Microorganisms for producing D-tagatose

Abstract

This disclosure relates to microorganisms useful for the biosynthesis of D-tagatose. Methods for producing the microorganisms of this disclosure and methods for producing D-tagatose are also provided.

Description

[Technical Field]

[0001] The subject matter of this disclosure is compositions and methods for producing low-calorie sugars in microorganisms. The subject matter of this disclosure further relates to compositions and methods for producing tagatose in microorganisms. [Background technology]

[0002] Current industrial production of D-tagatose relies on a complex, multi-step in vitro enzymatic synthesis. One method involves hydrolysis of lactose to galactose and glucose, and conversion of galactose to tagatose by L-arabinose isomerase (EC 5.3.1.4) at high temperatures. The main drawbacks of this in vitro synthesis include the need for multiple separation and purification processes, and the thermodynamically unfavorable nature of the isomerization of galactose to tagatose. Another method involves conversion of D-fructose-6-phosphate (F6P) to D-tagatose-6-phosphate (T6P) using fructose 1,6-bisphosphate aldolase (EC 4.1.2.13), followed by dephosphorylation to D-tagatose by a non-specific phosphatase. In vivo, this pathway is inhibited by intracellular regulators. In vitro, this requires the addition of expensive cofactors and other enzymes. Therefore, there is a need for novel methods and compositions for producing D-tagatose. [Overview of the project]

[0003] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 537,140, ​​filed on 7 September 2023, which is incorporated in its entirety by reference.

[0004] Sequence List This application includes a sequence listing submitted electronically in XML format, the entire sequence listing of which is incorporated into this disclosure by reference. The XML copy created on September 5, 2024, is named 081906-1460213_253710PC_SL and has a size of 148,479 bytes.

[0005] In one embodiment, the disclosure relates to a recombinant microorganism containing an exogenous epimerase, wherein the recombinant microorganism produces an increased amount of tagatose compared to the naturally occurring microorganism.

[0006] In some embodiments, the epimerase is the tagatose-1,6-bisphosphate aldolase subunit GatZ or the ketose diphosphate aldolase subunit KbaZ. In some embodiments, the epimerase contains an amino acid sequence that is at least about 80% identical to the amino acid sequence shown in SEQ ID NO: 58. In some embodiments, the epimerase contains or consists of the amino acid sequence shown in SEQ ID NO: 58.

[0007] In some embodiments, the epimerase is Escherichia coli (E. coli) KbaZ. In some embodiments, the epimerase contains an amino acid sequence that is at least about 80% identical to the amino acid sequence shown in SEQ ID NO: 60. In some embodiments, the epimerase contains or consists of the amino acid sequence shown in SEQ ID NO: 60.

[0008] In some embodiments, the microorganism further comprises a recombinant phosphatase. In some embodiments, the phosphatase is hexitol phosphatase A (HxpA). In some embodiments, the phosphatase is Escherichia coli (E. coli) HxpA. In some embodiments, the phosphatase comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence shown in SEQ ID NO: 62. In some embodiments, the phosphatase comprises or consists of the amino acid sequence shown in SEQ ID NO: 62.

[0009] In some embodiments, the recombinant microorganism further comprises an exogenous galactose:H+ symporter (GalP) and glucokinase (Glk). In some embodiments, the GalP is E. coli GalP, and the Glk is E. coli Glk. In some embodiments, the GalP comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence shown in SEQ ID NO: 38, and the Glk comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence shown in SEQ ID NO: 40. In some embodiments, the GalP comprises the amino acid sequence shown in SEQ ID NO: 38, and the Glk comprises the amino acid sequence shown in SEQ ID NO: 40.

[0010] In some embodiments, the recombinant microorganism further includes mutations in genes encoding enzymes of the pentose phosphate pathway compared to the naturally occurring microorganism. In some embodiments, the enzyme of the pentose phosphate pathway is glucose-6-phosphate 1-dehydrogenase (Zwf). In some embodiments, the recombinant microorganism further includes mutations in genes encoding enzymes of the glycolysis pathway compared to the naturally occurring microorganism.

[0011] In some embodiments, the glycolytic enzyme is phosphofructokinase-1 (PfkA), phosphofructokinase-2 (PfkB), or pyruvate kinase (PykF). In some embodiments, the glycolytic enzyme is phosphofructokinase-1 (PfkA).

[0012] In some embodiments, the recombinant microorganism further includes a mutation in a gene encoding an enzyme that converts fructose-6-phosphate to psicose-6-phosphate. In some embodiments, the enzyme that converts fructose-6-phosphate to psicose-6-phosphate is D-allulose-6-phosphate 3-epimerase (AlsE). In some embodiments, the recombinant microorganism further includes a mutation in a gene encoding an enzyme in the mannose biosynthesis pathway. In some embodiments, the enzyme in the mannose biosynthesis pathway is mannose-6-phosphate isomerase (ManA).

[0013] In some embodiments, the recombinant microorganism further comprises mutations in a gene encoding an enzyme for carbohydrate metabolism. In some embodiments, the enzyme for carbohydrate metabolism is the D-tagatose-1,6-bisphosphate aldolase subunit (GatY). In some embodiments, the enzyme for carbohydrate metabolism is the D-tagatose-1,6-bisphosphate aldolase subunit (KbaY).

[0014] In some embodiments, the recombinant microorganism further comprises mutations in genes encoding glycogen biosynthesis enzymes selected from phosphoglucumutase (Pgm), UDP-glucose pyrophosphorylase, glycogen synthase, glycogen branching enzyme, glycogenin, and combinations thereof. In some embodiments, the glycogen biosynthesis enzyme is phosphoglucumutase (Pgm).

[0015] In another embodiment, the disclosure relates to a microorganism comprising a recombinant polynucleotide encoding an epimerase, wherein the expression of the epimerase and the phosphatase results in increased tagatose production compared to a microorganism lacking the recombinant polynucleotide.

[0016] In some embodiments, the epimerase is the tagatose-1,6-bisphosphate aldolase subunit GatZ or the ketose diphosphate aldolase subunit KbaZ. In some embodiments, the epimerase is Escherichia coli (E. coli) GatZ. In some embodiments, the epimerase is Escherichia coli (E. coli) KbaZ.

[0017] In some embodiments, the microorganism further comprises a recombinant phosphatase. In some embodiments, the phosphatase is hexitol phosphatase A (HxpA). In some embodiments, the phosphatase is Escherichia coli (E. coli) HxpA.

[0018] In some embodiments, the microorganism further comprises a mutation in at least one gene. In some embodiments, the at least one gene is selected from glucose-6-phosphate 1-dehydrogenase (zwf), phosphofructokinase-1 (pfkA), phosphofructokinase-2 (pfkB), pyruvate kinase (pykF), D-allulose-6-phosphate 3-epimerase (alsE), mannose-6-phosphate isomerase (manA), phosphoglucomutase (pgm), D-tagatose-1,6-bisphosphate aldolase subunit (gatY), D-tagatose-1,6-bisphosphate aldolase subunit (kbaY), and combinations thereof.

[0019] In some embodiments, the at least one gene is glucose-6-phosphate 1-dehydrogenase (zwf), phosphofructokinase-1 (pfkA), D-allulose-6-phosphate 3-epimerase (alsE), mannose-6-phosphate isomerase (manA), phosphoglucomutase (pgm), D-tagatose-1,6-bisphosphate aldolase subunit (gatY), D-tagatose-1,6-bisphosphate aldolase subunit (kbaY), or a combination thereof.

[0020] In some embodiments, the mutation is a deletion. In some embodiments, the mutation reduces or eliminates the expression or activity of the enzyme. In some embodiments, the microorganism is Escherichia coli, Bacillus subtilis, or Lactococcus lactis.

[0021] In one embodiment, the disclosure also relates to a method for producing tagatose, comprising culturing a microorganism disclosed herein under conditions suitable for converting a substrate into tagatose. In some embodiments, the substrate comprises glucose.

[0022] Furthermore, this disclosure relates to a method for producing a tagatose-containing food, comprising culturing a microorganism disclosed herein under conditions suitable for converting a substrate to tagatose, and mixing the tagatose with at least one food to form a tagatose-containing food. In some embodiments, the food is a beverage, yogurt, ice cream, baked good, or nutrition bar. [Brief explanation of the drawing]

[0023] [Figure 1]Figures 1A and 1B illustrate the pathway for the biosynthetic production of D-tagatose. Glucose is taken up by the phosphotransferase system (PTS) or GalP / Glk and phosphorylated to glucose-6-phosphate (G6P). G6P is then isomerized to fructose-6-phosphate (F6P) by glucose-6-phosphate isomerase. F6P is epimerized to tagatose-6-phosphate by the tagatose-1,6-bisphosphate aldolase subunit GatZ or the ketose diphosphate aldolase subunit KbaZ, and then dephosphorylated to free tagatose by native phosphatases. Finally, free tagatose can diffuse across the cell membrane into the supernatant. Competitive pathways include the pentose phosphate pathway catalyzed by glucose-6-phosphate dehydrogenase (Zwf), glycolysis catalyzed by phosphofructokinases A and B (PfkA and B), the conversion of F6P to psicose-6-phosphate catalyzed by D-allulose-6-phosphate 3-epimerase, and the mannose biosynthesis pathway catalyzed by mannose-6-phosphate isomerase (ManA).

[0024] [Figure 2] Figure 2 shows the tagatose production capacity of Escherichia coli (E. coli). Cells were grown for 24 hours at 30°C in M9P medium containing 10 g / L glucose (M9 minimal medium containing 5 g / L yeast extract).

[0025] [Figure 3] Figure 3 shows the gene replacement experiment for D-tagatose production. The gatZ gene was deleted in AL4330 to create AL4386. Two plasmids, pAL2521 (empty) and pAL2490 (PLlacO1:gatZ) (Table 2), were constructed under the inducible promoter PLlacO1, as a negative control and to express gatZ, respectively. Cells were grown at 30°C in M9P medium containing 10 g / L glucose and induced with 1 mM IPTG.

[0026] [Figure 4] Figure 4 shows a comparison of endogenous phosphatases for D-tagatose production. Seven plasmids, pAL2490, pAL2491, pAL292, pAL2493, pAL2494, pAL2495, and pAL2496, were constructed to express additional hxpA, hxpB, ybiV, yidA, yigL, yihX, or yqaB under the inducible promoter PLlacO1 along with gatZ. AL4330, containing the phosphatase plasmids, was cultured at 30°C in M9P medium containing 10 g / L glucose and induced with 1 mM IPTG.

[0027] [Figure 5] Figures 5A–5C illustrate strategies for D-tagatose biosynthesis. Figure 5A shows the ismoring strategy for D-tagatose production. Figure 5B shows the phosphorylation and dephosphorylation strategies for D-tagatose. Figure 5C shows the proposed biosynthetic production of D-tagatose in Escherichia coli (E. coli). Deleted steps are located outside the tagatose production module box. Added and expressed steps are located inside the tagatose production module box. PTS is a phosphotransferase; GatZ is a putative tagatose-1,6-bisphosphate aldolase 2 chaperone; KbaZ is a putative tagatose-1,6-bisphosphate aldolase 1 chaperone; HxpA is hexitol phosphatase A; Zwf is NADP+-dependent glucose-6-phosphate dehydrogenase; Pgm is phosphoglucomutase; PfkA is 6-phosphofructokinase 1; PfkB is 6-phosphofructokinase 2; AlsE is D-allulose-6-phosphate 3-epimerase; ManA is mannose-6-phosphate isomerase; GatY is tagatose-1,6-bisphosphate aldolase 2; and KbaY is tagatose-1,6-bisphosphate aldolase 1.

[0028] [Figure 6]Figures 6A-6D show the D-tagatose production capacity of Escherichia coli (E. coli). Cells were grown in M9P medium containing 10 g L-1 glucose at 37°C until the OD600 was approximately 0.4, and then grown at 30°C for 24 hours. 1 mM IPTG was added when the OD600 was approximately 0.4. Figure 6A shows D-tagatose production in AL1050 (MG1655+lacIq tetR specR), AL3755 (AL1050 with ΔpfkA), and AL4240 (AL1050 with ΔpfkA Δzwf) (Table 4). Figure 6B shows the effect of each epimerase gene deletion on D-tagatose production in AL4240. Figure 6C shows D-tagatose production in AL4424 (AL1050 with ΔpfkA Δzwf ΔgatZ) with and without additional gatZ expression (Tables 4 and 5). Figure 6D shows the effect of additional expression of each phosphatase gene and gatZ on D-tagatose production in AL4424 (AL1050 with ΔpfkA Δzwf ΔgatZ). Error bars indicate sd (n=3 biological repeats).

[0029] [Figure 7] Figures 7A and 7B illustrate the regulation of gene expression for D-tagatose production. Cells were grown in M9P medium containing 10 g L-1 glucose at 37°C until the OD600 was approximately 0.4, and then at 30°C for 24 hours. When the OD600 was approximately 0.4, 1 mM IPTG was added as needed. Figure 7A shows that each candidate epimerase gene was expressed with hxpA under the PLlacO1 promoter (Table 5) in AL4424 (AL1050 with ΔpfkA Δzwf ΔgatZ, Table 4). Figure 7B shows that either gatZ or kbaZ and hxpA were expressed under PLlacO1 or PgadB. The start codon of hxpA was changed from GTG to ATG (hxpA*). Errors are shown as sd (n=3 biological repeats).

[0030] [Figure 8]Figure 8 shows the effect of gene deletion on D-tagatose production. Cells were grown in M9P medium containing 10 g L-1 glucose at 37°C until OD600 was approximately 0.4, and then at 30°C for 24 hours. Errors are indicated by sd. (n=3 biological replicates).

[0031] [Figure 9] Figures 9A to 9F show high cell density D-tagatose production. Cultures were grown in M9P medium with glucose concentrations of 40g L-1 (Figures 9A to 9C) and 15g L-1 (Figures 9D to 9F) at 37°C until the OD600 was approximately 0.4 to 0.6. If necessary, induction with 1 mM IPTG was performed, and the cultures were grown for a further 30 minutes. The cultures were then centrifuged and resuspended in M9P medium containing 40g L-1 (Figures 9A to 9C) and 15g L-1 (Figures 9D to 9F) glucose until the OD600 was approximately 10. If necessary, induction with 1 mM IPTG was performed, and the cultures were grown at 30°C. M9P medium containing 40g / L-1 (Figures 9A to 9C) and 15g / L-1 (Figures 9D to 9F) glucose was added to the production medium daily. Figures 9A and 9D show D-tagatose production. Figures 9B and 9E show D-fructose production. Figures 9D and 9F show D-mannitol production. Error bars indicate sd (n=3 biological repeats).

[0032] [Figure 10] Figure 10 shows the effect of gene deletion on D-mannose and D-psicose production. Cells were grown in M9P medium containing 10 g L-1 glucose at 37°C until OD600 was approximately 0.4, and then at 30°C for 24 hours. Each strain was transformed with pAL2606 (PgadB:gatZ-hxpA). Error bars indicate sd (n=3 biological repeats).

[0033] [Figure 11]Figure 11 shows glucose consumption of AL4534 under high cell density conditions. Glucose consumption of AL4534 with pAL2606 or pAL2607. Cultures were grown in M9P medium containing 40 g L-1 glucose at 37°C until the OD600 was approximately 0.4. The cultures were then centrifuged and resuspended in M9P medium containing 40 g L-1 glucose until the OD600 was approximately 10, and grown at 30°C for 24 hours. Error bars indicate sd(n=3 biological repeats). [Modes for carrying out the invention]

[0034] The market for rare sugars as food products, nutritional supplements, and health aids is expanding. Among rare sugars, D-tagatose is attracting particular attention. However, current methods for producing D-tagatose are costly, inefficient, thermodynamically unfavorable, and limit its potential for widespread use. Importantly, this disclosure addresses various obstacles in D-tagatose production, including thermodynamic barriers, limited yields, the need for purified enzymes, and the addition of cofactors. Another important finding disclosed herein is that *E. coli* naturally possesses a thermodynamically favorable pathway for D-tagatose production, which allows for improved D-tagatose production without the introduction of heterologous genes by increasing the expression of native genes and eliminating competing pathways. The subject of this disclosure is to facilitate the industrial-scale production of D-tagatose without requiring expensive enzyme purification or the difficult separation of raw materials and products.

[0035] This disclosure is based in part on the discovery that microorganisms can be created that have specific genetic modifications (e.g., gene deletions) to produce low-calorie sugars. In certain embodiments, the low-calorie sugar is tagatose. For clarity rather than limitation, a detailed description of the subject matter of this disclosure is divided into the following subsections. 1. Definition, 2. Microorganisms that produce tagatose, 3. Methods for producing and constructing microorganisms. 4. A method for manufacturing Tagatose, and 5.Food

[0036] ●1.Definition The terms used herein generally have the common meanings in the art within the context of the present invention and in the specific context in which each term is used. Certain terms are described below or elsewhere in this specification to provide additional guidance to practitioners when describing the methods and compositions of the present invention and the methods for preparing and using them.

[0037] As used herein, the use of the words "a" or "an" in conjunction with the term "comprising" in the claims and / or specification may mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more."

[0038] The terms “about” or “approximately” mean an acceptable range of error for a particular value as determined by those skilled in the art, which in part depends on how the value is measured or determined, i.e., the limits of the measuring system. For example, “about” can mean a difference of three standard deviations or more than three standard deviations, according to convention in the art. Alternatively, “about” can mean a range of up to 20% difference from a given value, preferably up to 10% difference, more preferably up to 5% difference, and more preferably up to 1% difference. Or, particularly with respect to biological systems or processes, the term can mean a difference of one order of magnitude from a given value, preferably up to five times the difference, and more preferably up to two times the difference.

[0039] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” and “contain(s)” as used herein, and their variations thereof, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. This disclosure also assumes other embodiments of the embodiments or elements presented herein, whether expressly described or not, that “comprising,” “consisting of,” and “consisting essentially of.”

[0040] As used herein, the term “microorganism” means any organism that exists as a microscopic cell and is contained within the realms of archaea, bacteria, or eukaryotes, where eukaryotes include yeasts and filamentous fungi, protozoa, algae, or higher protists. In certain embodiments, the term includes, but is not limited to, all species of bacteria, archaea, and bacteria, as well as eukaryotic microorganisms such as yeasts and fungi, and organisms having prokaryotic or eukaryotic or microscopic size. In certain embodiments, the term microorganism includes cells that can be cultured to produce chemical substances (e.g., sugars). In certain embodiments, the microorganism is a prokaryotic microorganism. In certain embodiments, the prokaryotic microorganism is a bacterium.

[0041] As used herein, the terms “bacterium,” “bacteria,” or “eubacteria” refer to the realm of prokaryotes. In certain embodiments, bacteria include Gram-negative bacteria, Gram-positive bacteria, proteobacteria, cyanobacteria, spirochetes and related species, Planctomyces, Bacteroides, Chlamydia, green sulfur bacteria, green non-sulfur bacteria, radiation-resistant micrococcuses, and Thermotoga and Thermosipho thermophiles.

[0042] As used herein, the term “Gram-negative bacteria” includes cocci, non-enterobacilli, and enterobacilli. Genera of Gram-negative bacteria include, but are not limited to, Neisseria, Spirillum, Pasteurella, Brucella, Yersinia, Francisella, Haemophilus, Bordetella, Escherichia, Salmonella, Shigella, Klebsiella, Proteus, Vibrio, and Pseudo Examples include the genera Pseudomonas, Bacteroides, Acetobacter, Aerobacter, Agrobacterium, Azotobacter, Spirilla, Serratia, Vibrio, Rhizobium, Chlamydia, Rickettsia, Treponema, and Fusobacterium.

[0043] As used herein, the term "Gram-positive bacteria" includes cocci, non-spore-forming bacilli, and spore-forming bacilli. Examples of Gram-positive bacteria include, but are not limited to, Actinomyces, Bacillus, Clostridium, Corynebacterium, Erysipelothrix, Lactobacillus, Listeria, Mycobacterium, Myxococcus, Nocardia, Staphylococcus, Streptococcus, and Streptomyces.

[0044] As used herein, the term “recombinant microorganism” refers to a microorganism containing one or more recombinant polynucleotides.

[0045] As used herein, the term “exogenous” refers to a molecule that is not naturally found in a given yeast, bacterium, organism, microorganism, or cell in nature, and / or is not produced by them. As used herein, the term “endogenous” refers to a molecule that is naturally found in a given yeast, bacterium, organism, microorganism, or cell in nature, and / or is produced by them.

[0046] As used herein, the terms “nucleic acid molecule,” “nucleotide sequence,” or “polynucleotide” refer to a single-stranded covalent sequence of nucleotides in which the 3' and 5' ends of each nucleotide are linked by phosphodiester bonds, or a double-stranded covalent sequence. Nucleic acid molecules may contain deoxyribonucleotide bases or ribonucleotide bases and may be produced synthetically in vitro or isolated from natural sources.

[0047] As used herein, “recombinant polynucleotide” means a polynucleotide whose exact nucleotide sequence is exotic (i.e., not found in nature) to a given host. In certain embodiments, the recombinant polynucleotide sequence is found in a given host in non-natural (e.g., more or less than expected) quantities, or further comprises two or more subsequences of the polynucleotide whose relationship to each other is not the same as that found in nature. For example, but not limited to, the recombinant polynucleotide may have two or more sequences from unrelated polynucleotides or from endogenous nucleotides arranged to create a new polynucleotide. In certain embodiments, the disclosure provides for the introduction of a recombinant polynucleotide encoding a polypeptide not normally found in the microorganism into a microorganism. Referring to the genome of a microorganism, the polynucleotide sequence encoding the polypeptide is recombinant or heterologous.

[0048] As used herein, “gene” refers to a DNA region (including exons and introns) that codes for a gene product, and all DNA regions that regulate the production of a gene product, whether or not such regulatory sequences are adjacent to coding sequences and / or transcription sequences. In certain non-limiting embodiments, a gene includes translation regulatory sequences such as promoter sequences, terminators, ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, origins of replication, matrix attachment sites, and locus regulatory regions.

[0049] The terms “polypeptide,” “peptide,” “amino acid sequence,” and “protein,” as used interchangeably herein, refer to molecules formed from the linkage of at least two amino acids. The linkage between one amino acid residue and the next is an amide bond, sometimes also called a peptide bond. Polypeptides can be obtained by suitable methods known in the art, including isolation from natural sources, expression in recombinant expression systems, chemical synthesis, or enzymatic synthesis. These terms can be applied to amino acid polymers, in which one or more amino acid residues are artificial chemical mimics of corresponding naturally occurring amino acids, and also to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0050] As used herein, the term “amino acid” may refer to natural and synthetic amino acids, as well as amino acid analogs and amino acid mimes that function similarly to natural amino acids. Natural amino acids are those encoded by the genetic code, as well as those subsequently modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs and amino acid derivatives may refer to compounds having the same basic chemical structure as natural amino acids, i.e., hydrogen, a carboxyl group, an amino group, and a carbon atom bonded to an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs may have a modified R group (e.g., norleucine) or a modified peptide skeleton, but retain the same basic chemical structure as natural amino acids. Amino acid mimes are chemical compounds that have a structure different from the general chemical structure of amino acids, but function similarly to naturally occurring amino acids. Non-exclusive examples of amino acids include tryptophan, phenylalanine, histidine, glycine, cysteine, alanine, tyrosine, serine, methionine, asparagine, leucine, asparagine, threonine, isoleucine, proline, glutamic acid, aspartic acid, hydroxylproline, arginine, cystine, glutamine, lysine, valine, ornithine, taurine, and combinations thereof.

[0051] As used herein, the term “isolated” refers to material that has been removed from at least one naturally associated component (for example, removed from its original environment).

[0052] As used herein, the terms “decrease” and “decrease” refer to a measurable decrease in an endpoint (e.g., enzyme activity, compound production, protein expression) of at least about 10%, at least about 50%, at least about 75%, or at least about 90%. In certain embodiments, the decrease may range from about 10% to about 100%.

[0053] As used herein, the terms “increase,” “rise,” and “rise” refer to a measurable increase in an endpoint (e.g., enzyme activity, compound production, protein expression) of at least about 10%, at least about 50%, at least about 75%, or at least about 90%. In certain embodiments, the increase may be between about 10% and about 100%. In certain embodiments, the increase may be at least about 10 times, at least about 100 times, or at least about 1000 times or more, or at least more than 1000 times. In certain embodiments, the increase may be at least about 100 times, at least about 1000 times or more, or at least about 10,000 times or more.

[0054] As used herein, the term "isomerase" refers to any enzyme of EC class 5 that catalyzes a geometric or structural change within a single molecule. Depending on the type of isomerization they catalyze, isomerases may be called racemases or epimerases (EC subclass 5.1), cis-trans isomerases (EC subclass 5.2), intramolecular oxidoreductases (EC subclass 5.3), intramolecular transferases (mutases) (EC subclass 5.4), or intramolecular lyases (EC subclass 5.5), while other isomerases are placed in EC subclass 5.99.

[0055] As used herein, the term “epimerase” refers to a class of enzymes that catalyze the inversion of chiral groups in substrates having several chiral centers.

[0056] As used herein, the term “dehydrogenase” refers to any enzyme that catalyzes the removal of hydrogen atoms (e.g., dehydrogenation) in biological reactions. Dehydrogenases are present in many biochemical pathways and are involved in driving electron transport chain reactions in cellular respiration. In certain embodiments, dehydrogenases act in conjunction with hydrogen-receptor coenzymes NAD and FAD.

[0057] As used herein, the term "phosphatase" refers to a class of enzymes that catalyze the removal of phosphate groups from organic compounds. In certain embodiments, the phosphatase catalyzes the removal of phosphate groups from sugars. In certain embodiments, the sugar is a hexose.

[0058] As used herein, the term "aldolase" refers to an enzyme of subclass EC 4.1.2 (i.e., an aldehyde lyase) that catalyzes aldol condensation and the reverse thereof.

[0059] Techniques for determining the sequence identity of nucleic acids and amino acids are known in the art. Typically, such techniques involve determining the nucleotide sequence of a gene's mRNA and / or the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide sequence or a second amino acid sequence. Genomic sequences can also be determined and compared in this manner. Generally, identity refers to the exact nucleotide-to-nucleotide correspondence of two polynucleotide sequences, or the exact amino acid-to-amino acid correspondence of two polypeptide sequences. Two or more sequences (polynucleotides or amino acids) can be compared by determining their percentage identity. Whether nucleic acid sequences or amino acid sequences, the percentage identity of two sequences is the number of exact matches between the two aligned sequences divided by the length of the shorter sequence, multiplied by 100. Unless otherwise indicated, percentage identity is determined for two sequences that have been compared and aligned for the maximum correspondence across a comparison window or specified region, as measured using the BLAST or BLAST 2.0 sequence comparison algorithm with default parameters. For example, see the NCBI website at ncbi.nlm.nih.gov / BLAST. For example, BLASTN and BLASTP can be used with the following default parameters: Genetic code = standard; filter = none; strand = both; cutoff = 60; expected value = 10; matrix = BLOSUM62; description = 50 sequences; sort criterion = high score; database = non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swiss protein+Spupdate+PIR. Details of these programs can be found on the GenBank website.

[0060] A “mutation” within a gene can include, for example, changes in nucleotides, deletions of one or more nucleotides (the nucleotides may include the entire coding sequence and / or the promoter or other regulatory sequences), and insertions of one or more nucleotides (which may occur in the coding sequence of a gene or its regulatory components (e.g., the gene's promoter)). Mutations can also include, for example, mutations that reduce or eliminate function (e.g., nonsense mutations), and genomic changes that reduce or knock out the expression of a gene product.

[0061] ●2. Microorganisms that produce Tagatose This disclosure provides genetically modified microorganisms. In certain embodiments, the microorganisms of this disclosure can produce increased amounts of tagatose, for example, compared to naturally occurring control microorganisms.

[0062] D-tagatose is a naturally occurring but rare monosaccharide. It is a ketohexose with the same empirical formula as common monosaccharides such as glucose and fructose. It is the 4-position epimer of fructose. Its enantiomer, L-tagatose, is not known in nature but has been synthesized. D-tagatose has the following formula: [ka]

[0063] D-tagatose is 92% as sweet as sucrose (e.g., table sugar), but has only about one-third the nutritional energy (e.g., calories). As a result, D-tagatose can be used as a substitute for sucrose and artificial sweeteners.

[0064] This disclosure provides genetically modified microorganisms with increased tagatose production compared to naturally occurring microorganisms. Figures 1 and 5C illustrate the biochemical pathways regulated in the exemplary microorganisms of this disclosure. In these exemplary microorganisms, glucose is taken up and phosphorylated to glucose-6-phosphate (G6P) by a phosphotransferase system (PTS) or GalP / Glk. G6P is then isomerized to fructose-6-phosphate (F6P) by glucose-6-phosphate isomerase. F6P is then epimerized to tagatose-6-phosphate by the tagatose-1,6-bisphosphate aldolase subunit GatZ and / or ketose diphosphate aldolase subunit KbaZ, which is then dephosphorylated by a native phosphatase to free tagatose. Finally, the free tagatose can diffuse across the cell membrane into the supernatant. In some embodiments, the microorganisms of the present disclosure also involve gene editing (e.g., knockout) of certain competitive pathways, such competitive pathways include the pentose phosphate pathway catalyzed by glucose-6-phosphate dehydrogenase (Zwf); glycolysis catalyzed by phosphofructokinase A and phosphofructokinase B (PfkA and PfkB); the conversion of fructose-6-phosphate to psicose-6-phosphate catalyzed by allulose-6-phosphate 3-epimerase (AlsE); and the mannose biosynthesis pathway catalyzed by mannose-6-phosphate isomerase (ManA).

[0065] 2.1. Tagatose-producing enzyme In certain embodiments, the microorganism of the Disclosure comprises the overexpression of at least one gene encoding an enzyme that catalyzes a reaction for producing tagatose. In certain embodiments, the microorganism of the Disclosure comprises a recombinant polynucleotide encoding at least one enzyme that catalyzes a reaction for producing tagatose. In certain embodiments, the enzyme is an epimerase, for example, an epimerase that converts fructose-6-phosphate (F6P) to tagatose-6-phosphate. Non-limiting examples of epimerases include tagatose-1,6-bisphosphate aldolase, ketose diphosphate aldolase, methylmalonyl-CoA epimerase, UDP-galactose 4-epimerase, UDP-glucose 4-epimerase, UDP-glucuronide 4-epimerase, UDP-glucuronide 5'-epimerase, ribose-5-phosphate epimerase, GDP-mannose 3,5-epimerase, L-ribulose phosphate 4-epimerase, UDP- Examples include N-acetylglucosamine 2-epimerase, UDP-N-acetylglucosamine 4-epimerase, UDP-galactose 4-epimerase, UDP-glucose 4-epimerase, UDP-glucuronic acid 4-epimerase, UDP-glucuronic acid 5'-epimerase, GDP-mannose 3,5-epimerase, methylmalonyl-CoA epimerase, ribose-5-phosphate epimerase, and UDP-N-acetylglucosamine 2-epimerase.

[0066] In certain embodiments, the epimerase is the tagatose-1,6-bisphosphate aldolase subunit GatZ (UniProt number P0C8J8). GatZ catalyzes the reversible epimerization of D-tagatose-6-phosphate to D-fructose-6-phosphate. In certain embodiments, GatZ is Escherichia coli GatZ. In certain embodiments, GatZ is Sigella sonnei GatZ, Sigella flexinelli GatZ, or Enterobacteriaceae GatZ. In certain embodiments, GatZ contains an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence shown in SEQ ID NO: 58. In certain embodiments, GatZ contains an amino acid sequence shown in SEQ ID NO: 58. In a particular embodiment, GatZ consists of the amino acid sequence shown in SEQ ID NO: 58. SEQ ID NO: 58 is shown below. MKTLIARHKAGEHIGICSVCSAHPLVIEAALAFDRNSTRKVLIEATSNQVNQFGGYTGMTPADFREFVFTIADKVGFARERIILGGDHLGPNCWQQENADAAMEKSVELVKEYVRAGFSKIHLDASMSCAGDPIPLAPETVAERAAVLCFAAESVATDCQREQLSYVIGTEVPVPGGEASAIQSVHITHVEDAANTLRTHQKAFIARGLTEALTRVIAIVVQPGVEFDHSNIIHYQPQEAQPLAQWIENTRMVYEAHSTDYQTRTAYWELVRDHFAILKVGPALTFALREAIFALAQIEQELIAPENRSGCLAVIEEVMLDEPQYWKKYYRTGFNDSLLDIRYSLSDRIRYYWPHSRIKNSVETMMVNLEGVDIPLGMISQYLPKQFERIQSGELSAIPHQLIMDKIYDVLRAYRYGCAE [Sequence ID 58]

[0067] In certain embodiments, the gene gatZ is encoded by a nucleotide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the nucleotide sequence shown in SEQ ID NO: 59. In certain embodiments, gatZ comprises the nucleotide sequence shown in SEQ ID NO: 59. SEQ ID NO: 59 is shown below.

[0068] In certain embodiments, the epimerase is the ketose diphosphate aldolase subunit KbaZ (UniProt No. P0C8K0). KbaZ catalyzes the reversible epimerization of D-tagatose-6-phosphate to D-fructose-6-phosphate. In certain embodiments, KbaZ is E. coli KbaZ. In certain embodiments, KbaZ contains an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence shown in SEQ ID NO: 60. In certain embodiments, KbaZ consists of the amino acid sequence shown in SEQ ID NO: 60. SEQ ID NO: 60 is shown below. MKHLTEMVRQHKAGKTNGIYAVCSAHPLVLEAAIRYASANQTPLLIEATSNQVDQFGGYTGMTPADFRGFVCQLADSLNFPQDALILGGDHLGPNRWQNLPAAQAM ANADDLIKSYVAAGFKKIHLDCSMSCQDDPIPLTDDIVAERAARLAKVAEETCLEHFGEADLEYVIGTEVPVPGGAHETLSELAVTTPDAARATLEAHRHAFEKQGL NAIWPRIIALVVQPGVEFDHTNVIDYQPAKASALSQMVENYETLIFEAHSTDYQTPQSLRQLVIDHFAILKVGPALTFALREALFSLAAIEEELVPAKACSGLRQV LEDVMLDRPEYWQSHYHGDGNARRLARGYSYSDRVRYYWPDSQIDDAFAHLVRRNLADSPIPLPLISQYLPLQYVKVRSGELQPTPRELIINHIQDILAQYHTACEGQ [Sequence ID 60]

[0069] In certain embodiments, the gene kbaZ is encoded by a nucleotide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the nucleotide sequence shown in SEQ ID NO: 61. In certain embodiments, kbaZ comprises the nucleotide sequence shown in SEQ ID NO: 61. SEQ ID NO: 61 is shown below.

[0070] In certain embodiments, the enzyme is a phosphatase, for example, a phosphatase that dephosphorylates tagatose-6-phosphate to free tagatose. In certain embodiments, the phosphatase catalyzes the removal of a phosphate group from a sugar. In certain embodiments, the sugar is a hexose.

[0071] In certain embodiments, the phosphatase is hexitol phosphatase A (HxpA) (UniProt number P77625). HxpA catalyzes the dephosphorylation of D-allose-6-phosphate. In certain embodiments, HxpA is E. coli HxpA. In certain embodiments, HxpA comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence shown in SEQ ID NO: 62. In certain embodiments, HxpA comprises the amino acid sequence shown in SEQ ID NO: 62. SEQ ID NO: 62 is shown below. MRCKGFLFDLDGTLVDSLPAVERAWSNWARRHGLAPEEVLAFIHGKQAITSLRHFMAGKSEADIAAEFTRLEHIEATETEGITALPGAIALLSHLNKAGIPWAIVTSGSMPVARARHKIAGLPAPEVFVTAERVKRGKPEPDAYLLGAQLLGLAPQECVVVEDAPAGVLSGLAAGCHVIAVNAPADTPRLNEVDLVLHSLEQITVTKQPNGDVIIQ [Sequence ID 62]

[0072] In certain embodiments, the gene hxpA is encoded by a nucleotide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the nucleotide sequence shown in SEQ ID NO: 63. In certain embodiments, hxpA comprises the nucleotide sequence shown in SEQ ID NO: 63. SEQ ID NO: 63 is shown below. GTGCGGTGCAAAGGTTTTCTGTTTGATCTTGATGGAACGCTGGTGGATTCCCTGCCTGCGGTAGAACGGGCGTGGAGCAACTGGGCCAGACGTCATGGGTTAGCGCCGGAAGAGGTGCTGGCTTTCATTCACGGTAAACAGGCGATCACCTCTCTGCGCCAT TTTATGGCGGGCAAATCCGAGGCTGATATTGCCGCCGAGTTTACGCGTCTGGAGCACATCGAGGCCACGGAACCGAAGGTATTACCGCGCTTCCGGGGGCAATCGCCTTACTCAGTCATTTGAATAAAGCAGGTATTCCGTGGGCCATTGTGACTTCTGGCT CCATGCCGGTAGCGCGAGCGCGCCATAAAATAGCTGGGCTTCCCGCACCAGAGGTGTTTGTAACCGCTGAGCGAGTGAAGCGCGGAAAACCAGAACCTGATGCGTATCTGTTAGGCGCGCAGCTGCTGGGGCTTGCGCCGCAGGAGTGTGTGGTGGTGGAAGA TGCTCCCGCTGGCGTGCTTTCTGGCCTGGCGGCGGGTTGTCATGTCATTGCGGTTAACGCTCCGGCAGATACCCCGCGCCTGAATGAGGTCGATTTGGTCCTCCACAGTCTGGAGCAAATTACTGTGACCAAACAGCCAAATGGCGATGTTATTATTCAGTGA [Sequence number 63]

[0073] In certain embodiments, the phosphatase is hexitol phosphatase B (HxpB) (UniProt number P77247 or UniProt number Q7ADF8). HxpB catalyzes the dephosphorylation of D-psicose-6-phosphate. In certain embodiments, HxpB is E. coli HxpB. In certain embodiments, HxpB comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence shown in SEQ ID NO: 3. In certain embodiments, HxpB comprises the amino acid sequence shown in SEQ ID NO: 3. SEQ ID NO: 3 is shown below. MSTPRQILAAIFDMDGLLIDSEPLWDRAELDVMASLGVDISRRNELPDTLGLRIDMVVDLWYARQPWNGPSRQEVVERVIARAISLVEETRPLLPGVREAVALCKEQGLLVGLASASPLHMLEKVLTMFDLRDSFDALASAEKLPYSKPHPQVYLDCAAKLGVDPLTCVALEDSVNGMIASKAARMRSIVVPAPEAQNDPRFVLADVKLSSLTELTAKDLLG [Sequence ID 3]

[0074] In certain embodiments, HxpB comprises the amino acid sequence shown in SEQ ID NO: 4. In certain embodiments, HxpB consists of the amino acid sequence shown in SEQ ID NO: 4. SEQ ID NO: 4 is shown below. MSTPRQILAAIFDMDGLLIDSEPLWDRAELDVMASLGVDISRRNELPDTLGLRIDMVVDLWYARQPWNGPSRQEVVERVIARAISLVEETRPLLPGVREAVALCKEQGLLVGLASASPLHMLEKVLTMFDLRDSFDALASAEKLPYSKPHPQVYLDCAAKLGVDPLTCVALEDSVNGMIASKAARMRSIVVPAPEAQNDPRFVLANVKLSSLTELTAKDLLG [Sequence ID 4]

[0075] In certain embodiments, the gene hxpB is encoded by a nucleotide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the nucleotide sequence shown in SEQ ID NO: 5. In certain embodiments, hxpB comprises the nucleotide sequence shown in SEQ ID NO: 5. In certain embodiments, hxpB consists of the nucleotide sequence shown in SEQ ID NO: 5. SEQ ID NO: 5 is shown below. ATGTCAACCCCGCGTCAGATTCTTGCTGCAATTTTTGATATGGATGGATTACTTATCGACTCAGAACCTTTATGGGATCGAGCCGAACTGGATGTGATGGCAAGCCTGGGGGTGGATATCTCCCGTCGTAACGAGCTGCCGGACACCTTAGGTTTACGCATCGATAT GGTGGTCGATCTTTGGTACGCCCGGCAACCGTGGAATGGGCCAAGCCGTCAGGAAGTAGTAGAACGGGTTATTGCCCGTGCCATTTCACTGGTTGAAGAGACACGTCCATTATTACCAGGCGTGCGCGAAGCCGTTGCGTTATGCAAAGAACAAGGTTTATTGGTGG GACTGGCCTCCGCGTCACCACTACATATGCTGGAAAAAAGTGTTGACCATGTTTGACTTACGCGACAGTTTCGATGCCCTCGCCTCGGCCGAAAAACTGCCTTACAGCAAGCCGCATCCGCAAGTATATCTCGACTGCGCAGCAAAACTGGGCGTTGACCCTCTGACC TGCGTAGCGCTGGAAGATTCGGTAAATGGCATGATCGCCTCTAAAGCAGCCCGCATGCGTTCCATCGTCGTTCCTGCGCCAGAAGCGCAAAATGATCCACGTTTTGTATTAGCAGACGTCAAACTTTCATCGCTGACAGAACTCACCGCAAAAGACCTTCTCGGTTGA [Sequence number 5]

[0076] In certain embodiments, one or more transporters that transport glucose into cells can be expressed (i.e., overexpressed) within the cell, thereby increasing intracellular glucose levels. In some embodiments, galactose:H + One or both of the cotransporter (GalP) and / or glucokinase (Glk) are expressed in the microorganism. For example, in some embodiments, GalP transports glucose to the cell, where it is phosphorylated to glucose-6-phosphate by Glk and assimilated into the central carbon metabolic pathway.

[0077] In a particular embodiment, the galactose:H + The cotransporter (GalP) is as described in EG12148 (EcoCyc) or P0AEP1 (UniProt). In certain embodiments, GalP is E. coli GalP. In certain embodiments, GalP comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence shown in SEQ ID NO: 38. In certain embodiments, GalP comprises the amino acid sequence shown in SEQ ID NO: 38. SEQ ID NO: 38 is shown below. MPDAKKQGRSNKAMTFFVCFLAALAGLLFGLDIGVIAGALPFIADEFQITSHTQEWVVSSMMFGAAVGAVGSGWLSFKLGRKKSLMIGAILFVAGSLFSAAAPNVEVLILSRVLLG LAVGVASYTAPLYLSEIAPEKIRGSMISMYQLMITIGILGAYLSDTAFSYTGAWRWMLGVIIIPAILLLIGVFFLPDSPRWFAAKRRFVDAERVLLRLRDTSAEAKRELDEIRESL QVKQSGWALFKENSNFRRAVFLGVLLQVMQQFTGMNVIMYYAPKIFELAGYTNTTEQMWGTVIVGLTNVLATFIAIGLVDRWGRKPTLTLGFLVMAAGMGVLGTMMHIGIHSPSAQYFAIAMLLMFIVGFAMSAGPLIWVLCSEIQPLKGRDFGITCSTATNWIANMIVGATFLTMLNTLGNANTFWVYAALNVLFILLTLWLVPETKHVSLEHIERNLMKGRKLREIGAHD (Sequence ID 38)

[0078] In certain embodiments, the gene galP is encoded by a nucleotide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the nucleotide sequence shown in SEQ ID NO: 39. In certain embodiments, galP comprises the nucleotide sequence shown in SEQ ID NO: 39. SEQ ID NO: 39 is shown below.

[0079] In certain embodiments, the glucokinase (Glk) is as described in EG12957 (EcoCyc) or P0A6V8 (UniProt). In certain embodiments, Glk is E. coli Glk. In certain embodiments, Glk comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence shown in SEQ ID NO: 40. In certain embodiments, Glk comprises the amino acid sequence shown in SEQ ID NO: 40. SEQ ID NO: 40 is shown below. MTKYALVGDVGGTNARLALCDIASGEISQAKTYSGLDYPSLEAVIRVYLEEHKVEVKDGCIAIACPITGDWVAMTNHTWAFSIAEMKKNLGFSHLEIINDFTAVSMAIPMLKKEHLIQFGGAEPVEGKPIAVYGAGTGLGVAHLVHVDKRWVSLPGEGGHVDFAPNSEEEAIILEILRAEIGHVSAERVLSGPGLVNLYRAIVKADNRLPENLKPKDITERALADSCTDCRRALSLFCVIMGRFGGNLALNLGTFGGVFIAGGIVPRFLEFFKASGFRAAFEDKGRFKEYVHDIPVYLIVHDNPGLLGSGAHLRQTLGHIL (Sequence ID 40)

[0080] In certain embodiments, the gene glk is encoded by a nucleotide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the nucleotide sequence shown in SEQ ID NO: 41. In certain embodiments, glk comprises the nucleotide sequence shown in SEQ ID NO: 41. SEQ ID NO: 41 is shown below. atgACAAAGTATGCATTAGTCGGTGATGTGGGCGGCACCAACGCACGTCTTGCTCTGTGTGATATTGCCAGTGGTGAAATCTCGCAGGCTAAGACCTATTCAGGGCTTGATTACCCCAGCTCGAAGCGGTCATTCGCGTTTATCTTGAAGAACATAAGGTCGAGGTGAAAGACGGCTGTATTGCCATCGCTTGCCCAATTACCGGTGACTGGGTGGCGATCAACCATACCTGGGCGT TCTCAATTGCCGAAATGAAAAAGAATCTCGGTTTTAGCCATCTGGAAATTATTAACGATTTTACCGCTGTATCGATGGCGATCCCGATGCTGAAAAAAGCATCTGATTCAGTTGCGTCAGAACCGGTCGAAGGTAAGCCTATTGCGGTTTACGGTGCCGGAACGGGGCTTGGGGTTGCGCATCTGGTCCATGTCGATAAGCGTTGGGGTAAGCTTGCCAGGCGAAGGCGTCACGTT GATTTTGCCCGAATAGTGAAGAAGAGGCCATTATCCTCGAAATATTGCGTGCGGAAATTGGTCATGTTTCGCGGAAGCGCGTGCTTTCTGGCCCTGGGCTGGTGAATTTGTATCGCGCCAATTGTGTGAAAGCTGACAACCGCCTGCCAGAAAATCTCAAGCCAAAAGATATTACCGAGCGCGCTGGCTGACACGCTGACACGATTGCCCGCCGCATTGTCGCGTTTTGCGTCATTATGGCCGTTTTGCGGGCAATCTGGCCGCTCAATCTCGGGACATTTGGCGCGGTGTTTATTGCGGCGGTATCGGTTCCTTGAGTTCCTTCGTGCCGCATTTGAAGATAAAGGGCGCTTTTAAAGAATATGTCCATGATATTCCGGTGTATCTCCATCGTCCATGACAATCCGGGCCTTTCTCGGTTCCGGTGGCACATTTACGCCAGACCTTAGGTCACATTCTgtaa (sequence number 41)

[0081] While not bound by any theory, the inventors of this disclosure believe that any enzyme performing a similar function to the enzymes described above may be used in the microorganisms of this disclosure. For example, but not limited to, the microorganisms of this disclosure may include any enzyme that catalyzes the reversible epimerization of D-fructose-6-phosphate to D-tagatose-6-phosphate. In another non-limiting example, the microorganisms of this disclosure may include any enzyme that dephosphorylates D-tagatose-6-phosphate to free tagatose.

[0082] 2.2. Competitive Paths In certain embodiments, the microorganisms of the Disclosure include mutations in one or more genes encoding one or more enzymes that modulate a biochemical pathway that can reduce tagatose production. In certain embodiments, the microorganisms of the Disclosure include reduced expression of genes encoding enzymes that modulate a biochemical pathway that can reduce tagatose production. Physiologically, cells catalyze sugars to produce energy (e.g., ATP) via the pentose phosphate pathway and glycolysis. The inventors of the Disclosure have found that deletion or reduced expression of genes encoding enzymes in certain metabolic pathways results in increased tagatose production.

[0083] In certain embodiments, the microorganism of the Disclosure includes a mutation in the gene encoding an enzyme that converts fructose-6-phosphate (F6P) to psicose-6-phosphate. In certain embodiments, the microorganism of the Disclosure includes reduced expression of the gene encoding an enzyme that converts fructose-6-phosphate (F6P) to psicose-6-phosphate. In certain embodiments, the enzyme that converts fructose-6-phosphate (F6P) to psicose-6-phosphate is D-allulose-6-phosphate 3-epimerase (AlsE) (UniProt No. P32719). In certain embodiments, AlsE is Escherichia coli AlsE. A representative nucleotide sequence of the gene alsE is represented by SEQ ID NO: 2 or is at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 2. SEQ ID NO: 2 is shown below. ATGAAAATCTCCCCCTCGTTAATGTGTATGGATCTGCTGAAATTTAAAGAACAGATCGAATTTATCGACAGCCATGCCGATTACTTCCACATCGATATCATGGACGGTCACTTTGTCCCCAATCTGACACTCTCACCGTTCTTCGTAAGTCAGGTTAAAAAACTGGCAACTAAA CCGCTCGACTGTCATCTGATGGTGACGCGGCCGCAGGATTACATTGCTCAACTGGCGCGTGCGGGAGCAGATTTCATCACTCTGCATCCGGAAACCATCAACGGCCAGGCGTTCCGCCTGATTGATGAAATCCGCCGTCATGACATGAAAGTGGGGCTGATCCTTAACCCGGAG ACGCCAGTTGAGGCCATGAAATACTATATCCATAAGGCCGATAAAATTACGGTCATGACTGTCGATCCCGGCTTTGCCGGACAACCGTTCATTCCTGAAATGCTGGATAAACTTGCCGAACTGAAGGCATGGCGTGAACGAGAAGGTCTGGAGTACGAAATTGAGGTGGACGGT TCCTGCAACCAGGCAACTTACGAAAAACTGATGGCGGCAGGGGCGGATGTCTTTATCGTCGGCACTTCCGGCCTGTTTAATCATGCGGAAAATATCGACGAAGCATGGAGAATTATGACCGCGCAGATTCTGGCTGCAAAAAGCGAGGTACAGCCTCATGCAAAAACAGCATAA [Sequence number 2]

[0084] In certain embodiments, *E. coli* AlsE contains an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence shown in SEQ ID NO: 1. SEQ ID NO: 1 is shown below. MKISPSLMCMDLLKFKEQIEFIDSHADYFHIDIMDGHFVPNLTLSPFFVSQVKKLATKPLDCHLMVTRPQDYIAQLARAGADFITLHPETINGQAFRLIDEIRRHDMKVGLILNPETPVEAMKYYIHKADKITVMTVDPGFAGQPFIPEMLDKLAELKAWREREGLEYEIEVDGSCNQATYEKLMAAGADVFIVGTSGLFNHAENIDEAWRIMTAQILAAKSEVQPHAKTA [Sequence ID 1]

[0085] In certain embodiments, the microorganisms of the Disclosure include mutations in genes encoding enzymes of the pentose phosphate pathway. In certain embodiments, the microorganisms of the Disclosure include reduced expression of genes encoding enzymes of the pentose phosphate pathway. In certain embodiments, the enzymes of the pentose phosphate pathway are selected from the group consisting of glucose-6-phosphate dehydrogenase, 6-phosphogluconolactose, phosphogluconate dehydrogenase, phosphopentose isomerase, phosphopentose epimerase, transketolase, and transaldolase. In certain embodiments, the enzymes of the pentose phosphate pathway are glucose-6-phosphate dehydrogenase (Zwf) (Entrez Gene ID:946370; EcoCyc ID:EG11221; UniProt ID:P0AC53). Zwf catalyzes the oxidation of glucose-6-phosphate to 6-phosphogluconolactone. In certain embodiments, Zwf is Escherichia coli Zwf. The typical nucleotide sequence of the zwf gene is shown in SEQ ID NO: 6, or is at least 90%, at least 95%, at least 95%, or at least 99% identical to SEQ ID NO: 6. SEQ ID NO: 6 is shown below.

[0086] In certain embodiments, E. coli Zwf contains an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence shown in SEQ ID NO: 20. SEQ ID NO: 20 is shown below. MAVTQTAQACDLVIFGAKGDLARRKLLPSLYQLEKAGQLNPDTRIIGVGRADWDKAAYTKVVREALETFMKETIDEGLWDTLSARLDFCNLDVNDTAAFSRLGAMLDQKNRITINYFAMPPS TFGAICKGLGEAKLNAKPARVVMEKPLGTSLATSQEINDQVGEYFEECQVYRIDHYLGKETVLNLLALRFANSLFVNNWDNRTIDHVEITVAEEVGIEGRWGYFDKAGQMRDMIQNHLLQILC MIAMSPPSDLSADSIRDEKVKVLKSLRRIDRSNVREKTVRGQYTAGFAQGKKVPGYLEEEGANKSSNTETFVAIRVDIDNWRWAGVPFYLRTGKRLPTKCSEVVVYFKTPELNLFKESWQDLPQNKLTIRLQPDEGVDIQVLNKVPGLDHKHNLQITKLDLSYSETFNQTHLADAYERLLLETMRGIQALFVRRDEVEEAWKWVDSITEAWAMDNDAPKPYQAGTWGPVASVAMITRDGRSWNEFE [Sequence ID 20]

[0087] In certain embodiments, Zwf is Bacillus subtilis Zwf. A typical amino acid sequence of Bacillus subtilis Zwf is found as P54547(Uniprot) / BSU23850(KEGG), or is shown in Sequence ID No. 10, or is at least 90%, at least 95%, at least 95%, or at least 99% identical to Sequence ID No. 10. Sequence ID No. 10 is shown below. MKTNQQPKAVIVIFGATGDLAKRKLYPSIHRLYQNGQIGEEFAVVGVGRRPWSNEDLRQTVKTSISSSADKHIDDFTSHFYYHPFDVTNPGSYQELNVLLNQLEDTYQIPNNRMFYLAMAPE FFGTIAKTLKSEGVTATTGWSRLVIEKPFGHDLPSAQALNKEIREAFTEDQIYRIDHYLGKQMVQNIEVIRFANAIFEPLWTNRYISNIQITSSESLGVEDRARYYEKSGALRDMVQNHIMQ MVALLAMEPPIKLNTEEIRSEKVKVLRALRPIAKDEVDEYFVRGQYHAGEIDGVPVPAYTDEDNVAPDSNTETFVAGKLLIDNFRWAGVPFYIRTGKRMKEKSTKIVVQFKDIPMNLYYGNENNMNPNLLVIHIQPDEGITLYLNAKKLGGAAHAQPIKLDYCSNCNDELNTPEAYEKLIHDCLLGDATNFAHWDEVALSWSFVDSISETWAANKTLSPNYESGSMGPKESDDLLVKDGLHWWNI (Sequence ID 10)

[0088] The typical nucleotide sequence of the Bacillus subtilis zwf gene is shown in SEQ ID NO: 26, or is at least 90%, at least 95%, at least 95%, or at least 99% identical to SEQ ID NO: 26. SEQ ID NO: 26 is shown below. gtgaaaacaaaccaacaaccaaaagcagtaattgtcatattcggtgcaactggagatttagcaaaacgaaaattgtatccgtctattcaccgtttatatcaaaacggacaaatcggagaagagtttgcagtggtaggagttggaagaagaccttggtctaatgaggatcttcgccaaactgttaaaacatccatttcctcatctgcagataagcatatagatgatttcacgtctcatttttactatcacccgtttgacgtgacaaaccctggttcttatcaagagctaaacgtattgcttaaccagctggaagatacatatcaaattcctaacaacagaatgttctacttggcaatggctcctgaattcttcggaacgattgcaaaaacattaaaatcagagggtgtaacagctacaaccggctggtcccgccttgtcatcgaaaaaccgttcggccatgatctgccaagcgcacaggcattgaataaagaaatccgcgaagcatttacggaagatcaaatttacagaatcgaccattatctaggcaaacaaatggttcagaacattgaagtgattcgatttgccaatgcgattttcgaaccgctttggacaaaccgctacatttcaaacattcaaatcacatctagcgaatcactaggc gttgaagaccgcgcaagatattacgaaaaatcaggcgcccttgcgacatggtgcaaaccatattatgcagatggttgcccttcttgcaatggagccgcctatcaaattgaacacagaagaaatccgcagcgagaaaagtgaaggtgctgagagcactgcgtcct attgcaaaagacgaagtggatgaatactttgtgcgcggacaatatcatgctggtgaaattgacggtgtaccggttcctgcttatacagatgaagataatgtcgctcctgactccaatacagaaacctttgttgccggcaagctcttgatcgacaacttcagatgg gctggtgttccattctacatcagaaccggaaaacgaatgaaagaaaagtccacaaaaattgtcgttcaatttaaggacattccgatgaacctgtactacggtaatgaaaacaacatgaatccgaacttgcttgtcattcatattcagcctgacgaaggcattacg ctttacttaaatgctaaaaagcttggcggagcagcacacgcacagccaatcaaactcgattattgcagcaattgcaatgacgagttgaacacccctgaagcatatgaaaaactaattcacgactgtcttcttggcgatgcaacaaactttgcacactgggatgaa gttgccctttcttggagctttgtcgactctatttctgaaacatgggcagcaaacaaaaccttatctcctaactacgaatcaggctcaatgggaccgaaagaatctgatgatcttttggtgaaagacggcttacactggtggaacatataa (sequence number 26)

[0089] In certain embodiments, Zwf is Lactococcus lactis Zwf. A typical amino acid sequence of Lactococcus lactis Zwf is found in GenBank HE92651.1:glucose-6-phosphate dehydrogenase, EC1.1.1.49, or is shown in SEQ ID NO: 11, or is at least 90%, at least 95%, at least 95%, or at least 99% identical to SEQ ID NO: 11. SEQ ID NO: 11 is shown below. MTEQKQALFTIFGATGDLAKRKLYPSLFRFLFKKGELADNFAVIGTARRPWTNEYYREVVLESIKDLMNSKTEAENFASHFYYQSHDVSDSSHYVNLKDLGEKLRKQYKTAGNQVFFLAMAPQFFG TIAEHLKSENILTGEGFERIVIEKPFGTSYDTAKSLNDSLAKVFSEEQIFRIDHYLGKEMIQAVSAVRFANPIFESLWNNQHIDNVQITFAEFIGVEDRGGYYETSGALKDMIQNHVLQVLSLIAM EKPEKFDESYIVKEKVKALNAIRQYSSEEALENFVRGQYIAGRFDGEDYLGYREEDSVATDSRTETFAAGKFVIDNERWSGVPFYVRSGKRMTEKGTRINIVFKKDKDNLFAENCDDQSVQNVLTIYIQPTEGFSLSVNGKAAGQGFHLEPLRLNFRHDSEFLGNSPEAYEKLFLDVLNGDGTNFSHWEEAARAWELIDVIREAWDKETSELPTYAARTMGPKAAFDLLEKNGHEWAWQPDLWYQERGYYNK (Sequence ID 11)

[0090] The representative nucleotide sequence of the Lactococcus lactis zwf gene is shown in SEQ ID NO: 33, or is at least 90%, at least 95%, at least 95%, or at least 99% identical to SEQ ID NO: 33. SEQ ID NO: 33 is shown below.

[0091] In certain embodiments, the microorganisms of the Disclosure include mutations in the genes encoding enzymes for glycogen biosynthesis. In certain embodiments, the microorganisms of the Disclosure include reduced expression of the genes encoding enzymes for glycogen biosynthesis. In certain embodiments, the enzymes for glycogen biosynthesis are selected from the group consisting of phosphoglucumutase (Pgm), UDP-glucose pyrophosphorylase, glycogen synthase, glycogen branching enzyme, and glycogenin. In certain embodiments, the enzyme for glycogen biosynthesis is phosphoglucumutase (Pgm) (Entrez Gene ID:946370; EcoCyc ID:EG12144; Uniprot ID:P36938). Pgm (EC 5.4.2.2) is an enzyme that transfers a phosphate group on an α-D-glucose monomer from position 1 to position 6 in the forward direction or from position 6 to position 1 in the reverse direction. In certain embodiments, Pgm is Escherichia coli Pgm. The typical nucleotide sequence of the gene pgm is shown in SEQ ID NO: 47, or is at least 90%, at least 95%, at least 95%, or at least 99% identical to SEQ ID NO: 47. SEQ ID NO: 47 is shown below.

[0092] In certain embodiments, E. coli Pgm contains an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence shown in SEQ ID NO: 48. SEQ ID NO: 48 is shown below. MAIHNRAGQPAQQSDLINVAQLTAQYYVLKPEAGNAEHAVKFGTSGHRGSAARHSFNEPHILAIAQAIAEERAKNGITGPCYVGKDTHALSEPAFISVLEVLAANGVDVIVQENNGFTPTPAVSNAILVHNKKGGP LADGIVITPSHNPPEDGGIKYNPPNGGPADTNVTKVVEDRANALLADGLKGVKRISLDEAMASGHVKEQDLVQPFVEGLADIVDMAAIQKAGLTLGVDPLGGSGIEYWKRIGEYYNLNLTIVNDQVDQTFRFMHLDK DGAIRMDCSSECAMAGLLALRDKFDLAFANDPDYDRHGIVTPAGLMNPNHYLAVAINYLFQHRPQWGKDVAVGKTLVSSAMIDRVVNDLGRKLVEVPVGFKWFVDGLFDGSFGFGGEESAGASFLRFDGTPWSTDKDGIIMCLLAAEITAVTGKNPQEHYNELAKRFGAPSYNRLQAAATSAQKAALSKLSPEMVSASTLAGDPITARLTAAPGNGASIGGLKVMTDNGWFAARPSGTEDAYKIYCESFLGEEHRKQIEKEAVEIVSEVLKNA [Sequence ID 48]

[0093] In certain embodiments, Pgm is Bacillus subtilis Pgm. A representative nucleotide sequence of the gene pgm is shown in SEQ ID NO: 49, or is at least 90%, at least 95%, at least 95%, or at least 99% identical to SEQ ID NO: 49. SEQ ID NO: 49 is shown below.

[0094] In certain embodiments, Bacillus subtilis Pgm contains an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence shown in SEQ ID NO: 50. SEQ ID NO: 50 is shown below. MSKKPAALIILDGFGLRNETVGNAVALAKPNFDRYWNQYPHQTLTASGEAVGLPEGQMGNSEVGHLNIGAGRIVYQSLTRVNVAIREGEFERNQTFLDAISNAKENNKALHLFGLLSDGGVHSHIN HLFALLKLAKKEGLTKVYIHGFLDGRDVGPQTAKTYINQLNDQIKEIGVGEIASISGRYYSMDRDKRWDRVEKAYRAMAYGEGPSYRSALDVVDDSYANGIYDEFVIPSVITKENGEPVAKIQDGDSV IFYNFRPDRAIQISNTFTNKDFRDFDRGENYPKNLYFVCLTHFSETVDGYVAFKPINLDNTVGEVLSQHGLKQLRIAETEKYPHVTFFMSGGREAEFPGEERILINSPKVATYDLKPEMSAYEVKDALVKEIEADKHDAIILNFANPDMVGHSGMVEPTIKAIEAVDECLGEVVDAILAKGGHAIITADHGNADILITESGEPHTAHTTNPVPVIVTKEGITLREGGILGDLAPTLLDLLGVEKPKEMTGTSLIQK (Sequence ID 50)

[0095] In certain embodiments, Pgm is Lactococcus lactis Pgm. A representative nucleotide sequence of the gene pgm is shown in SEQ ID NO: 51, or is at least 90%, at least 95%, at least 95%, or at least 99% identical to SEQ ID NO: 51. SEQ ID NO: 51 is shown below. ATGTTTAAAGCAGTATTGTTTGATTTAGATGGTGTAATTACAGATACCGCAGAGTATCATTTTAGAGCTTGGAAAGCTTTGGCTGAAGAAATTGGCATTAATGGTGTTGACCGCCAATTTAATGAGCAATTAAAAGGGGTCTCACGAGAAGACTCGCTTCAGAAAA TTCTAGATTTAGCTGATAAAAAAGTATCAGCTGAGGAATTTAAAGAACTTGCTAAGAGAAAAAATGATAACTATGTGAAAATGATTCAGGATGTGTCGCCAGCCGATGTCTATCCTGGAATTTTACAATTACTCAAAGATTTACGTTCAAATAAAATCAAAATTGCT TTAGCATCGGCTTCTAAGAATGGTCCATTTTTATTAGAGAGAATGAATTTAACTGGATATTTTGATGCAATTGCTGATCCGGCTGAAGTTGCAGCATCAAAACCAGCACCAGATATTTTTATTGCAGCAGCACATGCAGTGGGTGTTGCCCCCTCTGAATCAATTG GGTTAGAGGATTCTCAAGCTGGAATTCAAGCCATCAAAGATTCAGGGGCTTTACCAATTGGTGTAGGGCGCCCAGAAGATTTGGGAGATGATATCGTCATTGTGCCTGATACTTCACACTATACATTAGAATTTTTGAAAGAAGTTTGGCTTCAAAAGCAAAAATGA [Sequence number 51]

[0096] In certain embodiments, Lactococcus lactis Pgm contains an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence shown in SEQ ID NO: 52. SEQ ID NO: 52 is shown below. MFKAVLFDLDGVITDTAEYHFRAWKALAEEIGINGVDRQFNEQLKGVSREDSLQKILDLADKKVSAEEFKELAKRKNDNYVKMIQDVSPADVYPGILQLLKDLRSNKIKIALASASKNGPFLLEKMNLTGYFDAIADPAEVAASKPAPDIFIAAAHAVGVAPSESIGLEDSQAGIQAIKDSGALPIGVGRPEDLGDDIVIVPDTSYYTLEFLKEVWLQKQK (Sequence ID 52)

[0097] In certain embodiments, the microorganisms of the Disclosure include mutations in genes encoding glycolytic enzymes. In certain embodiments, the microorganisms of the Disclosure include reduced expression of genes encoding glycolytic enzymes. In certain embodiments, the glycolytic enzyme is selected from the group consisting of phosphofructokinase A, phosphofructokinase B, fructose diphosphate aldolase, triose phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase, and pyruvate kinase. In certain embodiments, the glycolytic enzyme is phosphofructokinase B (PfkB). In certain embodiments, the glycolytic enzyme is pyruvate kinase. In certain embodiments, the glycolytic enzyme is phosphofructokinase A (PfkA) (Entrez Gene ID:948412; EcoCyc ID:EG10699; UniProt ID:P0A796). PfkA catalyzes the phosphorylation of D-fructose-6-phosphate to fructose-1,6-bisphosphate by ATP, which is the first commitment step in glycolysis. In certain embodiments, PfkA is E. coli PfkA. A representative nucleotide sequence of the gene pfkA is shown in Sequence ID No. 7. Sequence ID No. 7 is shown below. ATGATTAAGAAAATCGGTGTGTTGACAAGCGGCGGTGATGGCCCAGGCATGAACGCCGCAATTCGCGGGGTTGTTCGTTCTGCGCTGACAGAGGTCTGGAAGTAATGGGTATTTATGACGGCTATCTGGGTCTGTATGAAGACCGTATGGTACAGCTAGACCGTTACAGCGTGTCGACATGATCAACCGTGGCGGTACGTTCCTCGGTTCTGCGCGTTCCCGGGAATTCCGCGACGAG AACATCCGCGCGTGCTATCGAAAACCTGAAAAAACGTGGTATCGACGCGCTGGTGGTTATCGGCGTGACGGTTCCTACATGGGTCAATGCGTCTGACCGAAATGGGCTTCCCGTGCATCGGTCTGCCGGGCACTATCGACAACGACATCAAAGGCACTGACTACACTATCGGTTTCTTCACTGCGCTGAGCACCGTTGTAGAAGCGATCGACCGTCTGCGTGACACCTCTTCTTC ACCAGCGTATTTCCGTGGTGGTGGAAGTGATGGCCGTTATTTGGAGAATCTGACGTTGGCTCGGCCATTGCCGGTGGCTGTGATTCGTTGTGGTTCCGGAAGTTGAATTCAGCCGTGGAAGACCCTGGTTAAACGAAATCAAAGCGGGTATCGCGAAAAGGTAAAAACACGCGATCGTGGCGATTACCGGAACATATGTGTGGATGTGTGTCGAACTGGCGCATTTCATCGAGAAAAAACCGGTCGTGTGCGCAACTTGTGGTGGCCCACATCCAGCGCGGTGGTTTCCCGGTGGCTTACGAACGAACAGCTGGTTCACCACGACATCATCGACGCTATCGAAAACAGCGTCCGTTCAAAGGTGACTGGCTGGACTGCGCGAAAAAACTGTATTAA [sequence number 7]

[0098] In certain embodiments, E. coli PfkA contains an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence shown in SEQ ID NO: 21. SEQ ID NO: 21 is shown below. MIKKIGVLTSGGDAPGMNAAIRGVVRSALTEGLEVMGIYDGYLGLYEDRMVQLDRYSVSDMINRGGTFLGSARFPEFRDENIRAVAIENLKKRGIDALVVIGGDGSYMGAMRLTEMGFPCIGLPGTIDNDIKGTDYTIGFFTALSTVVEAIDRLRDTSSSHQRISVVEVMGRYCGDLTLAAAIAGGCEFVVVPEVEFSREDLVNEIKAGIAKGKKHAIVAITEHMCDVDELAHFIEKETGRETRATVLGHIQRGGSPVPYDRILASRMGAYAIDLLLAGYGGRCVGIQNEQLVHHDIIDAIENMKRPFKGDWLDCAKKLY [Sequence ID 21]

[0099] In certain embodiments, PfkA is Bacillus subtilis PfkA. A typical amino acid sequence of Bacillus subtilis PfkA is found as O34529(Uniprot) / BSU29190(KEGG), or is shown in Sequence ID No. 12, or is at least 90%, at least 95%, at least 95%, or at least 99% identical to Sequence ID No. 12. Sequence ID No. 12 is shown below. MKRIGVLTSGGDSPGMNAAVRAVVRKAIYHDVEVYGIYNGYAGLISGKIEKLELGSVGDIIHRGGTKLYTARCPEFKTVEGREKGIANLKKLGIEGLVVIGGDGSYMGAKKLTEHGFPCVGVPGTIDNDIPGTDFTIGFDTALNTVIDAIDKIRDTATSHERTYVIEVMGRHAGDIALWAGLAGGAESILIPEADYDMHEIIARLKRGHERGKKHSIIIVAEGVGSGVEFGKRIEEETNLETRVSVLGHIQRGGSPSAADRVLASRLGAYAVELLLEGKGGRCVGIQNNKLVDHDIIEILETKHTVEQNMYQLSKELSI (Sequence ID 12)

[0100] The typical nucleotide sequence of the Bacillus subtilis gene pfkA is shown in SEQ ID NO: 27, or is at least 90%, at least 95%, at least 95%, or at least 99% identical to SEQ ID NO: 27. SEQ ID NO: 27 is shown below. atgaaacgaataggggtattaacgagcggcggggattccccgggaatgaacgcagcagttcgcgcagtgtcagaaaagcgatctatcatgacgttgaagtttacggtatttacaacggatacgcgggattgatcagcggaaagattgaaaagcttgaactcggatcagtaggcgatatattacatcgtggagggactaagctttatacggcgagatgtcctgaattcaaaacagttga ggccgtgaaaaagggatagcaaacttgaagaagcttggtattgaaggccttgttgttatcggtggagacggttcctatatgggtgcgaaaaaattaacggaaacgggtttccatgtgtaggtgtaccgggtacaattgataatgacattccgggcactgattttacaatcggtttcgatacagctttaaatacagtaattgacgcaattgataagattcgcgatacagcgacttctcat gaacgtacatatgtaatcgaagtaatgggccgtcatgccggcgatatcgcattgtgggccggtcttgcaggggcgcagaatcgatcttaatccctgaggcagactatgacatgcacgaaatcattgcccgcttaaaaacgcggccacgaacgcggcaagaagcacagtattattgttgccgaaggtgtaggcagcgggtgtgaattcgggaaacgcattgaagaagaaacaaaatcttgaaactaggtatctgtattggccatatccagcggggattctccgagtgctgctgaccgtgtgtgttggcaagccgtctcggcgcatatgcacaatgcacattgcaactgcagttgaactgctgcttgaaggaaaaggcggacgctgtgtaggttatacaaaacaataagcttgtagaccacatgatattatagagaaatacttgagacaaaacacacagttgagcaaaacatgtatcagctttcaaaagaactgtctatctaa (sequence number 27)

[0101] In certain embodiments, PfkA is Lactococcus lactis PfkA. A representative amino acid sequence of Lactococcus lactis PfkA is found in GenBank: WP_003131080.1:ATP-dependent 6-phosphofructokinase, EC2.7.1.11, or is shown in SEQ ID NO: 13 below, or is at least 90%, at least 95%, at least 95%, or at least 99% identical to SEQ ID NO: 13. MKRIAVLTSGGDAPGMNAAIRAVVRKAISEGIEVYGINHGYAGMVAGDIFPLTSASVGDKIGRGGTFLYSARYPEFAQVEGQLAGIEQLKKFGIEGVVVIGGDGSYHGAMRLTEHGFPAVGLPGTIDNDIVGTDFTIGFDTAVSTVVDALDKIRDTSSSHNRTFVVEVMGRNAGDIALNAGIAAGADDICIPEKEFKFENVVNNINKGYEKGKNHHIIVLAEGVMTGEEFATKLKEAGYKGDLRVSVLGHIQRGGSPTARDRVLASRMGARAVELLRDGIGGVAVGIRNEELVESPILGTAEEGALFSLTTEGGIKVNNPHKAGLELYRLNSALNNLNLN (Sequence ID 13)

[0102] The typical nucleotide sequence of the Lactococcus lactis gene pfkA is shown in SEQ ID NO: 32, or is at least 90%, at least 95%, at least 95%, or at least 99% identical to SEQ ID NO: 32. SEQ ID NO: 32 is shown below.

[0103] In certain embodiments, PfkB is E. coli PfkB. A representative nucleotide sequence of the PfkB gene is shown in Sequence ID No. 22. Sequence ID No. 22 is shown below. atgGTACGTATCTATACGTTGACACTTGCGCCCTCTCTGATAGCGCAACAATTACCCCGCAAATTTATCCCGAAGGAAAACTGCGCTGTACCGCACCGGTGTTCGAACCCGGGGGCGGCGCATCAACGTCGCCCGCGCCATTGCCCATCTTGGAGGCAGTGCCAGCGATCTTCCGGCGGGTGGCGCGACGGCGAACACCTGGTTTCACTGTTGGCGGATGAAAATG TCCCCGTCGCTACTGTAGAAGCCAAAGACTGGACCCGGCAGAATTTACACGTACATGTGGAAGCAAGCGGTGAGCAGTATCGTTTTGTTATGCCAGGCGCGGCATTAAATGAAGATGAGTTTCGCCAGCTTGAAGAGCAAGTTCTGGAAATTGAATCCGGGGCCATCCTGGTCATAAGCGGAAGCCTGCCGCCAGGTGGAAGCTGGAAAAATTAACCCAACTGATTTCCGCT GCGCAAAAAACAAGGGATCCGCTGCATCGTCGACAGTTCTGGCGAAGCGTTAAGTGCCAGCACTGGCAATTGGTAACATCGAGTTGGTAAGCCTAACCAAAAGAACTCAGTGCGCTGGTGAATCGCGGAACTCACCCAGCCGGAGAGTGCCGCAAAGCCGCGCGAGAAATCGTTAATAGCGGCAAGGCCAAACGGGTTGTCGTTCCCTGGGTCCACAAGGAGCGCTGGTGTTGATAGTGAAAACTGTATTCAGGTGGTGCCACCACCCGGTGAAAGCCAGATCACCGTTGGGCGCTGGTGGACAGCATGGTCGGCGCGGATGACACTGAAACTGGCAGAAAATGCCCTCTCTTGAAGAGATGGTTCGTTTTGGCGTAGCTGCGGGAGTGCAGCCACACTCAATCAGGGAAACGCTCTGTGCTCCCATGACGATACGCAAAAATTTACGCTTACCTTTCCCGCtaa [sequence number 22]

[0104] In certain embodiments, E. coli PfkB contains an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence shown in SEQ ID NO: 23. SEQ ID NO: 23 is shown below. MVRIYTLTLAPSLDSATITPQIYPEGKLRCTAPVFEPGGGGINVARAIAHLGGSATAIFPAGGATGEHLVSLLADENVPVATVEAKDWTRQNLHVHVEASGEQYRFVMPGAALNEDEFRQLEEQVLEIESGAILVISGSLPPGVKLEKLTQLIS AAQKQGIRCIVDSSGEALSAALAIGNIELVKPNQKELSALVNRELTQPDDVRKAAQEIVNSGKAKRVVVSLGPQGALGVDSENCIQVVPPPVKSQSTVGAGDSMVGAMTLKLAENASLEEMVRFGVAAGSAATLNQGTRLCSHDDTQKIYAYLSR [Sequence number 23]

[0105] In certain embodiments, the microorganisms of the Disclosure do not have a deficiency or reduced expression of hexokinase. In certain embodiments, the microorganisms of the Disclosure do not have a deficiency, disruption, or reduced expression of glucokinase. In certain embodiments, the microorganisms of the Disclosure do not have a deficiency or reduced expression of glucose-6-phosphate isomerase.

[0106] In certain embodiments, the microorganisms of this disclosure include mutations in the gene encoding the enzyme of the allose degradation pathway. In certain embodiments, the microorganisms of this disclosure include reduced expression of the gene encoding the enzyme of the allose degradation pathway. In certain embodiments, the enzyme of the allose degradation pathway is allose-6-phosphate isomerase (RpiB) (Entrez Gene ID:948602; EcoCyc ID:EG11827; UniProt ID:P37351). RpiB catalyzes the interconversion of ribulose-5-P and ribose-5-P, and the interconversion of D-allose-6-phosphate (All6P) and D-allose-6-phosphate. In certain embodiments, RpiB is Escherichia coli RpiB. A representative nucleotide sequence of the gene rpiB is shown in SEQ ID NO: 8, or is at least 90%, at least 95%, at least 95%, or at least 99% identical to SEQ ID NO: 8. SEQ ID NO: 8 is shown below. ATGAAAAAGATTGCATTTGGCTGTGATCATGTCGGTTTCATTTTAAAACATGAAATAGTGGCACATTTAGTTGAGCGTGGCGTTGAAGTGATTGATAAAGGAACCTGGTCGTCAGAGCGTACTGATTATCCACATTACGCCAGTCAAGTCGCACTGGCTGTTGCTGGCGGAGAGGTTGATGGCGGGATTTTGATTTGTGGTACTGGCGTCGGTATTTCGATAGCG GCGAACAAGTTTGCCGGAATTCGCGCGGTCGTCTGTAGCGAACCTTATTCCGCGCAACTTTCGCGGCAGCATAACGACACCAACGTGCTGGCTTTTGGTTCACGAGTGGTTGGCCTCGAACTGGCAAAAATGATTGTGGATGCGTGGCTGGGCGCACAGTACGAAGGCGGTCGTCATCAACAACGCGTGGAGGCGATTACGGCAATAGAGCAGCGGAGAAATTGA [Sequence number 8]

[0107] In certain embodiments, E. coli RpiB contains an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence shown in SEQ ID NO: 24. SEQ ID NO: 24 is shown below. MKKIAFGCDHVGFILKHEIVAHLVERGVEVIDKGTWSSERTDYPHYASQVALAVAGGEVDGGILICGTGVGISIAANKFAGIRAVVCSEPYSAQLSRQHNDTNVLAFGSRVVGLELAKMIVDAWLGAQYEGGRHQQRVEAITAIEQRRN [Sequence ID 24]

[0108] In certain embodiments, RpiB is the Bacillus subtilis gene rpiB. A typical amino acid sequence of RpiB is found as A0A6M4JQ63(Uniprot) / BSU36920(KEGG), or is shown in SEQ ID NO: 14, or is at least 90%, at least 95%, at least 95%, or at least 99% identical to SEQ ID NO: 14. SEQ ID NO: 14 is shown below. MKVAIASDHGGVHIRNEIKELMDELQIEYIDMGCDCGSGSVDYPDYAFPVAEKVVSGEVDRGILICGTGIGMSISANKVKGIRCALAHDTFSAKATREHNDTNILAMGERVIGPGLAREIAKIWLTTEFTGGRHQTRIGKISDYEEKNL (Sequence ID 14)

[0109] The typical nucleotide sequence of the Bacillus subtilis gene rpiB is shown in SEQ ID NO: 28, or is at least 90%, at least 95%, at least 95%, or at least 99% identical to SEQ ID NO: 28. SEQ ID NO: 28 is shown below. atgaaagtagccattgcatcggatcatggcggcgttcacattcgaaatgaaatcaaagagttaatggacgaattgcaaattgaatatattgatatgggctgtgactgcggca gcggctctgtcgattatccggattatgcttttccggtggccgaaaaagtggttagcggcgaagttgacagaggcattttaatttgcgggacaggcatcggcatgagcatttcc gctaataaagtaaaagggattcgctgcgcgctggcgcacgataccttcagcgcgaaggcgacgagggagcataatgacacaaacatccttgcgatgggtgaacgggtgatcg gacctggtttggctcgggaaatcgcaaaaatctggctgactactgagtttaccgggggaagacaccaaacgcgtattggaaaaatctccgattatgaagagaaaaacctgtag (Sequence number 28)

[0110] In certain embodiments, RpiB is Lactococcus lactis RpiB. A representative amino acid sequence of the gene rpiB is found as ribose-5-phosphate isomerase, EC 5.3.1.6, or as shown in SEQ ID NO: 15, or is at least 90%, at least 95%, at least 95%, or at least 99% identical to SEQ ID NO: 15. MDNLKKQVGIKAAEFVKSGMVVGLGTGSTAAYFVEELGRRIAEEQLEITGVTTSNVTSSQARALGIPLASIDEVDYVDLTVDGADEIDSSLNGIKGGGAALLMEKIVATYSKDYIWIVDESKLSENLGSFKIPVEVIPYGSQQVFKKFEAAGYAPTWRLNEENERLITDMHHFIIDLHISQIKEPEKLAEELDLMVGVVEHGLFNNMVKKVIVAGNEGVRIINK (Sequence ID 15)

[0111] The typical nucleotide sequence of the Lactococcus lactis gene rpiB is shown in SEQ ID NO: 34, or is at least 90%, at least 95%, at least 95%, or at least 99% identical to SEQ ID NO: 34. SEQ ID NO: 34 is shown below. atgGATAATTTAAAAAAACAAGTCGGCATAAAAGCTGCTGAATTTGTTAAATCAGGAATGGTCGTTGGTTTAGGAACTGGGTCAACAGCAGCCTATTTTGTCGAAGAATTGGGTCGAAGAATTGCCGAAGAACAATTGGAAATTACGGGTGTAACAACGTCCAATGTA ACAAGTAGCCAAGCCAGAGCTCTTGGAATTCCTTTAGCCTCTATTGACGAAGTAGATTATGTTGATTTAACAGTTGATGGCGCAGATGAAATTGATTCTTCACTAAATGGTATTAAAGGTGGTGGAGCAGCACTTCTAATGGAAAAAATTGTTGCAACCTACTCAAAAG ACTATATTTGGATTGTTGATGAAAGTAAATTATCAGAAAATCTAGGATCCTTTAAAATTCCTGTAGAAGTTATTCCTTATGGCTCACAACAAGTTTTTAAAAAATTCGAAGCGGCTGGCTATGCTCCAACTTGGCGTCTAAATGAGGAAAACGAGAGATTGATAACGGA TATGCATCACTTTATTATTGACCTTCATATCTCTCAAATTAAAGAACCAGAAAAACTTGCTGAAGAGCTTGATTTAATGGTTGGAGTTGTTGAACACGGCCTCTTTAATAACATGGTTAAAAAAGTGATTGTTGCTGGCAACGAAGGCGTAAGAATAATAAATAAGtaa (Sequence number 34)

[0112] In certain embodiments, the microorganism of the Disclosure includes mutations in the gene encoding an enzyme in the mannose biosynthesis pathway. In certain embodiments, the microorganism of the Disclosure includes reduced expression of the gene encoding an enzyme in the mannose biosynthesis pathway. In certain embodiments, the enzyme in the mannose biosynthesis pathway is mannose-6-phosphate isomerase (ManA) (Entrez Gene ID:944840; EcoCyc ID:EG10566; UniProt ID:P00946). ManA is involved in the synthesis of GDP-mannose and dolichol-phosphate-mannose, which are required for several important mannosyl translocation reactions. ManA also catalyzes the interconversion of fructose-6-phosphate and mannose-6-phosphate. In certain embodiments, ManA is Escherichia coli ManA. A representative nucleotide sequence of the gene manA is shown in SEQ ID NO: 9 below, or is at least 90%, at least 95%, at least 95%, or at least 99% identical to SEQ ID NO: 9.

[0113] In certain embodiments, E. coli ManA contains an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence shown in SEQ ID NO: 25. SEQ ID NO: 25 is shown below. MQKLINSVQNYAWGSKTALTELYGMENPSSQPMAELWMGAHPKSSSRVQNAAGDIVSLRDVIESDKSTLLGEAVAKRFGELPFLFKVLCAAQPLSIQVHPNKHNSEIGFAKENAAGIPMDAAERNYKDPNHKPELVFALTPFLAMNAFREFSEIVSLLQPVAGAHPAIAHFLQQPDAERLSELFASLLNMQGEEKSRALAILKSALDSQQGEPWQTIRLISEFYPEDSGLFSPLLLNVVKLNPGEAMFLFAETPHAYLQGVALEVMANSDNVLRAGLTPKYIDIPELVANVKFEAKPANQLLTQPVKQGAELDFPIPVDDFAFSLHDLSDKETTISQQSAAILFCVEGDATLWKGSQQLQLKPGESAFIAANESPVTVKGHGRLARVYNKL [Sequence ID 25]

[0114] In certain embodiments, ManA is Bacillus subtilis ManA. A typical amino acid sequence of ManA is found in O31646(Uniprot) / BSU12020(KEGG), or shown in SEQ ID NO: 16, or is at least 90%, at least 95%, at least 95%, or at least 99% identical to SEQ ID NO: 16. MTTEPLFFKPVFKERIWGGTALADFGYTIPSQRTGECWAFAAHQNGQSVVQNGMYKGFTLSELWEHHRHLFGQLEGDRFPLLTKILDADQDLSVQVHPNDEYANIHENGELGKTECWYIIDCQKDAEIIYGHNATTKEELTTMIERGEWDELLRRVKVKPGDFFYVPSGTVHAIGKGILALETQQNSDTTYRLYDYDRKDAEGKLRELHLKKSIEVIEVPSIPERHTVHHEQIEDLLTTTLIECAYFSVGKWNLSGSASLKQQKPFLLISVIEGEGRMISGEYVYPFKKGDHMLLPYGLGEFKLEGYAECIVSHL (Sequence ID 16)

[0115] The typical nucleotide sequence of the Bacillus subtilis gene manA is shown in SEQ ID NO: 29, or is at least 90%, at least 95%, at least 95%, or at least 99% identical to SEQ ID NO: 29. atgacgactgaaccgttatttttcaagcctgttttcaaagaaagaatttggggcgggaccgctttagctgattttggctataccattccgtcacaacgaacaggggagtgctgggcttttgccgcgcatcaaaatggtcaaagcgttgttcaaaacggaatgtat aaggggttcacgctcagcgaattatgggaacatcacagacatttattcggacagcttgaaggggaccgtttccctctgcttacaaaaaattattagatgctgaccaggacttatctgttcaggtgcatccgaatgaatgaatatgccaacatacatgaaaacggtgag cttggaaaaacagaatgctggtacattattgattgccaaaaaagatgccgagattattatggccacaatgcaacaacaaaggaagaactaactaccatgatagagcgtggagaatgggatgagctcttgcgccgtgtaaaggtaaagccgggggatttttctat gtgccaagcggtactgttcatgcgattggaaaaggaattcttgctttggagacgcagcagaactcagacacaacctacagattatatgattatgaccgaaaagatgcagaaggcaagctgcgcgagcttcatctgaaaaagagcattgaagtgatagaggtcccg tctattccagaacggcatacagttcaccatgaacaaattgaggattgcttacaacgacattgattgaatgcgctactttcggtgggaatggaacttatcaggatcagcaagcttaaagcagcaaaaaccattccttcttatcagtgtgattgaagggagaggccgtatgatctctggtgagtatgtctatcctttcaaaaaaggagatcatatgttgctgccttacggtcttggagaatttaaactcgaaggatatgcagaatgtatcgtctcccatctgtaa (sequence number 29)

[0116] In certain embodiments, in Bacillus subtilis, one or both of the following are also deleted, optionally in combination with the deletion of Bacillus subtilis manA:

[0117] (i) Bacillus subtilis yvyI. A typical amino acid sequence of YvyI is found in P39841 (Uniprot) / BSU35790 (KEGG), or shown in Sequence ID No. 17, or is at least 90%, at least 95%, at least 95%, or at least 99% identical to Sequence ID No. 17. Sequence ID No. 17 is shown below. MTQSPIFLTPVFKEKIWGGTALRDRFGYSIPSESTGECWAISAHPKGPSTVANGPYKGKTLIELWEEHREVFGGVEGDRFPLLTKLLDVKEDTSIKVHPDDYYAGENEEGELGKTECWYIIDCKENAEIIYGHTARSKTELVTMINSGDWEGLLRRIKIKPGDFYYVPSGTLHALCKGALVLETQQNSDATYRVYDYDRLDSNGSPRELHFAKAVNAATVPHVDGYIDESTESRKGITIKTFVQGEYFSVYKWDINGEAEMAQDESFLICSVIEGSGLLKYEDKTCPLKKGDHFILPAQMPDFTIKGTCTLIVSHI (Sequence ID 17)

[0118] The typical nucleotide sequence of the Bacillus subtilis gene yvyI is shown in SEQ ID NO: 30, or is at least 90%, at least 95%, at least 95%, or at least 99% identical to SEQ ID NO: 30. SEQ ID NO: 30 is shown below. atgacgcaatcaccgatttttctaacgcctgtgtttaaagaaaaaatctggggcggaaccgctttacgagatagatttggatacagtattccttcagaatcaacgggggaatgctggggccatttccgctcatccaaaaggaccgagcactgttgcaaatggccccgtataaaaggaaagacattgatcgagctttgggaagagcaccgtgaagtattcggcggcgtagaggggatcgg tttccgcttctgacaaagctgctggatgtgaaggaagatacgtcaattaaagttcaccctgatgattactatgccggagaaaacgaagagggagaactcggcaagacggaatgctggtacattatcgactgtaaggaaaacgcagaaatcatttacgggcatacggcccgctcaaaaaccgaacttgtcacaatgatcaacagcggtgactgggagggcctgctcgaagaatcaaaa ttaaaccgggtgatttctattatgtgccgagcggaacgctgcacgcattgtgcaagggggcccttgttttagactcagcaaaattcagatgccacataccgggtgtacgattatgaccgtcttgatagcaacggaagtccgagagagcttcatttgccaaagcggtcaatgccgccacggttccccatgtggagggtatagatagatgaatcgacagaatcaagaaaaaggaataaccattaaaacatttgtccaaggggaatattttcggtttataaatgggacatcaatggcgaagctgaaatggctcaggatgaatcctttctgatttgcagcgtgatagaaggaagcggtttgctcaagtatgaagtatgaagtatgaacttgacggctcaaatgcccgattttcgtgtctcatatttaa (sequence number 30)

[0119] (ii) gmuF of Bacillus subtilis. A typical amino acid sequence of gmuF is found in O05511 (Uniprot) / BSU05870 (KEGG), or shown in Sequence ID No. 18, or is at least 90%, at least 95%, at least 95%, or at least 99% identical to Sequence ID No. 18. Sequence ID No. 18 is shown below. MTHPLFLEPVFKERLWGGTKLRDAFGYAIPSQKTGECWAVSAHAHGSSSVKNGPLAGKTLDQVWKDHPEIFGFPDGKVFPLLVKLLDANMDLSVQVHPDDDYAKLHENGDLGKTECWYIIDCKDDAELILGHHASTKEEFKQRIESGDWNGLLRRIKIKPGDFFYVPSGTLHALCKGTLVLEIQQNSDTTYRVYDYDRCNDQGQKRTLHIEKAMEVITIPHIDKVHTPEVKEVGNAEIIVYVQSDYFSVYKWKISGRAAFPSYQTYLLGSVLSGSGRIINNGIQYECNAGSHFILPAHFGEFTIEGTCEFMISHP (Sequence ID 18)

[0120] The typical nucleotide sequence of the Bacillus subtilis gene gmuF is shown in SEQ ID NO: 31, or is at least 90%, at least 95%, at least 95%, or at least 99% identical to SEQ ID NO: 31. SEQ ID NO: 31 is shown below. atgacgcatccattatttttagagcctgtctttaaagaaagactatggggagggacgaagcttcgtgacgttttggctacgcaataccctcacaaaaaacaggtgagtgctggggccgtttctgcacatgcccatggctcgtcgtctgtaaaaaatggcccgctggcaggaaagacacttgatcaagtatggaaagatcatccagagatattcgggtttccggatggtaaggtgttt ccgctgctggtaaagctgctggaggccaatatggatctctccgtgcaagtccatcctgatgatgattatgcaaaactgcacgaaaatggcgaccttggtaaaacggagtgctggtatatcattgattgcaaagatgacgccgaactaattttgggacatcatgcaagcacaaaggaagagttcaaacaacgaatagaaagcggtgattggaacgggctgctgaggcgaatcaaaatc aagccaggagatttcttttatgtgccaagcggtacactccatgctttatgtaagggaacccttgtccttgaaatccagcaaaaactctgatacaacatatcgcgtatacgattatgaccgctgtaatgaccaggccaaaaaagaactcttcatagaaaaagccatggaagtcataacgataccgcatatcgataaagtgcatacaccggaagtaaaagaagttggtaacgctgagatcatgtttatgtgcaatcagattaatttctcagtgtacaaatggagaattagcggccgagctgctttccttcatatcaaacctatttgctggggagtgttctgagcggatcagcacgaatcataaataatggtattcagtatgaatgcaatgcaggctcacactttatctgcctgcgcatttggagaatttacaatagaaggaacatgtgaattcatgatatctcatccttaa (sequence number 31)

[0121] In certain embodiments, ManA is Lactococcus lactis ManA. A typical amino acid sequence of Lactococcus lactis ManA is mannose-6-phosphate isomerase, EC5.3.1.8, or is shown in SEQ ID NO: 19, or is at least 90%, at least 95%, at least 95%, or at least 99% identical to SEQ ID NO: 19. SEQ ID NO: 19 is shown below. MKEPLFLNSVLQEKIWGGDHLKEFGYDLPSDKVGEYWAISAHPHGVSTIANGEFKGQKLDQLYASHRELFGDSKKEVFPLLTKILDANDWLSVQVHPDDEYGQKHEGELGKTECWYIISAEPGAEIIYGHNAKSREELAEMIKSGDWDHLLRKVKVKTGDFFHVPSGTMHAIGAGIVILETQQSSDTTYRVYDFDRKDDQGNLRELHIQQSIDVLNIPGDKVPENQVKTEKFADAEITTLVKSDFFDVYKWQIHGDHEFTKVADYTLVSVLDGQGKLTVDGNEYPVEKGAHFILPSNIEKWNLSGQLEIIASNPA (Sequence ID 19)

[0122] The typical nucleotide sequence of the Lactococcus lactis gene manA is shown in SEQ ID NO: 35, or is at least 90%, at least 95%, at least 95%, or at least 99% identical to SEQ ID NO: 35. SEQ ID NO: 35 is shown below. atgAAAGAACCATTGTTTTTGAACTCAGTTTTGCAAGAAAAAATCTGGGGCGGCGACCATTTGAAAGAGTTTGGCTATGATTTGCCATCAGACAAAGTTGGTGAATATTGGGCTTATTCTGCTCATCCACATGGTGTGTCAACAATTGCTAATGGCGAATTTAAAGGTCAAAAACTTGACCAATTATACGCAAGTCACCGCGAATTGTTTGGTGATAGTAAAAAAGAAGTTTTTCCC TTACTAACTAAAATTTAGATGCCAATGACTGGCTTTCTGTGCAAGTTCATCCAGATGAGAATATGGACAAAAACATGAAGGTGAACTTGGAAAAACTGAATGTTGGTACATATTATTCAGCTGAACCAGGTGCTGAAATTATCTATGGACATAATGCTAAATCACGTGAAGAATTAGCAGAAATGATTAAATCTGGTGATTGGGATCATTGTTACGTAAGGTAAAAGTGAAAAACA GGAGATTTCTTCCATGTTCCGTCAGGAACAATGCACGCAATCGGTGCTGGAATTGTTATTCTGAAACACAACAATCTTCTGATACAACTTACCGTGTTTTATGATTTCGACCGTAAAGATGACCAAGGAAATCTACGTGAATTACATATTCAACAATCAATTGATGTATTGAATATTCCGGGCGACAAAGTTCCTGAAATCAAGTTAAAACTGAAAAAATTTGCTGATGCAGAAAATTACAACTCTTGTGGAAATCAGATTTCTTGATGTTATAAATGGCAAATTCATGGTGACCATGAATTTACCAAAGTTGCTGATTACACTTTAGTTTCTGTACTTGGATGGTCAAGGAAAATTAACAGTTGGAAAATGAATATCCAGTTGAAAAAGGAGCTCATTTCATCTACCAAGCAACATTGAAAAATGGAATTTGTCTGGTCAATTAGAAATTATTGCCAGCAATCCTGCCTgataa (sequence number 35)

[0123] In certain embodiments, the microorganisms of the disclosure include mutations in genes encoding enzymes for carbohydrate metabolism. In certain embodiments, the microorganisms of the disclosure include reduced expression of genes encoding enzymes for carbohydrate metabolism.

[0124] In certain embodiments, the enzyme involved in carbohydrate metabolism is the D-tagatose-1,6-bisphosphate aldolase subunit (GatY) (UniProt ID: P0C8J6). GatY catalyzes the reversible aldol condensation of dihydroxyacetone phosphate (DHAP or glyceron-phosphate) and glyceraldehyde 3-phosphate (G3P) to produce tagatose-1,6-bisphosphate (TBP). In certain embodiments, GatY is E. coli GatY. A representative nucleotide sequence of the gatY gene is shown in SEQ ID NO: 81, or is at least 90%, at least 95%, at least 95%, or at least 99% identical to SEQ ID NO: 81. In certain embodiments, E. coli GatY contains an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence described in SEQ ID NO: 82. SEQ ID NOs: 81 and 82 are shown below. ATGTACGTGGTATCGACAAAGCAGATGCTGAACAACGCACAGCGCGGCGGTTATGCGGTTCGGCATTCAATATTCACAATCTCGAAACGATGCAAGTGGTGGTAGAAACCGCTGCCAACCTGCATGCGCCGGTCATCATCGCCGGAACGCCTGGCACATTTACTCATGCTGGTACAGAAAATCTGTTGGCGCTGGTCAGCGCGATGGCGAAG CAATATCACCATCCACTGGCAATTCATCTCGACCATCACACGAAATTTGACGATATCGCTCAGAAGGTTCGTTCTGGCGTGCGCTCAGTCATGATTGACGCCTCGCATTTGCCTTTGCGCAAAATTTTCACGGGTCAAAGAGGTGGTGGATTTTTGCCATCGCTTTGATGTCAGCGTCGAGGCGGAGCTGGGGCAACTTGGCGGCCAGGAAG ATGATGTGCAAGTCAATGAAGCCGATGCGTTGTACACCAACCCCGCTCAGGCGCGTGAATTTGCCGAGGCAACCGGAATTGATTCCCTGGGCGGTCGCCATCGGCACGGCTCATGGGATGTATGCACGCGCACCGGCGCTTGATTTTTCTAGACTGGAAACATTCGCCAGTGGGTGTAACTTACCGCTGGTGCTGCATGGCGCTCAGGGTATCGACTAAGGATATCGAGCAAACCATCAAACTGGGGATATGCAAAATCAACGTTGCAACGGAGCTGAAAAATGCCTTCTCGCGAGGCGTTAAAAATTACCTGACCGACCCACCCTGAAGCGACCCCGGGATTATTTGCAGTCGGCTAAATCCGCAATGCGCGATGTGGTGTGGACAAAGTGATTGCCGGTGGCTGCGAGGCAGGGCATAAA [sequence number 81] MYVVSTKQMLNNAQRGGYAVPAFNIHNLETMQVVVETAANLHAPVIIAGTPGTFTHAGTENLLALVSAMAKQYHHPLAIHLDHHTKFDDIAQKVRSGVRSVMIDASHLPFAQNISRVKEVVDFCHRFDVSVEAELGQLGGQE DDVQVNEADALYTNPAQAREFAEATGIDSLAVAIGTAHGMYASAPALDFSRLENIRQWVNLPLVLHGASGLSTKDIQQTIKLGICKINVATELKNAFSQALKNYLTEHPEATDPRDYLQSAKSAMRDVVSKVIADCGCEGRA [Sequence number 82]

[0125] In certain embodiments, the enzyme involved in carbohydrate metabolism is the D-tagatose-1,6-bisphosphate aldolase subunit (KbaY) (UniProt ID: P0AB74). KbaY catalyzes the reversible aldol condensation of dihydroxyacetone phosphate (DHAP or glyceron-phosphate) and glyceraldehyde 3-phosphate (G3P) to produce tagatose-1,6-bisphosphate (TBP). In certain embodiments, KbaY is E. coli KbaY. A representative nucleotide sequence of the gene kbaY is shown in SEQ ID NO: 83, or is at least 90%, at least 95%, at least 95%, or at least 99% identical to SEQ ID NO: 83. In certain embodiments, E. coli KbaY contains an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence described in SEQ ID NO: 84. SEQ ID NOs: 83 and 84 are shown below. ATGAGCATTATCTCCACTAAATATCTGTTACAGGACGCCCAGGCCAATGGCTACGCGGTGCCTGCTTTTAACATTCATAACGCCGAGACGATCCAAGCGATCTCGAAGTGTGCAGTGAAATGCGATCGCCGGTGATCCTCGCCGGAACGCCGGGGACCTTTAAACACATCGGCCTGGAAGAGATCTACGCCCTGTGTAGCGCCTATTCCACAAC CTACAACATGCCACTGGCGCTGCATCTCGACCACCACGAATCGCTGGATGATATTCGCCGTAAAGTCCACGCAGGTGTGCGCAGTGCGATCGACGGCAGCCACTTCCCGTTTGCGAGAACGTGAAGCTGGTGAAATCGGTTGTTGACTTCTGCCACTCACAAGATTGCAGCGTGGAAGCAGAACTGGGCCGCCTGGGCGGTGTTGAAGAT ACATGAGCGTTGACGCCGAAAGTGCATTCCTGACCGATCCACAAGAAGCTAAACGCTTTGTCGAACTGACTGGCGTCGACAGCCTGGCGGTAGCGATTGGTACGGCCAGGCTTATACAGCAAAACCCGGAAGATTGATTTCCAGCGGCTGGCGGAAATTCGTGGAAGTGGTGGATGTTCCTCGGTGCTGCATGGTGCCAGCGATGTTCGGATGAATTTGTCGTGCACTATTGAACTTGGCGTCACAAAAGTGAACGTTGCCACAGAATTAAAAATAGCCTTCGCTGGCGCGGTTAAGCCTGGTTTGCGGAAAATCCGCAGGGTAATGATCCTCGTTATTATATGCGCGTCGGAATGGATGCGAATGAAGTTGTCAGAAATAAAATTAATGTCTGTGTTCAGCGGAATCGAATTTCAGCATAA [sequence number 83] MSIISTKYLLQDAQANGYAVPAFNIHNAETIQAILEVCSEMRSPVILAGTPGTFKHIALEEIYALCSAYSTTYNMPLALHLDHHESLDDIRRKVHAGVRSAMIDGSHFPFAENVKLVKSVVDFCHSQDCSVEAELGRLGGVEDDMSVDAESAFLTDPQEAKRFVELTGVDSLAVAIGTAHGLYSKTPKIDFQRLAEIREVVDVPLVLHGASDVPDEFVRRTIELGVTKVNVATELKIAFAGAVKAWFAENPQGNDPRYYMRVGMDAMKEVVRNKINVCGSANRISA [Sequence ID 84]

[0126] In certain embodiments, the gene deletion includes non-frameshift deletions, frameshift deletions, or combinations thereof. In certain embodiments, the gene deletion can be achieved by insertions (e.g., non-frameshift insertions, frameshift insertions, or combinations thereof). In certain embodiments, the gene deletion includes nonsense mutations.

[0127] ·2.3.Cells This disclosure provides recombinant microorganisms. Any cultureable microorganism is suitable for use in the compositions and methods described herein. In some embodiments, the microorganism is a bacterium. In some embodiments, the microorganism is Aceiobacter aceti, Achromobacter, Acidiphilium, Acinetobacter, Actinomadura, Actinoplanes, Aeropyrumpernix, Agrobacterium, Alcaligenes, Ananas comosus (M), Arthrobacter, Bacillus alcalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulars, Bacillus clausii, Bacillus lentus, Bacillus lichenifirmis, Bacillus macerans, Bacillus stearothermophilus, Bacillus subtilis, Bifidobacterium, Brevibacillus brevis, Burkholderia cepacia, Candida cylindracea, Carica papaya (L), Cellulosimicrobium, Cephalosporium, Chaetomium erraticum, Chaetomium gracile, Clostridium, Clostridium butyricum, Clostridium acetobutylicum, Clostridium thermocellum, Corynebacterium (glutamicum), Corynebacterium efficiens, Escherichia coli, Enterococcus, Erwina chrysanthemi, Gliconobacter, Gluconacetobacter, Haloarcula, Humicola insolens, Kitasatospora setae, Klebsiella, Klebsiella oxytoca, Kocuria, Lactlactis, Lactobacillus, Lactobacillusfermentum, Lactobacillus sake, Lactococcus, Lactococcus lactis, Leuconostoc, Methylocystis, Methanolobus siciliae, Methanogenium organophilum, Methanobacterium bryantii, Microbacterium imperiale, Micrococcus lysodeikticus, Microlunatus, Mucorjavanicus, Mycobacterium, Myrothecium, Nitrobacter, Nitrosomonas, Nocardia, Papaya carica, Pediococcus, Pediococcus halophilus, Paracoccus pantotrophus, Propionibacterium, Pseudomonas, Pseudomonas fluorescens, Pseudomonas denitrificans, Pyrococcus, Pyrococcus furiosus, Pyrococcus horikoshii, Rhizobium, Rhizomucor miehei, Rhizomucor pusillus Lindt, Rhizopus, Rhizopus delemar, Rhizopus japonicas, Rhizopus niveus, Rhizopus oryzae, Rhizopus oligosporus. Rhodococcus, Sckroiina libertina, Sphingobacterium multivorum, Sphingobium, Sphingomonas, Streptococcus, Streptococcus thermophilus Y-1, Streptomyces, Streptomyces griseus, Streptomyces lividans, Streptomyces murinus, Streptomyces ruhiginosus, Streptomyces violaceoruber, Streptoverticillium mobaraense, Tetragenococcus, Thermus, ThiosphaeraThe microorganism is selected from the group consisting of pantotropha, Trametes, Vibrio alginolyticus, Xanthomonas, Zymomonas, and Zymomonus mobilis. In certain embodiments, the microorganism is Escherichia coli (E. coli). In certain embodiments, the microorganism is Bacillus subtilis. In certain embodiments, the microorganism is Lactococcus latillus.

[0128] In certain embodiments, Escherichia coli (E. coli) is selected from the group consisting of enterotoxigenic Escherichia coli (ETEC), enteropathogenic Escherichia coli (EPEC), enteroinvasive Escherichia coli (EIEC), enterohemorrhagic Escherichia coli (EHEC), urinary tract pathogenic Escherichia coli (UPEC), verotoxin-producing Escherichia coli, Escherichia coli O157:H7, Escherichia coli O104:H4, Escherichia coli O121, Escherichia coli O104:H21, Escherichia coli K1, and Escherichia coli NC101. In certain embodiments, Escherichia coli is Escherichia coli K12. In certain embodiments, Escherichia coli is Escherichia coli B. In certain embodiments, Escherichia coli is Escherichia coli C.

[0129] In certain embodiments, the Escherichia coli (E. coli) is NCTC 12757, NCTC 12779, NCTC 12790, NCTC 12796, NCTC 12811, ATCC 11229, ATCC 25922, ATCC 8739, DSM 30083, BC 5849, BC 8265, BC 8267, BC 8268, BC 8270, BC 8271, BC 8272, BC 8273, BC 8276, BC 8277, BC 8278, BC 8279, BC 8312, BC 8317, BC 8319, BC 8320, BC 8321, BC 8322, BC 8326, BC 8327, BC 8331, BC It is derived from strains selected from the group consisting of 8335, BC 8338, BC 8341, BC 8344, BC 8345, BC 8346, BC 8347, BC 8348, BC 8863, and BC 8864.

[0130] BC 4734 (O26:H11), BC 4735 (O157:H-) BC 4735 (O157:H-) BC 4735 (O157:H-) (nd)、BC 4738 (O157:H7)、BC 4945 (O26:H-)、BC 4946 (O157:H7)、BC 4947 (O111:H-)、BC 4948 (O157:H)、BC 4948 (O157:H)、BC 4948 (O157:H-)、BC 4948 (O157:H-)49) (O157:H7)、BC 5580 (O157:H7)、BC 5582 (O3:H)、BC 5643 (O2:H5)、BC 5644 (O128)、BC 5645 (O55:H-)、BC 5645 (O55:H-)、BC 5645 (O157:H-6) (O101:H9)、BC 5648 (O103:H2)、BC 5850 (O22:H8)、BC 5851 (O55:H-)、BC 5852 (O48:H21)、BC 5853 (O26:H11)854BC (O157:H7)、BC 5855 (O157:H-)、BC 5856 (O26:H-)、BC 5857 (O103:H2)、BC 5858 (O26:H11)、BC 7832、BC 7833(O153:H-78) (ONT:H-)、BC 7835 (O103:H2)、BC 7836 (O57:H-)、BC 7837 (ONT:H-)、BC 7838、BC 7839 (O128:H2)、BC 7840 (O157:H-78) (O23:H-)、BC 7842 (O157:H-)、BC 7843、BC 7844 (O157:H-)、BC 7845 (O103:H2)、BC 7846 (O26:H11)、BC 7846 (O157:H-8) (O157:H-)、BC 7849 (O156:H47)、BC 7850、BC 7851 (O157:H-)、BC 7852 (O157:H-)、BC 7853 (O5:H-)、BC 7853 (O5:H-)、BC 7853 (O157:H-7) (O157:H7)、BC 7856 (O26:H-)、BC 7857、BC 7858、BC 7859 (ONT:H-)、BC 7860 (O129:H-)、BC 7861 (O129:H-)、BC 7861、BC 7862 (BCO138383) 7864 (Oraw type:H-), BC 7865, BC 7866 (O26:H-), BC 7867 (O raw type:H-), BC 7868, BC 7869 (ONT:H-), BC 7870 (O113:H-), BC 7871 (ONT:H-), BC 7872 (ONT:H-), BC 7873, BC 7874 (O raw type: H-), BC 7875 (O157:H-), BC 7876 (O111:H-), BC 7877 (O146:H21), BC 7878 (O145:H-), BC 7879 (O22:H8), BC 7880 (O raw type: H-), BC 7881 (O145:H-), BC It is derived from strains selected from the group consisting of 8275 (O157:H7), BC 8318 (O55:K-:H-), BC 8325 (O157:H7), BC 8332 (ONT), and BC 8333.

[0131] In a particular embodiment, the E. coli strains are BC 8246 (O152:K-:H-), BC 8247 (O124:K(72):H3), BC 8248 (O124), BC 8249 (O112), BC 8250 (O136:K(78):H-), BC 8251 (O124:H-), BC 8252 (O144:K-:H-), BC 8253 (O143:K:H-), BC 8254 (O143), BC 8255 (O112), BC 8256 (O28a.e), BC 8257 (O124:H-), BC 8258 (O143), BC 8259 (O167:K-:H5), BC 8260 It is derived from strains selected from the group consisting of (O128a. c.:H35), BC 8261 (O164), BC 8262 (O164:K-:H-), BC 8263 (O164), and BC 8264 (O124).

[0132] In a particular embodiment, the E. coli strains are BC 5581 (O78:H11), BC 5583 (O2:K1), BC 8221 (O118), BC 8222 (O148:H-), BC 8223 (O111), BC 8224 (O110:H-), BC 8225 (O148), BC 8226 (O118), BC 8227 (O25:H42), BC 8229 (O6), BC 8231 (O153:H45), BC 8232 (O9), BC 8233 (O148), BC 8234 (O128), BC 8235 (O118), BC 8237 (O111), BC 8238 It is derived from strains selected from the group consisting of (O110:H17), BC 8240 (O148), BC 8241 (O6H16), BC 8243 (O153), BC 8244 (O15:H-), BC 8245 (O20), BC 8269 (O125a.c:H-), BC 8313 (O6:H6), BC 8315 (O153:H-), BC 8329, BC 8334 (O118:H12), and BC 8339.

[0133] The details of this range are BC 7567 (O86), BC 7568 (O128), BC 7571 (O114), BC 7572 (O119), and BC 7572 (O119). 7573 (O125), BC 7574 (O124), BC 7576 (O127a), BC 7577 (O126), BC 7578 (O142), BC 7579 (O26), BC 7580 (OK26), BC 7581 (O142) BC 7582 (O55) BC 7583 (O158) BC 7584 (O-) BC 7585 (O-) BC 7586 (O-) BC 8330 BC 8550 (O26) BC 8551 (O55) BC 8552 (O158) BC 8553 (O26) BC 8554 (O158) BC 8555 (O86) BC 8556 (O128) BC 8557 (OK26) BC 8558 (O55) BC 8558 (O55) BC 8560 (O158) BC 8561 (O158) BC 8562 (O114) BC 8563 (O86) BC 8564 (O128) BC 8565 (O158) BC 8566 (O158) BC 8567 (O158) BC 8568 (O111) BC 8569 (O128) BC 8570 (O114) BC 8571 (O128) BC 8572 (O128) BC 8573 (O158) BC 8574 (O158) BC 8575 (O158) BC 8576 (O158) BC 8577 (O158) BC 8578 (O158) BC 8583 (O158) BC 8583 (O128) BC 8584 (O158) BC 8585 (O128) BC (O158) BC 8588 (O26) BC 8589 (O86) BC 8590 (O127) BC 8591 (O128) BC 8592 (O114) BC 8593 (O114) BC 8594 (O114) BC (O125) BC 8596 (O158) BC 8597 (O26) BC 8598 (O26) BC 8599 (O158) BC 8605 (O158) BC 8606 (O158) BC 8607 (O158) BC 8608 BCIt is derived from strains selected from the group consisting of (O128), BC 8609 (O55), BC 8610 (O114), BC 8615 (O158), BC 8616 (O128), BC 8617 (O26), BC 8618 (O86), BC 8619, BC 8620, BC 8621, BC 8622, BC 8623, BC 8624 (O158), and BC 8625 (O158).

[0134] In a particular embodiment, the Bacillus subtilis bacteria are derived from 168 strains.

[0135] In a particular embodiment, the Lactococcus latylus is derived from strain A12.

[0136] In certain embodiments, the microorganism is a fungal cell. In some embodiments, the fungal cells are Aspergillus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryze, Aspergillus melleus, Aspergillus pulverulentus, Aspergillus saitoi, Aspergillus sojea, Aspergillus terreus, Aspergillus pseudoterreus, Aspergillus usamii, Candida rugosa, Issatchenkia orientalis, Kluyveromyces, Kluyveromyces fragilis, Kluyveromyces lactis, Kluyveromyces marxianas, Penicillium, Penicillium camemberti, Penicillium citrinum, Penicillium emersonii, Penicillium roqueforti, Penicillium lilactinum, Penicillium multicolor, and Rhodosporidium. The species is selected from the group consisting of toruloides, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma, Trichoderma longibrachiatum, Trichoderma reesei, Trichoderma viride, Trichosporon penicillaium, Yarrowia lipolytica, and Zygosaccharomyces rouxii.

[0137] In certain embodiments, the microorganism is a yeast cell. In certain embodiments, the yeast cell is a budding yeast (Saccharomyces cerevisiae).

[0138] 2.4. Exemplary Microorganisms In certain embodiments, the disclosure provides recombinant microorganisms having increased D-tagatose production compared to naturally occurring microorganisms. In certain embodiments, the recombinant microorganism comprises an exogenous epimerase. In certain embodiments, the exogenous epimerase is the tagatose-1,6-bisphosphate aldolase subunit GatZ and / or the ketose diphosphate aldolase subunit KbaZ. In certain embodiments, the recombinant microorganism comprises an exogenous phosphatase. In certain embodiments, the exogenous phosphatase is hexitol phosphatase A (HxpA). In certain embodiments, the recombinant microorganism is a bacterium. In certain embodiments, the bacterium is Escherichia coli (E. coli).

[0139] In certain embodiments, the disclosure provides recombinant microorganisms having increased D-tagatose production compared to naturally occurring microorganisms. In certain embodiments, the recombinant microorganism comprises an exogenous epimerase and deletions of 1, 2, 3, or 4 genes. In certain embodiments, the exogenous epimerase is the tagatose-1,6-bisphosphate aldolase subunit GatZ and / or the ketose diphosphate aldolase subunit KbaZ. In certain embodiments, the exogenous phosphatase is hexitol phosphatase A (HxpA). In certain embodiments, the four deletion genes are glucose-6-phosphate 1-dehydrogenase, phosphofructokinase-1, D-allulose-6-phosphate 3-epimerase, and mannose-6-phosphate isomerase. In certain embodiments, the recombinant microorganism further comprises the exogenous phosphatase. In certain embodiments, the recombinant microorganism is a bacterium. In certain embodiments, the bacteria are Escherichia coli (E. coli).

[0140] In certain embodiments, the disclosure provides a microorganism comprising a recombinant polynucleotide having increased D-tagatose production compared to a naturally occurring microorganism. In certain embodiments, the recombinant polynucleotide comprises a nucleotide sequence encoding an exogenous epimerase. In certain embodiments, the exogenous epimerase is the tagatose-1,6-bisphosphate aldolase subunit GatZ and / or the ketose diphosphate aldolase subunit KbaZ. In certain embodiments, the recombinant polynucleotide further comprises a nucleotide sequence encoding an exogenous phosphatase. In certain embodiments, the exogenous phosphatase is hexitol phosphatase A (HxpA). In certain embodiments, the recombinant microorganism is a bacterium. In certain embodiments, the bacterium is Escherichia coli (E. coli).

[0141] In certain embodiments, the disclosure provides a microorganism comprising a recombinant polynucleotide having increased D-tagatose production compared to a naturally occurring microorganism. In certain embodiments, the recombinant polynucleotide comprises a nucleotide sequence encoding an exogenous epimerase. In certain embodiments, the exogenous epimerase is the tagatose-1,6-bisphosphate aldolase subunit GatZ and / or the ketose diphosphate aldolase subunit KbaZ. In certain embodiments, the recombinant polynucleotide further comprises an exogenous phosphatase. In certain embodiments, the exogenous phosphatase is hexitol phosphatase A (HxpA). In certain embodiments, the microorganism further comprises a deletion of a first gene. In certain embodiments, the first gene is glucose-6-phosphate 1-dehydrogenase. In certain embodiments, the microorganism further comprises a deletion of a second gene. In certain embodiments, the second gene is phosphofructokinase-1. In certain embodiments, the microorganism further comprises a deletion of a third gene. In certain embodiments, the third gene is D-allulose-6-phosphate 3-epimerase. In certain embodiments, the microorganism further comprises a deletion of a fourth gene. In certain embodiments, the fourth gene is mannose-6-phosphate isomerase. In certain embodiments, the recombinant microorganism is a bacterium. In certain embodiments, the bacterium is Escherichia coli (E. coli). In certain embodiments, the bacterium is Bacillus subtilis (B. subtilis). In certain embodiments, the bacterium is Lactococcus lactis (L. lactis).

[0142] In certain embodiments, the disclosure provides a recombinant microorganism having increased D-tagatose production compared to a naturally occurring microorganism. In certain embodiments, the recombinant microorganism comprises an exogenous epimerase, an exogenous nuclease, an sgRNA, and four gene deletions. In certain embodiments, the exogenous epimerase is the tagatose-1,6-bisphosphate aldolase subunit GatZ and / or the ketose diphosphate aldolase subunit KbaZ. In certain embodiments, the recombinant microorganism further comprises an exogenous phosphatase. In certain embodiments, the exogenous phosphatase is hexitol phosphatase A (HxpA). In certain embodiments, the four deletion genes are zwf, pfkA, alsE, and manA. In certain embodiments, the sgRNA targets pfkB. In certain embodiments, the recombinant microorganism is a bacterium. In certain embodiments, the bacterium is Escherichia coli (E. coli). In certain embodiments, the bacterium is Bacillus subtilis (B. subtilis). In certain embodiments, the bacterium is Lactococcus lactis (L. lactis).

[0143] In certain embodiments, the disclosure provides a microorganism comprising a recombinant polynucleotide having increased production of tagatose compared to a naturally occurring microorganism. In certain embodiments, the recombinant polynucleotide comprises a nucleotide sequence encoding an exogenous epimerase and a nucleotide sequence encoding a nuclease. In certain embodiments, the exogenous epimerase is the tagatose-1,6-bisphosphate aldolase subunit GatZ and / or the ketose diphosphate aldolase subunit KbaZ. In certain embodiments, the recombinant polynucleotide further comprises a nucleotide sequence encoding an exogenous phosphatase. In certain embodiments, the exogenous phosphatase is hexitol phosphatase A (HxpA). In certain embodiments, the microorganism further comprises a deletion of a first gene. In certain embodiments, the first gene is zwf. In certain embodiments, the microorganism further comprises a deletion of a second gene. In certain embodiments, the second gene is pfkA. In certain embodiments, the microorganism further comprises a deletion of a third gene. In certain embodiments, the third gene is alsE. In certain embodiments, the microorganism further comprises a deletion of a fourth gene. In certain embodiments, the fourth gene is manA. In certain embodiments, the microorganism further comprises sgRNA. In certain embodiments, the sgRNA targets pfkB. In certain embodiments, the recombinant microorganism is a bacterium. In certain embodiments, the bacterium is Escherichia coli (E. coli). In certain embodiments, the bacterium is Bacillus subtilis (B. subtilis). In certain embodiments, the bacterium is Lactococcus lactis (L. lactis).

[0144] In certain embodiments, the disclosure provides recombinant microorganisms having increased D-tagatose production compared to naturally occurring microorganisms. In certain embodiments, the recombinant microorganism comprises an exogenous epimerase. In certain embodiments, the exogenous epimerase is the tagatose-1,6-bisphosphate aldolase subunit GatZ and / or the ketose diphosphate aldolase subunit KbaZ. In certain embodiments, the recombinant microorganism comprises an exogenous phosphatase. In certain embodiments, the exogenous phosphatase is hexitol phosphatase A (HxpA). In certain embodiments, the recombinant microorganism further comprises mutations in at least one gene selected from glucose-6-phosphate 1-dehydrogenase (zwf), phosphofructokinase-1 (pfkA), phosphofructokinase-2 (pfkB), pyruvate kinase (pykF), D-allulose-6-phosphate 3-epimerase (alsE), mannose-6-phosphate isomerase (manA), phosphoglucomutase (pgm), D-tagatose-1,6-bisphosphate aldolase subunit (gatY), D-tagatose-1,6-bisphosphate aldolase subunit (kbaY), and combinations thereof. In certain embodiments, the recombinant microorganism is a bacterium. In certain embodiments, the bacterium is Escherichia coli (E. coli).

[0145] In certain embodiments, the disclosure provides recombinant microorganisms having increased D-tagatose production compared to naturally occurring microorganisms. In certain embodiments, the recombinant microorganism comprises an exogenous epimerase. In certain embodiments, the exogenous epimerase is the tagatose-1,6-bisphosphate aldolase subunit GatZ and / or the ketose diphosphate aldolase subunit KbaZ. In certain embodiments, the recombinant microorganism comprises an exogenous phosphatase. In certain embodiments, the exogenous phosphatase is hexitol phosphatase A (HxpA). In certain embodiments, the recombinant microorganism further comprises mutations in at least one gene selected from glucose-6-phosphate 1-dehydrogenase (zwf), phosphofructokinase-1 (pfkA), phosphofructokinase-2 (pfkB), pyruvate kinase (pykF), D-allulose-6-phosphate 3-epimerase (alsE), mannose-6-phosphate isomerase (manA), phosphoglucomutase (pgm), D-tagatose-1,6-bisphosphate aldolase subunit (gatY), D-tagatose-1,6-bisphosphate aldolase subunit (kbaY), and combinations thereof. In certain embodiments, the recombinant microorganism is a bacterium. In certain embodiments, the bacterium is Bacillus subtilis.

[0146] In certain embodiments, the disclosure provides recombinant microorganisms having increased D-tagatose production compared to naturally occurring microorganisms. In certain embodiments, the recombinant microorganism comprises an exogenous epimerase. In certain embodiments, the exogenous epimerase is the tagatose-1,6-bisphosphate aldolase subunit GatZ and / or the ketose diphosphate aldolase subunit KbaZ. In certain embodiments, the recombinant microorganism comprises an exogenous phosphatase. In certain embodiments, the exogenous phosphatase is hexitol phosphatase A (HxpA). In certain embodiments, the recombinant microorganism further comprises mutations in at least one gene selected from glucose-6-phosphate 1-dehydrogenase (zwf), phosphofructokinase-1 (pfkA), phosphofructokinase-2 (pfkB), pyruvate kinase (pykF), D-allulose-6-phosphate 3-epimerase (alsE), mannose-6-phosphate isomerase (manA), phosphoglucomutase (pgm), D-tagatose-1,6-bisphosphate aldolase subunit (gatY), D-tagatose-1,6-bisphosphate aldolase subunit (kbaY), and combinations thereof. In certain embodiments, the recombinant microorganism is a bacterium. In certain embodiments, the bacterium is Lactococcus lactis (L. lactis).

[0147] ●3. Method for preparing and constructing Tagatose microorganisms This disclosure also provides methods for producing and / or constructing any of the microorganisms disclosed herein. One or more recombinant polynucleotides of this disclosure can be introduced into microorganisms using, but are not limited to, many recombinant techniques commonly known in the art, including protoplast fusion, transfection, transformation, conjugation, and transduction. These techniques include conventional molecular biology techniques (e.g., recombinant techniques), microbiology, cell biology, and biochemistry, which are within the scope of the skills of those skilled in the art. Further information on these techniques can be found in: Molecular Cloning: A Laboratory Manual, 2nd edition (Sambrook et al., 1989); Oligonucleotide Synthesis (Gait, ed., 1984); Animal Cell Culture (Freshney, ed., 1987); Gene Transfer Vectors for Mammalian Cells (Miller and Calos, eds., 1987); Current Protocols in Molecular Biology (Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); and Current Protocols in Immunology (Coligan et al., ed., 1991).

[0148] 3.1 Recombinant Polynucleotides In certain embodiments, the recombinant polynucleotides disclosed herein can be stably integrated into microbial chromosomes. In certain embodiments, the recombinant polynucleotides disclosed herein are stably integrated into microbial chromosomes using homologous recombination, transposition-based chromosome integration, recombinase-mediated cassette exchange (RMCE; e.g., using the Cre-lox system), or integration plasmids (e.g., yeast integration plasmids). Various integration techniques suitable for various microorganisms are known in the art (see, for example, Griffiths, AJF, Miller, JH, Suzuki, DT et al., An Introduction to Genetic Analysis. 7th edition, New York: WHFreeman; 2000). In certain embodiments, the recombinant polynucleotides disclosed herein are maintained on an extrachromosomal plasmid (e.g., an expression plasmid or vector) in the recombinant microorganism of this disclosure. Various extrachromosomal plasmids suitable for various microorganisms are known in the art, including, but are not limited to, replication plasmids (e.g., yeast replication plasmids containing autonomous replication sequences, ARS), centromere plasmids (e.g., yeast centromere plasmids containing autonomous replication sequences, CEN), episomal plasmids (e.g., 2-pm plasmids), and / or artificial chromosomes (e.g., yeast artificial chromosomes, YAC, or bacterial artificial chromosomes, BAC).

[0149] ·3.1.1. Vector In certain embodiments, this disclosure provides vectors comprising nucleotide sequences disclosed herein. As used herein, the term “vector” refers to a polynucleotide construct designed to introduce nucleic acids into one or more microorganisms. Vectors may include, but are not limited to, cloning vectors, expression vectors, shuttle vectors, plasmids, and cassettes. As used herein, the term “plasmid” refers to a circular double-stranded DNA construct used as a cloning vector and / or expression vector. In certain embodiments, the plasmid may be an extrachromosomal self-replicating genetic element (e.g., an episomal plasmid) when introduced into a microorganism. In certain embodiments, the plasmid may be integrated into a microbial chromosome. In certain embodiments, the vector may direct the expression of a operably linked coding region, e.g., an “expression vector”. These expression vectors enable the expression of exogenous polynucleotides and / or exogenous polypeptides in microorganisms. In certain embodiments, the vector enables the integration of one or more polynucleotides into the genome of a microorganism.

[0150] In certain embodiments, the vector disclosed herein includes a promoter. In certain embodiments, the vector is a bacterial expression vector or a prokaryotic expression vector. In certain embodiments, the vector is a yeast expression vector or a fungal cell expression vector.

[0151] In certain embodiments, the vectors disclosed herein include a nucleotide sequence(plural) in a single operon.

[0152] 3.1.2. Promoter In certain non-limiting embodiments, the recombinant polynucleotides disclosed herein include a control sequence, enhancer, or promoter. For example, but not limited to, nucleotide sequences encoding the gatZ gene and / or the hxpB gene may be operably ligated to a control sequence, enhancer, or promoter.

[0153] As used herein, the term “promoter” refers to any nucleotide sequence that regulates the initiation of transcription of a particular coding sequence under its control. Biologically, a promoter does not transcribe itself, but regulates an assembly of components that initiate the transcription of other nucleotide sequences. Furthermore, a promoter can restrict this assembly and subsequent transcription to specific preconditions. For example, but not limited to, a promoter can enable transcription in response to one or more environmental, temporal, or developmental stimuli. Bacterial and fungal cells have numerous proteins that sense external or internal conditions and initiate signaling cascades that end with the binding of a protein to a particular promoter and the subsequent initiation of nucleic acid transcription under the control of that promoter. In certain embodiments, promoters are endogenous. In certain embodiments, promoters are exogenous. In certain embodiments, promoters are artificially designed for expression in a particular species.

[0154] In certain embodiments, the promoter is a constitutive promoter. A constitutive promoter is a promoter that drives the continuous expression of a nucleotide sequence without interruption in response to internal or external stimuli. Constitutive promoters are commonly used in recombinant engineering to ensure the continuous expression of a desired nucleotide sequence. Constitutive promoters result in robust levels of nucleic acid expression and are therefore used in many recombinant engineering applications to achieve high levels of recombinant protein and enzyme activity. A non-limiting example of a constitutive promoter included in this disclosure is the E. coli promoter P spc , P bla , P RNAI , P RNAII , P1 and P2 of rrnB, and lambda phage promoter P Lexamples include (Liang, S.T. et al., JMoi. Biol. 292(1):19-37(1999)). In some embodiments, the promoter is active during the stationary phase of a microorganism. Exemplary stationary phase promoters can be found, for example, in Shimada et al., JOURNAL OF BACTERIOLOGY, November 2004, pp. 7112-7122; Pletnev et al., ACTA NATURAE|VOL.7 No.4(27) 2015.

[0155] In certain embodiments, the promoter is an inducible promoter. An inducible promoter is a promoter that drives expression of a nucleotide sequence in response to a stimulus. An inducible promoter drives sustained expression upon exposure to a specific stimulus (e.g., IPTG). In certain embodiments, the inducible promoter drives a graded level of expression that correlates with the amount of the stimulus. Non-limiting examples of stimuli for inducible promoters include heat shock, an exogenous compound or the absence thereof (e.g., a sugar, a metal, a drug, or phosphate), salt or osmotic shock, oxygen, and a biological stimulus (e.g., a growth factor or a pheromone). Non-limiting examples of inducible promoters include the E. coli promoter P lac , P taq ), P tac , P T7 , P BAD , and P Lacuv are mentioned.

[0156] In certain embodiments, the recombinant polynucleotide may include a plurality of promoters. In certain embodiments, the plurality of promoters may be the same. For example, but not limited to, the recombinant polynucleotide may include a nucleotide sequence encoding a gatZ gene operably ligated to a first promoter, and a nucleotide sequence encoding an hpxB gene operably ligated to a second promoter, wherein the first and second promoters are the same. In certain embodiments, the plurality of promoters may be different. For example, but not limited to, the recombinant polynucleotide may include a nucleotide sequence encoding a gatZ gene operably ligated to a first promoter, and a nucleotide sequence encoding an hxpB gene operably ligated to a second promoter, wherein the first and second promoters are different. In certain embodiments, the promoter is P LlacO1 It is a promoter. In a particular embodiment, P LlacO1 The promoter contains the nucleotide sequence shown in SEQ ID NO: 42. In a particular embodiment, P LlacO1 The promoter consists of the nucleotide sequence shown in SEQ ID NO: 42. LlacO1 The promoter is the promoter P of phage lambda, in which the CI binding site is replaced with lacO1. L This is a hybrid regulatory region that includes [a specific component]. The hybrid design enables potent promotion that can be inhibited by LacI, a Lac inhibitor (i.e., a repressor), or induced by IPTG.

[0157] In a particular embodiment, the promoter is P LtetO1 It is a promoter. In a particular embodiment, P LTETO1 The promoter contains the nucleotide sequence shown in SEQ ID NO: 43. In a particular embodiment, P LTETO1 The promoter consists of the nucleotide sequence shown in SEQ ID NO: 43.

[0158] In a particular embodiment, the promoter is PT7 It is a promoter. In a particular embodiment, P T7 The promoter contains the nucleotide sequence shown in SEQ ID NO: 44. In a particular embodiment, P T7 The promoter consists of the nucleotide sequence shown in SEQ ID NO: 44.

[0159] In a particular embodiment, the promoter is P tet It is a promoter. In a particular embodiment, P tet The promoter contains the nucleotide sequence shown in SEQ ID NO: 45. In a particular embodiment, P tet The promoter consists of the nucleotide sequence shown in SEQ ID NO: 45.

[0160] In a particular embodiment, the promoter is P gadB It is a promoter. In a particular embodiment, P gadB The promoter contains the nucleotide sequence shown in SEQ ID NO: 46. In a particular embodiment, P gadB The promoter consists of the nucleotide sequence shown in SEQ ID NO: 46.

[0161] P LlacO1 Promoter nucleotide sequence: AATTGTGAGCGGATAACAATTGACATTGTGAGCGGATAACAAGATACTGAGCACATCAGCAGGACGCACTGACCGAATTCATTAAAGAGGAGAAAAGATATACC (SEQ ID NO: 42)

[0162] P LtetO1 Promoter nucleotide sequence: tccctatcagtgatagagatgacatccctatcagtgatagagatactgagcacatcagcaggacgcactgaccgaattcattaaagaggagaaaggtacc (SEQ ID NO: 43)

[0163] P T7 Promoter nucleotide sequence: taatacgactcactataggggaattgtgagcggataacaattcccctctagaaataattttgtttaactttaagaaggagatatacc (SEQ ID NO: 44)

[0164] P tet Promoter nucleotide sequence: gttgacactctatcgttgatagagttattttaccactccctatcagtgatagagaaaagaattcaaaagatctaaagaggagaaaggatct (Sequence ID 45)

[0165] P gadB Promoter nucleotide sequence: GTAATAATTTTATAAATGCGTTCAAAATAATAATCAAGTACTAATAGTGATATTTTAAGGTCTGATTTTTACGTGATAATTCAGGAGACACAGAATGCGCATAAAAATAACAGCATAAAACACCTTACCACCACCCAAGAATTTCATATTGTATTGTTTTTCAATGAAAAAATAT TATTCGCGTAATATCTCACGATAAATAACATTAGGATTTTGTTATTTAAACACGAGTCCTTTGCACTTGCTTACTTTATCGATAAATCCTACTTTTTTAATGCGATCCAATCATTTTAAGGAGTTTAAAATGGATAAGAAGCAAGTGAATTCATTAAAGAGGAGAAAAGATATACC (Sequence number 46)

[0166] In certain embodiments, the promoter is a quiescent promoter. As used herein, the term “quiescent promoter” refers to an upstream promoter of a gene that is transcribed during the quiescent phase of microbial growth. The life cycle of an E. coli culture consists of five distinct phases: induction, logarithmic, quiescent, death, and long quiescent. The induction phase occurs when cells are inoculated into a culture medium and adjust their metabolic processes according to their new environment. The cells then rapidly grow and divide, entering the logarithmic phase. It is at this point that enzymes related to central carbon metabolism are most important, and the transcription of corresponding genes is upregulated. When cells sense environmental stressors such as a deficiency of culture medium nutrients, their growth and division slow down, and the culture enters the quiescent phase. The use of a quiescent promoter prevents the production pathway from competing with central carbon metabolism for carbon flux during the logarithmic growth phase, when cells strictly require carbon to grow and divide.

[0167] In a particular embodiment, the stationary promoter is P gadB In a particular embodiment, P gadB The promoter contains the nucleotide sequence shown in SEQ ID NO: 46 or SEQ ID NO: 53. In certain embodiments, P gadB The promoter consists of the nucleotide sequence shown in SEQ ID NO: 53.

[0168] In a particular embodiment, the stationary promoter is P cbpA2 In a particular embodiment, P cbpA2 The promoter contains the nucleotide sequence shown in SEQ ID NO: 54. In a particular embodiment, P cbpA2 The promoter consists of the nucleotide sequence shown in SEQ ID NO: 54.

[0169] In a particular embodiment, the stationary promoter is P ihfA4 In a particular embodiment, P ihfA4 The promoter contains the nucleotide sequence shown in SEQ ID NO: 55. In a particular embodiment, P ihfA4The promoter consists of the nucleotide sequence shown in SEQ ID NO: 55.

[0170] In a particular embodiment, the stationary promoter is P dps In a particular embodiment, P dps The promoter contains the nucleotide sequence shown in SEQ ID NO: 56. In a particular embodiment, P dps The promoter consists of the nucleotide sequence shown in SEQ ID NO: 56. GTAATAATTTTATAAATGCGTTCAAAATAATAATCAAGTACTAATAGTGATATTTTAAGGTCTGATTTTTACGTGATAATTCAGGAGACACAGAATGCGCATAAAAATAACAGCATAAAACACCTTACCACCACCCAAGAATTTCATATTGTATTGTTTTTCAATGAAAAAATATT ATTCGCGTAATATCTCACGATAAATAACATTAGGATTTTGTTATTTAAACACGAGTCCTTTGCACTTGCTTACTTTATCGATAAATCCTACTTTTTTAATGCGATCCAATCATTTTAAGGAGTTTAAAATGGATAAGAAGCAAGTCGAATTCATTAAAGAGGAGAAAGGTACCATG (Sequence number 53) TTTGCAGTGCAACTAATTCCATGTATATTACTACCCATATAGCGTCTATAAAATTTAATAAATAATGACGCCCTAGTTAAACTTAAAGTGCCTGGTTCAACTATCAAAAATCGCTCACCCTTTTCACCTGTTTAAAATATGTTCAGCAACCCATCTTGATG GCGACCTCCTCTCCGCGATGATTTCAATAACATATTCTGTGTTGGCATATGAAATTTTGAGGATTACCCTACACTTATAGGAGTTACCTTACAGGGGTTCCTTCAATTTGTGTTGATTTACGCGAGATAACGCTCGAATTCATTAAAGAGGAGAAAGGTACCATG (Sequence number 54) TATCCGAATGTAAGAAAGTTGGCGTAAATCAGGTAGTTGGCGTAAACTTATTTGACGTGTACCGCGTAAGGGTGTTGCGGAGGGGTATAAGAGCCTCGCCATAAGCCTGATCCTGCAAGATACCAGCCGTACACTCGAAGAAGGAGATTGCCGCTACCGTCGCCAAATGTGTAGAGGCATTAAAAAGAGCGATTCCAGGCATCATTGAGGGATTGAACCTCGAATTCATTAAAAGAGGAGAAAGGTACCATG (sequence number 55) TCATTGAATCTTTATTAGTTTTGTTTTCACGCTTGTTACCACTATTAGTGTGTAGGAACAGCCAGAATAGCGGAACACATAGCGGAGACAGCCGGTGCTATACTTAATCTCGTTAATTACTGGGACATAACATAACAAGGATATGAATTCGAATTCATTAAAAGAGGAGAAAGGTACCATG (sequence number 56)

[0171] ·3.1.3 gene markers In certain embodiments, the recombinant polynucleotides of the Disclosure include genetic markers. These genetic markers enable the selection of microorganisms having one or more desired polynucleotides (e.g., recombinant polynucleotides). In certain embodiments, the genetic markers are antibiotic resistance markers selected from the group consisting of apramycin resistance, ampicillin resistance, kanamycin resistance, spectinomycin resistance, tetracycline resistance, neomycin resistance, chloramphenicol resistance, gentamicin resistance, erythromycin resistance, carbenicillin resistance, actinomycin D resistance, neomycin resistance, polymyxin resistance, zeosin resistance, and streptomycin resistance. In certain embodiments, the genetic markers include a coding sequence for an antibiotic resistance protein (e.g., β-lactamase in the case of a certain ampicillin resistance marker) and a promoter or enhancer element that drives the expression of the coding sequence in the microorganisms of the Disclosure. In certain embodiments, the microorganisms of the Disclosure are grown under conditions in which the antibiotic resistance marker is expressed and confers resistance to the microorganisms, thereby enabling selection of the microorganisms in which the marker has been successfully incorporated. In certain embodiments, the gene marker is a nutritional requirement marker. In certain embodiments, the nutritional requirement marker is a gene involved in vitamin synthesis, amino acid synthesis, fatty acid synthesis, or carbohydrate metabolism. In certain embodiments, the nutritional requirement marker is a gene for synthesizing amino acids. In certain embodiments, the nutritional requirement marker is a gene for synthesizing glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, tyrosine, tryptophan, serine, threonine, cysteine, methionine, asparagine, glutamine, lysine, arginine, histidine, aspartic acid, or glutamic acid. In certain embodiments, the nutritional requirement marker is a gene for synthesizing adenosine, biotin, thiamine, leucine, glucose, lactose, or maltose.In certain embodiments, the microorganisms of the Disclosure are grown under conditions in which a nutrient requirement tolerance marker is expressed in an environment or culture medium lacking the corresponding nutrient, thereby conferring growth to the microorganism (which lacks the endogenous capacity to produce the nutrient), and thereafter, microorganisms in which the marker has been successfully incorporated are selected.

[0172] 3.2. Gene deletion and reduced expression In certain embodiments, the Disclosure also provides methods for introducing any deletion of a gene or enzyme disclosed herein. These deletions can be induced by any suitable gene editing method. In certain embodiments, the deletions are induced by methods including homologous recombination, zinc finger nucleases, meganucleases, activator-like effector nucleases (TALENs), clustered, regularly spaced short palindromic repeats (CRISPR) systems, or combinations thereof.

[0173] In certain embodiments, deletions are created by the CRISPR system. The clustered, regularly spaced, short palindromic repeat (CRISPR) system is a genome editing tool discovered in prokaryotic cells. When used for genome editing, this system includes Cas9 (a protein that can modify DNA using crRNA as its guide), CRISPR RNA (a crRNA containing a region that binds to tracrRNA (generally in a hairpin loop form) that forms an active complex with Cas9, along with the RNA used by Cas9 to guide Cas9 to the correct part of host DNA), transactivating crRNA (a tracrRNA that binds to crRNA and also forms an active complex with Cas9), and optionally included, a DNA repair template region (DNA that guides a cellular repair process that enables the insertion of a specific DNA sequence). Multiple crRNAs and the tracrRNA can be packaged together to form a single guide RNA (sgRNA). This sgRNA can be ligated together with the Cas9 gene and constructed in a plasmid for transfecting cells. In certain embodiments, the CRISPR system includes a base editing agent. In certain embodiments, the CRISPR system includes a transposase / recombinase. In certain embodiments, the CRISPR system includes a prime editor. In certain embodiments, the CRISPR system includes an epigenetic modulator. In certain embodiments, the CRISPR system includes a CRISPRoff system. Further details relating to the CRISPR systems of this disclosure can be found in Anzalone et al., Nature biotechnology 38.7(2020):824-844, and Nunez et al., Cell 184.9(2021):2503-2519, and Jiang et al., Appl Environ Microbiol. April 2015;81(7):2506-14, each of which is incorporated by reference.

[0174] In certain embodiments, deletions are produced by zinc finger nucleases. Zinc finger nucleases (ZFNs) are artificial restriction enzymes produced by combining a zinc finger DNA-binding domain and a DNA-cleaving domain. The zinc finger domain can be manipulated to target specific DNA sequences, allowing zinc finger nucleases to target desired sequences within the genome. The DNA-binding domain of individual ZFNs typically contains multiple individual zinc finger repeats, each capable of recognizing multiple base pairs. The most common way to create novel zinc finger domains is by combining known zinc finger "modules" with less specificity. The most common cleaving domain in ZFNs is the nonspecific cleaving domain derived from the IIs-type restriction endonuclease FokI.

[0175] In certain embodiments, deletions are created by the TALEN system. Activator-like effector nucleases (TALENs) are restriction enzymes that can be designed to cleave specific sequences of DNA. The TALEN system operates on much the same principle as ZFNs. They are created by combining an activator-like effector DNA-binding domain with a DNA-cleaving domain. Activator-like effectors (TALEs) consist of 33-34 amino acid repeat motifs with two variable positions that have strong recognition of specific nucleotides. By assembling arrays of these TALEs, the TALE DNA-binding domain can be designed to bind to a desired DNA sequence, thereby inducing the nuclease to cleave at a specific location in the genome.

[0176] In certain embodiments, deletions are produced by meganucleases. Meganucleases are endodeoxyribonucleases that recognize double-stranded DNA sites of approximately 12 to 40 base pairs that exist only once in the genome. Meganucleases are among the most specific naturally occurring restriction enzymes. Because meganucleases can replace, eliminate, or modify sequences in a highly targeted manner, they are also defined as molecular DNA scissors. Protein engineering makes it possible to modify their recognition and target sequences.

[0177] In certain embodiments, the disclosure also provides a method for reducing the expression of any of the genes or enzymes disclosed herein. In certain embodiments, the reduction of gene and enzyme expression disclosed herein involves using oligonucleotides having sequences complementary to the mRNA of the genes disclosed herein (e.g., zwf, manA, alsE, pfkA, pfkB, etc.). Non-limiting examples of these oligonucleotides include small interfering RNA (siRNA), short hairpin RNA (shRNA), and microRNA (miRNA). In certain embodiments, these oligonucleotides may be at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to at least a portion of the zwf mRNA sequence. In certain embodiments, these oligonucleotides may be identical to at least a portion of the zwf mRNA sequence. In certain embodiments, these oligonucleotides may be at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, and at least about 99% identical to at least a portion of the pfkA mRNA sequence. In certain embodiments, these oligonucleotides may be identical to at least a portion of the pfkA mRNA sequence. In certain embodiments, these oligonucleotides may be at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, and at least about 99% identical to at least a portion of the pfkB mRNA sequence. In certain embodiments, these oligonucleotides may be identical to at least a portion of the pfkB mRNA sequence.In certain embodiments, these oligonucleotides may be at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, and at least about 99% identical to at least a portion of the alsE mRNA sequence. In certain embodiments, these oligonucleotides may be at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, and at least about 99% identical to at least a portion of the manA mRNA sequence. In certain embodiments, these oligonucleotides may be at least about a portion of the manA mRNA sequence. In certain embodiments, the antisense nucleic acid molecule, shRNA molecule, miRNA molecule, or siRNA molecule may contain DNA or atypical or non-naturally occurring residues, such as, but not limited to, phosphorothioate residues.

[0178] In some embodiments, the reduction of gene and enzyme expression disclosed herein may involve the use of CRISPR, which can mutate coding sequences or promoters to reduce or eliminate the expression of gene products, or CRISPRi may be targeted to genes disclosed herein to reduce the expression of one or more genes. See, for example, Arroya-Olarte et al., Microorganisms, April 2021;9(4):844. Zhang et al., Front.Microbiol. (March 31, 2021).

[0179] In certain embodiments, the reduction of gene and enzyme expression disclosed herein involves the use of a CRISPR system. In certain embodiments, the CRISPR system includes Cas9. In certain embodiments, Cas9 includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence shown in SEQ ID NO: 85. In certain embodiments, Cas9 consists of the amino acid sequence shown in SEQ ID NO: 85. SEQ ID NO: 85 is shown below.

[0180] In certain embodiments, the gene and enzyme expression reduction disclosed herein involves the use of a CRISPRi system. A CRISPRi system can silence genes at the transcriptional level and may have fewer sequence-specific off-target effects than RNAi. In certain embodiments, the CRISPRi system includes a catalytically dead Cas9 (dCas9). dCas9 is a programmable transcription factor that can target a promoter via sgRNA and function as a repressor. In certain embodiments, dCas9 includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence shown in SEQ ID NO: 57. In certain embodiments, dCas9 comprises the amino acid sequence shown in SEQ ID NO: 57. SEQ ID NO: 57 is shown below.

[0181] In certain embodiments, a nuclease (e.g., Cas9 or dCas9) is regulated by a promoter (e.g., described in Section 3.1.2). In certain embodiments, the promoter is an inductive promoter. In certain embodiments, the promoter is a quiescent promoter. In certain embodiments, the CRISPR / CRISPRi system includes a small guide RNA (sgRNA). In certain embodiments, the sgRNA of the CRISPR / CRISPRi system targets a gene encoding an enzyme in a competing pathway. For example, but not limited to, the sgRNA may target the zwf gene, pgm gene, pfkA gene, pfkB gene, ManA gene, or AlsE gene. In certain embodiments, the sgRNA can target any portion of a gene. For example, but not limited to, the sgRNA can target a promoter, operator, or protein-coding sequence.

[0182] In certain embodiments, the CRISPR / CRISPRi system includes a nuclease (e.g., Cas9 or dCas9) and sgRNA. In certain embodiments, the nuclease (e.g., Cas9 or dCas9) is regulated by an inductive promoter. In certain embodiments, the inductive promoter is P tet In certain embodiments, the sgRNA targets the pfkB gene. In certain embodiments, the sgRNA targets the promoter of the pfkB gene.

[0183] 3.3. Transformation and gene editing In certain embodiments, the Disclosure provides the use of transformations of the plasmids and vectors disclosed herein. The vectors and plasmids disclosed herein can be used to transform cells via any system known in the Art. For example, but not limited to, the microorganisms of the Disclosure can be transformed by particle impact, chemical transformation, Agrobacterium transformation, nanospike transformation, electroporation, and viral transformation.

[0184] In certain embodiments, the vectors of this disclosure can be introduced into microorganisms using a variety of techniques, including transformation, transfection, transduction, viral infection, gene gun, or Ti-mediated gene transfer. Non-limiting examples of these methods include calcium phosphate transfection, DEAE-dextran-mediated transfection, lipofection, and electroporation (see, for example, Davis, L., Dibner, M., and Battey, I., 1986 "Basic Methods in Molecular Biology"; Gietz et al., Nucleic Acids Res. 27:69~74 (1992); Ito et al., J. Bacterol. 153:163~168 (1983); and Becker and Guarente, Methods in Enzymology 194:182~187 (1991)). In certain embodiments, transformed microorganisms are referred to as recombinant microorganisms.

[0185] In certain embodiments, the disclosure provides a method for introducing exogenous proteins (e.g., nucleases), RNA (e.g., gRNA), and DNA (e.g., recombinant polynucleotides disclosed herein) into microorganisms. Various methods for achieving this have been previously described, including direct transfection of protein and nucleotide sequences or DNA transformation, followed by intracellular expression of RNA and proteins (see, for example, Dicarlo, JE et al., "Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems." Nucleic Acids Res (2013).doi:10.1093 / nar / gkt135; Ren, ZJ, Baumann, RG and Black, LW, "Cloning of linear DNAs in vivo by overexpressed T4 DNA ligase: construction of a T4 phage hoc gene display vector." Gene 195, 303~311 (1997); Lin, S., Staahl, BT, Alla, RK and Doudna, JA, "Enhanced homology-directed human genome engineering by controlled timing of CRISPR / Cas9 delivery." Elife 3, e04766 (2014)).

[0186] 3.4. Recombination Systems In certain embodiments, the disclosure also provides homologous recombination systems for editing (e.g., insertions, deletions) in microorganisms. In certain embodiments, the homologous recombination system may be native to the host cell or introduced into the cell host. For example, but not limited to, the genes of the homologous recombination system can be introduced on a plasmid, or on a linear DNA fragment, or as RNA or a set of RNAs and translated therefrom, or as a protein or a set of proteins. In certain embodiments, the method includes recombinant polynucleotides disclosed herein. In certain embodiments, the polynucleotide includes sequences (e.g., left and right homologous arms) homologous to a region in a nucleic acid (e.g., genome, plasmid, etc.) such that the left and right homologous arms are separated by designed gene editing (e.g., promoters, insertions, substitutions, SNPs, terminators, degrons, tag sequences, degradation signal sequences, or deletions). In certain embodiments, the recombinant polynucleotide includes a gene marker, a counter-selectable gene marker (e.g., SacB or PheS), and an origin of replication (e.g., R6K).

[0187] In certain embodiments, the recombinant polynucleotide containing the homologous arm and the sequence for gene editing is introduced into a microorganism using any of the methods disclosed herein (e.g., transformation via electroporation, conjugation, etc.). In certain embodiments, after transformation, the resulting transformants may be seeded in a culture medium to select transformants that express a selectable gene marker. Recombination between the plasmid containing the homologous arm and the target gene locus in the nucleic acid (e.g., genome, plasmid, etc.) may occur at one of two homologous sites on the plasmid that are targeted by the homologous arm and adjacent to the designed gene edit. In certain embodiments, the resulting transformants may grow as colonies on a selective medium, be selected, and seeded in a second type of selective medium (e.g., a counter-selectable medium). In certain embodiments, the second type of selective medium allows for the selection of cells containing the desired gene edit.

[0188] In certain embodiments, the methods disclosed herein involve using proteins from one or more recombinant systems. The recombinant systems may be endogenous or exogenous to the microorganism. In certain embodiments, proteins from one or more recombinant systems may be introduced as nucleic acids (e.g., plasmids, linear DNA or RNA, or integrons), integrated into the genome of a host cell, or stably expressed from extrachromosomal elements. In certain embodiments, proteins from one or more recombinant systems may be introduced as RNA and translated by the host cell. In certain embodiments, proteins from one or more recombinant systems may be introduced into the host cell as proteins. Non-limiting examples of recombinant systems include homologs, orthologues, or paralogs of any of the proteins derived from the λ-red recombinant system, RecET recombinant system, Red / ET recombinant system, λ-red recombinant system, RecET recombinant system, Red / ET recombinant system, or λ-red mediated recombinant system, or any combination thereof.Further details regarding the recombination system from the RecET recombination system are incorporated herein by reference in their entirety: Zhang Y., Buchholz F., Muyrers JPP and Stewart AF, "A new logic for DNA engineering using recombination in E. coli." Nature Genetics 20(1998)123~128; Muyrers, JPP, Zhang, Y., Testa, G., and Stewart AF, "Rapid modification of bacterial artificial chromosomes by ET-recombination." Nucleic Acids Res. 27(1999)1555~1557; Zhang Y., Muyrers JPP, Testa G. and Stewart AF, "DNA cloning by homologous recombination in E. coli." Nature Biotechnology 18(2000)1314~1317; and Muyrers JP et al., "Techniques: Recombinogenic engineering--new options for cloning and Any method described in "manipulating DNA" Trends Biochem Sci. May 2001;26(5):325-31 may be used.

[0189] ●4. Method for manufacturing D-Tagatose This disclosure also provides a method for producing D-tagatose. Cell-free methods (e.g., in vitro synthesis) utilize thermodynamically undesirable enzymatic reactions. In certain embodiments, the method of this disclosure for producing D-tagatose comprises culturing a microorganism (e.g., one disclosed in Section 2) and purifying the D-tagatose.

[0190] ·4.1.Cell culture This disclosure provides methods for culturing the microorganisms disclosed herein. As used herein, “culturing” cells means introducing a suitable culture medium under suitable conditions to promote cell growth. In certain embodiments, culturing is carried out using liquid or solid growth media. In certain embodiments, culturing is carried out under aerobic or anaerobic conditions, based on the requirements of the microorganism and the desired metabolic state of the microorganism. In certain embodiments, culturing includes specific conditions such as temperature, pressure, light, pH, and cell density.

[0191] In certain embodiments, a method for producing tagatose includes a culture medium for culturing recombinant bacteria. As used herein, “culture medium” refers to any composition or broth that supports the growth of microorganisms disclosed herein. The culture medium may be liquid or solid. In certain embodiments, the culture medium includes nutrients, salts, buffers, elements, and other compounds that support cell growth and viability. Furthermore, the culture medium may include sources of nitrogen, carbon, amino acids, carbohydrates, trace elements, vitamins, and minerals. In certain embodiments, the culture medium includes a complex extract (e.g., yeast extract). In certain embodiments, the culture medium is concentrated to support rapid growth. In certain embodiments, the culture medium is modified to support slower growth. In certain embodiments, the culture medium includes an agent (e.g., an antibiotic) that can inhibit the growth of contaminated organisms or kill contaminated organisms. In certain embodiments, the culture medium includes an agent (e.g., IPTG) that can activate an inducible promoter or enzyme. Non-limiting examples of culture media included in this disclosure include M9 medium, lysogenic medium (LB), Terrific broth (TB), and YT broth. In certain embodiments, the culture medium includes a substrate that is converted to tagatose by recombinant microorganisms.

[0192] In certain embodiments, the substrate is a sugar (e.g., glucose or fructose) that can be phosphorylated by bacteria via a kinase (e.g., hexokinase) and converted to fructose-6-phosphate. In certain embodiments, the substrate is glucose. In certain embodiments, glucose may be derived from cellulose, C5 sugars, hemicellulose, and / or xylose. In certain embodiments, the substrate is a component of the culture medium. In certain embodiments, the substrate is replenished in the culture medium. In certain embodiments, the substrate is continuously present in the culture medium. In certain embodiments, the substrate is replenished during the growth phase. In certain embodiments, the substrate is replenished during the quiescent phase.

[0193] 4.2. Purification of Tagathos In certain embodiments, the method of the Disclosure further includes purifying tagatose produced by the microorganism of the Disclosure from, for example, a cell culture or cell culture medium. Products can be purified from microorganisms or microbial cultures using various methods known in the Art. In certain embodiments, one or more products may be purified sequentially from, for example, a continuous culture. In certain embodiments, one or more products may be purified separately from fermentation, for example, from a batch culture or a fed-batch culture. Those skilled in the art will understand that the specific purification method used may depend, among other things, on the microorganism, the culture conditions, and / or the specific product.

[0194] In certain embodiments, the purification of D-tagatose includes separating or filtering microorganisms from cell culture medium, separating D-tagatose from culture medium (e.g., by chromatography), concentrating water (e.g., by evaporation), and freeze-drying D-tagatose.

[0195] ●5.Food This disclosure also provides a delivery system method for use in food products containing D-tagatose prepared and / or produced by any of the microorganisms disclosed herein.

[0196] As used herein, the term “food” includes all food products, e.g., those described in 21 CFR 101.12. Non-limiting examples of such food products include frozen desserts, baked goods, fillings, nutritional beverages, drinks, salad dressings or similar dressings, sauces, icings, puddings and custards, batters, and the like. Various baked goods are disclosed in U.S. Patent No. 6,536,599, the entire disclosure of which is incorporated herein by reference. Non-limiting examples of bakery products include cookies, cakes, rolls, pastries, pie crusts, brownies, bread, bagels, and the like. D-tagatose prepared and / or produced by any of the microorganisms disclosed herein is also suitable as an ingredient in frozen foods.

[0197] In certain embodiments, the food is prepared by mixing D-tagatose in an ingestible vehicle with any optional ingredient to form a homogeneous mixture. The final composition is readily prepared using standard methods and apparatus commonly known to those skilled in the art in the corresponding field, such as the confectionery field. Apparatus useful in accordance with the subject matter of this disclosure includes mixing apparatuses well known in the art, and therefore the selection of specific apparatus will be obvious to those skilled in the art.

[0198] As used herein, “mixing,” for example, “mixing D-tagatose with food,” refers to a process in which a flavor composition is mixed with or added to a finished product, or mixed with some or all of the components of a product during product manufacturing, or any combination of these processes. As used in the context of mixing, the term “product” refers to either the product or any of its components. The mixing process may include processes selected from adding D-tagatose to a product, spraying D-tagatose onto a product, coating a product with D-tagatose, suspending a product in D-tagatose, coating a product with D-tagatose, attaching D-tagatose to a product, encapsulating a product with D-tagatose, mixing D-tagatose with a product, and any combination thereof. D-tagatose may be a liquid, a dry powder, a spray, a paste, a suspension, or any combination thereof.

[0199] In certain embodiments, this application relates to a modified edible food produced by a method disclosed herein. In certain embodiments, the food can be produced by a method for producing edible products that is well known to those skilled in the art.

[0200] In certain embodiments, D-tagatose prepared and / or produced by any of the microorganisms disclosed herein can be dissolved or dispersed in one of many known edible, acceptable liquids, solids, or other carriers, such as water of neutral, acidic, or basic pH; natural water / fat emulsions such as fruit or vegetable juices, vinegar, marinades, beer, wine, milk, or condensed milk; whey or whey products; edible oils and shortening; fatty acids; certain low molecular weight oligomers of propylene glycol; glyceryl esters of fatty acids; and dispersions or emulsions of such hydrophobic substances in aqueous media; salts such as sodium chloride; solvents such as vegetable powders or ethanol; and solid edible diluents such as vegetable powders or flours, which can then be combined with precursors of edible or medicinal products, or applied directly to edible or medicinal products.

[0201] Those skilled in the art of preparing and selling food products are well aware of the wide variety of classes, subclasses, and species of edible compositions and, in their efforts to prepare and sell a variety of edible compositions, utilize well-known and recognized technical terms to refer to those edible compositions. A list of such technical terms is enumerated below, and it is particularly intended that the flavors of the edible compositions listed below can be modified or enhanced, either alone or in any reasonable combination or mixture thereof, using D-tagatose prepared and / or produced by any of the microorganisms disclosed herein.

[0202] In certain embodiments, foods to which D-tagatose prepared and / or produced by any of the microorganisms disclosed herein is mixed include, for example, the wet soup category, the dried and cooked food category, the beverage category, the frozen food category, the snack food category, and the seasoning or seasoning blend as described herein.

[0203] In certain embodiments, D-tagatose prepared and / or produced by any of the microorganisms disclosed herein may be used in one or more confectionery, chocolate candies, tablets, countlines, selfmie / softlines in bags, boxed goods, standard boxed goods, twist-wrapped miniatures, seasonal chocolates, chocolates with toys, assortments, other chocolate candies, mints, standard mints, power mints, hard candies, lozenges, gummies, jellies and chews, toffees, caramels and nougat, medicinal candies, lollipops, and more. Coris, other candies, gum, chewing gum, sugared gum, sugar-free gum, functional gum, bubble gum, bread, packaged / mass-produced bread, unpackaged / artisan bread, pastries, cakes, packaged / mass-produced cakes, unpackaged / artisan cakes, cookies, chocolate-covered biscuits, sandwich biscuits, filled biscuits, savory biscuits and savory crackers, bread substitutes, breakfast cereals, RTE cereals, family breakfast cereals, flakes, Muse Lee, other RTE cereals, kids' breakfast cereals, hot cereals, ice cream, impulse ice cream, single serving dairy ice cream, single serving frozen desserts, multi-pack dairy ice cream, multi-pack frozen desserts, takeaway ice cream, takeaway dairy ice cream, ice cream desserts, bulk ice cream, takeaway frozen desserts, frozen yogurt, artisan ice cream, dairy products, milk, fresh milk / pasteurized milk, fresh whole milk / pasteurized milk, fresh semi-skimmed milk / pasteurized milk, long-life / UHT milk, whole Fat-long life milk (UHT), semi-skimmed long life milk (UHT), non-fat long life milk (UHT), goat milk, concentrated / evaporated milk, plain condensed / evaporated milk, flavored condensed milk, functional condensed milk and other condensed milks, flavored milk beverages, dairy-only flavored milk beverages, flavored milk beverages containing fruit juice, soy milk, sour milk beverages, fermented milk beverages, coffee whiteners, powdered milk, flavored powdered milk beverages, cream, cheese, processed cheese, spreadable processed cheese,Unspreadable processed cheese, raw cheese, spreadable raw cheese, hard cheese, packaged hard cheese, unpackaged hard cheese, yogurt, plain / natural yogurt, flavored yogurt, fruit yogurt, probiotic yogurt, drinking yogurt, regular drinking yogurt, probiotic drinking yogurt, chilled desserts and shelf-stable desserts, dairy desserts, soy-based desserts, chilled snacks, fromage frais and quark, plain fromage frais and quark, flavored fromage frais and quark, savory fromage frais and quark, sweet snacks and savory snacks, fruit snacks, chips / crisps, extruded snacks, tortilla / corn chips, popcorn, pretzels, nuts, other sweet snacks and savory snacks, snack bars, granola bars, breakfast bars, energy bars, fruit bars, other snack bars, meal replacements, diet products, recovery drinks, ready meals, canned ready meals, frozen ready meals Dried ready meals, chilled ready meals, dinner mixes, frozen pizzas, chilled pizzas, soups, canned soups, dried soups, instant soups, chilled soups, UHT soups, frozen soups, pasta, canned pasta, dried pasta, chilled / fresh pasta, noodles, plain noodles, instant ramen, cup noodles / bowl instant ramen, pouch instant ramen, cold noodles, snack noodles, canned foods, canned meat and meat products, canned fish / seafood, canned vegetables, canned tomatoes, canned beans, canned fruit, canned ready meals, canned soups, Canned pasta, other canned foods, frozen foods, frozen processed lean meat, frozen processed poultry, frozen processed fish / seafood, frozen processed vegetables, frozen meat substitutes, frozen potatoes, oven-baked potato chips, other oven-baked potato products, non-oven-frozen potatoes, frozen bakery products, frozen desserts, frozen ready meals, frozen pizza, frozen soup, frozen noodles, other frozen foods, dried foods, dessert mixes, dried ready meals, dried soup, instant soup, dried pasta, plain noodles, instant ramen, cup noodles / bowl instant ramen,Instant noodles in pouches, chilled foods, chilled processed meats, chilled fish / seafood products, chilled processed fish, chilled coated fish, chilled smoked fish, chilled lunch kits, chilled ready meals, chilled pizzas, chilled soups, chilled / fresh pasta, cold noodles, oils and fats, olive oil, vegetable oils and seed oils, cooking oils, butter, margarine, spreadable oils and fats, functional spreadable oils and fats, sauces, dressings and seasonings, tomato paste and tomato puree, bouillon / solid soup stocks, solid soup stocks, gravy mixes, liquid soup stocks and stocks, herbs and spices, fermented sauces, soy sauce-based sauces, pasta sauces, Mixed with wet sauces, dry sauce / powder mixes, ketchup, mayonnaise, regular mayonnaise, mustard, salad dressings, regular salad dressings, low-fat salad dressings, vinaigrettes, dips, pickles, other sauces, dressings and condiments, baby food, formula, standard formula, follow-up formula, infant formula, hypoallergenic formula, prepared baby food, dried baby food, other baby food, spreads, jams and preserves, honey, chocolate spread, nut-based spreads, and yeast-based spreads.

[0204] 5.1. Chewing Gum In certain embodiments, D-tagatose prepared and / or produced by any of the microorganisms disclosed herein can be used in low-calorie gum formulations and can also be used in sugared chewing gum. Various details of chewing gum compositions are disclosed in U.S. Patent No. 6,899,911, the entire disclosure of which is incorporated herein by reference. The chewing gum compositions of the subject matter of this disclosure follow the general pattern outlined below. Generally, chewing gum compositions typically contain a chewable gum base portion that is essentially water-free and water-insoluble, a water-soluble bulk portion, and a flavor that is typically water-insoluble. The water-soluble portion dissipates along with some of the flavor over a period of time during chewing. The gum base portion remains in the mouth throughout chewing. The insoluble gum base generally includes elastomers, elastomer solvents, plasticizers, waxes, emulsifiers, and inorganic fillers. It often also includes plastic polymers such as polyvinyl acetate that behave somewhat as plasticizers. Other plastic polymers that can be used include polyvinyl laurate, polyvinyl alcohol, and polyvinylpyrrolidone. Elastomers can include polyisobutylene, butyl rubber (isobutylene-isoprene copolymer), styrene-butadiene rubber, and natural latex such as chicle. Elastomer solvents are often resins such as terpene resins. Plasticizers, sometimes called softeners, are typically fats and oils, including animal fats, hydrogenated and partially hydrogenated vegetable oils, and cocoa butter. Commonly used waxes include paraffin, microcrystalline waxes, and natural waxes such as beeswax and carnauba wax. Microcrystalline waxes, especially those with high crystallinity, can be considered thickeners or texture modifiers.

[0205] In certain embodiments, the insoluble gum base constitutes about 5% to about 95% by weight of the gum. More preferably, the insoluble gum base constitutes 10% to 50% by weight of the gum, most preferably about 20% to 35% by weight of the gum. The gum base typically also includes a filler component. The filler component may be calcium carbonate, magnesium carbonate, talc, dicalcium phosphate, etc. The filler can constitute about 5% to about 60% by weight of the gum base. Preferably, the filler constitutes about 5% to 50% by weight of the gum base.

[0206] Gum bases typically also contain softening agents, including glycerol monostearate and glycerol triacetate. Gum bases may also contain optional components such as antioxidants, colorants, and emulsifiers. The subject matter of this disclosure intends to use any commercially available gum base.

[0207] The water-soluble portion of chewing gum may further include softeners, sweeteners, flavors, physiological coolants, and combinations thereof. Sweeteners often act as bulking agents in the gum. Bulking agents typically make up about 5% to 95% of the gum composition.

[0208] Softeners are added to chewing gum to optimize its chewability and texture. Softeners, also known in the art as plasticizers or plasticizers, generally constitute about 0.5% to about 15% of chewing gum. Softeners intended by the subject of this disclosure include glycerin, lecithin, and combinations thereof. Furthermore, aqueous solutions of sweeteners, such as aqueous solutions of sweeteners containing sorbitol, hydrolyzed hydrogenated starch, corn syrup, and combinations thereof, can be used as softeners and binders in the gum.

[0209] As described above, D-tagatose prepared and / or produced by any of the microorganisms disclosed herein can be used in low-calorie gum formulations. However, formulations containing sugars are also within the scope of the present invention. Sugar sweeteners generally include, but are not limited to, sucrose, dextrose, maltose, dextrin, dried invert sugar, fructose, galactose, corn syrup solids, etc., either alone or in any combination, and include sugar-containing components commonly known in the chewing gum field. D-tagatose prepared and / or produced by any of the microorganisms disclosed herein can also be used in combination with sugarless sweeteners. Generally, sugarless sweeteners include components that have sweet properties but do not contain commonly known sugars, and include, but are not limited to, sugar alcohols such as sorbitol, hydrogenated isomaltulose, mannitol, xylitol, lactitol, erythritol, hydrolyzed hydrogenated starch, and maltitol, either alone or in any combination.

[0210] Depending on the required specific sweetness release profile and storage stability, coated or uncoated high-intensity sweeteners can be used in chewing gum compositions or in coatings applied to centers made from these gum compositions. High-intensity sweeteners, preferably aspartame, can be used at levels of about 0.01% to about 3.0%. Encapsulated aspartame is a high-intensity sweetener with improved stability and release properties compared to free aspartame. Free aspartame can also be added, and when using aspartame, a combination of some free aspartame and encapsulated aspartame is preferred. Other high-intensity sweeteners that can be used in gum centers are saccharin, thaumatin, alitame, saccharin salts, sucralose, stevia, and acesulfame K. Overall, the chewing gum composition preferably contains about 0.5% to about 90% sweeteners. Most typically, the sweeteners include at least one bulk sweetener and at least one high-intensity sweetener. Optional ingredients such as colorants, emulsifiers, and pharmaceuticals may also be added as separate components of the chewing gum composition or as part of the gum base.

[0211] Aqueous syrups such as corn syrup and hydrogenated corn syrup can be used, especially when their water content is reduced. This can preferably be done by co-evaporating the aqueous syrup with a plasticizer such as glycerin or propylene glycol to a water content of less than 10%. Preferred compositions include hydrolyzed hydrogenated starch solids and glycerin. Such syrups and methods for preparing them are discussed in detail in U.S. Patent No. 4,671,967.

[0212] A method for producing chewing gum according to the subject matter of this disclosure involves the sequential addition of various chewing gum components to any commercially available mixer known in the art. After the components are completely mixed, the gum is discharged from the mixer and formed into a desired form, such as by being rolled into a sheet and cut into sticks, extruded into a mass, or poured into a mold to form pellets. Generally, the components are mixed by first melting the gum base and adding it to a running mixer. The base can also be melted in the mixer itself. At this time, colorants or emulsifiers may also be added along with the syrup and part of the filler. Further parts of the filler can then be added to the mixer. Flavorings are typically added along with the final part of the filler. If the flavorings are coated or otherwise modified in such a way that they are incorporated into the delivery system to alter their release rate, they are preferably added after the final part of the filler has been added. The entire mixing procedure typically takes 5 to 20 minutes, but longer mixing times may be required. Those skilled in the art will recognize that many variations of the above procedure can be followed.

[0213] When formed into pellets or balls, chewing gum compositions can be coated. The coating initially exists as a liquid syrup containing about 30% to about 80% or 85% sugar or sugar alcohol and a solvent such as about 15% or 20% to about 70% water. Generally, the coating process is carried out using a conventional panning apparatus. The gum center tablets to be coated are placed in the panning apparatus to form a moving mass.

[0214] The material or syrup that ultimately forms the coating is applied or distributed onto the gum center tablet. D-tagatose can be added before, during, and after application of the syrup to the gum center. Once the coating has dried and formed a hard surface, additional syrup can be added to create multiple coatings or multiple layers of coating. D-tagatose may be added to any of the coatings and / or layers, or not to any of them.

[0215] In the panning procedure, the syrup is added to the gum center tablets at a temperature range of approximately 100°F to approximately 240°F. Preferably, the syrup temperature is approximately 140°F to approximately 200°F. Most preferably, the syrup temperature should be kept constant throughout the process to prevent the polyol in the syrup from crystallizing. The syrup can be mixed with the gum center tablets, sprayed onto the gum center tablets, poured onto the gum center tablets, or added to the gum center tablets in any way known to those skilled in the art.

[0216] In certain embodiments, a flexible coating is formed by adding a powder coating after a liquid coating. The powder coating may include natural carbohydrate gum hydrolysates, maltodextrin, gelatin, cellulose derivatives, starch, modified starch, sugars, sugar alcohols, natural carbohydrate gums, and fillers such as talc and calcium carbonate.

[0217] Each component of the coating on the gum center can be applied in a single layer or in multiple layers. Generally, multiple layers are obtained by applying a single coating, drying the layer, and then repeating this process. The amount of solids added by each coating step primarily depends on the concentration of the coating syrup. Any number of coatings can be applied to gum center tablets. Preferably, no more than about 75 coatings are applied to the gum center. More preferably, less than about 60 coatings are applied, and most preferably, from about 30 to about 60 coatings are applied. In any event, the subject matter of the present disclosure contemplates applying a sufficient amount of syrup to obtain a coated chewing gum product containing from about 10% to about 65% of coating. Preferably, the final product contains from about 20% to about 50% of coating.

[0218] Those skilled in the art will recognize that multiple pre-measured aliquots of coating syrup can be applied to the gum center to obtain a plurality of coated layers. However, it is also contemplated that the volume of the aliquot of syrup applied to the gum center can be varied throughout the entire coating procedure.

[0219] Once the syrup coating has been applied to the gum center, the syrup is dried in an inert medium. A preferred drying medium includes air. Preferably, forced drying air contacts the wet syrup coating within a temperature range of from about 70°F to about 110°F. More preferably, the drying air is within a temperature range of from about 80°F to about 100°F. The present invention also contemplates that the drying air has a relative humidity of less than about 15 percent. Preferably, the relative humidity of the drying air is less than about 8%.

[0220] Dry air can pass through and mix with the syrup-coated gum center in any manner commonly known in the art. Preferably, the dry air is blown over and around the syrup-coated gum center at a flow rate of about 2800 cubic feet / minute in large-scale operation. Lower flow rates will be used when smaller amounts of material are being processed or when smaller equipment is used. If flavor is applied after the syrup coating has dried, one assumption of the subject matter of this disclosure is that the flavor is dried with or without a drying medium.

[0221] The amount of D-tagatose used herein is generally a matter of preference and depends on factors such as the type of final chewing gum composition, the individual flavors, the gum base used, and the desired flavor intensity. Therefore, the amount of D-tagatose can be varied to obtain the desired results in the final product, and such variation is within the capabilities of those skilled in the art without requiring excessive experimentation. In the gum composition, D-tagatose prepared and / or produced by any of the microorganisms disclosed herein is generally present in an amount of about 0.02% to about 5% by weight, preferably about 0.1% to about 2% by weight, and more preferably about 0.8% to about 1.8% by weight of the chewing gum composition.

[0222] 5.2. Confectionery Another important aspect of the subject matter of this disclosure includes confectionery compositions incorporating D-tagatose prepared and / or produced by any of the microorganisms disclosed herein, and methods for preparing confectionery compositions. The preparation of confectionery formulations is well known in the art. Confectionery has been classified into “hard” confectionery and “soft” confectionery. D-tagatose prepared and / or produced by any of the microorganisms disclosed herein can be incorporated into confectionery by mixing the compositions of the subject matter of this disclosure with conventional hard and soft confectionery.

[0223] Hard confectionery can be processed and formulated by conventional means. Generally, hard confectionery has a base consisting of a mixture of sugars and other carbohydrate fillers kept in an amorphous or vitreous state. Hard confectionery may also be sugar-free. Hard confectionery may also be low in calories. This form is generally considered to be a solid sugar syrup having about 0.5% to about 1.5% moisture. Such materials typically contain up to about 92% by weight of sugar, up to about 55% by weight of corn syrup, and about 0.1% to about 5% by weight of water in the final composition. The syrup component is generally prepared from sucrose and corn syrup, but may contain other materials. In certain embodiments, the syrup component contains D-tagatose prepared and / or produced by any of the microorganisms disclosed herein. Further ingredients such as flavorings, sweeteners, acidulants, and colorants may also be added.

[0224] Such confections can be routinely prepared by conventional methods, including but not limited to, those of a scraping surface cooker, also known as a flame cooker, a vacuum cooker, and a high-speed atmospheric pressure cooker. More useful apparatus for the subject of this disclosure includes cooking and mixing apparatuses well known in the field of confectionery making, and therefore the selection of specific apparatuses will be obvious to those skilled in the art.

[0225] The baking method includes traditional methods for making candy bases. In this method, the desired amount of carbohydrate extender is dissolved in water by heating it in a kettle until the extender dissolves. Additional extenders can then be added, and the mixture can be cooked until a final temperature of 145°C to 156°C is achieved. The batch is then cooled and processed into a plastic-like mass, incorporating additives such as flavorings and colorings.

[0226] High-speed atmospheric pressure cookers use a heat exchange surface, which involves spreading the candy film onto the heat exchange surface, and the candy is heated to 165°C-170°C within seconds. The candy is then rapidly cooled to 100°C-120°C, processed into a plastic-like mass, which allows for the incorporation of additives such as flavorings and colorings. In a vacuum cooker, the carbohydrate extender is boiled to 125°C-132°C, and vacuum is applied to remove additional water by boiling without further heating. Once cooking is complete, the mass is semi-solid and has a plastic-like consistency. At this point, flavorings, colorings, and other additives are mixed into the mass by routine mechanical mixing operations.

[0227] The optimal mixing time required to uniformly combine flavorings, colorants, and other additives during the conventional manufacturing of hard candies is determined by the time needed to achieve a uniform distribution of the ingredients. Generally, mixing times of 2 to 10 minutes have been found to be acceptable.

[0228] Once the candy mass has been properly temperature-controlled, it can be cut into workable portions or molded into the desired shape. Various molding techniques can be used depending on the desired shape and size of the final product. General considerations regarding the composition and preparation of hard candies can be found in HALieberman, Pharmaceutical Dosage Forms: Tablets, Vol. 1 (1989), Marcel Dekker, Inc., New York, NY, pp. 419–582, whose disclosure is incorporated herein by reference.

[0229] Compressed tablet confections contain specific ingredients and are formed into a structure under pressure. These confections generally contain sugar in amounts up to about 95% by weight of the composition, as well as typical tablet excipients such as binders, lubricants, flavorings, and colorants. These confections may also be sugar-free.

[0230] Similar to hard confectionery, soft confectionery can also be utilized in embodiments of the subject matter of this disclosure. Preparation of soft confectionery such as nougat includes conventional methods such as the combination of two main components: (1) a high-boiling point syrup such as corn syrup, and (2) a relatively lightweight textured frappé prepared from a mixture of these, generally consisting of a plant protein such as egg albumin, gum arabic, gelatin, soy-derived compounds, a sugar-free milk-derived compound such as milk protein, and a mixture thereof. The frappé is generally relatively lightweight and may have a density in the range of, for example, about 0.5 grams / cc to about 0.7 grams / cc.

[0231] High-boiling point syrups for soft confectionery, or "bob syrups," are relatively viscous, denser than the aforementioned frappé components, and often contain a considerable amount of carbohydrate extenders. Traditionally, the final nougat composition is prepared by adding "bob syrup" to the frappé under stirring to form a basic nougat mixture. Additional ingredients such as flavorings, additional carbohydrate extenders, colorants, preservatives, pharmaceuticals, or mixtures thereof may then be added, again under stirring. Soft confectionery can also be prepared without sugar. A general consideration of the composition and preparation of nougat confectionery can be found in BWMinifie, Chocolate, Cocoa and Confectionery: Science and Technology, 2nd edition, AVI Publishing Co., Inc., Westport, Conn. (1983), pp. 576-580, the disclosure of which is incorporated herein by reference.

[0232] Generally, the frappé components are prepared first, and then the syrup components are slowly added under stirring at a temperature of at least about 65°C, preferably at least about 100°C. The mixture of these components is continued to form a homogeneous mixture, and then this mixture is cooled to a temperature below 80°C, at which point flavors can be added. This mixture is further mixed for an additional period until it is ready to be removed and molded into the appropriate confectionery shape.

[0233] According to this disclosure, a certain amount of D-tagatose prepared and / or produced by any of the microorganisms disclosed herein can be mixed into hard and soft confectionery. The exact amount of D-tagatose used is usually a matter of preference and depends on factors such as the specific type of confectionery being prepared, the type of filler or carrier used, the type of flavor used, and the desired intensity of breath-freshening sensation. Thus, the amount of D-tagatose can be varied to obtain the desired result in the final product, and such variation is within the capabilities of those skilled in the art without requiring excessive experimentation. Generally, the amount of D-tagatose typically present in hard or soft confectionery is about 0.001% to about 20% by weight of the confectionery, preferably about 0.01% to about 15% by weight, more preferably about 0.01% to about 10% by weight, more preferably about 0.01% to about 5% by weight, and more preferably 0.01% to about 0.5% by weight.

[0234] The subject matter of this disclosure extends to methods for producing improved confectionery. D-tagatose prepared and / or produced by any of the microorganisms disclosed herein can be incorporated into conventional hard or soft confectionery compositions using standard techniques and equipment known to those skilled in the art. Useful equipment relating to the subject matter of this disclosure includes mixing and heating equipment well known in the field of confectionery production, and therefore the selection of specific equipment will be obvious to those skilled in the art.

[0235] In this method, the composition is prepared by mixing D-tagatose with other components of the confectionery composition. The other components are incorporated into the composition as indicated by the properties of the desired composition, as is usually well known to those skilled in the art. The final confectionery composition is easily prepared using methods commonly known in food technology and pharmaceutical technology. The confectionery mixture can then be molded into the desired confectionery shape.

[0236] D-tagatose prepared and / or produced by any of the microorganisms disclosed herein can be formulated with conventional ingredients that provide a variety of textures suitable for specific applications. Such ingredients may be in the form of hard and soft confectionery, tablets, toffee, nougat, chewing candy, chewing gum, and centered candies (both sugared and sugarless). Acceptable ingredients can be selected from a wide range of materials. Such materials include, but are not limited to, diluents, binders and adhesives, lubricants, disintegrants, fillers, wetting agents, buffers, and adsorbents. The preparation of such confectionery and chewing gum products is well known.

[0237] 5.3. Chocolate and Fillings The subject matter of this disclosure also includes chocolate products, chocolate-flavored confectionery, and chocolate-flavored compositions used together with and / or in them. Chocolate also includes those containing crumb solids or solids made entirely or partially by a crumb process. Various types of chocolate are disclosed, for example, in U.S. Patent No. 7,968,140 and U.S. Patent No. 8,263,168, whose entire contents are incorporated herein by reference. A general consideration of the composition and preparation of chocolate confectionery can be found in BWMinifie, Chocolate, Cocoa and Confectionery: Science and Technology, 2nd edition, AVI Publishing Co., Inc., Westport, Conn. (1982), whose disclosure is incorporated herein by reference.

[0238] As used herein, the term “chocolate” refers to solid or semi-plastic food products and is intended to refer to all chocolate compositions or chocolate-like compositions containing a fat-based component phase or fat-like composition. This term is intended to include standardized or non-standardized compositions that conform to U.S. Identification Standards (SOI), Codex Alimentarius and / or other international standards, as well as compositions that do not conform to U.S. Identification Standards or other international standards. Unless otherwise specified, this term includes dark chocolate, baking chocolate, sweet chocolate, bittersweet chocolate or semisweet chocolate, milk chocolate, buttermilk chocolate, skim milk chocolate, mixed dairy chocolate, white chocolate, sweet cocoa and vegetable fat coatings, sweet chocolate and vegetable fat coatings, milk chocolate and vegetable fat coatings, vegetable fat-based coatings, pastels including white chocolate or coatings made from cocoa butter or vegetable fat or a combination thereof, nutritionally modified chocolate-like compositions (chocolate or coatings made from calorie-reducing components), and low-fat chocolate, aerated chocolate, compound coatings, off-spec chocolate, and chocolate-like compositions.

[0239] Off-spec chocolate is obtained, for example, when nutritional carbohydrates and sweeteners are partially or completely replaced; or when cocoa butter, cocoa butter substitutes, cocoa butter equivalents, cocoa butter extenders, cocoa butter replacements, cocoa butter substitutes, or milk fat are partially or completely replaced; or when ingredients having flavors that mimic milk, butter, or chocolate are added; or when other additions or deletions to the formulation are made outside the FDA identification standards for chocolate or combinations thereof. Chocolate-like compositions are fat-based compositions, such as carob, that can be used as chocolate substitutes in applications such as panning, molding, or enrobing.

[0240] In the United States, chocolate is subject to identity standards promulgated by the U.S. Food and Drug Administration (FDA) under the Federal Food, Drug, and Cosmetic Act. Definitions and standards for various types of chocolate are well established in the United States. Non-standard chocolate is chocolate having a composition outside the specified range of standardized chocolate.

[0241] In certain specific embodiments, the chocolate may contain D-tagatose prepared and / or produced by any of the microorganisms disclosed herein. Furthermore, the chocolate may contain sugar syrup / solids, invert sugar, hydrolyzed lactose, maple sugar, brown sugar, molasses, honey, sugar substitutes, and the like. Nutritional carbohydrate sweeteners having varying degrees of sweetness intensity may be any of those typically used in the art, including but not limited to sucrose, for example sucrose derived from sugar cane or beets, dextrose, fructose, lactose, maltose, glucose syrup solids, corn syrup solids, invert sugar, hydrolyzed lactose, honey, maple sugar, brown sugar, molasses, and the like. Sugar substitutes can partially replace nutritional carbohydrate sweeteners. High-intensity sweeteners include aspartame, cyclamate, saccharin, acesulfame-K, neohesperidin dihydrochalcone, sucralose, alitame, stevia sweeteners, glycyrrhizin, thaumatin, and mixtures thereof. Preferred high-intensity sweeteners are aspartame, cyclamate, saccharin, and acesulfame-K. Examples of sugar alcohols may be any of those typically used in the art, and include sorbitol, mannitol, xylitol, maltitol, isomalt, lactitol, and the like.

[0242] Chocolate may also contain bulking agents. As defined herein, the term "bulking agent" may be any of those typically used in the art, and includes polydextrose, cellulose and derivatives thereof, maltodextrin, gum arabic, and the like.

[0243] Chocolate products may contain emulsifiers. Examples of safe and appropriate emulsifiers may be any of those typically used in the art, including lecithin derived from plant sources such as soy, safflower, and corn; phosphatidylcholine or phosphatidylethanolamine; or fractional lecithin, monoglycerides and diglycerides, diacetyltartrate esters of monoglycerides and diglycerides (also known as DATEM); monosodium phosphate derivatives of monoglycerides and diglycerides from edible oils and fats; sorbitan monostearate; hydroxylated lecithin; lactyl fatty acid esters of glycerol and propylene glycol; polyglycerol esters of fatty acids; propylene glycol monoesters and propylene glycol diesters of fats and fatty acids; or emulsifiers that may be approved for the soft candy category as defined by the U.S. FDA. Furthermore, other emulsifiers that can be used include polyglycerol polyricinoleate (PGPR), ammonium salts of phosphatidic acid, (e.g., YN) sucrose ester, or oat extract, or any emulsifier that has been found to be suitable for chocolate or similar fat / solid systems or any blend.

[0244] The term "chocolate-flavored confectionery" refers to food products that have the flavor / aroma of chocolate and contain a cocoa fraction, but exclude "chocolate" itself. These products are characterized by being stable for extended periods at ambient temperature (e.g., more than a week) and being microbiologically stable at 18-30°C under normal atmospheric conditions. Examples include chocolate-flavored hard candies, chocolate-flavored chewables, and chocolate-flavored chewing gum.

[0245] The term "chocolate-flavored composition" refers to a chocolate-flavored composition that contains a cocoa fraction and has the flavor / aroma of chocolate, but excludes "chocolate." Examples include chocolate-flavored cake mix, chocolate-flavored ice cream, chocolate-flavored syrup, and chocolate-flavored baking products. This term includes chocolate-flavored compositions (e.g., cakes, nougat, pudding, etc.) as well as compositions that do not have a chocolate flavor (e.g., caramel, etc.).

[0246] 5.4. Savory Foods and Other Foods In certain embodiments, D-tagatose prepared and / or produced by any of the microorganisms disclosed herein is incorporated into a savory food. In certain embodiments, the savory food is a food having a savory flavor, including, but not limited to, spicy flavors, pepper flavors, dairy flavors, vegetable flavors, tomato flavors, dill flavors, meat flavors, poultry flavors, chicken flavors, and reaction flavors added or generated during heating of the food.

[0247] In certain embodiments, D-tagatose prepared and / or produced by any of the microorganisms disclosed herein is incorporated into a wet soup category of food, which includes wet soups / liquid soups, regardless of concentration or container, and also includes frozen soups. In certain embodiments, soup food means food prepared from meat, poultry, fish, vegetables, grains, fruits, and / or other ingredients, which may contain visible portions of some or all of these ingredients. Soup food may be clear (as broth) or thick (as chowder), smooth or pureed or chunky, ready to eat, semi-concentrated or concentrated, and may be served hot or cold as the first course or main course of a meal, or as a snack between meals (sipped like a drink). Soup may be used as an ingredient to prepare other meal ingredients and may range from broth (consommé) to sauce (cream or cheese-based soup).

[0248] In certain embodiments, D-tagatose prepared and / or produced by any of the microorganisms disclosed herein is incorporated into the dry food category and the prepared food category of foods, which include (i) cooking aids such as powders, granules, pastes, concentrated liquid products (including concentrated bouillons, bouillons and bouillon-like products in compressed cube, tablet or powder or granular form, sold separately as finished products or as ingredients in products), sauces and recipe mixes (not technical); and (ii) dried soup mixes, dried instant soups and dried ready-to-cook soups. (iii) Convenience foods for cooking, including dried soups and freeze-dried soups, pasta dishes, potato dishes and rice dishes, as well as dried or shelf-stable preparations of meals and single-serving meals; and (iii) condiments, marinades, salad dressings, salad toppings, dips, breadings, batter mixes, storage-stable spreads, barbecue sauces, liquid recipe mixes, concentrates, sauces or sauce mixes (including salad recipe mixes), sold as finished products or as ingredients in products, whether dried, liquid or frozen.

[0249] In certain embodiments, D-tagatose prepared and / or produced by any of the microorganisms disclosed herein is incorporated into meat foods. In certain embodiments, meat foods include foods made by processing the edible residues of any dead animal, including birds, fish, crustaceans, shellfish, and mammals. Examples of meat foods include, but are not limited to, cooked beef, lamb, pork, poultry, or seafood. Examples of such meat foods include, for example, bologna, frankfurter sausages, sausages, luncheons, deli slices, loaf, bacon, meatballs, fish sticks, chicken fingers, and ground meat, such as meatloaf, meatballs, and hamburgers. Meat foods may be combined with artificial meat foods. Examples of artificial meat foods include, but are not limited to, meat substitutes, meat imitation products, soy burgers, soy bologna, soy frankfurter sausages, soy sausages, soy luncheon loaf, soy bacon, and soy meatballs. Artificial meat products may be combined with meat products.

[0250] In certain embodiments, D-tagatose prepared and / or produced by any of the microorganisms disclosed herein is incorporated into foods in the snack food category. In certain embodiments, snack foods include, but are not limited to, sweet snacks and sweet snack bars, and savory snacks and savory snack bars, and may be any foods that are light, casual meals. Examples of snack foods include, but are not limited to, fruit snacks, chips / crisps, extruded snacks, tortillas / corn chips, popcorn, pretzels, nuts, and other sweet and savory snacks. Examples of snack bars include, but are not limited to, granola / muesli bars, breakfast bars, energy bars, fruit bars, and other snack bars.

[0251] In certain embodiments, D-tagatose prepared and / or produced by any of the microorganisms disclosed herein is incorporated into frozen foods, which include chilled or frozen foods, for example, but are not limited to, ice cream, impulse ice cream, single-serving dairy ice cream, single-serving frozen desserts, multi-pack dairy ice cream, multi-pack frozen desserts, takeaway ice cream, takeaway dairy ice cream, ice cream desserts, bulk ice cream, takeaway frozen desserts, frozen yogurt, artisan ice cream, frozen ready meals, frozen pizza, chilled pizza, frozen soup, frozen pasta, frozen processed red meat, frozen processed poultry, frozen processed fish / seafood, frozen processed vegetables, frozen meat substitutes, frozen potatoes, frozen bakery products, and frozen desserts.

[0252] 5.4. Pharmaceuticals D-tagatose prepared and / or produced by any of the microorganisms disclosed herein may also be in pharmaceutical form. One non-limiting example of a pharmaceutical form is a suspension. Pharmaceutical suspensions can be prepared by conventional formulation methods. Suspensions may contain auxiliary materials used in the preparation of suspensions of the art. Suspensions of the subject matter of this disclosure may include preservatives, buffers, suspending agents, defoaming agents, sweeteners, flavoring agents, colorants or decolorizers, solubilizers, and combinations thereof.

[0253] Flavorings such as natural and artificial flavors, as well as flavors well known to those skilled in the art, such as mint (including peppermint), menthol, citrus flavors (including orange and lemon), artificial vanilla, cinnamon, and fruit flavors (both individual and mixed), can be used in an amount of about 0.01% to about 5% by weight, more preferably 0.01% to about 0.5% by weight, of the suspension.

[0254] A pharmaceutical suspension of the subject matter of this disclosure can be prepared as follows: (i) mix a thickener with water heated to about 40°C to about 95°C, preferably about 40°C to about 70°C to form a dispersion if the thickener is not water-soluble, and form a solution if the thickener is water-soluble; (ii) mix D-tagatose prepared and / or produced by any of the microorganisms disclosed herein with water to form a solution; (iii) optionally mix a flavoring agent with the thickener-water mixture to form a homogeneous thickener-flavoring agent; (iv) combine a sweetener solution with the thickener-flavoring agent and mix until homogeneous; (v) mix optional auxiliary materials such as colorants, flavoring agents, decolorizing agents, solubilizers, defoamers, buffers and additional water with the mixture from step (iv) to form a suspension.

[0255] D-tagatose prepared and / or produced by any of the microorganisms disclosed herein may also be in chewable form. Several considerations are important for achieving acceptable stability and quality as well as good taste and texture in chewable formulations. These considerations include the amount of active substance per tablet, the flavoring agents used, the degree of compressibility of the tablets, and additional properties of the composition. Chewable pharmaceutical candies are prepared by procedures similar to those used to make soft confectionery. General considerations of confectionery in the form of lozenges and chewable tablets can be found in HALieberman and L. Lachman, Pharmaceutical Dosage Forms: Tablets Volume 1, Marcel Dekker, Inc., New York, NY (1989), pp. 367–418, whose disclosure is incorporated herein by reference. A typical procedure involves forming a boiled sugar-corn syrup blend to which a frappé mixture is added. The boiled sugar-corn syrup blend can be prepared from sugar and corn syrup blended in a parts-by-weight ratio of about 90:10 to about 10:90. The sugar-corn syrup blend is heated to a temperature above about 120°C to remove water and form a molten mass. The frappé is generally prepared from milk proteins such as gelatin, egg albumin, and casein, and plant proteins such as soy protein, and is added to a gelatin solution and rapidly mixed at ambient temperature to form an aerated spongy mass. The frappé is then added to the molten candy mass and mixed until homogeneous at a temperature of about 65°C to about 120°C. Then, as the temperature is lowered to about 65°C to 95°C, D-tagatose prepared and / or produced by any of the microorganisms disclosed herein is added to the homogeneous mixture, at which point additional components such as flavorings and colorings may be added. This formulation is further cooled and molded into pieces of the desired size.

[0256] In other pharmaceutical embodiments, flavoring agents are incorporated into an ingestible topical vehicle, which may be in the form of mouthwash, rinse, ingestible spray, suspension, or dental gel. Typical non-toxic ingestible vehicles known in the pharmaceutical field can be used in the subject matter of this disclosure. Preferred ingestible vehicles are water, ethanol, and water-ethanol mixtures. Water-ethanol mixtures are generally used in weight ratios of about 1:1 to about 20:1, preferably about 3:1 to about 20:1, and most preferably about 3:1 to about 10:1. The pH value of the ingestible vehicle is generally about 4 to about 7, preferably about 5 to about 6.5. Ingested topical vehicles with a pH value of less than about 4 generally irritate the mouth, and ingestible vehicles with a pH value greater than about 7 generally result in an unpleasant mouthfeel.

[0257] Topical flavorings for ingestion may also contain conventional additives commonly used in such products. Conventional additives include fluorine-providing compounds, sweeteners, flavorings, colorants, humectants, buffers, and emulsifiers, provided that the additives do not interfere with the flavor characteristics of the composition. The amounts of colorants and humectants and these additives mentioned above can be used in topical flavoring compositions. Flavorings that can be used include those known to those skilled in the art, such as natural and artificial flavors. Suitable flavorings include mint such as peppermint, citrus flavors such as orange and lemon, artificial vanilla, cinnamon, and various fruit flavors (both individual and mixed). The amount of flavoring used in topical flavoring compositions is usually a matter of preference and depends on factors such as the type of final topical flavoring composition, the individual flavors used, and the desired flavor intensity. Therefore, the amount of flavoring can be varied to obtain the desired result in the final product, and such variation is within the capabilities of those skilled in the art without the need for excessive experimentation. When used, flavoring agents are generally used in amounts that may be, for example, about 0.05% to about 6% by weight of the ingestible topical composition.

[0258] 5.5. Pet Food Products D-tagatose prepared and / or produced by any of the microorganisms disclosed herein can be used in a wide variety of pet food products.

[0259] As used herein, the terms “pet food” or “pet food product” refer to products or compositions intended for consumption by companion animals such as cats, dogs, guinea pigs, rabbits, birds, and horses. For example, but not limited to, companion animals may be “house” dogs, such as Canis lupus familiaris. “Pet food” or “pet food product” includes any food, feed, snacks, food supplements, liquids, beverages, treats, toys (chewable toys and / or consumable toys), meal substitutes or meal replacements.

[0260] In certain embodiments, D-tagatose prepared and / or produced by any of the microorganisms disclosed herein is added directly to the pet food product. In certain embodiments, D-tagatose prepared and / or produced by any of the microorganisms disclosed herein may be added before, during, or after the formulation or packaging of the pet food product.

[0261] Non-exclusive examples of suitable pet food products include wet food products, dry food products, moist food products, pet food supplements (e.g., vitamins), pet beverage products, snacks and treats, and the pet food categories described herein.

[0262] In certain embodiments, the pet food product is a dry food product. A dry or low-moisture complete nutrition pet food product may contain less than about 15% moisture. In certain embodiments, the pet food product is a wet food product. A wet or high-moisture complete nutrition pet food product may contain more than about 50% moisture. In certain embodiments, the pet food product is a complete nutrition moist food product. A moist, for example, semi-moist, semi-dry, soft-dry, soft-moist, or medium-moisture complete nutrition pet food product contains about 15% to about 50% moisture.

[0263] In certain embodiments, the pet food product is a pet food snack product. Non-limiting examples of pet food snack products include snack bars, pet chews, crunchy treats, cereal bars, snacks, biscuits, and sweets products.

[0264] 5.6. Delivery System In certain embodiments, D-tagatose can be incorporated into a delivery system for use in edible compositions. In certain embodiments, the composition includes other flavor or taste modifiers such as salty substances, umami substances, bitter substances, astringent substances, and / or savory substances. The delivery system may be liquid or solid, aqueous or non-aqueous. The delivery system is generally adapted to suit the needs of the flavor and / or edible composition into which the D-tagatose is incorporated.

[0265] D-tagatose can be used in liquid, dry, and / or solid forms. When used in dry form, appropriate drying methods such as spray drying can be used. Alternatively, D-tagatose may be encapsulated or absorbed into water-soluble materials, including but not limited to cellulose, starch, sugar, maltodextrin, and gum arabic. Practical techniques for preparing such dry forms are well known in the art and can be applied to the subject matter of this disclosure.

[0266] D-tagatose can be used in many different physical forms known in the art to provide an initial burst of taste, flavor and / or texture; and / or a long-lasting sensation of taste, flavor and / or texture. Such physical forms include, but are not limited to, free forms such as spray-dried forms, powdered forms and beaded forms, and encapsulated forms, as well as mixtures thereof.

[0267] In certain embodiments, D-tagatose is encapsulated. Encapsulation materials and / or encapsulation techniques can be selected to improve the stability of D-tagatose and / or the food. In certain embodiments, the encapsulation materials and / or encapsulation techniques are selected to modify the release profile of D-tagatose.

[0268] Suitable encapsulation materials include, but are not limited to, hydrophilic colloids such as alginates, pectins, agars, guar gums, and cellulose, proteins, polyvinyl acetate, polyethylene, cross-linked polyvinylpyrrolidone, polymethyl methacrylate, polylactidic acid, polyhydroxyalkanoates, ethylcellulose, polyvinyl acetate phthalate, polyethylene glycol esters, methyl methacrylate-co-methacrylate, ethylene-vinyl acetate (EVA) copolymers, and combinations thereof. Suitable encapsulation techniques, though not limited to, spray coating, spray drying, spray cooling, absorption, adsorption, inclusion complex formation (e.g., preparation of flavor / cyclodextrin complexes), coacervation, fluidized bed coating, or other processes that can be used to encapsulate the components with the encapsulation material.

[0269] An encapsulation delivery system for flavoring agents or sweeteners (e.g., D-tagatose) contains a hydrophobic matrix of fat or wax surrounding a sweetener core or flavoring agent core. The fat can be selected from any number of conventional materials, such as fatty acids, glycerides or polyglycerol esters, sorbitol esters, and mixtures thereof. Examples of fatty acids include, but are not limited to, hydrogenated and partially hydrogenated vegetable oils such as palm oil, palm kernel oil, peanut oil, rapeseed oil, rice bran oil, soybean oil, cottonseed oil, sunflower oil, safflower oil, and mixtures thereof. Examples of glycerides include, but are not limited to, monoglycerides, diglycerides, and triglycerides.

[0270] Useful waxes can be selected from the group consisting of natural waxes, synthetic waxes, and mixtures thereof. Non-limiting examples include paraffin wax, petrolatum, carbowx, microcrystalline wax, beeswax, carnauba wax, candelilla wax, lanolin, bayberry wax, sugarcane wax, whale wax, rice bran wax, and mixtures thereof.

[0271] The fats and waxes can be used individually or in combination in varying amounts of about 10% to 70% by weight of the encapsulated system, or in amounts of about 30% to 60% by weight. When used in combination, the fats and waxes are preferably present in a ratio of about 70:10 to 85:15, respectively.

[0272] Typical encapsulated compositions, flavoring agents, or sweetener delivery systems are disclosed in U.S. Patent No. 4,597,970 and U.S. Patent No. 4,722,845, the entire disclosure of which is incorporated herein by reference.

[0273] Examples of liquid delivery systems include, but are not limited to, systems having a dispersion of D-tagatose, such as carbohydrate syrups and / or emulsions. Liquid delivery systems may also include extracts in which D-tagatose is solubilized in a solvent. Solid delivery systems can be prepared by spray drying, spray coating, spray cooling, fluidized bed drying, absorption, adsorption, coacervation, complexing, or any other standard technique. In some embodiments, the delivery system may be selected to be compatible with or to function in an edible composition. In some embodiments, the delivery system includes an oily material such as a fat or oil. In some embodiments, the delivery system includes a confectionery fat such as cocoa butter, a cocoa butter substitute, a cocoa butter substitute, or a cocoa butter equivalent.

[0274] When used in a dry form, appropriate drying methods such as spray drying can be used. Alternatively, D-tagatose may be adsorbed or absorbed onto a substrate such as cellulose, starch, sugar, maltodextrin, or gum arabic, or it may be encapsulated. Practical techniques for preparing such dry forms are well known in the art. [Examples]

[0275] The subject matter of this disclosure can be better understood by referring to the following. The following examples are illustrative and should not be construed as limiting.

[0276] ●Example 1 - Biosynthesis of D-tagatose from glucose Escherichia coli (E. coli) can naturally produce trace amounts of D-tagatose. In this embodiment, D-tagatose production is improved by overexpressing key genes, eliminating competing pathway genes, and optimizing production conditions.

[0277] • Evaluation of D-tagatose production capacity in Escherichia coli (E. coli) First, it was tested whether Escherichia coli (E. coli) possessed the enzyme capable of producing tagatose. Cultures were grown at 30°C on M9P medium containing 10 g / L glucose (M9 minimal medium containing 5 g / L yeast extract), and supernatant samples were taken 24 hours after inoculation and analyzed using HPLC. Gene knockout (KO) was constructed in AL3601 (Table 1). The double KO strain AL4240, possessing ΔpfkA and Δzwf, produced tagatose (Figure 2). The gene zwf encodes the enzyme glucose-6-phosphate dehydrogenase (Zwf) (EC 1.1.1.363), which converts G6P to 6-phospho-D-glucono-1,5-lactone as the first committed step in the pentose phosphate pathway (PPP). pfkA encodes phosphofructokinase A (EC 2.7.1.11), which converts F6P to fructose-1,6-bisphosphate (F16BP) as part of the first committed step of glycolysis. A dual knockout strain (AL4240) containing ΔpfkA and Δzwf produced 0.4 g / L of tagatose, demonstrating that Escherichia coli (E. coli) possesses the enzyme capable of producing D-tagatose (Figure 2). Further deletion of competing pathways resulted in increased tagatose production.

[0278] Table 1. Stock List [Table 1]

[0279] • Determination of the Tagatose-producing enzyme Next, we hypothesized that fructose-6-phosphate (F6P) is an intermediate for D-tagatose in Escherichia coli (E. coli). The production pathway begins with the native assimilation of glucose into E. coli via the phosphotransferase system (PTS) or GalP / Glk, which converts glucose to glucose-6-phosphate (G6P). G6P is then isomerized to F6P via glucose-6-phosphate isomerase (Gpi) (EC 5.3.1.9). F6P can be converted to tagatose-6-phosphate by epimerase. Phosphatases can dephosphorylate tagatose-6-phosphate to free tagatose, which is then excreted from the cell. A tagatose production system using phosphorylation and dephosphorylation steps as the driving force should be more efficient than the pathways currently used in the industrial production of tagatose.

[0280] Literature searches and genome mining identified one epimerase and seven candidate phosphatases. The tagatose-1,6-bisphosphate aldolase subunit (GatZ) showed activity against D-tagatose. The seven phosphatases (HxpA, HxpB, YbiV, YidA, YigL, YihX, and YqaB) exhibited broad substrate specificity for various hexoses.

[0281] The gatZ gene was deleted in AL4330 (ΔpfkA Δzwf ΔmanA ΔalsE Δpgm) to create AL4386. AL4386 did not produce detectable amounts of D-tagatose (Figure 3), while gatZ expression from the AL4386 expression plasmid restored D-tagatose production (Figure 3). These results indicate that GatZ is responsible for the conversion of F6P to tagatose-6-phosphate.

[0282] Seven E. coli phosphatases (HxpA, HxpB, YbiV, YidA, YigL, YihX, YqaB) with broad substrate specificity to various hexoses were tested for D-tagatose production in AL4330. Seven plasmids, pAL2490, pAL2491, pAL292, pAL2493, pAL2494, pAL2495, and pAL2496, were used with an inducible promoter P LlacO1 Cells were constructed to further express hxpA, hxpB, ybiV, yidA, yigL, yihX, or yqaB along with gatZ under the same conditions (Table 2). Cells were cultured at 30°C in M9P medium containing 10 g / L glucose and induced with 1 mM IPTG. Strains containing pAL2490(hxpA) produced the most D-tagatose (Figure 4).

[0283] Table 2. Plasmid List [Table 2]

[0284] Table 3 [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] [Table 3-9] [Table 3-10] [Table 3-11] [Table 3-12] [Table 3-13] [Table 3-14]

[0285] ●Example 2 - Activation of D-tagatose production ability in Escherichia coli Growing awareness of the health impact of dietary habits is fueling a significant demand for healthier food choices (Alsubhi et al., 2023). In particular, there is increasing scrutiny of the role of dietary sugars in non-communicable diseases (Bluher, 2019; Lustig et al., 2012; Prinz, 2019). In response to this public health issue, the food industry is adopting artificial sweeteners as a means of reducing the calorie content of sugars without compromising consumer satisfaction, as evidenced by the projected growth of the sugar substitutes market, which is expected to reach US$20.6 billion by 2025 ("Sugar Substitutes Market Size and Forecast," 2017).

[0286] The current sweetener market is largely comprised of non-sugar substitutes, despite recent guidelines issued by the World Health Organization advising against the use of non-sugar sweeteners to prevent weight loss or non-communicable diseases (Rizas et al., 2023). Rare sugars, characterized by slight variations in chemical structure compared to common sugars like glucose, are increasingly sought after as alternative sweeteners (Ahmed et al., 2022). D-tagatose, a C4 epimer of the rare sugar and fructose, has received "generally recognized safe" status and is 92% as sweet as sucrose (Xu et al., 2014). D-tagatose is marketed as a low-calorie sweetener, although its calorie value remains controversial, ranging from 1.5 kcal / g to 3 kcal / g (Ahmed et al., 2022). D-tagatose is an ideal sugar substitute in beverages due to its high solubility [58% (w / w) at 21°C] and lower viscosity than sucrose (Oh, 2007). Furthermore, its favorable flavor profile and desirable browning effect further enhance its suitability for use as a sugar substitute (Oh, 2007). D-tagatose has been reported to aid in weight loss and exhibits additional beneficial effects such as anti-plaque, non-cariogenic, anti-halitosis, prebiotic, and anti-biofilm properties (Oh, 2007).

[0287] D-tagatose is found in trace amounts in sterilized milk powder, hot cocoa, cheese, yogurt, and other dairy products (Roy et al., 2018). However, extraction of D-tagatose from natural sources is not economically feasible due to complex purification processes and low yields (Oh, 2007). Synthetic methods for D-tagatose production also suffer from similar drawbacks, along with the additional use of acids, bases, and catalysts (Roy et al., 2018). A potential solution to this problem is the "Ismoring" strategy, which utilizes enzymes and functions as a framework for the interconversion of aldohexoses, ketohexoses, and hexitols (Granstrom et al., 2004). In this regard, two such enzymes have been widely studied (Figure 5A): 1) L-arabinose isomerase (EC 5.3.1.4) interconverting D-galactose and D-tagatose, and 2) galactitol 2-dehydrogenase (EC 1.1.1.16) interconverting galactitol and D-tagatose. However, these methods face challenges such as the need for purified enzymes, cofactors like NAD+, low thermal stability, or lack of thermodynamic drive, resulting in low yields and high production costs (Dai and Jin, 2024; Noor et al., 2012). To address these challenges, recent studies have encapsulated L-arabinose isomerase in Lactobacillus plantarum, achieving high yields of D-tagatose. However, this method relies on high-cost D-galactose as a starting material and requires separation from D-tagatose after production, further increasing costs (Baumgartner et al., 2015). Alternatively, microbial production has been applied to convert lactose to D-tagatose in Saccharomyces cerevisiae, but this method utilizes only D-galactose from lactose, and the yield is low because D-glucose is used to maintain cell growth (Liu et al., 2019).As a result, the final culture medium contained galactose, galactitol, and D-tagatose, leading to higher separation and purification costs (Liu et al., 2019).

[0288] D-fructose has potential as a starting material for D-tagatose production, but a significant gap exists in nature: there is no C4 epimerase that can directly convert D-fructose to D-tagatose (Jeon et al., 2023; Lee et al., 2017b; Shin et al., 2020). The C4-epimerization activity of tagaturonic acid-fructuronic acid epimerase from Thermotoga petrophila has been identified (Shin et al., 2020). Recently, phosphorylation and dephosphorylation strategies have emerged as promising pathways for the production of rare sugars from glucose, providing economically advantageous raw materials (Dai et al., 2022; Taylor et al., 2023). This pathway involves a crucial C4 epimerization reaction that converts D-fructose-6-phosphate (F6P) to D-tagatose-6-phosphate (T6P). Subsequent dephosphorylation of T6P provides substantial thermodynamic driving force, promoting efficient D-tagatose production. Using this pathway, a whole-cell biocatalyst was developed, yielding 3.38 g / L of D-tagatose from 10 g / L of maltodextrin (Dai et al., 2022). However, due to the inefficiency of the conversion, glucose and fructose remain in the solution after production (Dai et al., 2022).

[0289] In this disclosure, it is discovered that Escherichia coli (E. coli) possesses all the enzymes necessary to convert D-glucose to D-tagatose (Figure 5B). D-glucose is converted to F6P via glycolysis, and epimerization of F6P to T6P is achieved by the native enzyme, namely GatZ, a putative tagatose-1,6-bisphosphate aldolase 2-chaperone (Nobelmann and Lengeler, 1995), or KbaZ, a putative tagatose-1,6-bisphosphate aldolase 1-chaperone (Brinkkotter et al., 2000). Although these enzymes are annotated as aldolase chaperones (Brinkkotter et al., 2002), this example demonstrates the C4 epimerization activity of these enzymes in Escherichia coli (E. coli). Furthermore, dephosphorylation of T6P can be achieved by hexitol phosphatase A (HxpA). Production of D-tagatose from D-glucose in Escherichia coli (E. coli) was achieved solely by the native genes of E. coli. D-tagatose production was further enhanced by expressing additional genes for this pathway and eliminating competing pathways, including the pentose phosphate pathway, glycogen biosynthesis, glycolysis, the D-psicose production pathway, and the D-mannose degradation pathway.

[0290] • Natural D-tagatose production capacity of Escherichia coli (E. coli) The innate production capacity of *E. coli* to produce D-tagatose from D-glucose was investigated. The production strain derived from MG1655 (Table 4) did not produce D-tagatose under production conditions (Figure 6A). To accumulate the important intermediate F6P, the pfkA gene encoding phosphofructokinase A was deleted, but the ΔpfkA strain did not produce D-tagatose (Figure 6A). The zwf gene encoding glucose-6-phosphate dehydrogenase was deleted to further increase the F6P pool. The ΔpfkA Δzwf strain (AL4240, Table 4) produced 0.21 g / L in 24 hours. -1D-tagatose was produced (Figure 6A), indicating that Escherichia coli naturally possesses all the enzymes necessary to produce D-tagatose from D-glucose.

[0291] Table 4. Strains used in this embodiment. [Table 4]

[0292] • Elucidation of the D-tagatose production pathway in Escherichia coli (E. coli) While Escherichia coli (E. coli) has been used to produce D-tagatose, these efforts have relied primarily on heterologous enzymes (Dai et al., 2022; Liu et al., 2023; Zhang et al., 2020). The natural D-tagatose pathway was hypothesized to involve the conversion of D-glucose to F6P via glycolysis, followed by the action of epimerase to convert F6P to T6P. Subsequently, dephosphorylation of T6P by phosphatase yields D-tagatose (Figure 5B).

[0293] Five endogenous enzymes were identified as candidates possessing C4 epimerase activity for converting F6P to T6P. First, D-fructose-1,6-bisphosphate aldolase encoded by fbaA has been shown to exhibit C4 epimerase activity in vitro (Lee et al., 2017b, 2017a). Next, GatZ, annotated as a putative tagatose-1,6-bisphosphate aldolase 2-chaperone, has been used to promote the growth of Agrobacterium tumefaciens on galactitol, suggesting that GatZ can perform the reverse reaction used in this embodiment, the conversion from T6P to F6P (Kohlmeier et al., 2019). Furthermore, KbaZ, annotated as a tagatose-1,6-bisphosphate aldolase 2-chaperone, was found to have high structural similarity to GatZ. Finally, the enzymes GatY (tagatose-1,6-bisphosphate aldolase 2) and KbaY (tagatose-1,6-bisphosphate aldolase 1) have a high structural similarity to FbaA (Brinkkotter et al., 2002).

[0294] To elucidate the C4 epimerase involved in D-tagatose production, each candidate gene was deleted in AL4240 (ΔpfkA Δzwf, Table 4). The fbaA gene is essential and therefore could not be deleted (Goodall et al., 2018). Tagatose production disappeared with gatZ deletion alone (Figure 6B), suggesting that GatZ converts F6P to T6P in Escherichia coli (E. coli). However, expression of gatZ from an expression plasmid under the IPTG-inducible promoter PLlacO1 (Lutz and Bujard, 1997) in AL4424 (ΔpfkA Δzwf ΔgatZ, Table 4) did not compensate for D-tagatose production (Figure 6C). It has been demonstrated that gatZ expression enhances the activity of tagatose-1,6-bisphosphate aldolase (Brinkkotter et al., 2002), suggesting a potential diversion of T6P, shifting its focus away from D-tagatose production towards glycolysis. It was hypothesized that additional expression of T6P phosphatase is necessary to redirect the carbon flux back towards D-tagatose production. We selected phosphatases with broad substrate specificity (Kuznetsova et al., 2006) and screened AL4424 (ΔpfkA Δzwf ΔgatZ, Table 4) by expressing GatZ along with hexitol phosphatase B (HxpB), sugar phosphatase YbiV, sugar phosphatase YidA, hexitol phosphatase A (HxpA), α-D-glucose-1-phosphate phosphatase YihX, phosphate sugar phosphatase YigL, or fructose-1-phosphate phosphatase YqaB. Co-expression of GatZ and hexitol phosphatase A (HxpA) restored D-tagatose production in AL4424 (Figure 4D).

[0295] Other C4 epimerase candidate genes may not be expressed under these culture conditions. Therefore, in AL4424 (ΔpfkA Δzwf ΔgatZ, Table 4), each epimerase candidate was expressed together with hxpA from the expression plasmid. Additional expression of kbaZ restored D-tagatose production, but expression of fbaA, kbaY, or gatY did not (Figure 7A). gatZ and kbaZ were used for further modification.

[0296] Analysis of structural similarity of C4 epimerase The search for C4 epimerases to facilitate the conversion of D-fructose to D-tagatose has been a key effort in the sugar industry (Shin et al., 2020). The C4-epimerization ability of tagaturonic acid-fructuronic acid epimerase from Thermotoga petrophila was identified (Shin et al., 2020). However, this reaction is thermodynamically unfavorable, resulting in a mixture of D-fructose and D-tagatose. An enzyme from Agrobacterium tumefaciens C58 capable of converting F6P to T6P was identified (Wichelecki et al., 2015). This finding has enabled a phosphorylation-dephosphorylation pathway for D-tagatose production (Dai et al., 2022). Another C4 epimerase showing structural similarity to GatZ from Escherichia coli (E. coli) was identified in Sinorhizobium meliloti (Kohlmeier et al., 2019).

[0297] GatZ and KbaZ were identified as having similar structures. Clustal Omega was used to align the structures of GatZ and KbaZ from Escherichia coli (E. coli) with other published C4 epimerase enzymes. The structures of these epimerases were obtained from Alphafold and analyzed for structural similarity.

[0298] • Regulation of D-tagatose production pathway gene expression The native hxpA gene has a GTG start codon. To improve the translation efficiency of hxpA, the start codon was changed to ATG(hxpA*) (Sussman et al., 1996). This modification is achieved by additional expression of gatZ, which is 0.48 gL. -1 And, through the additional expression of kabZ, 0.60 gL -1 This improved the D-tagatose titer (Figure 7B). The PLlacO1 promoter was replaced with the PgadB promoter, a stationary-phase promoter that is approximately 100 times stronger than PLlacO1 (Taylor et al., 2023). The D-tagatose productivity using PgadB was similar to that of PLlacO1 (Figure 7B).

[0299] • Elimination of conflicting paths AL4240 (ΔpfkA Δzwf, Table 4) produced using the plasmid was administered in 0.8 gL doses together with D-tagatose. -1 D-mannose and 1.1 gL -1 D-psicose was produced (Figure 10). The manA gene encodes mannose-6-phosphate isomerase, which interconverts F6P with mannose-6-phosphate (Gao et al., 2005), and diverts the carbon flux to D-mannose production. The alsE gene encodes D-allulose-6-phosphate 3-epimerase, which interconverts F6P with D-psicose-6-phosphate and is responsible for D-psicose production (Taylor et al., 2023). By deleting manA in AL4240 (ΔpfkA Δzwf, Table 4), AL4290 (ΔpfkA Δzwf ΔmanA, Table 4) was created. This deletion increased D-tagatose production to 0.6 gL-1 and eliminated D-mannose production (Figure 5). Similarly, by deleting alsE in AL4240 (ΔpfkA Δzwf, Table 1), AL4314 (ΔpfkA Δzwf ΔalsE, Table 4) was obtained. This deletion resulted in a D-tagatose production of 0.67 gL. -1This improved the process and eliminated D-psicose production (Figure 8). Both of these genes were deleted in AL4240 (ΔpfkA Δzwf, Table 4) to create AL4315 (ΔpfkA Δzwf ΔalsE ΔmanA, Table 4). 0.53 gL of AL4315 (ΔpfkA Δzwf ΔalsE ΔmanA, Table 4) using the production plasmid was produced. -1 It produced D-tagatose (Figure 8) but did not produce D-psicose or D-mannose.

[0300] Escherichia coli (E. coli) can store excess glucose in the form of glycogen (Sekar et al., 2020). The pgm gene, which encodes phosphoglucumutase, converts G6P to glucose-1-phosphate (Eydallin et al., 2007). By deleting the pgm gene in AL4315 (ΔpfkA Δzwf ΔalsE ΔmanA, Table 4), AL4330 (ΔpfkA Δzwf ΔalsE ΔmanA Δpgm, Table 4) was constructed. 0.82 gL of AL4330 using the production plasmid was obtained. -1 D-tagatose was produced (Figure 8).

[0301] T6P can be converted by tagatose-6-phosphate kinase (PfkB) to D-tagatose 1,6-bisphosphate, which is subsequently converted to glyceron phosphate and D-glyceraldehyde 3-phosphate by aldolases (GatY and KbaY) (Brinkkotter et al., 2002). In those strains, the pfkA gene is deleted, so the pfkB gene cannot be deleted. Overexpression of gatZ has been shown to enhance tagatose-1,6-bisphosphate aldolase activity in Escherichia coli (Brinkkotter et al., 2002). Activated tagatose-1,6-bisphosphate aldolase activity diverts the carbon flux away from D-tagatose production and towards glycolysis. By deleting the kbaY gene in AL4330 (ΔpfkA Δzwf ΔalsE ΔmanA Δpgm, Table 1), AL4493 (ΔpfkA Δzwf ΔalsE ΔmanA Δpgm ΔkbaY, Table 4) was generated. Similarly, by deleting gatY in AL4330 (ΔpfkA Δzwf ΔalsE ΔmanA Δpgm, Table 4), AL4533 (ΔpfkA Δzwf ΔalsE ΔmanA Δpgm ΔgatY, Table 4) was generated. AL4534 (ΔpfkA Δzwf ΔalsE ΔmanA Δpgm ΔgatY ΔkbaY, Table 4) was prepared by deleting both gatY and kbaY from AL4330 (DpfkA Δzwf ΔalsE ΔmanA Δpgm ΔgatY ΔkbaY, Table 4). However, these deletions did not improve D-tagatose production (Figure 8).

[0302] • Production of D-tagatose under high culture density conditions Ensuring production performance under high cell density conditions is essential for gaining a comprehensive understanding of production capacity (Theisen and Liao, 2017) (Crater and Lievense, 2018). To address potential production constraints arising from the amount of available glucose, AL4534 strains having pAL2606 (PgadB:gatZ-hxpA*) or pAL2607 (PgadB:kbaZ-hxpA*, Tables 4 and 5) were cultured under high cell density conditions for 24 hours. Both of these strains produced 40 gL in 24 hours. -1 This consumed glucose (Figure 11). Based on these results, tagatose production under high cell density conditions was reduced to 40 gL daily for two days. -1 The test was conducted by supplying glucose. After the first 24 hours, AL4534 using pAL2606 (gatZ) and pAL2607 (kbaZ) each consumed 3.2 gL. -1 and 3.4gL -1 These strains produced D-tagatose (Figure 9A). These strains also produced D-fructose and D-mannitol as byproducts (Figures 9B and 9C). A glucose bolus was added to each culture to increase the glucose concentration in the medium to 40 gL. -1 It was restored to this state. After further incubation for 24 hours, AL4534 using pAL2606 (gatZ) and pAL2607 (kbaZ) was cultured at 7.0 gL each. -1 and 8.7 gL -1 D-tagatose was produced (Figure 9A). D-fructose and D-mannitol production also increased (Figure 9a). This observation suggests that, due to the low C4 epimerase activity of GatZ and KbaZ, accumulated F6P redirects the carbon flux to D-fructose and D-mannitol. D-mannitol production is made possible by the conversion of F6P to D-mannitol-1-phosphate (Mtl1P) via the enzyme mannitol-1-phosphate 5-dehydrogenase, encoded by the gene mtlD.

[0303] HxpA exhibits substrate promiscuity to F6P and Mtl1P (Kuznetsova et al., 2006; Sevin et al., 2017). To minimize hxpA expression, this example reverted to the weaker PLlacO1 promoter plasmid systems pAL2574 (PLlacO1:gatZ-hxpA*) and pAL2575 (PLlacO1:kbaZ-hxpA*, Table 5). AL4534 containing pAL2574 (gatZ) and pAL2575 (kbaZ) was expressed at 4.5 gL each after 24 hours. -1 and 3.5gL -1 These strains produced D-tagatose (Figure 9A). After a bolus of glucose administration, and 24 hours later, each strain produced 8.1 gL -1 and 7.6gL -1 D-tagatose was produced. In addition, 8.8 gL -1 and 7.8gL -1 D-fructose, and 7.3 gL -1 and 7.1 gL -1 D-mannitol was produced (Figures 9B and 9C). Reducing hxpA expression levels resulted in similar D-tagatose titers, but D-fructose and D-mannitol were still produced.

[0304] Table 5. Plasmids used in this example. [Table 5]

[0305] • Carbon starvation strategies to eliminate byproduct formation The AL4534 strain was shown to be unable to assimilate D-tagatose under production conditions. Furthermore, although Escherichia coli (E. coli) can redirect phosphorylated D-tagatose to glycolysis (Ha et al., 2022), various E. coli strains have been shown to be unable to grow on D-tagatose (Joo et al., nd).

[0306] Carbon starvation is an effective approach to reduce byproduct formation for D-tagatose production. Under D-glucose-deficient conditions, strains cannot utilize the produced D-tagatose but can use other byproducts as a carbon source. AL4534, which has pAL2574 (PLlacO1, gatZ), pAL2575 (PLlacO1, kbaZ), pAL2606 (PgadB, gatZ), and pAL2607 (PgadB, kbaZ, Table 5), is 15 gL -1 It was used for D-tagatose production under high-density conditions with daily addition of glucose (Figures 9D-9F). Within 24 hours, the strains each produced 1.5 gL -1 , 1.2gL -1 , 1.4gL -1 and 1.5gL -1 D-tagatose was produced (Figure 9D). In particular, AL4534, which had undetectable levels of D-fructose and contained pAL2574 (PLlacO1, gatZ) and pAL2575 (PLlacO1, kbaZ), was produced in quantities of 1.2 gL each. -1 and 2.1 gL -1 D-mannitol was produced (Figures 9E and 9F). Further glucose supplementation increased the D-tagatose titer to 4.6 gL after 48 hours. -1 , 4.5gL -1 , 4.7gL -1 and 4.3gL -1 The amount increased to (Figure 9D). AL4534 containing pAL2574 (PLlacO1, gatZ) and pAL2575 (PLlacO1, kbaZ) utilized the produced D-mannitol. However, AL4534 containing pAL2606 (PgadB, gatZ) and pAL2607 (PgadB, kbaZ) did not produce D-mannitol, and 2.0 gL was produced. -1 and 0.7 gL -1 D-fructose remained in the culture medium after 48 hours (Figures 9E and 9F). Furthermore, 15 g L -1 After glucose addition and 78 hours of incubation, they were each 7.3 gL -1 6.9gL -1 7.3gL-1 and 7.3 g L -1 of D-tagatose was produced (Figure 9D). In AL4534 harboring pAL2574 (PLlacO1, gatZ) and pAL2575 (PLlacO1, kbaZ), no by-product was detectable, while AL4534 harboring pAL2606 (PgadB, gatZ) and pAL2607 (PgadB, kbaZ) produced 5.2 g L -1 and 5.4 g L -1 of D-fructose, respectively (Figure 9E).

[0307] • Conclusion Herein, the present example reveals that E. coli only having knockouts of pfkA and zwf genes has an intrinsic ability to convert D-glucose into D-tagatose. Furthermore, it is demonstrated that GatZ and KbaZ can convert F6P into D-tagatose-6-phosphate. By exploiting the substrate promiscuity of native phosphatases to increase D-tagatose production, and further through elimination of competing pathways and overexpression of native E. coli genes, the present disclosure constructed a strain capable of producing D-tagatose from readily available feedstocks via a thermodynamically favorable biosynthetic pathway. Thanks to the inability of the strain of the present disclosure to utilize D-tagatose and the efficiency of said strain in consuming all D-glucose and by-products present in the medium, the need for downstream purification is reduced. These results suggest that during production, addition that maintains low glucose concentration and carbon starvation prevents the formation of by-products. The final strain of the present disclosure achieved conversion of 45 g L -1 of D-glucose into D-tagatose, which reached a titer of 7.3 g L -1 and a productivity of 0.1 g L -1 h -1 , with no formation of major by-products.

[0308] • Methods Reagents. All enzymes involved in the molecular cloning experiments were purchased from New England Biolabs (NEB). All synthetic oligonucleotides were synthesized by Integrated DNA Technologies. Sanger sequencing was provided by Genewiz. D-mannitol and D-tagatose were purchased from Tokyo Chemical Industry. D-psicose and D-mannose were purchased from Sigma-Aldrich. D-glucose and D-fructose were purchased from Fisher Scientific.

[0309] Strains and plasmids. All strains and plasmids used in this study are listed in Tables 4 and 5, respectively. Plasmids for D-tagatose production were constructed using sequence and ligation-independent cloning (SLIC) (Li and Elledge, 2007). The constructed plasmids were validated by sequencing.

[0310] Genome modifications, such as gene deletions and insertions, were constructed using CRISPR-Cas9-mediated homologous recombination (Jiang et al., 2015). Linear DNA repair fragments for gene deletions and insertions were constructed by amplifying genomic or plasmid DNA by PCR assembly. Plasmids encoding sgRNA for CRISPR-Cas9-mediated homologous recombination were constructed using the pTargetF plasmid (Addgene #62226) as a template and Q5 site-directed mutagenesis (New England Biolabs). All genome modifications were validated by sequencing. The guidelines for CRISPR-Cas9-mediated gene modifications used in this study are detailed in Table 6.

[0311] Table 6. Guideline for CRISPR-Cas9-mediated gene deletion and insertion. [Table 6]

[0312] Culture conditions. Overnight cultures were grown at 37°C in 3 mL of Luria-Bertani (LB) medium supplemented with appropriate antibiotics. Antibiotic concentrations were as follows: spectinomycin (50 μg mL -1 ), ampicillin (200 μg mL -1 ), kanamycin (50 μg mL -1 ). M9 minimal medium consists of 33.7 mM Na2HPO4, 22 mM KH2PO4, 8.6 mM NaCl, 9.4 mM NH4Cl, 2 mM MgSO4, 0.1 mM CaCl2, A5 trace metal mixture (2.86 mg L -1 H3BO3, 1.81 mg L -1 MnCl2·4H2O, 0.079 mg L -1 CuSO 4· ·5H2O, 49.4 μg L -1 Co(NO3) 2· ·6H2O), various concentrations of glucose, and appropriate antibiotics. M9P medium for D-tagatose production consists of M9 minimal medium supplemented with 5 g L -1 yeast extract and appropriate antibiotics. Inducer concentration is as follows: isopropyl-β-D-1-thiogalactopyranoside (IPTG) (1 mM). Optical density at 600 nm (OD 600 ) was measured using a Synergy H1 hybrid plate reader (BioTek Instruments, Inc.).

[0313] Production of D-tagatose. For normal cell density production experiments, overnight cultures were inoculated at 1% into 3 mL of M9P medium. Cells were grown at 37°C to the stated OD 600 , then induced with IPTG if required, and grown at 30°C for 24 hours. For high cell density production experiments in M9P medium, overnight cultures were inoculated at 1% into 150 mL of M9P medium. Cells were grown at 37°C until the OD 600 reached approximately 0.4 to 0.6. The cultures were then induced with IPTG if required, and grown for an additional 30 minutes. Cultures were centrifuged at 2,200 g for 5 minutes, and resuspended in M9P medium containing IPTG if required to reach the target OD600 The adjustments were made up to this point. The culture was grown at 30°C.

[0314] HPLC analysis. Analysis of D-tagatose, D-glucose, D-mannose, D-psicose, D-mannitol, and D-fructose concentrations was performed using HPLC (Shimadzu) equipped with a refractive index detector (RID) 10 A and a Rezex® RCU-USP sugar alcohol column (Phenomenex). The mobile phase consisted of 100% MilliQ water. Samples were injected at a rate of 1 μL at 0.5 mL min, with the column oven set to 83°C and the RID cell temperature to 40°C. -1 The sample was analyzed at the specified flow rate for 7.5 minutes. To prepare the sample for HPLC analysis, 300 μL of the culture was centrifuged at 17,000 g for 5 minutes. The supernatant was applied to a 0.2 μm PVDF hydrophilic membrane 96-well filter plate and centrifuged into a polystyrene 96-well filter at 2,000 rpm for 2 minutes.

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[0359] While the subject matter and its advantages have been described in detail, it should be understood that various changes, substitutions, and modifications can be made herein without departing from the spirit and scope of the invention as defined by the appended claims. Furthermore, the scope of this application is not intended to be limited to specific embodiments of the processes, machines, articles, compositions of substances, means, methods, and steps described herein. As will be readily apparent to those skilled in the art from the disclosure of the subject matter, existing or future-developed processes, machines, articles, compositions of substances, means, methods, or steps that perform substantially the same function or achieve substantially the same results as the corresponding embodiments described herein can be utilized in accordance with the subject matter of this disclosure. Accordingly, the appended claims are intended to include within their scope such processes, machines, articles, compositions of substances, means, methods, or steps.

[0360] Patents, patent applications, publications, product descriptions, and protocols are referenced throughout this application, and their entirety is incorporated herein by reference for all purposes.

Claims

1. Recombinant microorganisms containing exogenous epimerase that produce increased amounts of D-tagatose compared to naturally occurring microorganisms.

2. The recombinant microorganism according to claim 1, wherein the epimerase is the tagatose-1,6-bisphosphate aldolase subunit GatZ or the ketose diphosphate aldolase subunit KbaZ.

3. The recombinant microorganism according to claim 1 or claim 2, wherein the epimerase is Escherichia coli (E. coli) GatZ.

4. The recombinant microorganism according to claim 3, wherein the epimerase contains an amino acid sequence that is at least about 80% identical to the amino acid sequence shown in SEQ ID NO:

58.

5. The recombinant microorganism according to claim 3 or claim 4, wherein the epimerase contains or consists of the amino acid sequence shown in SEQ ID NO:

58.

6. The recombinant microorganism according to claim 1 or claim 2, wherein the epimerase is Escherichia coli (E. coli) KbaZ.

7. The recombinant microorganism according to claim 6, wherein the epimerase contains an amino acid sequence that is at least about 80% identical to the amino acid sequence shown in SEQ ID NO:

60.

8. The recombinant microorganism according to claim 6 or claim 7, wherein the epimerase contains or consists of the amino acid sequence shown in SEQ ID NO:

60.

9. The recombinant microorganism according to any one of claims 1 to 8, wherein the recombinant microorganism further comprises an exogenous phosphatase.

10. The recombinant microorganism according to claim 9, wherein the phosphatase is hexitol phosphatase A (HxpA).

11. The recombinant microorganism according to claim 9 or 10, wherein the phosphatase is Escherichia coli (E. coli) HxpA.

12. The recombinant microorganism according to any one of claims 9 to 11, wherein the phosphatase comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence shown in SEQ ID NO:

62.

13. The recombinant microorganism according to any one of claims 9 to 12, wherein the phosphatase comprises or consists of the amino acid sequence shown in SEQ ID NO:

62.

14. The recombinant microorganism contains exogenous galactose:H ＋ A recombinant microorganism according to any one of claims 1 to 13, further comprising a symporter (GalP) and glucokinase (Glk).

15. The recombinant microorganism according to claim 14, wherein the GalP is Escherichia coli (E. coli) GalP and the Glk is Escherichia coli (E. coli) Glk.

16. The recombinant microorganism according to claim 14 or claim 15, wherein the GalP comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence shown in SEQ ID NO: 38, and the Glk comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence shown in SEQ ID NO:

40.

17. The recombinant microorganism according to any one of claims 14 to 16, wherein the GalP comprises or consists of the amino acid sequence shown in SEQ ID NO: 38, and the Glk comprises or consists of the amino acid sequence shown in SEQ ID NO:

40.

18. A recombinant microorganism according to any one of claims 1 to 17, further comprising mutations in the gene encoding an enzyme in the pentose phosphate pathway compared to a naturally occurring microorganism.

19. The recombinant microorganism according to claim 18, wherein the enzyme in the pentose phosphate pathway is glucose-6-phosphate 1-dehydrogenase (Zwf).

20. A recombinant microorganism according to any one of claims 1 to 19, further comprising mutations in genes encoding glycolytic enzymes compared to naturally occurring microorganisms.

21. The recombinant microorganism according to claim 20, wherein the glycolytic enzyme is phosphofructokinase-1 (PfkA), phosphofructokinase-2 (PfkB), or pyruvate kinase (PykF).

22. The recombinant microorganism according to claim 20 or claim 21, wherein the glycolytic enzyme is phosphofructokinase-1 (PfkA).

23. The recombinant microorganism according to any one of claims 1 to 22, further comprising a mutation in a gene encoding an enzyme that converts fructose-6-phosphate to psicose-6-phosphate.

24. The recombinant microorganism according to claim 23, wherein the enzyme that converts fructose-6-phosphate to psicose-6-phosphate is D-allulose-6-phosphate 3-epimerase (AlsE).

25. A recombinant microorganism according to any one of claims 1 to 24, further comprising a mutation in a gene encoding an enzyme in the mannose biosynthesis pathway.

26. The recombinant microorganism according to claim 25, wherein the enzyme in the mannose biosynthesis pathway is mannose-6-phosphate isomerase (ManA).

27. A recombinant microorganism according to any one of claims 1 to 26, further comprising a mutation in a gene encoding an enzyme for carbohydrate metabolism.

28. The recombinant microorganism according to claim 27, wherein the enzyme for carbohydrate metabolism is the D-tagatose-1,6-bisphosphate aldolase subunit (GatY).

29. The recombinant microorganism according to claim 27, wherein the enzyme for carbohydrate metabolism is the D-tagatose-1,6-bisphosphate aldolase subunit (KbaY).

30. A recombinant microorganism according to any one of claims 1 to 29, further comprising mutations in genes encoding enzymes of glycogen biosynthesis selected from phosphoglucumutase (Pgm), UDP-glucose pyrophosphorylase, glycogen synthase, glycogen branching enzyme, glycogenin, and combinations thereof.

31. The recombinant microorganism according to claim 30, wherein the enzyme for glycogen biosynthesis is phosphoglucumutase (Pgm).

32. A microorganism comprising a recombinant polynucleotide encoding epimerase, wherein the expression of the epimerase results in increased production of D-tagatose compared to a microorganism lacking the recombinant polynucleotide.

33. The microorganism according to claim 32, wherein the epimerase is a tagatose-1,6-bisphosphate aldolase subunit GatZ or a ketose diphosphate aldolase subunit KbaZ.

34. The microorganism according to claim 32 or claim 33, wherein the epimerase is Escherichia coli (E. coli) GatZ.

35. The recombinant microorganism according to claim 32 or claim 33, wherein the epimerase is Escherichia coli (E. coli) KbaZ.

36. The recombinant microorganism according to any one of claims 32 to 35, wherein the recombinant microorganism further comprises an exogenous phosphatase.

37. The recombinant microorganism according to claim 36, wherein the phosphatase is hexitol phosphatase A (HxpA).

38. The recombinant microorganism according to claim 37, wherein the phosphatase is Escherichia coli (E. coli) HxpA.

39. A microorganism according to any one of claims 32 to 38, further comprising a mutation in at least one gene.

40. The microorganism according to claim 39, wherein the at least one gene is selected from glucose-6-phosphate 1-dehydrogenase (zwf), phosphofructokinase-1 (pfkA), phosphofructokinase-2 (pfkB), pyruvate kinase (pykF), D-allulose-6-phosphate 3-epimerase (alsE), mannose-6-phosphate isomerase (manA), phosphoglucomutase (pgm), D-tagatose-1,6-bisphosphate aldolase subunit (gatY), D-tagatose-1,6-bisphosphate aldolase subunit (kbaY), and combinations thereof.

41. The microorganism according to claim 39, wherein the at least one gene is glucose-6-phosphate 1-dehydrogenase (zwf), phosphofructokinase-1 (pfkA), D-allulose-6-phosphate 3-epimerase (alsE), mannose-6-phosphate isomerase (manA), phosphoglucomutase (pgm), D-tagatose-1,6-bisphosphate aldolase subunit (gatY), D-tagatose-1,6-bisphosphate aldolase subunit (kbaY), or a combination thereof.

42. The microorganism according to any one of claims 18 to 31 and 39 to 41, wherein the mutation is a deletion.

43. The microorganism according to any one of claims 18 to 31 and 39 to 41, wherein the mutation reduces or eliminates the expression or activity of the enzyme.

44. The microorganism according to any one of claims 1 to 43, wherein the microorganism is Escherichia coli, Bacillus subtilis, or Lactococcus lactis.

45. A method for producing D-tagatose, comprising culturing a microorganism according to any one of claims 1 to 44 under conditions suitable for converting a substrate to D-tagatose.

46. The method according to claim 45, wherein the substrate comprises D-glucose.

47. A method for producing food containing D-tagatose, a) Culturing the microorganism described in any one of claims 1 to 45 under conditions suitable for converting the substrate to D-tagatose, and b) Mixing the tagatose with one or more food products to form a food product containing tagatose. Methods that include...

48. The method according to claim 47, wherein the food is a beverage, yogurt, ice cream, baked good, or nutrition bar.

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