as DSM 34147);

[0731] Streptococcus thermophilus strain ST2-ABU3 (deposited as DSM 34146).

[0732] - A Streptococcus thermophilus strain deposited with the DSMZ [German Collection of Microorganisms and Cell Cultures, Inhofenstrasse 7B, D-38124 Braunschweig, Germany] on February 16, 2022 under the deposit number DSM 34172 (referred to herein as ST1 or DSM34172).

[0733] The applicant requires that samples of the deposited microorganisms described herein be provided to experts only prior to the date of grant of the patent.

[0734] In the case of those designations for which European patent protection is sought, samples of those deposited microorganisms may be obtained before the publication referring to the grant of the European patent or before the date on which the said application is refused or withdrawn, or is deemed to be withdrawn, and only to experts nominated by the person requesting access to the said samples and with the consent of i) the applicant and / or ii) the European Patent Office, as the case may be.

Claims

1. A lactose-positive, sucrose-negative Streptococcus thermophilus strain carrying one or more mutations selected from the group consisting of: 1) one or more mutations in one or more genes of a sucrose regulator; the mutations significantly reduce the ability of the strain to grow on sucrose and / or transport sucrose; and the mutations at least comprise i) a mutation in one or more of the scrA gene and / or the scrB gene and / or the scrR gene, and ii) optionally further mutations in the promoter region regulating the expression of the scrA, scrB and / or scrR genes, e.g. mutations that protect the strain from reversion to a sucrose-negative phenotype; and 2) optionally one or more further mutations affecting a glucose transporter, such as glcU and / or its expression, which restore or improve glucose consumption by the strain when the strain is grown on glucose as sole carbohydrate source.

2. The lactose-positive, sucrose-negative thermophilic Streptococcus strain according to claim 1, which is a glucose-positive or slow-growing strain on glucose.

3. The lactose-positive, sucrose-negative thermophilic Streptococcus strain of claim 1 or 2, having one or more mutations in one or more of the scrA and scrB genes of the sucrose regulon.

4. The lactose-positive, sucrose-negative thermophilic Streptococcus strain of claim 3, wherein the mutation in one or more genes of the sucrose regulon actually disrupts gene function in the scrA gene, for example, resulting in a mutation in the gene encoding EIIABC. 蔗糖 Insertion or deletion of the open reading frame of the protein gene results in translation of a truncated EIIABC encoded by the scrA gene. 蔗糖 Protein, for example, by inserting or deleting nucleotides in the open reading frame (ORF) of the scrA gene, such as a mutation by inserting or deleting nucleotides at or around the nucleotide corresponding to position 133 of SEQ ID NO:

212.

5. The lactose-positive, sucrose-negative thermophilic Streptococcus strain according to any one of claims 1 to 4, having a mutation in the promoter region regulating the expression of any one of the scrA, scrB and / or scrR genes.

6. The lactose-positive, sucrose-negative thermophilic Streptococcus strain according to claim 5, wherein the mutation is a SNP in the promoter region of the scrA and / or scrB gene, such as a SNP in the promoter region of the scrB gene, such as a mutation near the -10 and -35 sequences of the promoter, such as a mutation from T to G at position 38 of SEQ ID NO: 213 corresponding to the upstream of the scrB promoter, such as a mutation that will comprise the entire sequence identified by SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto (pscrB 829 ).

7. A lactose-positive, sucrose-negative thermophilus Streptococcus strain according to any one of claims 1 to 6, wherein the strain has a mutation that affects a glucose transporter, and when the strain is grown on glucose as a sole carbohydrate source, the mutation restores or improves the glucose consumption of the strain, such as when compared to a lactose-positive and sucrose-positive thermophilus Streptococcus parent strain, the growth of the strain on glucose is fully restored.

8. The lactose-positive, sucrose-negative thermophilic Streptococcus strain according to claim 7, wherein the mutation affecting the glucose transporter affects glcU and / or its expression, such as a mutation located in the promoter region of glcU, such as a mutation selected from the following a) Insertion of a T nucleotide at position -75 of the glcU promoter identified as SEQ ID NO: 216 in the poly-T region (pglcU IN_T ), for example a mutation containing a T nucleotide at position 102 of SEQ ID NO: 217, for example a mutation comprising the entire sequence identified by SEQ ID NO: 217 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto; b) Insertion of a C nucleotide at position -75 of the glcU promoter identified as SEQ ID NO: 216 in the poly-T region (pglcU IN_C ), for example a mutation that will contain a C nucleotide at position 102 of SEQ ID NO: 218, for example a mutation that will comprise the entire sequence identified by SEQ ID NO: 218 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto; c) a mutation of a T to C nucleotide at the position corresponding to position -81 of the glcU promoter identified as SEQ ID NO: 216 in the poly-T region (pglcU SNP_C ), for example a mutation that would contain a C nucleotide at position 96 of SEQ ID NO: 219, for example a mutation that would comprise the entire sequence identified by SEQ ID NO: 219 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto; d) inserting a sequence at a position corresponding to nucleotides 110 and 111 of the glcU promoter identified as SEQ ID NO: 216, for example inserting a sequence identified by nucleotides 1 to 1321 of SEQ ID NO: 221, for example a mutation (pglcU) comprising the entire sequence identified by SEQ ID NO: 220 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, or SEQ ID NO: 221 or a variant sequence having at least about 90%, such as at least about 95% identity thereto IS ).

9. The lactose-positive, sucrose-negative thermophilic Streptococcus strain according to any one of claims 1 to 8, further comprising one or more mutations selected from the group consisting of: a) a mutation in the glcK gene encoding glucokinase, the glucokinase activity of which in the strain is significantly reduced but not zero; and / or b) Generate code EIIC Man A mutation of the open reading frame shift of the gene for the protein, resulting in a stop codon at position 180 of SEQ ID NO: 222, for example, by inserting a nucleotide C at any of positions 438, 439 or 440 of SEQ ID NO: 223 in the manM gene (manM 12997 ); and / or a mutation comprising a substitution of nucleotide G to T at position 916 of SEQ ID NO: 79 of the manL gene (manL 13740 ), resulting in a stop codon (IIAB) at position 306 of the protein of SEQ ID: 80 Man ); and / or a mutation comprising a substitution of nucleotide G at position 625 of manM with nucleotide T (manM 16404 ), resulting in a stop codon at position 209; and / or a mutation comprising the insertion of a nucleotide A at a stretch of 5 nucleotides A at positions 37-41, resulting in a frameshift in the manN gene (resulting in manN 13741 mutant allele), making IID Man The protein is truncated at position 28 of SEQ ID NO:

167.

10. The lactose-positive, sucrose-negative Streptococcus thermophilus strain according to any one of claims 1 to 9, which is a galactose-negative strain, for example by mutations in the glcK gene and / or the ccpA gene, optionally in combination with a mutation in one of the genes encoding a protein of a mannose-glucose-specific PTS.

11. The lactose-positive, sucrose-negative thermophilic Streptococcus strain according to claims 9-10, which has a mutation in the glcK gene encoding glucokinase, wherein the glucokinase activity in the strain is significantly reduced but not zero.

12. The lactose-positive, sucrose-negative thermophilic Streptococcus strain according to any one of claims 9 to 11, wherein the mutated glcK gene encodes a glucokinase selected from the group consisting of: a) a glucokinase having an amino acid at position 275 which is not glutamic acid, in particular not an acidic amino acid, in particular lysine, b) The amino acid at position 275 is not glutamic acid, in particular not an acidic amino acid, in particular lysine (glcK 378 ), and a glucokinase with arginine at position 278 and / or serine at position 279; and c) a glucokinase having an amino acid at position 144 which is not glycine, in particular not an aliphatic amino acid, in particular serine.

13. The lactose-positive, sucrose-negative thermophilic Streptococcus strain according to any one of claims 9 to 12, wherein the mutated glcK gene encodes a glucokinase whose sequence is selected from the group consisting of: a) a sequence as defined in SEQ ID NO: 25, wherein the amino acid at position 275 is not glutamic acid, in particular is not an acidic amino acid, in particular is lysine; and b) a GlcK variant sequence having at least 90% similarity or identity to SEQ ID NO: 25, wherein the amino acid corresponding to position 275 of SEQ ID NO: 25 (or the amino acid at position 275 of the glucokinase) is not glutamic acid, in particular not an acidic amino acid, in particular lysine; c) a sequence as defined in SEQ ID NO: 46, wherein the amino acid at position 144 is not glycine, in particular is not an aliphatic amino acid, in particular is serine; and d) a GlcK variant sequence having at least 90% similarity or identity to SEQ ID NO: 46, wherein the amino acid at position 144 of the glucose kinase corresponding to SEQ ID NO: 46 (or the amino acid at position 144 of the glucose kinase) is not glycine, in particular is not an aliphatic amino acid, in particular is serine.

14. The lactose-positive, sucrose-negative Streptococcus thermophilus strain according to any one of claims 1 to 13, further carrying a mutation in the ccpA gene.

15. The lactose-positive, sucrose-negative Streptococcus thermophilus strain according to any one of claims 1 to 14, further carrying a mutation in a gene encoding a mannose-glucose specific PTS protein, such as a mutation in a gene selected from the group consisting of a manL gene, a manM gene and a manN gene.

16. The lactose-positive, sucrose-negative thermophilic Streptococcus strain according to claim 14 or 15, wherein the ccpA gene carries a mutation selected from the group consisting of: a nonsense mutation located between the 1st nucleotide and the 270th nucleotide of the coding sequence of the ccpA gene, and a mutation located in the first quarter of the coding sequence of the ccpA gene resulting in a frameshift of the open reading frame of the ccpA gene.

17. The lactose-positive, sucrose-negative thermophilic Streptococcus strain of claim 16, wherein the sequence of the mutated ccpA gene is selected from the group consisting of: a) a sequence as defined in SEQ ID NO: 71; and b) a ccpA variant sequence that is at least 90%, such as at least 95%, identical to SEQ ID NO:

71.

18. The lactose-positive, sucrose-negative Streptococcus thermophilus strain according to any one of claims 1 to 17, wherein when the strain is capable of fermenting milk as described in Example 5 during a 24-hour fermentation period: a) the concentration of residual lactose in the fermented milk is less than 60 mM; and / or b) the pH of the milk is reduced to a pH (pH) at which the acidification rate becomes clearly less than 0.1 mUpH / min 终止 ), wherein the pH 终止 Between 4.4 and 5.3, and optionally between pH 6 and pH 5.5, the slope is at least -0.008 UpH / min.

19. The lactose-positive, sucrose-negative Streptococcus thermophilus strain according to any one of claims 1 to 18, wherein the strain has the ability to consume lactose in excess compared to a parent strain without the one or more mutations, for example wherein the residual amount of lactose when fermenting sweet milk as described and determined according to Example 6 is less than 100 mM, for example less than 90 mM, 80 mM, 70 mM, 60 mM, 50 mM, 40 mM, 30 mM, 20 mM, 10 mM, 8 mM, 6 mM, 4 mM, 2 mM or 1 mM.

20. The lactose-positive, sucrose-negative Streptococcus thermophilus strain according to any one of claims 1-19, wherein the strain has the ability to release increased amounts of glucose compared to a parent strain without the one or more mutations, for example wherein the glucose release as described and determined according to Example 6 is above 10 mM, for example above 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM or 90 mM.

21. A lactose-positive, sucrose-negative thermophilic Streptococcus strain according to any one of claims 1-20, wherein the strain releases a glucose concentration determined as described in Example 6 of at least 50 mM, such as at least 60, 70, 80 or 90 mM, or an increase of at least 150% or at least 200%, such as 300%, 400%, 500% compared to the glucose concentration released by the parent strain, when both strains are determined as described in Example 6.

22. A composition comprising at least one, in particular one, lactose-positive, sucrose-negative Streptococcus thermophilus strain according to any one of claims 1 to 21, in particular in combination with another lactic acid bacterium, in particular in combination with one or more strains selected from the group consisting of: a strain of the genus Lactobacillus, such as a strain of Lactobacillus delbrueckii subsp bulgaricus; a strain of the genus Lactococcus, such as a strain of Lactococcus lactis; or a strain of the genus Bifidobacterium.

23. A method for producing a fermented milk product, in particular fermented milk, comprising inoculating a milk substrate with a lactose-positive, sucrose-negative Streptococcus thermophilus strain according to any one of claims 1 to 21 or a composition according to claim 19, and fermenting the inoculated milk to obtain a fermented milk product.

24. Use of a lactose-positive, sucrose-negative Streptococcus thermophilus strain according to any one of claims 1 to 21 or a composition according to claim 22 for the manufacture of a fermented milk product.

25. A fermented milk product comprising at least one, in particular one, lactose-positive, sucrose-negative Streptococcus thermophilus strain according to any one of claims 1 to 21 or a lactose-positive, sucrose-negative Streptococcus thermophilus strain obtained by the method according to claim 23.

26. A thermophilic Streptococcus strain, e.g., a lactose-positive, sucrose-negative thermophilic Streptococcus strain selected from the group consisting of: - strain deposited on January 18, 2022 in the name of DuPont Nutrition Biosciences under the number DSM34132 at the Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures in accordance with the Budapest Treaty; - strain deposited on January 18, 2022 in the name of DuPont Nutrition Biosciences under the number DSM34134 at the Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures in accordance with the Budapest Treaty; - strain deposited on January 18, 2022 in the name of DuPont Nutrition Biosciences under the number DSM34138 at the Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures in accordance with the Budapest Treaty; - strain deposited on January 18, 2022 in the name of DuPont Nutrition Biosciences under the number DSM34139 at the Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures in accordance with the Budapest Treaty; - strains deposited on January 18, 2022 in the name of DuPont Nutrition Biosciences under the number DSM34140 at the Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures in accordance with the Budapest Treaty; - the strain deposited on January 18, 2022 at the Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures under the number DSM34146 in the name of DuPont Nutrition Biosciences under the Budapest Treaty; - the strain deposited on January 18, 2022 at the Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures under the number DSM34147 in the name of DuPont Nutrition Biosciences under the Budapest Treaty; and -The strain deposited on February 16, 2022 at the Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures under the number DSM34172 in the name of DuPont Nutrition Biosciences under the Budapest Treaty.

27. A lactose-positive, sucrose-negative thermophilic Streptococcus strain selected from the group consisting of: a strain corresponding to the Streptococcus thermophilus strain deposited at the Leibniz Institute DSMZ under the Budapest Treaty on January 14, 2014 under the number DSM34172 in the name of Danisco Deutschland GmbH [referred to herein as ST1 or DSM34172 strain], wherein the sequence of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, resulting in a frameshift, such that a truncated EIIABC is translated 蔗糖 (scrA 829 FS ); a strain corresponding to the Streptococcus thermophilus strain deposited on January 14, 2014 at the Leibniz Institute DSMZ under the Budapest Treaty in the name of Danisco Deutschland GmbH under the number DSM34172 [referred to herein as ST1 or DSM34172 strain], wherein the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto (pscrB 829 ); a strain corresponding to the Streptococcus thermophilus strain deposited on January 14, 2014 at the Leibniz Institute DSMZ under the Budapest Treaty under the number DSM34172 in the name of Danisco Deutschland GmbH [referred to herein as ST1 or DSM34172 strain], wherein the locus (pglcU) as the promoter region upstream of the glcU gene is replaced by the corresponding sequence as shown in any one of SEQ ID NOs: 217 to 220 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pglcU SNP_C ,pglcU IN_T ,pglcU IN_C ,pglcU IS ); a strain corresponding to the Streptococcus thermophilus strain deposited at the Leibniz Institute DSMZ under the Budapest Treaty on January 14, 2014 under the number DSM34172 in the name of Danisco Deutschland GmbH [referred to herein as ST1 or DSM34172 strain], wherein the sequence of the open reading frame (ORF) of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, resulting in a frameshift, such that a truncated EIIABC is translated 蔗糖 (scrA 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pscrB 829 ); a strain corresponding to the Streptococcus thermophilus strain deposited at the Leibniz Institute DSMZ under the Budapest Treaty on January 14, 2014 under the number DSM34172 in the name of Danisco Deutschland GmbH [referred to herein as ST1 or DSM34172 strain], wherein the sequence of the open reading frame (ORF) of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, resulting in a frameshift, such that a truncated EIIABC is translated 蔗糖 (scrA 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pscrB 829 ), and the gene locus (pglcU) as the promoter region upstream of the glcU gene is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217-220 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pglcU SNP_C ,pglcU IN_T ,pglcU IN_C ,pglcU IS ); a strain corresponding to the Streptococcus thermophilus strain deposited at the Leibniz Institute DSMZ under the Budapest Treaty on January 14, 2014 under the number DSM34172 in the name of Danisco Deutschland GmbH [referred to herein as ST1 or DSM34172 strain], wherein the sequence of the open reading frame (ORF) of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, resulting in a frameshift, such that a truncated EIIABC is translated 蔗糖 (scrA 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pscrB 829 ), and the gene locus (pglcU) as the promoter region upstream of the glcU gene is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217-220 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pglcU SNP_C ,pglcU IN_T ,pglcU IN_C ,pglcU IS ), and optionally wherein the glcK gene encoding glucokinase comprises a sequence as defined by SEQ ID NO: 25 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, wherein the amino acid at position 275 is lysine (glcK 378 ), and optionally further comprising generating an encoding said EIIC Man A mutation of the open reading frame shift of the gene for the protein, resulting in a stop codon at position 180 of SEQ ID NO: 222, for example, by inserting a nucleotide C at any of positions 438, 439 or 440 of SEQ ID NO: 223 in the manM gene (manM 12997 ), and / or a mutation comprising a substitution of nucleotide G to T at position 916 of SEQ ID NO: 79 of the manL gene (manL 13740 ), resulting in a stop codon (IIAB) at position 306 of the protein of SEQ ID: 80 Man ); and / or a mutation comprising a substitution of nucleotide G at position 625 of manM with nucleotide T (manM 16404 ), resulting in a stop codon at position 209; and / or a mutation comprising the insertion of a nucleotide A at a stretch of 5 nucleotides A at positions 37-41, resulting in a frameshift in the manN gene (resulting in manN 13741 mutant allele), making IID Man The protein is truncated at position 28 of SEQ ID NO: 167; and optionally further comprises the ccpA sequence of SEQ ID NO: 71 or a ccpA variant sequence having at least about 90%, such as at least 95%, identity to SEQ ID NO: 71; a strain of Streptococcus thermophilus, such as the strain deposited on 14 January 2014 at the Leibniz Institute DSMZ under the Budapest Treaty in the name of Danisco Deutschland GmbH under the number DSM34172 [referred to herein as strain ST1 or DSM34172], which strain comprises one or more of the following characteristics: 1) The sequence of the open reading frame (ORF) of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, resulting in a frame shift, so that a truncated EIIABC is translated 蔗糖 (scrA 829 FS ), 2) The sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pscrB 829 ), 3) the locus (pglcU) as the promoter region upstream of the glcU gene is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217-220 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pglcU SNP_C ,pglcU IN_T ,pglcU IN_C ,pglcU IS ), 4) The glcK gene encoding glucokinase comprises a sequence as defined in SEQ ID NO: 25 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, wherein the amino acid at position 275 is lysine (glcK 378 ), 5) Having generated the EIIC encoding Man A mutation of the open reading frame shift of the gene for the protein, resulting in a stop codon at position 180 of SEQ ID NO: 222, for example, by inserting a nucleotide C at any of positions 438, 439 or 440 of SEQ ID NO: 223 in the manM gene (manM 12997 ), and / or a mutation comprising a substitution of nucleotide G to T at position 916 of SEQ ID NO: 79 of the manL gene (manL 13740 ), resulting in a stop codon (IIAB) at position 306 of the protein of SEQ ID: 80 Man ); and / or a mutation comprising a substitution of nucleotide G at position 625 of manM with nucleotide T (manM 16404 ), resulting in a stop codon at position 209; and / or a mutation comprising the insertion of a nucleotide A at a stretch of 5 nucleotides A at positions 37-41, resulting in a frameshift in the manN gene (resulting in manN 13741 mutant allele), making IID Man The protein is truncated at position 28 of SEQ ID NO:167; 6) a ccpA sequence having the sequence of SEQ ID NO:71 or a ccpA variant sequence having at least about 90%, such as at least 95%, identity to SEQ ID NO:71; a strain corresponding to the Streptococcus thermophilus strain deposited at the Leibniz Institute DSMZ under the Budapest Treaty on January 14, 2014 under the number DSM28255 in the name of Danisco Deutschland GmbH [referred to herein as ST4 or DSM28255 strain], wherein the sequence of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, resulting in a frameshift, such that a truncated EIIABC is translated 蔗糖 (scrA 829 FS ); a strain corresponding to the Streptococcus thermophilus strain deposited at the Leibniz Institute DSMZ under the Budapest Treaty on January 14, 2014 under the number DSM28255 in the name of Danisco Deutschland GmbH [referred to herein as ST4 or DSM28255 strain], wherein the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto (pscrB 829 ); a strain corresponding to the Streptococcus thermophilus strain deposited on January 14, 2014 at the Leibniz Institute DSMZ under the Budapest Treaty under the number DSM28255 in the name of Danisco Deutschland GmbH [referred to herein as ST4 or DSM28255 strain], wherein the locus (pglcU) as the promoter region upstream of the glcU gene is replaced by the corresponding sequence as shown in any one of SEQ ID NOs: 217 to 220 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pglcU SNP_C ,pglcU IN_T ,pglcU IN_C ,pglcU IS ); a strain corresponding to the Streptococcus thermophilus strain deposited on January 14, 2014 at the Leibniz Institute DSMZ under the Budapest Treaty under the number DSM28255 in the name of Danisco Deutschland GmbH [referred to herein as ST4 or DSM28255 strain], wherein the sequence of the open reading frame (ORF) of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, resulting in a frameshift, such that a truncated EIIABC is translated 蔗糖 (scrA 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pscrB 829 ); a strain corresponding to the Streptococcus thermophilus strain deposited on January 14, 2014 at the Leibniz Institute DSMZ under the Budapest Treaty under the number DSM28255 in the name of Danisco Deutschland GmbH [referred to herein as ST4 or DSM28255 strain], wherein the sequence of the open reading frame (ORF) of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, resulting in a frameshift, such that a truncated EIIABC is translated 蔗糖 (scrA 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pscrB 829 ), and the gene locus (pglcU) as the promoter region upstream of the glcU gene is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217-220 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pglcU SNP_C ,pglcU IN_T ,pglcU IN_C ,pglcU IS ); a strain corresponding to the Streptococcus thermophilus strain deposited on January 14, 2014 at the Leibniz Institute DSMZ under the Budapest Treaty under the number DSM28255 in the name of Danisco Deutschland GmbH [referred to herein as ST4 or DSM28255 strain], wherein the sequence of the open reading frame (ORF) of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, resulting in a frameshift, such that a truncated EIIABC is translated 蔗糖 (scrA 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pscrB 829 ), and the gene locus (pglcU) as the promoter region upstream of the glcU gene is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217-220 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pglcU SNP_C ,pglcU IN_T ,pglcU IN_C ,pglcU IS ), and optionally wherein the glcK gene encoding glucokinase comprises a sequence as defined by SEQ ID NO: 25 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, wherein the amino acid at position 275 is lysine (glcK 378 ), and optionally further comprising generating an encoding said EIIC Man A mutation of the open reading frame shift of the gene for the protein, resulting in a stop codon at position 180 of SEQ ID NO: 222, for example, by inserting a nucleotide C at any of positions 438, 439 or 440 of SEQ ID NO: 223 in the manM gene (manM 12997 ), and / or a mutation comprising a substitution of nucleotide G to T at position 916 of SEQ ID NO: 79 of the manL gene (manL 13740 ), resulting in a stop codon (IIAB) at position 306 of the protein of SEQ ID: 80 Man ); and / or a mutation comprising a substitution of nucleotide G at position 625 of manM with nucleotide T (manM 16404 ), resulting in a stop codon at position 209; and / or a mutation comprising the insertion of a nucleotide A at a stretch of 5 nucleotides A at positions 37-41, resulting in a frameshift in the manN gene (resulting in manN 13741 mutant allele), making IID Man The protein is truncated at position 28 of SEQ ID NO: 167; and optionally further comprises the ccpA sequence of SEQ ID NO: 71 or a ccpA variant sequence having at least about 90%, such as at least 95%, identity to SEQ ID NO: 71; a strain of Streptococcus thermophilus, such as the strain deposited on 14 January 2014 at the Leibniz Institute DSMZ under the Budapest Treaty in the name of Danisco Deutschland GmbH under the number DSM 28255 [referred to herein as strain ST4 or DSM 28255], which strain comprises one or more of the following characteristics: 1) The sequence of the open reading frame (ORF) of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, resulting in a frame shift, so that a truncated EIIABC sucrose (scrA) is translated. 829 FS ), 2) The sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pscrB 829 ), 3) the locus (pglcU) as the promoter region upstream of the glcU gene is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217-220 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pglcU SNP_C ,pglcU IN_T ,pglcU IN_C ,pglcU IS ), 4) The glcK gene encoding glucokinase comprises a sequence as defined in SEQ ID NO: 25 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, wherein the amino acid at position 275 is lysine (glcK 378 ), 5) Having generated the EIIC encoding Man A mutation of the open reading frame shift of the gene for the protein, resulting in a stop codon at position 180 of SEQ ID NO: 222, for example, by inserting a nucleotide C at any of positions 438, 439 or 440 of SEQ ID NO: 223 in the manM gene (manM 12997 ), and / or a mutation comprising a substitution of nucleotide G to T at position 916 of SEQ ID NO: 79 of the manL gene (manL 13740 ), resulting in a stop codon (IIAB) at position 306 of the protein of SEQ ID: 80 Man ); and / or a mutation comprising a substitution of nucleotide G at position 625 of manM with nucleotide T (manM 16404 ), resulting in a stop codon at position 209; and / or a mutation comprising the insertion of a nucleotide A at a stretch of 5 nucleotides A at positions 37-41, resulting in a frameshift in the manN gene (resulting in manN 13741 mutant allele), making IID Man The protein is truncated at position 28 of SEQ ID NO:167; 6) a ccpA sequence having the sequence of SEQ ID NO:71 or a ccpA variant sequence having at least about 90%, such as at least 95%, identity to SEQ ID NO:71; - A strain corresponding to the Streptococcus thermophilus strain deposited at the Leibniz Institute DSMZ under the number DSM34132 on January 18, 2022 in the name of DuPont Nutrition Biosciences under the Budapest Treaty [referred to herein as ST2 or DSM34132 strain], wherein the sequence of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, resulting in a frameshift, such that a truncated EIIABC is translated 蔗糖 (scrA 829 FS ); - A strain corresponding to the Streptococcus thermophilus strain deposited at the Leibniz Institute DSMZ under the number DSM34132 on January 18, 2022 in the name of DuPont Nutrition Biosciences under the Budapest Treaty [referred to herein as ST2 or DSM34132 strain], wherein the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto (pscrB 829 ); - A strain corresponding to the Streptococcus thermophilus strain deposited at the Leibniz Institute DSMZ under the number DSM34132 on January 18, 2022 in the name of DuPont Nutrition Biosciences under the Budapest Treaty [referred to herein as ST2 or DSM34132 strain], wherein the locus (pglcU) as the promoter region upstream of the glcU gene is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217-220 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pglcU SNP_C ,pglcU IN_T ,pglcU IN_C ,pglcU IS ); A strain corresponding to the Streptococcus thermophilus strain deposited at the Leibniz Institute DSMZ under the number DSM34132 on January 18, 2022 in the name of DuPont Nutrition Biosciences under the Budapest Treaty [referred to herein as ST2 or DSM34132 strain], wherein the sequence of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, resulting in a frameshift, such that a truncated EIIABC sucrose (scrA) is translated 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pscrB 829 ); - A strain corresponding to the Streptococcus thermophilus strain deposited at the Leibniz Institute DSMZ under the number DSM34132 on January 18, 2022 in the name of DuPont Nutrition Biosciences under the Budapest Treaty [referred to herein as ST2 or DSM34132 strain], wherein the sequence of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, resulting in a frameshift, such that a truncated EIIABC is translated 蔗糖 (scrA 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pscrB 829 ), and the gene locus (pglcU) as the promoter region upstream of the glcU gene is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217-220 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pglcU SNP_C ,pglcU IN_T ,pglcU IN_C ,pglcU IS ); - A strain corresponding to the Streptococcus thermophilus strain deposited at the Leibniz Institute DSMZ under the number DSM34132 on January 18, 2022 in the name of DuPont Nutrition Biosciences under the Budapest Treaty [referred to herein as ST2 or DSM34132 strain], wherein the sequence of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, resulting in a frameshift, such that a truncated EIIABC is translated 蔗糖 (scrA 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pscrB 829 ), and the gene locus (pglcU) as the promoter region upstream of the glcU gene is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217-220 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pglcU SNP_C ,pglcU IN_T ,pglcU IN_C ,pglcU IS ), and optionally wherein the glcK gene encoding glucokinase comprises a sequence as defined by SEQ ID NO: 25 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, wherein the amino acid at position 275 is lysine (glcK 378 ), and optionally further comprising generating an encoding said EIIC Man A mutation of the open reading frame shift of the gene for the protein, resulting in a stop codon at position 180 of SEQ ID NO: 222, for example, by inserting a nucleotide C at any of positions 438, 439 or 440 of SEQ ID NO: 223 in the manM gene (manM 12997 ), and / or a mutation comprising a substitution of nucleotide G to T at position 916 of SEQ ID NO: 79 of the manL gene (manL 13740 ), resulting in a stop codon (IIAB) at position 306 of the protein of SEQ ID: 80 Man ); and / or a mutation comprising a substitution of nucleotide G at position 625 of manM with nucleotide T (manM 16404 ), resulting in a stop codon at position 209; and / or a mutation comprising the insertion of a nucleotide A at a stretch of 5 nucleotides A at positions 37-41, resulting in a frameshift in the manN gene (resulting in manN 13741 mutant allele), making IID Man The protein is truncated at position 28 of SEQ ID NO: 167; and optionally further comprises the ccpA sequence of SEQ ID NO: 71 or a ccpA variant sequence having at least about 90%, such as at least 95%, identity to SEQ ID NO: 71; - a strain of Streptococcus thermophilus, such as the strain deposited on January 18, 2022 at the Leibniz Institute DSMZ under the number DSM34132 in the name of DuPont Nutrition Biosciences under the Budapest Treaty [referred to herein as ST2 or DSM34132 strain], which strain comprises one or more of the following characteristics: 1) The sequence of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, resulting in a frame shift, so that a truncated EIIABC is translated 蔗糖 (scrA 829 FS ), 2) The sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pscrB 829 ), 3) the locus (pglcU) as the promoter region upstream of the glcU gene is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217-220 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pglcU SNP_C ,pglcU IN_T ,pglcU IN_C ,pglcU IS ), 4) The glcK gene encoding glucokinase comprises a sequence as defined in SEQ ID NO: 25 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, wherein the amino acid at position 275 is lysine (glcK 378 ), 5) Having generated the EIIC encoding Man A mutation of the open reading frame shift of the gene for the protein, resulting in a stop codon at position 180 of SEQ ID NO: 222, for example, by inserting a nucleotide C at any of positions 438, 439 or 440 of SEQ ID NO: 223 in the manM gene (manM 12997 ), and / or a mutation comprising a substitution of nucleotide G to T at position 916 of SEQ ID NO: 79 of the manL gene (manL 13740 ), resulting in a stop codon (IIAB) at position 306 of the protein of SEQ ID: 80 Man ); and / or a mutation comprising a substitution of nucleotide G at position 625 of manM with nucleotide T (manM 16404 ), resulting in a stop codon at position 209; and / or a mutation comprising the insertion of a nucleotide A at a stretch of 5 nucleotides A at positions 37-41, resulting in a frameshift in the manN gene (resulting in manN 13741 mutant allele), making IID Man The protein is truncated at position 28 of SEQ ID NO:167; 6) a ccpA sequence having the sequence of SEQ ID NO:71 or a ccpA variant sequence having at least about 90%, such as at least 95%, identity to SEQ ID NO:71; - A strain corresponding to the Streptococcus thermophilus strain deposited with the DSMZ [German Collection of Microorganisms and Cell Cultures, Inhofenstrasse 7B, D-38124 Braunschweig, Germany] on September 29, 2020 under the accession number DSM33651 (referred to herein as ST3 or DSM33651), wherein the sequence of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, resulting in a frameshift, such that a truncated EIIABC is translated 蔗糖 (scrA 829 FS ); - A strain corresponding to the Streptococcus thermophilus strain deposited with the DSMZ [German Collection of Microorganisms and Cell Cultures, Inhofenstrasse 7B, Braunschweig, Germany, D-38124] on September 29, 2020 under the accession number DSM33651 (referred to herein as ST3 or DSM33651), wherein the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto (pscrB 829 ); - A strain corresponding to the Streptococcus thermophilus strain deposited on September 29, 2020 with the accession number DSM33651 at DSMZ [German Collection of Microorganisms and Cell Cultures, Inhofenstrasse 7B, Braunschweig, Germany, D-38124] (referred to herein as ST3 or DSM33651), wherein the locus (pglcU) as the promoter region upstream of the glcU gene is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217-220 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pglcU SNP_C ,pglcU IN_T ,pglcU IN_C ,pglcU IS ); - A strain corresponding to the Streptococcus thermophilus strain deposited with the DSMZ [German Collection of Microorganisms and Cell Cultures, Inhofenstrasse 7B, D-38124 Braunschweig, Germany] on September 29, 2020 under the accession number DSM33651 (referred to herein as ST3 or DSM33651), wherein the sequence of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, resulting in a frameshift, such that a truncated EIIABC is translated 蔗糖 (scrA 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pscrB 829 ); - A strain corresponding to the Streptococcus thermophilus strain deposited with the DSMZ [German Collection of Microorganisms and Cell Cultures, Inhofenstrasse 7B, D-38124 Braunschweig, Germany] on September 29, 2020 under the accession number DSM33651 (referred to herein as ST3 or DSM33651), wherein the sequence of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, resulting in a frameshift, such that a truncated EIIABC is translated 蔗糖 (scrA 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pscrB 829 ), and the gene locus (pglcU) as the promoter region upstream of the glcU gene is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217-220 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pglcU SNP_C ,pglcU IN_T ,pglcU IN_C ,pglcU IS ); - A strain corresponding to the Streptococcus thermophilus strain deposited with the DSMZ [German Collection of Microorganisms and Cell Cultures, Inhofenstrasse 7B, D-38124 Braunschweig, Germany] on September 29, 2020 under the accession number DSM33651 (referred to herein as ST3 or DSM33651), wherein the sequence of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, resulting in a frameshift, such that a truncated EIIABC is translated 蔗糖 (scrA 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pscrB 829 ), and the gene locus (pglcU) as the promoter region upstream of the glcU gene is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217-220 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pglcU SNP_C ,pglcU IN_T ,pglcU IN_C ,pglcU IS ), and optionally wherein the glcK gene encoding glucokinase comprises a sequence as defined by SEQ ID NO: 25 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, wherein the amino acid at position 275 is lysine (glcK 378 ), and optionally further comprising generating an encoding of the EIIC Man A mutation of the open reading frame shift of the gene for the protein, resulting in a stop codon at position 180 of SEQ ID NO: 222, for example, by inserting a nucleotide C at any of positions 438, 439 or 440 of SEQ ID NO: 223 in the manM gene (manM 12997 ), and / or a mutation comprising a substitution of nucleotide G to T at position 916 of SEQ ID NO: 79 of the manL gene (manL 13740 ), resulting in a stop codon (IIAB) at position 306 of the protein of SEQ ID: 80 Man ); and / or a mutation comprising a substitution of nucleotide G at position 625 of manM with nucleotide T (manM 16404 ), resulting in a stop codon at position 209; and / or a mutation comprising the insertion of a nucleotide A at a stretch of 5 nucleotides A at positions 37-41, resulting in a frameshift in the manN gene (resulting in manN 13741 mutant allele), making IID Man The protein is truncated at position 28 of SEQ ID NO: 167; and optionally further comprises the ccpA sequence of SEQ ID NO: 71 or a ccpA variant sequence having at least about 90%, such as at least 95%, identity to SEQ ID NO: 71; - A strain of Streptococcus thermophilus deposited on September 29, 2020 with the accession number DSM33651 at DSMZ [German Collection of Microorganisms and Cell Cultures, Inhofenstrasse 7B, D-38124 Braunschweig, Germany] (hereinafter referred to as ST3 or DSM33651), comprising one or more of the following characteristics: 1) The sequence of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, resulting in a frame shift, so that a truncated EIIABC is translated 蔗糖 (scrA 829 FS ), 2) The sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pscrB 829 ), 3) the locus (pglcU) as the promoter region upstream of the glcU gene is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217-220 or a variant sequence having at least about 90%, such as at least about 95% identity thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence having at least about 90%, such as at least about 95% identity thereto (pglcU SNP_C ,pglcU IN_T ,pglcU IN_C ,pglcU IS ), 4) The glcK gene encoding glucokinase comprises a sequence as defined in SEQ ID NO: 25 or a variant sequence having at least about 90%, such as at least about 95%, identity thereto, wherein the amino acid at position 275 is lysine (glcK 378 ), 5) Having generated the EIIC encoding Man A mutation of the open reading frame shift of the gene for the protein, resulting in a stop codon at position 180 of SEQ ID NO: 222, for example, by inserting a nucleotide C at any of positions 438, 439 or 440 of SEQ ID NO: 223 in the manM gene (manM 12997 ), and / or a mutation comprising a substitution of nucleotide G to T at position 916 of SEQ ID NO: 79 of the manL gene (manL 13740 ), resulting in a stop codon (IIAB) at position 306 of the protein of SEQ ID: 80 Man ); and / or a mutation comprising a substitution of nucleotide G at position 625 of manM with nucleotide T (manM 16404 ), resulting in a stop codon at position 209; and / or a mutation comprising the insertion of a nucleotide A at a stretch of 5 nucleotides A at positions 37-41, resulting in a frameshift in the manN gene (resulting in manN 13741 mutant allele), making IID Man The protein is truncated at position 28 of SEQ ID NO:167; 6) a ccpA sequence having the ccpA sequence of SEQ ID NO:71 or a ccpA variant sequence having at least about 90%, such as at least 95%, identity to SEQ ID NO:

71.

28. A polynucleotide comprising one or more mutations in one or more genes of a sucrose regulon, said one or more mutations significantly reducing the ability of a strain selected from the group consisting of: a) a strain corresponding to the strain of Streptococcus thermophilus deposited on January 14, 2014 at the Leibniz Institute DSMZ under the number DSM 28255 in the name of Danisco Deutschland GmbH under the Budapest Treaty, wherein the polynucleotide replaces the corresponding sequence in DSM 28255; b) a strain corresponding to the strain of Streptococcus thermophilus deposited at the Leibniz Institute DSMZ under the number DSM34132 on January 18, 2022 in the name of DuPont Nutrition Biosciences under the Budapest Treaty, wherein the polynucleotide replaces the corresponding sequence in DSM34132; c) a strain corresponding to the strain of Streptococcus thermophilus deposited on September 29, 2020 under the accession number DSM33651 in the DSMZ [German Collection of Microorganisms and Cell Cultures, Inhofenstrasse 7B, Braunschweig, Germany, postal code D-38124], wherein the polynucleotide replaces the corresponding sequence in DSM33651; d) A strain corresponding to the Streptococcus thermophilus strain deposited at the Leibniz Institute DSMZ under the number DSM34172 on February 16, 2022 in the name of DuPont Nutrition Biosciences under the Budapest Treaty, wherein the polynucleotide replaces the corresponding sequence in DSM34172.

29. A polynucleotide comprising one or more mutations affecting a glucose transporter, said one or more mutations restoring or improving glucose consumption in a strain selected from: a) a strain corresponding to the strain of Streptococcus thermophilus deposited on January 14, 2014 at the Leibniz Institute DSMZ under the number DSM 28255 in the name of Danisco Deutschland GmbH under the Budapest Treaty, wherein the polynucleotide replaces the corresponding sequence in DSM 28255; b) a strain corresponding to the strain of Streptococcus thermophilus deposited at the Leibniz Institute DSMZ under the number DSM34132 on January 18, 2022 in the name of DuPont Nutrition Biosciences under the Budapest Treaty, wherein the polynucleotide replaces the corresponding sequence in DSM34132, c) a strain corresponding to the strain of Streptococcus thermophilus deposited on September 29, 2020 under the accession number DSM33651 in the DSMZ [German Collection of Microorganisms and Cell Cultures, Inhofenstrasse 7B, Braunschweig, Germany, postal code D-38124], wherein the polynucleotide replaces the corresponding sequence in DSM33651; d) A strain corresponding to the Streptococcus thermophilus strain deposited at the Leibniz Institute DSMZ under the number DSM34172 on February 16, 2022 in the name of DuPont Nutrition Biosciences under the Budapest Treaty, wherein the polynucleotide replaces the corresponding sequence in DSM34172.

30. The polynucleotide of any one of claims 28 or 29, wherein the one or more mutations are as identified in any one of claims 3-8.

31. A method for selecting a lactose-positive, sucrose-negative thermophilic Streptococcus strain, the method comprising the steps of: 1) providing a strain of thermophilic Streptococcus, comprising a) one or more mutations in one or more genes of a sucrose regulator; said mutations significantly reduce the ability of said strain to grow on sucrose and / or transport sucrose; and said mutations at least comprise i) a mutation in one or more of the scrA gene and / or the scrB gene and / or the scrR gene, and ii) optionally further mutations in the promoter region regulating the expression of the scrA, scrB and / or scrR genes, e.g. mutations that protect the strain from reversion to a sucrose-negative phenotype; 2) screening the thermophilic Streptococcus strain from step 1), wherein the ability of the strain to grow on sucrose and transport sucrose is significantly reduced; as well as 3) optionally before, simultaneously with or after any of steps 1 and 2, screening for S. thermophilus strains having restored or improved glucose consumption, optionally from strains having one or more further mutations affecting a glucose transporter, e.g., affecting glcU and / or its expression.

32. The method according to claim 31, wherein the strain is as defined in any one of claims 1-21.

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