Use of foldases for improving heterologous expression of secretory molecules

Abstract

The invention relates to a bacillus licheniformis host cell for increasing production of secretory protein. In particular, the present invention relates to a Bacillus licheniformis host having a genetic modification that results in an increased production of heterologous, non-natural, secretory enzymes. Specifically, the present invention relates to a Bacillus licheniformis host cell which expresses a) a first polypeptide having peptidyl-prolyl cis-trans isomerase (EC 5.2. 1.8) activity comprising the amino acid sequence as shown in SEQ ID NO: 1, or at least 81% identical to SEQ ID NO: 1, and b) a second polypeptide having amylase activity comprising the amino acid sequence as shown in SEQ ID NO: 1, or at least 81% identical to SEQ ID NO: 2. Wherein the second polypeptide is heterologous to the Bacillus licheniformis host cell.

Description

Technical Field

[0001] The present invention relates to a Bacillus licheniformis host cell for increased production of secreted proteins. Specifically, the present invention relates to a Bacillus licheniformis host cell having a genetic modification that results in increased production of a heterologous, non-native, secreted enzyme. Specifically, the present invention relates to a Bacillus licheniformis host cell that expresses a) a first polypeptide having peptidyl-prolyl cis-trans isomerase (EC 5.2.1.8) activity, the first polypeptide comprising the amino acid sequence as set forth in SEQ ID NO: 1, or an amino acid sequence that is at least 81% identical to SEQ ID NO: 1, and b) a second polypeptide having amylase activity, wherein the second polypeptide is heterologous to the Bacillus licheniformis host cell. Background Art

[0002] Bacillus microorganisms are widely used as the industrial workhorse for producing valuable compounds, such as chemicals, polymers and proteins, particularly as proteins of enzymes for washing and / or cleaning activity enzymes or for feeding and food applications. The biotechnology production of these enzymes is carried out via the fermentation of this bacillus strain and the purification of the product subsequently. The bacillus strain can secrete a significant amount of protein into the fermentation liquid. Compared with production in the cell, this allows a simple product purification process, and has explained the success of bacillus in industrial applications. Therefore, bacillus cells are continuously optimized to increase the generation of these enzymes with a high degree of relevance. Particularly in a large-scale industrial production environment, even very little improvement also can have a great impact on production costs.

[0003] Extracellular proteins and enzymes are secreted by Bacillus cells. The major pathway for protein transport across the cell membrane is the general secretory "Sec" (SecA-YEG) pathway, in which the unfolded polypeptide chain is translocated by the SecYEG translocation complex into the cell wall-associated space where the signal peptide is cleaved and the polypeptide folding occurs.

[0004] The essential extracytoplasmic folding enzyme PrsA is a lipoprotein with peptidyl-prolyl cis-trans isomerase activity that assists polypeptide folding into a stable mature protein conformation. PrsA expression is regulated by feedback from the secretory stress response, and the PrsA folding enzyme itself is a substrate for multiple extracytoplasmic proteases (Krishnappa L, Monteferrante CG, Neef J, Dreisbach A, van Dijl JM. Degradation of extracytoplasmic catalysts for protein folding in Bacillus subtilis. Appl Environ Microbiol. 2014 Feb; 80(4): 1463-8. doi: 10.1128 / AEM.02799-13. Electronic publication on December 20, 2013. PMID: 24362423; PMCID: PMC3911040.).

[0005] Additional expression of the host's native PrsA foldase has been shown to improve the production of secreted proteins, such as heterologous subtilisins, lipases and especially amylases in Bacillus subtilis (WO94 / 019471) or Bacillus licheniformis (WO2021 / 146411).

[0006] WO2020 / 156903 discloses that both the PrsA protein and the amylase should be from the same bacterial species, and that at least some such homologous combinations show improved amylase expression in Bacillus subtilis. This means that the PrsA foldase is particularly beneficial for improving the production of polypeptides from the same bacterial species.

[0007] The identification of such a homologous PrsA protein and amylase combination is not necessarily given. EP 1 307 547 A1 discloses an amylase from Bacillus sp. A7-7 (DSM 12368) with an unknown genomic sequence. Bacillus cereus ATCC 14579 contains three prsA genes (genomic accession number AE016877), so it is not obvious which PrsA foldase would be the best combination for improving expression of an amylase from Bacillus cereus.

[0008] In Bacillus pumilus SAFR-032, a prsA gene is encoded on the chromosome (Genbank accession number CP000813.4, Stepanov VG, Tirumalai MR, Montazari S, Checinska A, Venkateswaran K, Fox GE. Bacillus pumilus SAFR-032 Genome Revisited: Sequence Update and Re-Annotation. PLoS One. 2016 Jun 28;11(6):e0157331. doi:10.1371 / journal.pone.0157331. PMID:27351589; PMCID:PMC4924849).

[0009] In addition, amylases have been engineered to meet application-related functions, such as improved stability at higher temperatures, in formulations such as detergents, under denaturing conditions such as pH, or stability against proteases, and therefore the sequence of the amylase deviates from the native sequence. The concept of homologous PrsA-amylase combinations of WO2020 / 156903 cannot be applied to chimeric amylases, such as hybrid amylases in which different domains of the protein are derived from different bacterial species.

[0010] However, improving the aforementioned product properties with engineered polypeptide variants is not necessarily accompanied by optimal expression of such non-natural proteins.

[0011] Thus, there remains a need for host systems that result in the overall enhanced production of polypeptides of interest, particularly amylases. Summary of the Invention

[0012] Advantageously, in studies underlying the present invention, it was discovered that Bacillus licheniformis host cells expressing the prsA foldase from Bacillus pumilus (SEQ ID NO: 1, also referred to herein as "peptidyl-prolyl cis-trans isomerase") resulted in increased production of a polypeptide of interest compared to control cells. Specifically, it was shown that expression of an amylase or variant thereof having an amino acid sequence as set forth in SEQ ID NO: 29, 35, 38, 40, 42, 48, 50, 53, 59, or 61 can be increased by co-expressing the PrsA foldase from Bacillus pumilus in Bacillus licheniformis host cells. This effect is surprising because Bacillus pumilus SAFR-032, the organism from which the prsA foldase is derived, does not naturally contain an amylase. Notably, the increased production of the Bacillus pumilus PrsA foldase was observed only in Bacillus licheniformis cells, and not in Bacillus subtilis cells.

[0013] Therefore, the present invention relates to a Bacillus licheniformis host cell expressing

[0014] a) a first polypeptide having peptidyl-prolyl cis-trans isomerase (EC 5.2.1.8) activity, the first polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence at least 81% identical to SEQ ID NO: 1, and

[0015] b) a second polypeptide having amylase activity, wherein the second polypeptide is heterologous to the Bacillus licheniformis host cell.

[0016] The present invention also relates to a method for producing a polypeptide having amylase activity, the method comprising

[0017] a) providing a Bacillus licheniformis host cell of the present invention, and

[0018] b) cultivating the Bacillus licheniformis host cell under conditions allowing expression of the polypeptide having amylase activity, and optionally,

[0019] c) obtaining or purifying the polypeptide having amylase activity.

[0020] The present invention also relates to a method for producing the Bacillus licheniformis host cell of the present invention, the method comprising

[0021] a) providing a Bacillus licheniformis host cell, and

[0022] b) introducing the following into the host cell provided in step a)

[0023] b1) a first polynucleotide encoding a first polypeptide having peptidyl-prolyl cis-trans isomerase (EC 5.2.1.8) activity, the first polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence that is at least 81% identical to SEQ ID NO: 1, and

[0024] b2) a second polynucleotide encoding a second polypeptide having amylase activity.

[0025] The present invention also relates to the following uses:

[0026] i) a first polypeptide having peptidyl-prolyl cis-trans isomerase (EC 5.2.1.8) activity, the first polypeptide comprising the amino acid sequence as shown in SEQ ID NO: 1, or an amino acid sequence at least 81% identical to SEQ ID NO: 1, and / or

[0027] ii) a polynucleotide encoding the first polypeptide,

[0028] Used to increase the production of a second polypeptide having alpha-amylase activity in a Bacillus licheniformis host cell.

[0029] Furthermore, the present invention relates to the use of the Bacillus licheniformis host cell of the invention for producing a second polypeptide, such as a polypeptide having alpha amylase activity.

[0030] In one embodiment of the host cells, methods and uses of the invention, the first polypeptide comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% or at least 100% identical to SEQ ID NO: 1.

[0031] In a preferred embodiment of the host cells, methods and uses of the invention, the second polypeptide has alpha amylase activity (EC 3.2.1.1).

[0032] In another preferred embodiment of the host cells, methods and uses of the invention, the second polypeptide has maltogenic alpha-amylase activity (EC 3.2.1.133).

[0033] In a preferred embodiment of the host cells, methods and uses of the invention, the second polypeptide comprises an amino acid sequence that is at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% identical to SEQ ID NO: 29, 35, 38, 40, 42, 48, 50, 53, 59 or 61.

[0034] In a preferred embodiment of the host cells, methods and uses of the present invention, the second polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 29, 35, 38, 40, 42, 48, 50, 53, 59 or 61.

[0035] In a preferred embodiment of the host cells, methods and uses of the present invention, the second polypeptide, e.g., an amylase, is secreted. Thus, the second polypeptide is a secreted polypeptide. Thus, the second polypeptide typically comprises a signal peptide, preferably at the N-terminus.

[0036] In a preferred embodiment of the host cells, methods and uses of the present invention, the host cell comprises:

[0037] a) a first expression cassette for said first polypeptide, said first expression cassette comprising a first promoter operably linked to a first polynucleotide encoding said first polypeptide, and optionally a terminator, and

[0038] b) a second expression cassette for a second polypeptide, said second expression cassette comprising a second promoter operably linked to a second polynucleotide encoding said second polypeptide, and optionally a terminator.

[0039] In a preferred embodiment of the host cells, methods and uses of the present invention, the first promoter and / or the second promoter is an inducer-independent promoter, such as a constitutive promoter, for example, the promoter of the aprE gene of Bacillus, such as the promoter of the aprE gene of Bacillus licheniformis. In one embodiment, the promoter comprises the nucleic acid sequence as shown in SEQ ID NO: 3, or a variant of said promoter having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 3.

[0040] In one embodiment, the first promoter and the second promoter are both inducer-independent promoters, such as constitutive promoters.

[0041] In another preferred embodiment of the host cells, methods and uses of the invention, the first promoter and / or the second promoter is an inducible promoter.

[0042] In one embodiment of the host cells, methods and uses of the invention, the first expression cassette and / or the second expression cassette is present on a plasmid in the host cell or is stably integrated into the chromosomal DNA of the host cell.

[0043] Typically, the host cell is from a strain of Bacillus licheniformis selected from the group consisting of Bacillus licheniformis strains ATCC 14580, ATCC 31972, ATCC 53757, ATCC 53926, ATCC 55768, DSM 13, DSM 394, DSM 641, DSM 1913, DSM 11259, and DSM 26543. DETAILED DESCRIPTION

[0044] It should be understood that "a" or "an" as used in the specification and claims may mean one or more, depending on the context in which it is used. Thus, for example, reference to "a cell" may mean that at least one cell can be utilized.

[0045] Furthermore, it should be understood that the term "at least one" as used herein means that one or more of the items mentioned after the term can be used according to the present invention. For example, if the term indicates that at least one feed solution should be used, this can be understood to mean one feed solution or more than one feed solution, i.e., two, three, four, five, or any other number of feed solutions. Depending on the item to which the term refers, the skilled artisan understands the upper limit (if any) to which the term may refer.

[0046] As used herein, the term "about" means that with respect to any number cited after the term, there is an interval of accuracy within which a technical effect can be achieved. Thus, as referred to herein, about preferably refers to the exact numerical value or a range around the exact numerical value of ±20%, preferably ±15%, more preferably ±10%, even more preferably ±5%.

[0047] As used herein, the term "comprising" should not be construed as limiting. Rather, the term indicates that there may be more than the actual items being referred to; for example, if the term refers to a method that includes certain steps, the presence of additional steps should not be excluded. However, the term "comprising" also encompasses embodiments in which only the items being referred to are present, i.e., the term merely has the limiting meaning of "consisting of."

[0048] The terms "polynucleotide," "nucleic acid sequence," "nucleotide sequence," "nucleic acid," and "nucleic acid molecule" are used interchangeably herein and refer to a polymeric linear chain of nucleotides, typically deoxynucleotides, of any length. The terms "polypeptide" and "protein" are used interchangeably herein and refer to a polymeric chain of amino acids linked together by peptide bonds of any length.

[0049] The terms "encode for" and "encode" are used interchangeably herein. Generally, the terms refer to the property of a specific nucleotide sequence in a polynucleotide (such as a gene, cDNA, or mRNA) to serve as a template for the synthesis of other macromolecules (such as a defined amino acid sequence). Thus, a gene encodes a protein if transcription and translation of the mRNA corresponding to the gene produces the protein in a cell or other biological system.

[0050] According to the present invention, the first polypeptide and the second polypeptide as defined elsewhere herein, and variants thereof, shall be expressed in a host cell.

[0051] Variants of a parent molecule can have an amino acid sequence that is at least n percent identical to the amino acid sequence of a corresponding parent enzyme having enzymatic activity, wherein n is an integer between 50 and 100, preferably 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99, compared to the full-length polypeptide sequence. The n percent identical variant enzymes described herein have enzymatic activity when compared to the parent enzyme.

[0052] In some embodiments, a variant of a parent polypeptide comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%, but less than 100% identical to the amino acid sequence of the parent polypeptide.

[0053] Therefore, variants can be defined by their sequence identity when compared with the parent enzyme.Sequence identity is usually provided with " sequence identity % " or " identity % ".In order to determine the percent identity between two amino acid sequences in the first step, a paired sequence alignment is generated between the two sequences, wherein the two sequences are compared (that is, paired global alignment) over their full length. This alignment is generated by a program implementing Needleman and Wunsch algorithm (J.Mol.Biol. (1979) 48, 443-453 pages), preferably by using the program " NEEDLE " (European Molecular Biology Open Software Suite (EMBOSS)) with program default parameters (gap opening=10.0, gap extension=0.5 and matrix=EBLOSUM62). For purposes of the present invention, a preferred alignment is an alignment from which the highest sequence identity can be determined.

[0054] After the two sequences have been compared, in a second step, an identity value should be determined based on the comparison. Thus, according to the present invention, the following calculation of percent identity applies:

[0055] % identity = (identical residues / length of the aligned region showing the corresponding sequences of the invention over its entire length) * 100. Thus, the sequence identity relevant to the comparison of two amino acid sequences according to this embodiment is calculated by dividing the number of identical residues by the length of the aligned region showing the corresponding sequences of the invention over its entire length. This value is multiplied by 100 to obtain "% identity".

[0056] In order to calculate the percent identity of two DNA sequences, be equally applicable to calculating the percent identity of two amino acid sequences with some specifications.For the DNA sequence of coded proteins, pairwise comparison should be carried out on the complete length of coding region from start codon to stop codon (not including introns).For non-protein coding DNA sequence, pairwise comparison should be carried out on the complete length of sequence of the present invention, therefore the zone outside full sequence of the present invention and another sequence or another sequence is compared.In addition, the preferred comparison program implementing Needleman and Wunsch algorithm (J.Mol.Biol. (1979) 48, 443-453 pages) is " NEEDLE " (European Molecular Biology Open Software Suite (EMBOSS)) with program default parameters (gap open=10.0, gap extension=0.5 and matrix=EDNAFULL).

[0057] Variant polypeptides can also be defined by their sequence similarity when compared to another sequence. Sequence similarity is usually provided as "% sequence similarity" or "% similarity". In order to calculate sequence similarity in a first step, a sequence alignment must be generated as described above. In a second step, percent similarity must be calculated, and percent sequence similarity takes into account that the defined amino acid groups share similar properties, for example, by their size, by their hydrophobicity, by their charge or by other characteristics. In this article, an amino acid exchanged by a similar amino acid is referred to as a "conservative mutation". Enzyme variants comprising conservative mutations appear to have minimal impact on protein folding, resulting in certain enzyme properties being maintained substantially compared to the enzyme properties of the parent enzyme.

[0058] For the determination of % similarity according to the present invention, the following applies, which also corresponds to the BLOSUM62 matrix, which is one of the most commonly used amino acid similarity matrices for database searching and sequence alignment:

[0059] Amino acid A is similar to amino acid S Amino acid D is similar to amino acid E; N Amino acid E is similar to amino acids D; K; Q Amino acid F is similar to amino acid W; Y Amino acid H is similar to amino acid N; Y Amino acid I is similar to amino acids L; M; V Amino acid K is similar to amino acids E; Q; R Amino acid L is similar to amino acids I; M; V Amino acid M is similar to amino acids I; L; V Amino acid N is similar to amino acids D; H; S Amino acid Q is similar to amino acids E; K; R Amino acid R is similar to amino acid K; Q Amino acid S is similar to amino acids A; N; T Amino acid T is similar to amino acid S Amino acid V is similar to amino acid I; L; M Amino acid W is similar to amino acid F; Y Amino acid Y is similar to amino acids F; H; W.

[0060] Conservative amino acid substitutions can occur in the full-length sequence of the polypeptide sequence of a functional protein (such as an enzyme). In one embodiment, such mutations do not belong to the functional domain of the enzyme. In another embodiment, conservative mutations do not belong to the catalytic center of the enzyme.

[0061] Therefore, the following calculation of percentage similarity applies:

[0062] % similarity = [(identical residues + similar residues) / length of the aligned region showing the corresponding sequences of the invention over their full length] * 100. Thus, sequence similarity relevant to a comparison of two amino acid sequences herein is calculated by adding the number of identical residues to the number of similar residues divided by the length of the aligned region showing the corresponding sequences of the invention over their full length. This value is multiplied by 100 to obtain "% similarity."

[0063] In particular, variant enzymes comprising conservative mutations having at least m percent similarity to the corresponding parent sequence, compared to the full-length polypeptide sequence, wherein m is an integer from 50 to 100, preferably 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99, are expected to have substantially unchanged enzymatic properties.

[0064] Variants of a parent polypeptide may have the activity or function of the parent enzyme. Thus, variants of amylases should have amylase activity, typically within the same EC class. Thus, when compared to the parent enzyme, variant enzymes described herein having m percent similarity have enzymatic activity.

[0065] A "variant" enzyme differs from a "parent" enzyme by certain amino acid changes, preferably amino acid substitutions at one or more amino acid positions.

[0066] When describing polypeptide variants, single amino acid abbreviations are used according to the generally accepted IUPAC single-letter or three-letter amino acid abbreviations.

[0067] As used herein, "amino acid change" refers to an amino acid substitution, deletion, or insertion.

[0068] "Substitutions" are described by providing the original amino acid, followed by the position number within the amino acid sequence, followed by the amino acid that replaces the original amino acid. For example, a substitution of histidine at position 120 with alanine is referred to as "His120Ala" or "H120A." A substitution may also be described by naming only the resulting amino acid in the variant without specifying the parent amino acid at that position, for example, "X120A" or "120A" or "Xaa120Ala" or "120Ala."

[0069] "Deletions" are described by providing the original amino acid, followed by the position number within the amino acid sequence, followed by an *. Thus, a deletion of glycine at position 150 is designated as "Gly150*" or "G150*." Alternatively, a deletion is indicated by, for example, "deletion of D183 and G184."

[0070] An "insertion" is described by providing the original amino acid, followed by the position number within the amino acid sequence, followed by the original amino acid and the additional amino acids. For example, the insertion of a lysine immediately adjacent to a glycine at position 180 is designated "Gly180GlyLys" or "G180GK". When more than one amino acid residue is inserted, such as, for example, the insertion of a Lys and an Ala after Gly180, this can be designated as: "Gly180GlyLysAla" or "G195GKA".

[0071] Where the substitution and insertion occur at the same position, this can be expressed as "S99SD+S99A" or simply "S99AD". Variants comprising multiple changes are separated by "+", for example, "Arg170Tyr+Gly195Glu", "R170Y+G195E" or "X170Y+X195E" indicate that the arginine and glycine at positions 170 and 195 are replaced by tyrosine and glutamic acid, respectively. Alternatively, multiple changes can be separated by spaces or commas, for example, "R170Y G195E" or "R170Y, G195E", respectively. When different alternative changes can be introduced at one position, the different changes are separated by commas, for example, "Arg1 70Tyr, Glu" and "R170T, E" indicate that the arginine at position 170 is replaced by tyrosine or glutamic acid, respectively. Alternative substitutions at specific positions can also be indicated as "X120A, G, H", "120A, G, H", "X120A / G / H" or "120A / G / H". Alternatively, different changes or optional substitutions can be indicated in brackets, for example, "Arg170[Tyr, Gly]" or "Arg170{Tyr, Gly}" or simply "R170[Y, G]" or "R170{Y, G}".

[0072] host cells

[0073] The Bacillus licheniformis host cell of the present invention comprises

[0074] a) a first polypeptide having peptidyl-prolyl cis-trans isomerase (EC 5.2.1.8) activity as defined elsewhere herein, and

[0075] b) a second polypeptide heterologous to the Bacillus licheniformis host cell, such as an amylase.

[0076] Therefore, the Bacillus licheniformis host cell of the present invention preferably comprises

[0077] a) a first polynucleotide encoding a first polypeptide having peptidyl-prolyl cis-trans isomerase (EC 5.2.1.8) activity, and

[0078] b) a second polynucleotide encoding a second polypeptide heterologous to the Bacillus licheniformis host cell, such as an amylase.

[0079] Preferably, the first and second polynucleotides are comprised by an expression cassette. Thus, the Bacillus licheniformis host cell of the present invention comprises

[0080] a) a first expression cassette for said first polypeptide, said first expression cassette comprising a first promoter operably linked to a first polynucleotide encoding said first polypeptide, and optionally a terminator, and

[0081] b) a second expression cassette for the second polypeptide, the second expression cassette comprising a second promoter operably linked to a second polynucleotide encoding the second polypeptide, and optionally a terminator.

[0082] The term "host cell" according to the present invention is a Bacillus licheniformis host cell. Preferably, the Bacillus licheniformis host cell belongs to Bacillus licheniformis strain ATCC 14580, ATCC 31972, ATCC 53757, ATCC 53926, ATCC 55768, DSM 13, DSM 394, DSM 641, DSM 1913, DSM 11259 or DSM 26543. In one embodiment, the host cell belongs to a Bacillus licheniformis strain, such as a host cell of the Bacillus licheniformis strain deposited under American Type Culture Collection No. ATCC 14580 (same as DSM 13, see Veith et al. "The complete genome sequence of Bacillus licheniformis DSM 13, an organism with great industrial potential." J. Mol. Microbiol. Biotechnol. (2004) 7: 204-211). Alternatively, the host cell is a host cell of Bacillus licheniformis strain ATCC31972. Alternatively, the host cell is a host cell of Bacillus licheniformis strain ATCC53757. Alternatively, the host cell is a host cell of Bacillus licheniformis strain ATCC53926. Alternatively, the host cell is a host cell of Bacillus licheniformis strain ATCC55768. Alternatively, the host cell is a host cell of Bacillus licheniformis strain DSM394. Alternatively, the host cell is a host cell of Bacillus licheniformis strain DSM641. Alternatively, the host cell is a host cell of Bacillus licheniformis strain DSM1913. Alternatively, the host cell is a host cell of Bacillus licheniformis strain DSM11259. Alternatively, the host cell is a host cell of Bacillus licheniformis strain DSM26543.

[0083] In addition, it is envisioned that the host cell of the modification as described herein does not produce poly-gamma-glutamic acid (pga) or produces the pga of reduced amount. Therefore, at least one gene related to poly-gamma-glutamic acid (pga) production has been inactivated (such as deletion). Preferably, at least one gene related to poly-gamma-glutamic acid (pga) is at least one gene selected from ywsC (pgsB), ywtA (pgsC), ywtB (pgsA) and ywtC (pgsE). Preferably, all the aforementioned genes, i.e. ywsC (pgsB), ywtA (pgsC), ywtB (pgsA) and ywtC (pgsE) have been inactivated (such as deletion).

[0084] In addition, it is envisioned that the modified host cell cannot form spores. This can be achieved by inactivating (such as deleting) at least one gene involved in spore formation. Genes involved in spore formation are well known in the art (EP 1391502) and include but are not limited to sigE, sigF, spoIIGA, spoIIE, sigG, spoIVCB, yqfD. In a preferred embodiment, the sigF gene is deleted.

[0085] In addition, it is envisioned that the modified host cells have reduced proteolytic activity (compared to control cells). This can be achieved by inactivating (such as deleting) at least one protease encoding gene, including but not limited to aprE, mpr, bpr, vpr, epr, wprA, ispA, aprX. Preferably, the aprE and mpr genes are deleted, most preferably the aprE gene is deleted.

[0086] In addition, it is envisioned that the modified host cell has reduced glycosidase activity. This can be achieved by inactivating (such as deleting) at least one gene encoding a glycosidase, including but not limited to α-amylase (EC 3.2.1.1), β-amylase (EC 3.2.1.2), and glucan 1,4-α-maltohydrolase (EC 3.2.1.133), cellulase (EC 3.2.1.4), endo-1,3-β-xylanase xylanase (EC 3.2.1.32), endo-1,4-β-xylanase (EC 3.2.1.8), lactase (EC 3.2.1.108), galactosidase (EC 3.2.1.23 and EC 3.2.1.24), mannanase (EC 3.2.1.24 and EC 3.2.1.25).

[0087] In a preferred embodiment, the gene encoding the endogenous α-amylase polypeptide (as shown in SEQ ID NO: 35) is deleted.

[0088] First polypeptide (peptidyl-prolyl cis-trans isomerase)

[0089] As referred to herein, the first polypeptide has peptidyl-prolyl cis-trans isomerase activity (EC 5.2.1.8). Thus, the first polypeptide is a peptidyl-prolyl cis-trans isomerase (also commonly referred to as a "foldase", "peptidyl-prolyl isomerase", "peptide bond isomerase" or "PPIase"). The terms "foldase", "peptidyl-prolyl cis-trans isomerase" and "PrsA" are used interchangeably herein.

[0090] As used herein, the term "peptidyl-prolyl cis-trans isomerase" refers to an enzyme that interconverts the cis- and trans-isomers of a peptide bond with the amino acid proline. Thus, it interconverts the cis- and trans-isomers of peptidyl-prolyl bonds within proteins. In Bacillus, peptidyl-prolyl cis-trans isomerases are membrane-bound lipoproteins that are thought to assist in the post-translocation folding of secretory proteins and stabilize them in the compartment between the cytoplasmic membrane and the cell wall.

[0091] Typically, the active form of the enzyme is a dimer of two monomers, i.e., a dimer formed by two monomers of the first polypeptide. Therefore, it will be understood by those skilled in the art that the dimer has peptidyl-prolyl cis-trans isomerase activity. Typically, peptidyl-prolyl cis-trans isomerase has two domains, a peptidyl-prolyl cis-trans isomerase (EC 5.2.1.8) domain and a chaperone domain that supports protein folding.

[0092] According to the present invention, the first polypeptide is a PrsA protein from Bacillus pumilus or a variant thereof (SEQ ID NO: 1). Preferably, the first polypeptide comprises the amino acid sequence as shown in SEQ ID NO: 1, or an amino acid sequence that is at least 81% identical to SEQ ID NO: 1. More preferably, the first polypeptide comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% identical to SEQ ID NO: 1. In addition, the first polypeptide may comprise or consist of the amino acid sequence as shown in SEQ ID NO: 1.

[0093] Furthermore, the first polypeptide (ie, the dimer formed by two monomers of the first polypeptide) preferably has peptidyl-prolyl cis-trans isomerase activity. Whether a polypeptide has this activity can be assessed by well-known assays, for example, by assays as described in Jakob et al., 2009 (Proc Natl Acad Sci US A. 2009 Dec 1; 106(48): 20282-7. doi: 10.1073 / pnas.0909544106. Electronically published on Nov 17, 2009. PMID: 19920179; PMCID: PMC2787138) and Jakob. 2014 (J Biol Chem. 2015 Feb 6; 290(6): 3278-92. doi: 10.1074 / jbc.M114.622910. Electronically published on Dec 17, 2014. PMID: 25525259; PMCID: PMC4319002).

[0094] Also preferably, the first polypeptide is capable of being transported and bound to the cell membrane of the host cell.Thus, the first polypeptide comprises a suitable signal peptide, such as the signal peptide of a natural enzyme.

[0095] Thus, the first polypeptide is associated with the cell membrane of the host cell, ie, it is present in the host cell as a membrane-bound polypeptide.

[0096] In one embodiment, the polypeptide variant has at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or 98% of the peptidyl-prolyl cis-trans isomerase activity of the parent polypeptide (i.e., the polypeptide having the sequence shown in SEQ ID NO: 2).

[0097] According to the present invention, a first polypeptide assists in the folding of a second polypeptide (eg, an amylase) that is co-expressed with the first polypeptide in a host.

[0098] In one embodiment of the present invention, the host cell expresses an endogenous PrsA polypeptide, namely, a Bacillus licheniformis PrsA polypeptide. The amino acid sequence of the Bacillus licheniformis PrsA polypeptide is shown in SED ID NO:12.

[0099] In an alternative embodiment of the present invention, the host cell does not express endogenous PrsA polypeptide. Therefore, the endogenous prsA gene is deleted. The amino acid sequence of the endogenous prsA gene is shown in SED ID NO: 13.

[0100] Second polypeptide ("polypeptide of interest")

[0101] The second polypeptide is also referred to herein as a "polypeptide of interest." These terms are used interchangeably herein.

[0102] Preferably, the second polypeptide is an amylase, ie a polypeptide having amylase activity. The amylase may be a naturally occurring amylase or a non-naturally occurring amylase.

[0103] As used herein, the term "amylase" generally refers to an enzyme having "amylolytic activity" or "amylase activity." "Amylolytic activity" or "amylase activity" describes the ability to hydrolyze glycosidic bonds in polysaccharides. Amylase activity can be determined by assays known to those skilled in the art for measuring amylase activity. Examples of assays for measuring amylase activity are the Phadebas assay or the EPS assay ("Infinity reagent"). In the Phadebas assay, amylase activity is determined by using Phadebas tablets as a substrate (Phadebas amylase test, provided by Magle Life Science). Starch is hydrolyzed by the amylase, producing soluble blue fragments. The absorbance of the resulting blue solution, measured spectrophotometrically at 620 nm, is a function of the amylase activity. The measured absorbance is directly proportional to the specific activity (activity / mg of pure amylase protein) of the amylase in question under the given conditions.

[0104] Alternatively, amylase activity can also be determined by using a method using ethylidene-4-nitrophenyl-α-D-maltoheptaglycoside (EPS). D-maltoheptaglycoside is a closed oligosaccharide that can be cleaved by endoamylase. After cleavage, the α-glucosidase enzyme included in the test kit digests the substrate to release free PNP molecules, which have a yellow color and can therefore be measured by visible spectrophotometry at 405nm. A test kit containing EPS substrate and α-glucosidase is, for example, manufactured by Roche Costum Biotech (catalog number 10880078103). The slope of the time-dependent absorption curve is directly proportional to the specific activity (activity per mg of enzyme) of the amylase under given conditions.

[0105] Typically, an amylase as referred to herein is an alpha amylase (EC 3.2.1.1), a beta amylase (EC 3.2.1.2) or a maltogenic alpha amylase (EC 3.2.1.133).

[0106] In a preferred embodiment, the amylase is an α-amylase (EC 3.2.1.1). α-Amylases are enzymes that catalyze the endohydrolysis of (1→4)-α-D-glucosidic bonds in polysaccharides containing three or more (1→4)-α-linked D-glucose units. The enzyme acts on, for example, starch or glycogen in a random manner, releasing reducing groups in the α-configuration, i.e., the initial anomeric configuration of the released free glycosyl groups. Other names are glycogenase, endoamylase, 4-α-D-glucan glucanohydrolase, and 1,4-α-D-glucan glucanohydrolase. The systematic name is "4-α-D-glucan glucanohydrolase." For example, polypeptides having the amino acid sequences shown in SEQ ID NOs: 29, 35, 38, 40, 42, 48, 50, 59, and 61, respectively, have α-amylase activity (EC 3.2.1.1). Variants of these amylases should also have α-amylase activity.

[0107] In another preferred embodiment, the amylase is a β-amylase (EC 3.2.1.2). β-amylases are enzymes that catalyze the hydrolysis of (1->4)-α-D-glucosidic bonds in polysaccharides to remove consecutive maltose units from the non-reducing end of the chain. The enzyme acts on, for example, starch or glycogen by conversion, thereby producing β-maltose. Other names are glycogen amylase, glycogenase, β-amylase, or 1,4-α-D-glucan maltohydrolase. The systematic name is "1,4-α-D-glucan maltohydrolase."

[0108] In another preferred embodiment, the amylase is a maltogenic α-amylase (EC 3.2.1.133). Maltogenic α-amylase (also referred to herein as "glucan 1,4-α-maltohydrolase") is an enzyme that catalyzes the hydrolysis of (1→4)-α-D-glucosidic bonds in polysaccharides to remove consecutive α-maltose residues from the non-reducing end of the chain. The enzyme acts on, for example, starch and related polysaccharides and oligosaccharides. The product is α-maltose. Other names are glucan 1,4-α-maltohydrolase or 1,4-α-D-glucan α-maltohydrolase. The systematic name is "1,4-α-D-glucan α-maltohydrolase". For example, a polypeptide having the amino acid sequence shown in SEQ ID NO: 53 has maltogenic α-amylase activity (EC 3.2.1.133). Variants of this amylase should also have maltogenic α-amylase activity.

[0109] In a preferred embodiment, the amylase as referred to herein comprises an amino acid sequence that is at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% identical to the amino acid sequence shown in SEQ ID NO: 29, 35, 38, 40, 42, 48, 50, 53, 59 or 61.

[0110] For example, an amylase as referred to herein comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% identical to the amino acid sequence shown in SEQ ID NO: 29, 35, 38, 40, 42, 48, 50, 53, 59 or 61.

[0111] For example, an amylase as referred to herein comprises an amino acid sequence as shown in SEQ ID NO: 29, 35, 38, 40, 42, 48, 50, 53, 59 or 61.

[0112] In one embodiment, an amylase as referred to herein comprises an amino acid sequence that is at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% or 100% identical to SEQ ID NO: 29.

[0113] In another embodiment, an amylase as referred to herein comprises an amino acid sequence that is at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% identical to SEQ ID NO: 35. In some embodiments, the amylase has less than 99% sequence identity to SEQ ID NO: 35, such as less than 98% or less than 97% sequence identity.

[0114] In another embodiment, an amylase as referred to herein comprises an amino acid sequence that is at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO: 38.

[0115] In another embodiment, an amylase as referred to herein comprises an amino acid sequence that is at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% or 100% identical to SEQ ID NO: 40.

[0116] In another embodiment, an amylase as referred to herein comprises an amino acid sequence that is at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% or 100% identical to SEQ ID NO: 42.

[0117] In another embodiment, an amylase as referred to herein comprises an amino acid sequence that is at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% or 100% identical to SEQ ID NO: 48.

[0118] In another embodiment, an amylase as referred to herein comprises an amino acid sequence that is at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% or 100% identical to SEQ ID NO: 50.

[0119] In another embodiment, an amylase as referred to herein comprises an amino acid sequence that is at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% or 100% identical to SEQ ID NO: 59.

[0120] In another embodiment, an amylase as referred to herein comprises an amino acid sequence that is at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% or 100% identical to SEQ ID NO: 61.

[0121] In another embodiment, an amylase as referred to herein comprises an amino acid sequence that is at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% identical to SEQ ID NO: 53.

[0122] As described above, the amylase may comprise the amino acid sequence shown in SEQ ID NO: 35 (or a variant thereof). The amylase having SEQ ID NO: 35 is an amylase from Bacillus licheniformis. This amylase has been described in WO 95 / 10603 (e.g., SEQ ID NO: 2). Suitable variants that can be used in the context of the present invention are described in WO 95 / 10603, comprising one or more substitutions at the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 178, 179, 181, 188, 190, 197, 201, 202, 207, 208, 209, 211, 243, 264, 304, 305, 391, 408, and 444, which variants have amylolytic activity. Variants are described in SEQ ID NO: 4 of WO 94 / 02597, WO 94 / 018314, WO 97 / 043424 and WO 99 / 019467.

[0123] As mentioned above, the amylase may comprise the amino acid sequence shown in SEQ ID NO: 59 (or a variant thereof). The amylase with SEQ ID NO: 59 is an amylase from Bacillus halmapalus, also known as "SP-722 amylase". In WO 96 / 23872, this amylase is also described as SEQ ID NO: 2 or SEQ ID NO: 7. Preferred variants that can be used in the context of the present invention are described in WO 97 / 3296, WO 99 / 194671 and WO 2013 / 001078.

[0124] As described above, the amylase may comprise the amino acid sequence shown in SEQ ID NO: 38 (or a variant thereof). The amylase having SEQ ID NO: 38 is an amylase from Bacillus sp. A 7-7 (DSM 12368). In one embodiment, the amylase comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 2, particularly in the region from amino acids 32 to 516 of SEQ ID NO: 2 as disclosed in WO 02 / 10356. SEQ ID NO: 2 as disclosed in WO 02 / 10356 is identical to SEQ ID NO: 38 of the present invention.

[0125] As described above, the amylase may comprise the amino acid sequence shown in SEQ ID NO: 48 (or a variant thereof). The amylase having SEQ ID NO: 48 is an amylase from Bacillus sp. strain TS-23 having SEQ ID NO: 48 of the present invention or having SEQ ID NO: 2 and variants thereof as disclosed in WO 2009 / 061380.

[0126] As described above, the amylase may comprise the amino acid sequence shown in SEQ ID NO: 40 (or a variant thereof). An amylase having SEQ ID NO: 40 (sometimes also referred to as a "dynamase") is an amylase from a Bacillus species having SEQ ID NO: 40 of the present invention or comprising amino acids 1 to 485 of SEQ ID NO: 2 as described in WO 00 / 60060 and at least 95% variants thereof.

[0127] In a preferred embodiment of the present invention the amylase is a hybrid amylase.

[0128] As described above, the amylase may be a hybrid amylase as described in WO 2006 / 066594. For example, the hybrid amylase may comprise the amino acid sequence as set forth in SEQ ID NO: 61 (or a variant thereof), or may be according to WO 2014 / 183920, wherein the A and B domains are at least 90% identical to SEQ ID NO: 2 of WO 2014 / 183920, and the C domain is at least 90% identical to SEQ ID NO: 6 of WO 2014 / 183920, wherein the hybrid amylase has amylolytic activity; preferably, the hybrid α-amylase is at least 95% identical to SEQ ID NO: 23 of WO 2014 / 183920 and has amylolytic activity. SEQ ID NO: 61 of the present invention is 99.4% identical to SEQ ID NO: 23 of WO 2014 / 183920.

[0129] The hybrid amylase may be according to WO 2014 / 183921, wherein the A and B domains are at least 75% identical to SEQ ID NO: 2, SEQ ID NO: 15, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 26, SEQ ID NO: 32 and SEQ ID NO: 39 as disclosed in WO 2014 / 183921, and the C domain is at least 90% identical to SEQ ID NO: 6 of WO 2014 / 183921, wherein the hybrid amylase has amylolytic activity; preferably, the hybrid alpha-amylase comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 6 of WO 2014 / 183921.

[0130] As described above, the amylase may be a hybrid amylase as disclosed in WO 2021 / 032881 (incorporated herein by reference), comprising the A and B domains of an alpha-amylase derived from Bacillus sp. A 7-7 (DSM 12368) and the C domain of an alpha-amylase derived from Bacillus cereus; preferably, the A and B domains are at least 75% identical to the amino acid sequence of SEQ ID NO: 42, and the C domain is at least 75% identical to the amino acid sequence of SEQ ID NO: 44, both sequences being as disclosed in WO 2021 / 032881; more preferably, the hybrid amylase is at least 80% identical to SEQ ID NO: 54 as disclosed in WO 2021 / 032881. SEQ ID NO: 54 of WO 2021 / 032881 corresponds to SEQ ID NO: 29 of the present application.

[0131] According to the present invention, the amylase is preferably a variant of the amylase having the sequence shown in SEQ ID NO: 29, such as the amylase designated Amy031 or Amy033 (see Examples).

[0132] In a preferred embodiment, the amylase variant has the following substitutions compared to the amylase comprising the amino acid sequence shown in SEQ ID NO: 29 (generally using the numbering of SEQ ID NO: 30): G4Q, N25H, R176K, G186E, T251E, L405M and Y482W.

[0133] In another preferred embodiment, the amylase variant has the following substitutions compared to the amylase comprising the amino acid sequence shown in SEQ ID NO: 29 (generally using the numbering of SEQ ID NO: 30): N25H, W116K, R176K, R181T, G186E, N195F, T225A, R320K and Y482W.

[0134] As described above, variants of a parent amylase as referred to herein (i.e., SEQ ID NO: 29, 35, 38, 40, 42, 48, 50, 53, 59, or 61) should have amylase activity. Furthermore, it is contemplated that production of the variant by the host cells of the present invention is increased compared to production of the variant by a control cell expressing the first polypeptide and the parent amylase. Thus, production of the variant amylase should be increased compared to production of the parent amylase.

[0135] The amylase used according to the present invention is not limited to the above-mentioned amylases.

[0136] In some embodiments of the invention, the amylase is an amylase from Geobacillus stearothermophilus comprising the amino acid sequence of SEQ ID NO: 6 as disclosed in WO 02 / 10355, or optionally an amylase having a C-terminal truncation beyond the wild-type sequence. Suitable variants of SEQ ID NO: 6 include those comprising a deletion at position 179 and / or 181 and / or 182 and / or a substitution at position 193.

[0137] In some embodiments of the invention, the amylase is an amylase from Bacillus sp. 707 comprising the amino acid sequence of SEQ ID NO: 6 and at least 95% variants thereof as disclosed in WO 99 / 19467. Preferred variants of SEQ NO: 6 are those having a substitution, deletion or insertion in one or more of the following positions: R181, G182, H183, G184, N195, I206, E212, E216, and K269.

[0138] In some embodiments of the invention, the amylase is an amylase from Bacillus sp. DSM 12649 having SEQ ID NO: 4 as disclosed in WO 00 / 22103 and at least 95% variants thereof.

[0139] In some embodiments of the invention, the amylase is an amylase from Bacillus megaterium DSM 90 having SEQ ID NO: 1 as disclosed in WO 2010 / 104675 and variants thereof of at least 95%.

[0140] In some embodiments of the invention, the amylase is an amylase from Bacillus amyloliquefaciens or a variant thereof, preferably selected from the amylase according to SEQ ID NO: 3 as described in WO 2016 / 092009.

[0141] In some embodiments of the invention, the amylase comprises the amino acid sequence as shown in SEQ ID NO: 12 as described in WO 2006 / 002643 or an amylase variant thereof comprising the substitutions Y295F and M202LITV within said SEQ ID NO: 12.

[0142] In some embodiments of the invention, the amylase comprises the amino acid sequence as shown in SEQ ID NO: 6 as described in WO 2011 / 098531, or amylase variants comprising a substitution at one or more positions selected from the group consisting of: 193 [G, A, S, T or M], 195 [F, W, Y, L, I or V], 197 [F, W, Y, L, I or V], 198 [Q or N], 200 [F, W, Y, L, I or V], 203 [F, W, Y, L, I or V], 206 [F, W, Y, N, L, I, V, H, Q, D or E], 210 [F, W, Y, L, I or V], 212 [F, W, Y, L, I or V], 213 [G, A, S, T or M] and 243 [F, W, Y, L, I or V].

[0143] The amylase may have SEQ ID NO: 1 as described in WO 2013 / 001078, or an amylase variant comprising alterations at two or more (several) positions corresponding to positions G304, W140, W189, D 134, E260, F262, W284, W347, W439, W469, G476, and G477 within said SEQ ID NO: 1.

[0144] In some embodiments of the invention, the amylase comprises an amino acid sequence as shown in SEQ ID NO: 2 as described in WO 2013 / 001087, or an amylase variant comprising a deletion of positions 181+182, or 182+183, or 183+184 within said SEQ ID NO: 2, optionally comprising one or two or more modifications at any of the positions corresponding to W140, W159, W167, Q169, W189, E194, N260, F262, W284, F289, G304, G305, R320, W347, W439, W469, G476, and G477 within said SEQ ID NO: 2.

[0145] In a preferred embodiment of the present invention the amylase is a commercially available amylase including but not limited to amylases available under the trade name Duramyl TM Termamyl TM 、Fungamyl TM 、Stainzyme TM 、Stainzyme Plus TM 、Natalase TM , Liquozyme X and BAN TM 、Amplift TM 、Amplify Prime TM (from Novozymes A / S) and Rapidase TM 、Purastar TM 、Powerase TM 、Effectenz TM (M100 from DuPont), Preferenz TM (S1000DuPont), PrimaGreen TM (ALL; DuPont), Optisize TM Products sold by (DuPont).

[0146] The present invention is not limited to amylases as polypeptides of interest. In some embodiments, the polypeptide of interest (i.e., the second polypeptide) is an enzyme other than amylase, such as an extracellular enzyme (other than amylase). In a specific embodiment, the enzyme is classified as an oxidoreductase (EC 1), a transferase (EC 2), a hydrolase (EC 3), a lyase (EC 4), an isomerase (EC 5), or a ligase (EC 6). In a preferred embodiment, the protein of interest is an enzyme suitable for use in detergents, feeds, and food applications.

[0147] More preferably, the enzyme is a hydrolase (EC 3), preferably a glycosidase (EC 3.2) or a peptidase (EC 3.4). Particularly preferred enzymes are enzymes selected from the group consisting of amylases, cellulases (EC 3.2.1.4), endo-1,3-β-xylanases xylanases (EC 3.2.1.32), endo-1,4-β-xylanases (EC 3.2.1.8), lactases (EC 3.2.1.108), galactosidases (EC 3.2.1.23 and EC 3.2.1.24), mannanases (EC 3.2.1.24 and EC 3.2.1.25), lipases (EC 3.1.1.3), phytases (EC 3.1.3.8), nucleases (EC 3.1.11 to EC 3.1.31) and proteases (EC 3.1.10). 3.4); in particular an enzyme selected from the group consisting of amylase, protease, lipase, mannanase, phytase, xylanase, phosphatase, β-galactosidase, lactase, glucoamylase, nuclease and cellulase, preferably amylase, mannanase, xylanase or protease, preferably protease.

[0148] In some embodiments, the second polypeptide is a xylanase.

[0149] In some embodiments, the second polypeptide is a mannanase.

[0150] Signal peptide - secretion signal for the polypeptide of interest

[0151] As referred to herein, a polypeptide of interest, such as an amylase polypeptide, is secreted, i.e., it is secreted by the Bacillus licheniformis host cells of the present invention. Thus, the polypeptide of interest, preferably an amylase, typically comprises a secretion signal at its N-terminus, i.e., a signal peptide that allows the polypeptide to be secreted from the host cell into the fermentation broth. Typically, the signal peptide is a Sec secretory pathway-specific signal peptide. Thus, the polypeptide of interest is secreted via the Sec pathway. Typically, the signal peptide is present at the N-terminus of the polypeptide of interest.

[0152] The terms "signal peptide," "secretion sequence," and "secretion signal peptide" are used interchangeably herein.

[0153] Signal peptides are well known in the art and can typically be found at the N-terminus of secreted Bacillus proteins, such as AmyB, AmyE, AmyL AmyM, AmyQ, AmyS, AprE, AprH, AspB, BglC, BglS, Bpr, CelA, CelA, Csn, Epr, ForD, GGT, LacZ, LipA, LytB, LytD, Pel, PhoD, PhrK, Vpr, YbdN, YckD, YddT, YfhK, YfjS, YhfM, YjfA, YkwD, YncM, YnfF, YobB, YvcE, or YvfO polypeptides.

[0154] The present invention also encompasses that the signal peptide sequence can be engineered by replacing amino acids or creating chimeric sequences to optimize secretion of the polypeptide of interest (EP2689015B1).

[0155] The signal peptide can also be selected from signal peptides comprising Sec secretory pathway and TAT secretory pathway sequence elements (Freudl, R. Signal peptides for recombinant protein secretion in bacterial expression systems. Microb Cell Fact 17, 52 (2018)), such as but not limited to the signal peptide of WprA, WapA, SpoIIP or YwbN polypeptide.

[0156] Exemplary signal peptides are shown below in Table A. In a preferred embodiment, the polypeptide comprises a signal peptide at the N-terminus as shown in Table A. Thus, the signal peptide preferably comprises or consists of an amino acid sequence selected from SEQ ID NOs: 67 to 109.

[0157] Table A: Exemplary signal peptides from various Bacillus polypeptides

[0158]

[0159]

[0160]

[0161]

[0162] *Used in the Examples section

[0163] In one embodiment, the signal peptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 69, 73 or 107.

[0164] As mentioned above, preferably, the polypeptide carries a functional signal peptide.

[0165] Whether a peptide acts as a secretion signal peptide can be assessed by bioinformatics using the SignalP signal peptide prediction tool (Almagro Armenteros JJ, Nielsen H.; SignalP 5.0 improves signal peptide predictions using deep neural networks. Nat Biotechnol. 2019 Apr; 37(4): 420-423); or by measuring the secretion capacity of a given polypeptide when fused to a potential secretion signal peptide, as previously shown (Brockmeier U, Eggert T. Systematic screening of all signal peptides from Bacillus subtilis: a powerful strategy in optimizing heterologous protein secretion in Gram-positive bacteria. J Mol Biol. 2006 Sep 22; 362(3): 393-402).

[0166] Expression constructs

[0167] The host cell of the present invention preferably comprises a polynucleotide encoding a first polypeptide and a polynucleotide encoding a second polypeptide.

[0168] a) a first expression cassette for said first polypeptide, said first expression cassette comprising a promoter operably linked to a first polynucleotide encoding said first polypeptide, and optionally a terminator, and

[0169] b) a second expression cassette for expressing the second polypeptide, the second expression cassette comprising a promoter operably linked to a second polynucleotide encoding the second polypeptide, and optionally a terminator.

[0170] Preferably, the two polynucleotides, i.e. the polynucleotide encoding the first polypeptide and the polynucleotide encoding the second polypeptide, the polynucleotide encoding at least one polypeptide of interest are heterologous to the host cell. The term "heterologous" (or exogenous or external or recombinant or non-natural) generally refers to a polynucleotide that is not native to the host cell. In some embodiments, a "heterologous" polynucleotide is an additional copy of a gene that is naturally present in the host. The term "heterologous" (or exogenous or external or recombinant or non-natural) polypeptide or protein as used throughout the specification is defined herein as a polypeptide or protein that is not native to the host cell.

[0171] In a preferred embodiment, the first polynucleotide and the second polynucleotide, and therefore the first expression cassette and the second expression cassette, are present on a plasmid. The term "plasmid" refers to an extrachromosomal circular DNA, i.e., a vector that replicates autonomously in a host cell. Therefore, a plasmid is understood to be an extrachromosomal vector.

[0172] In another preferred embodiment, the first polynucleotide and the second polynucleotide, and thus the first expression cassette and the second expression cassette, are stably integrated into the bacterial chromosome.

[0173] promoter

[0174] The first and second polynucleotides should be operably linked to a promoter.

[0175] As used herein, the term "operably linked" refers to a functional linkage between a promoter sequence and a polynucleotide encoding a polypeptide of interest, such that the promoter sequence is able to initiate transcription of the polynucleotide encoding the polypeptide of interest (also referred to herein as a gene of interest).

[0176] A "promoter" or "promoter sequence" is a nucleotide sequence located upstream of a gene, on the same strand as the gene, that enables transcription of the gene. The promoter is followed by the gene's transcription start site. The promoter is recognized by RNA polymerase (and any required transcription factors) to initiate transcription. A functional fragment or functional variant of a promoter is a nucleotide sequence that is recognized by RNA polymerase and capable of initiating transcription.

[0177] "Active promoter fragment," "active promoter variant," "functional promoter fragment," or "functional promoter variant" describes a fragment or variant of a promoter nucleotide sequence that still has promoter activity.

[0178] The promoter can be an "inducer-dependent promoter" or an "inducer-independent promoter," including constitutive promoters or promoters that are controlled by other cellular regulatory factors.

[0179] Those skilled in the art can select appropriate promoters to express the first polynucleotide and the second polynucleotide. For example, the polynucleotide encoding the first polypeptide of interest is preferably operably linked to an "inducer-dependent promoter" or an "inducer-independent promoter." In addition, the polynucleotide encoding the second polypeptide of interest is preferably operably linked to an "inducer-independent promoter" such as a constitutive promoter.

[0180] An "inducer-dependent promoter" is understood herein to be a promoter whose activity increases upon addition of an "inducer molecule" to the fermentation medium to effect transcription of a gene operably linked to the promoter. Thus, for an inducer-dependent promoter, the presence of the inducer molecule triggers, via signal transduction, an increase in expression of the gene operably linked to the promoter. Gene expression need not be absent prior to activation by the inducer molecule, but rather may be present at a low level of baseline gene expression that increases upon addition of the inducer molecule. An "inducer molecule" is a molecule whose presence in the fermentation medium is capable of affecting an increase in gene expression by increasing the activity of the inducer-dependent promoter operably linked to the gene. Preferably, the inducer molecule is a carbohydrate or an analog thereof. In one embodiment, the inducer molecule is a minor carbon source for the Bacillus cell. In the presence of a mixture of carbohydrates, the cell selectively utilizes the carbon source that provides it with the most energy and growth advantage (the major carbon source). At the same time, the inducer molecule inhibits various functions involving the catabolism and uptake of less preferred carbon sources (the minor carbon source). Typically, the primary carbon source for Bacillus is glucose and various other sugars and sugar derivatives that are used as secondary carbon sources by Bacillus. Secondary carbon sources include, but are not limited to, mannose or lactose.

[0181] Examples of inducer-dependent promoters are given in the table below with reference to the corresponding operon:

[0182]

[0183]

[0184] In contrast, the activity of a promoter that is not dependent on the presence of an inducer molecule (referred to herein as an "inducer-independent promoter") is constitutively active, or can be increased regardless of the presence of an inducer molecule added to the fermentation medium.

[0185] Constitutive promoters are independent of other cellular regulatory factors and transcription initiation is dependent on sigma factor A (sigA). The sigA-dependent promoter includes the "-35" region and the "-10" region, which are specific recognition sites for sigma factor A.

[0186] Preferably, the sequence of the inducer-independent promoter is selected from the group consisting of constitutive promoters, which are not limited to promoters Pveg, PlepA, PserA, PymdA, Pfba and their derivatives with different gene expression strengths (Guiziou et al., (2016): Nucleic Acids Res. 44 (15), 7495-7508), the aprE promoter of the subtilisin encoding the aprE gene of Bacillus, phage SPO1 promoters P4, P5, P15 (WO15118126), the cryIIIA promoter from Bacillus thuringiensis (WO 9425612), the amyQ promoter from Bacillus amyloliquefaciens, the amyL promoter and promoter variants from Bacillus licheniformis (US 5698415) and combinations thereof, or active fragments or variants thereof, preferably the aprE promoter sequence.

[0187] "aprE promoter" or "aprE promoter sequence" is a nucleotide sequence (or a portion or variant thereof) located upstream of the aprE gene, i.e., the gene encoding Bacillus subtilisin Carlsberg protease, which is on the same chain as the aprE gene, enabling transcription of the aprE gene.

[0188] In one embodiment of the present invention, the promoter is a promoter of an aprE gene, such as the promoter of the aprE gene of Bacillus licheniformis (which is used in the Examples section). For example, the promoter comprises a nucleic acid sequence as shown in SEQ ID NO: 3 or a nucleic acid sequence that is at least 80%, 85%, 90%, 93%, 95%, 98% or 99% identical to SEQ ID NO: 3.

[0189] For co-expression of the prsA gene in a Bacillus host, the native 5' prsA gene regulatory region of the prsA gene (WO9419471, WO2021146411) and a heterologous promoter (WO2020156903) can be used. Inducible promoters such as the IPTG-inducible promoter Pspac and the xylose-inducible promoter PxylA have been used to titrate the PrsA expression level in cells (Chen J et al. Biotechnol Lett. 2015 Apr; 37(4): 899-906. doi: 10.1007 / s10529-014-1755-3. Electronic publication on December 17, 2014. PMID: 25515799.).

[0190] The term "transcription start site" or "transcriptional start site" is understood to be the location where transcription begins at the 5' end of a gene sequence. In prokaryotes, the first nucleotide, designated +1, is typically an adenosine (A) or guanosine (G) nucleotide. In this context, the terms "site" and "signal" are used interchangeably herein.

[0191] The term "expression" or "gene expression" means the transcription of one or more specific genes or specific nucleic acid constructs. The term "expression" or "gene expression" specifically means the transcription of one or more genes or genetic constructs into structural RNA (e.g., rRNA, tRNA) or mRNA, with or without subsequent translation of the structural RNA or mRNA into protein. This process includes transcription of DNA and processing of the resulting mRNA product.

[0192] Optionally, the promoter includes a 5'UTR. This is a transcribed but untranslated region downstream of the -1 promoter position. For example, this untranslated region should contain a ribosome binding site to promote translation if the target gene encodes a peptide or polypeptide.

[0193] Regarding the 5'UTR, the present invention particularly teaches combining the promoter of the present invention with a 5'UTR comprising one or more stabilizing elements. In this way, mRNA synthesized from the promoter region can be processed to generate mRNA transcripts having a stabilizing sequence at the 5' end of the transcript. Preferably, such a stabilizing sequence at the 5' end of the mRNA transcript increases their half-life, as described by Hue et al., 1995, Journal of Bacteriology 177: 3465-3471. Suitable mRNA stabilizing elements are described in the following literature

[0194] - SEQ ID NOs. 1 to 5 of WO 8148575, preferably WO08140615, or fragments of these sequences that retain the mRNA stabilizing function, and

[0195] - WO08140615, preferably the Bacillus thuringiensis Cryll1A mRNA stabilizing sequence or the bacteriophage SP82 mRNA stabilizing sequence, more preferably the mRNA stabilizing sequence according to SEQ ID NO. 4 or 5 of WO08140615, more preferably the mRNA stabilizing sequence according to SEQ ID NO. 6 of WO08140615, or fragments of these sequences that retain the mRNA stabilizing function.

[0196] The preferred mRNA stabilizing element is selected from the group consisting of prE, grpE, cotG, SP82, RSBgsiB, and Cryll1A mRNA stabilizing elements, or fragments of these sequences that retain mRNA stabilizing function. The preferred mRNA stabilizing element is the grpE mRNA stabilizing element (corresponding to SEQ ID NO. 2 of WO 08148575).

[0197] 5'UTR also preferably includes the rib leader sequence of the modification positioned at promoter downstream and ribosome bind site (RBS) upstream.In the context of the present invention, rib leader sequence is defined as the leader sequence positioned at riboflavin biosynthesis gene (rib operon) upstream in bacillus cell, more preferably bacillus subtilis cell here.In bacillus subtilis, the rib operon including the gene related to riboflavin biosynthesis includes ribG (ribd) gene, ribB (ribE) gene, ribA gene and ribH gene. In bacillus subtilis, the transcription of riboflavin operon is controlled by riboswitch from rib promoter (Prib), and the riboswitch is related to the 5'-region of rib operon, the translation regulation leader region (rib leader) of almost 300 nucleotides between the translation start codon of the first gene ribG in transcription initiation and operon. WO2015 / 1181296, particularly page 23 to page 25, describes suitable rib leader sequence, which is incorporated herein by reference.

[0198] The definitions and explanations provided above apply mutatis mutandis below.

[0199] Method for producing a polypeptide of interest

[0200] The present invention also relates to a method for producing a polypeptide of interest (such as a polypeptide having amylase activity). Preferably, the method comprises the following steps:

[0201] a) providing a Bacillus licheniformis host cell of the present invention, and

[0202] b) cultivating the Bacillus licheniformis host cell under conditions allowing expression of the polypeptide having amylase activity, and optionally,

[0203] c) obtaining or purifying the polypeptide of interest, such as a polypeptide having amylase activity.

[0204] As used herein, the term "culturing" refers to keeping the modified host cells included in the culture alive and / or proliferating for at least a predetermined time. The term encompasses the exponential cell growth phase at the start of growth after inoculation as well as the stationary growth phase. The culture conditions should allow expression, i.e., production of the polypeptide of interest. Such conditions can be selected by those skilled in the art without further fuss. Exemplary conditions for culturing modified host cells are described in WO2020169564A1 or Example 1 or Example 2 of the Examples section. In one embodiment of the method of the present invention, the cultivation in step b) is performed as a fed batch culture.

[0205] If the method of the present invention is applied, the production of at least one polypeptide of interest is allowed to be increased. Preferably, compared with the expression in an unmodified control cell, i.e., a Bacillus licheniformis control cell that does not express the first polypeptide as indicated herein, production increases. In a preferred embodiment, compared with the expression in the control cell, the production of the polypeptide of interest increases by at least 20%, such as at least 50%, particularly at least 100% or at least 200%. For example, compared with the control cell, the production of the polypeptide of interest can increase by 20% to 300%, such as 100% to 300%. Expression can be measured by measuring the amount of the polypeptide in the host cell and / or culture medium. In addition, production can be assessed by measuring enzyme activity. For example, enzyme assays can be used to measure the activity of the polypeptide of interest.

[0206] The polypeptide of interest can be obtained or purified using methods known in the art. For example, the polypeptide can be obtained from the culture medium by methods such as centrifugation, filtration, extraction, spray drying or precipitation.

[0207] The polypeptide of interest may be purified by any method deemed appropriate, such as ion exchange chromatography, electrophoretic procedures, SDS-PAGE or extraction.

[0208] Methods for producing the Bacillus licheniformis host cells of the present invention

[0209] The present invention also relates to a method for producing the Bacillus licheniformis host cell of the present invention, the method comprising

[0210] a) providing a Bacillus licheniformis host cell, and

[0211] b) introducing the following into the host cell provided in step a)

[0212] b1) a first polynucleotide encoding a first polypeptide having peptidyl-prolyl cis-trans isomerase (EC 5.2.1.8) activity, the first polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence that is at least 81% identical to SEQ ID NO: 1, and

[0213] b2) a second polynucleotide encoding a second polypeptide having amylase activity.

[0214] The introduction in step b) can be carried out by any method deemed appropriate, such as for example by transformation with one or more plasmids comprising the first polynucleotide and / or the second polynucleotide. The plasmid preferably comprises a selectable marker gene.

[0215] The present invention also relates to the following uses:

[0216] i) a first polypeptide having peptidyl-prolyl cis-trans isomerase (EC 5.2.1.8) activity, the first polypeptide comprising the amino acid sequence as shown in SEQ ID NO: 1, or an amino acid sequence at least 81% identical to SEQ ID NO: 1, and / or

[0217] ii) a polynucleotide encoding the first polypeptide,

[0218] Used to increase the production of a second polypeptide having alpha-amylase activity in a Bacillus licheniformis host cell.

[0219] Finally, the present invention relates to the use of the Bacillus licheniformis host cell of the present invention for producing a polypeptide having alpha-amylase activity.

[0220] Throughout this application, various publications are referenced. The disclosures of all of these publications and those references cited within these publications are hereby incorporated by reference into this application in their entireties in order to more fully describe the state of the art to which this invention pertains.

[0221] Example

[0222] Materials and methods

[0223] The following examples are intended to illustrate the present invention only. Many possible variations that are obvious to those skilled in the art also fall within the scope of the present invention.

[0224] Unless otherwise stated, the following experiments were performed by applying standard equipment, methods, chemicals and biochemicals used in genetic engineering, molecular biology and the production of compounds by microbial culture and fermentation. See also Sambrook et al. (Sambrook, J. and Russell, DW molecular cloning. A laboratory manual, 3rd edition, Cold Spring Harbor, New York, Cold Spring Harbor Laboratory Press. 2001).

[0225] Electrocompetent Bacillus licheniformis cells and electroporation

[0226] DNA was transformed into Bacillus licheniformis (US5352604) via electroporation. Preparation of electrocompetent Bacillus licheniformis cells and transformation of DNA were essentially as described by Brigidi et al. (Brigidi, P., Mateuzzi, D. (1991). Biotechnol. Techniques 5, 5) with the following modifications: After DNA transformation, cells were recovered in 1 ml of LBSPG buffer and plated on selective LB-agar plates and incubated at 37°C for 60 minutes ( J., 1989, FEMS Microbio. Lett., 61: 165-170).

[0227] To overcome the B. licheniformis-specific restriction modification system of B. licheniformis strains, plasmid DNA was isolated from Ec#098 cells or B. subtilis Bs#056 cells as described below.

[0228] Plasmid isolation

[0229] Plasmid DNA was isolated from Bacillus and Escherichia coli cells by standard molecular biology methods as described in (Sambrook, J. and Russell, DW Molecular cloning. A laboratory manual, 3rd edition, Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press. 2001) or alkaline lysis (Birnboim, H.C., Doly, J. (1979). Nucleic Acids Res 7(6): 1513-1523). Bacillus cells were compared with Escherichia coli treated with 10 mg / ml lysozyme at 37° C. for 30 minutes prior to cell lysis.

[0230] plasmids

[0231] Plasmid p689-T2A-lac

[0232] The E. coli plasmid p689-T2A-lac contains the lacZ-α gene flanked by BpiI restriction sites, which is in turn flanked 5′ by the T1 terminator of the E. coli rrnB gene and 3′ by the T0λ terminator, and was sequenced as a gene synthesis construct (SEQ ID NO: 6).

[0233] Plasmid pEC 194RS-Bacillus temperature-sensitive deletion plasmid (WO2022018260) was used for cloning gene deletion and gene integration constructs.

[0234] Plasmid pBIL013: Bacillus subtilis integration destination plasmid

[0235] Plasmid pBIL013 is a gene integration plasmid with homology regions to the 5′ and 3′ regions of the aprE gene of Bacillus subtilis and a type II cloning cassette with a chloramphenicol resistance gene as a selectable marker. The plasmid backbone of plasmid BIL009 (WO2019016051) was PCR amplified with oligonucleotides SEQ ID NO: 18 and SEQ ID NO: 19. The 5′ homology and 3′ homology regions of the aprE gene were PCR amplified with oligonucleotides SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 23, respectively. Gene synthesis constructs (BioCat GmbH, Heidelberg) SEQ ID NO: 24 were provided for each type of cloning cassette. Individual genetic elements were assembled via type II cloning and BsaI restriction endonuclease as described (Radeck et al., 2017; Sci. Rep. 7: 14134), and the reaction mixture was subsequently transformed into Escherichia coli DH10B cells (Life technologies). Transformants were plated and incubated overnight at 37°C on LB-agar plates containing 25 μg / ml kanamycin. Plasmid DNA was isolated from individual clones and analyzed for accuracy by restriction enzyme digestion. The resulting plasmid, pBIL013, was sequence-verified.

[0236] Gene integration plasmid Bacillus licheniformis-pInt

[0237] pInt020-Cat::PaprE-prsA_Bpu integration plasmid :

[0238] The Cat::PaprE-prsA_Bpu integration plasmid for integration of the Bacillus pumilus prsA gene (prsA_Bpu; SEQ ID NO: 2) into the chloramphenicol-acetyltransferase (Cat) locus under the control of the Bacillus licheniformis aprE gene promoter (SEQ ID NO: 3) was constructed by a two-step cloning strategy. First, the prsA_Bpu expression cassette containing the Bacillus licheniformis aprE gene promoter and the Bacillus pumilus prsA gene was ordered as a gene synthesis fragment with flanking BpiI restriction endonuclease sites (SEQ ID NO: 4 and SEQ ID NO: 5, respectively) and subcloned into p689-T2A-lac with the restriction endonuclease BpiI in a type II assembly as described (Radeck et al., 2017; Sci. Rep. 7: 14134), and the reaction mixture was subsequently transformed into Escherichia coli DH10B cells (Life technologies). Transformants were plated and incubated overnight at 37°C on LB-agar plates containing 25 μg / ml kanamycin. Plasmid DNA was isolated from individual clones and analyzed for accuracy by restriction enzyme digestion. The resulting plasmid, p689-PaprE-prsA_Bpu, was sequence verified.

[0239] In the second type II assembly reaction, a plasmid for exchanging the chloramphenicol acetyltransferase (Cat) locus (SEQ ID NO: 7) with the prsA_Bpu expression cassette was constructed using the restriction endonuclease BsaI, using the plasmids pEC194RS and p689-PaaR-prsA_Bpu. 5' and 3' homology regions of the Cat locus (SEQ ID NO: 8 and SEQ ID NO: 9) were provided as gene synthesis constructs flanked by BsaI restriction sites compatible with pEC194RS and p689-PaaR-prsA_Bpu to allow directional cloning. Type II assembly with the restriction endonuclease BsaI was performed as described, and the reaction mixture was subsequently transformed into E. coli Ec#098. Transformants were plated and incubated overnight at 37°C on LB-agar plates containing 100 μg / ml ampicillin and 30 μg / ml chloramphenicol. Plasmid DNA was isolated from individual clones and analyzed for accuracy by sequencing. The resulting sequence-verified plasmid was designated pInt020_prsA_Bpu.

[0240] Gene integration plasmids for the prsA gene from other bacterial species were constructed as described for plasmid pInt20 and are listed in Table 1.

[0241] Table 1

[0242]

[0243] Plasmid pUK57: Type II assembly target Bacillus plasmid

[0244] Plasmid pUK57 (described in WO2022018260) is a derivative of plasmid pUB110 that contains a type II cloning cassette for assembly of gene expression vectors.

[0245] Amylase expression plasmid

[0246] Each amylase expression plasmid consists of 3-4 genetic elements, which are the plasmid backbone of pUK57, the promoter fragment of the aprE gene from Bacillus licheniformis (SEQ ID NO: 3), or the signal peptide-amylase gene fragment or the signal peptide fragment and amylase gene fragment. As described, the pUK57 vector, promoter fragment (SEQ ID NO: 4), signal peptide-amylase gene fragment or signal peptide gene fragment and amylase gene fragment, each containing a compatible type II restriction endonuclease BpiI site (see Table 2) were assembled with restriction endonuclease BpiI in an in vitro type II assembly reaction (Radeck et al., 2017; Sci. Rep. 7: 14134), and then the reaction mixture was transformed into Bs#056 cells to make them competent according to the method of Spizizen (Anagnostopoulos, C. and Spizizen, J. (1961). J. Bacteriol. 81, 741-746). The correct cloning of the final amylase plasmid was analyzed by restriction enzyme digestion and sequencing. Table 2 summarizes the amylase expression plasmids.

[0247] Table 2 - Amylase expression plasmids

[0248]

[0249]

[0250] Plasmid pAmy031

[0251]

[0252] (*) Amylase variants of SEQ ID NO: 29 having the mutations given in the numbering of SEQ ID NO: 30

[0253] Plasmid pAmy033

[0254]

[0255] (*) Amylase variants of SEQ ID NO: 29 having the mutations given in the numbering of SEQ ID NO: 30

[0256] strain

[0257] Escherichia coli strain Ec#098

[0258] E. coli strain Ec#098 is an E. coli INV 110 strain carrying the DNA-methyltransferase encoding expression plasmid pMDS003 WO2019016051 (Life technologies).

[0259] Bacillus subtilis strain Bs#056

[0260] Prototrophic Bacillus subtilis strain KO-7S (BGSCID: 1S145; Zeigler DR) was made competent according to the following method: Spizizen (Anagnostopoulos, C. and Spizizen, J. (1961). J. Bacteriol. 81, 741-746.) and transformed with linearized DNA-methyltransferase expression plasmid pMIS012 for integration of DNA-methyltransferase into the amyE gene, as described in WO2019 / 016051 for the production of Bacillus subtilis Bs#053. The cells were spread and incubated overnight at 37°C on LB-agar plates containing 10 μg / ml chloramphenicol. After incubation overnight at 37°C, grown colonies were picked and stroked on LB-agar plates containing 10 μg / ml chloramphenicol and LB-agar plates containing 10 μg / ml chloramphenicol and 0.5% soluble starch (Sigma). Starch plates were covered with Lugols solution containing iodine, and positive integrated clones were identified by negative amylase activity. After treatment with lysozyme (10 mg / ml) at 3°C for 30 minutes, genomic DNA of positive clones was isolated by standard phenol / chloroform extraction and subsequently analyzed by PCR for the correct integration of the MTase expression cassette. The resulting Bacillus subtilis strain was designated Bs#056.

[0261] Bacillus subtilis strains

[0262] The prototrophic Bacillus subtilis strain KO-7S (BGSCID: 1S145; Zeigler DR) was used for integration of the various prsA genes. Bacillus subtilis strain KO-7S and its derivatives were made competent as described for Bacillus subtilis Bs#056.

[0263] Bacillus subtilis strains with an integrated prsA expression cassette

[0264] The prsA genes listed in Table 3 were cloned into the pBIL013 plasmid along with the promoter of the Bacillus licheniformis secA gene (WO2019016051, SEQ ID NO: 25) via type II cloning with the BpiI restriction endonuclease as described. The assembled plasmid was linearized with the restriction endonuclease BsmbI as described for Bs#056, and the reaction mixture was then transformed into competent Bacillus subtilis strain KO-7S. Correct gene integration was screened for growth on selective chloramphenicol LB-agar plates and kanamycin sensitivity. Finally, the correct integration of the prsA gene expression cassette was confirmed by PCR amplification and Sanger sequencing. The resulting Bacillus subtilis prsA gene integrated strains are summarized in Table 3.

[0265] Table 3

[0266]

[0267] "D" stands for missing

[0268] Bacillus subtilis amylase expression strain

[0269] As described above for Bacillus subtilis Bs#056, the Bacillus subtilis strains listed in Table 3 were made competent. Amylase expression plasmids pAMY029, pAMY031, and pAMY035 were isolated from Bacillus subtilis Bs#056 strain and transformed into Bacillus subtilis strains PY79 KO-S (control strain) and Bs#112-Bs#115 and plated on LB-agar plates containing 20 μg / μl kanamycin. The correctness of the plasmid DNA of the individual clones was analyzed by restriction digestion, and functional enzyme expression was assessed by transferring individual clones to LB plates with 2% soluble starch to eliminate zone formation in amylase-producing strains. The resulting Bacillus subtilis expression strains are listed in Table 4.

[0270] Table 4: Overview of Bacillus subtilis amylase expression strains

[0271]

[0272] Bacillus licheniformis strains

[0273] Bacillus licheniformis strain Bli#008 (WO2022018260), which contains deletions in the subtilisin aprE gene, the amylase amyB gene, the sporulation factor sigF gene (spoIIAC), and the poly-gamma-glutamic acid synthesis gene, was used to integrate the prsA gene expression cassette into Bacillus licheniformis and thereby replace the chloramphenicol resistance gene (SEQ ID NO: 7) of Bacillus licheniformis.

[0274] For gene deletion / integration in the genes of Bacillus licheniformis strains, after selection on LB-agar plates containing 100 μg / ml ampicillin and 30 μg / ml chloramphenicol at 37°C, the deletion plasmid was transformed into competent Escherichia coli strain Ec#098 according to the following method: Chung (Chung, CT, Niemela, SL and Miller, RH (1989). One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proc. Natl. Acad. Sci. USA 86, 2172-2175). Plasmid DNA was isolated from individual clones and used for subsequent transfer into Bacillus licheniformis strains. The isolated plasmid DNAs each carried the DNA methylation pattern of Bacillus licheniformis and were protected from degradation when transferred into Bacillus licheniformis.

[0275] Bacillus licheniformis strains with integrated expression cassettes for the prsA gene of various bacterial species.

[0276] Electrocompetent Bacillus licheniformis cells were prepared as described above and transformed with 1 μg of pInt20_prsA_Bpu integrating plasmid isolated from E. coli Ec#098 after plating on LB agar plates containing 5 μg / ml erythromycin at 30°C.

[0277] The gene integration procedure was performed as follows :

[0278] Bacillus licheniformis cells carrying the plasmid were grown at 45°C on LB-agar plates containing 5 μg / ml erythromycin, driving the integration of the deletion plasmid into the chromosome via Campbell recombination. The chromosome contained one of the p pInt20_prsA_Bpu homology regions, which was homologous to the sequence 5' or 3' of the chloramphenicol cat gene. Clones were picked and plated on LB-agar plates containing 5 μg / ml erythromycin overnight at 30°C, followed by incubation in LB-medium without selection pressure at 45°C for 6 hours. Individual clones were picked and screened for successful genomic integration of the prsA expression construct at the cat locus by colony PCR analysis using oligonucleotides SEQ ID NO: 10 and SEQ ID NO: 11. Putative integration-positive individual clones were picked and incubated twice in LB medium without antibiotics overnight at 45°C to solidify the plasmid, and then plated on LB-agar plates and incubated overnight at 37°C. Single clones were analyzed by colony PCR for successful genomic integration of the prsA expression cassette at the cat locus. A single erythromycin-sensitive clone with correctly integrated prsA-B. pumilus expression cassette was isolated and designated B. licheniformis Bli#208.

[0279] Construction of additional prsA integrated strains was performed as described for B. licheniformis Bli#208. Table 5 summarizes the B. licheniformis strains with an integrated prsA expression cassette.

[0280] Table 5: Bacillus licheniformis strains with integrated prsA expression cassette

[0281]

[0282]

[0283] "D" stands for missing

[0284] Bacillus licheniformis amylase expression strain

[0285] As described above, the Bacillus licheniformis strains listed in Table 5 were made competent. Amylase expression plasmids pAMY029, pAMY031, pAMY033, and pAMY035 were isolated from Bacillus subtilis Bs#056 to carry the Bacillus licheniformis-specific DNA methylation pattern. The plasmids were transformed into the Bacillus licheniformis Bli#008 control strain (containing only the native prsA gene) and Bacillus licheniformis strains, with the prsA gene expression cassettes of Bacillus pumilus, Bacillus licheniformis, Bacillus lentus, and Geobacillus stearothermophilus integrated, respectively. The transformed strains were then plated on LB agar plates with 20 μg / μl kanamycin. The correctness of the plasmid DNA of the individual clones was analyzed by restriction digestion, and functional enzyme expression was assessed by transferring individual clones to LB plates with 2% soluble starch to eliminate zone formation in the amylase-producing strains. The resulting Bacillus licheniformis expression strains are listed in Table 6.

[0286] Table 6: Bacillus licheniformis amylase expressing strains with prsA genes from different strains

[0287]

[0288]

[0289] In the next step, Bacillus licheniformis amylase expression strains were further constructed as described above. The amylase expression plasmids shown in Table 7 below were transformed into Bacillus licheniformis Bli#008 control strain and Bacillus licheniformis Bli#208 strain, and the additional expression cassette of Bacillus pumilus prsA was integrated into the chromosome.

[0290] Table 7: Bacillus licheniformis amylase expressing strains with and without the prsA gene of Bacillus pumilus.

[0291]

[0292]

[0293] Example 1: Cultivation of Bacillus subtilis amylase-expressing strains co-expressing prsA genes from various strains

[0294] Bacillus subtilis amylase expression strains without additional prsA gene (control strain) and Bacillus subtilis amylase expression strains with additional psrA gene as listed in Table 4 were cultured in a microtiter plate based fed batch process (Habicher et al., 2019 Biotechnol J.; 15(2)).

[0295] All cultures were carried out at 30°C and 400 rpm in an orbital shaker with a diameter of 25 mm (Innova 42, New Brunswick Scientific, Eppendorf AG; Hamburg, Germany). The strains were cultured in two subsequent precultures in flower trays (MTP-48-OFF, m2p-labs GmbH) for synchronous growth. The first preculture was performed in 800 μl TB medium inoculated with a fresh single colony of the strain from streaked LB agar plates. After 20 hours at 30°C, a second preculture containing 800 μl V3 minimal medium (Meissner et al., 2015, Journal of industrial microbiology & biotechnology 42 (9): 1203-1215) was inoculated with 8 μl of the first preculture and cultured at 30°C for 24 hours. Use 48-well circular and deep-well microtiter plates to carry out microtiter plate-based feed batch main culture, wherein each well has a polymer containing glucose (FeedPlate, product number: SMFP08004, Kuhner Shaker GmbH; Herzogenrath, Germany). Use 70 μ l second pre-culture to inoculate 700 μ l supplemented with 5 mM CaCl2 without glucose V3-FP minimal medium. The main culture is incubated at 35 ℃ for 72 hours. The pre-culture is covered with sterile breathable sealing foil (AeraSeal film, Sigma-Aldrich) to avoid contamination. Seal the feed plate with sterile breathable evaporation-reducing foil (F-GPR48-10, m2p-labs GmbH) to reduce evaporation and avoid contamination.

[0296] At the end of the fermentation process, the culture sample was removed and the supernatant was prepared by centrifugation and sterile filtration with a 0.2 μm filter. For poorly soluble amylases, a suitable dilution step is required before filtration or sterile filtration. Amylase activity is determined by a method using the substrate ethylidene-4-nitrophenyl-α-D-maltoheptaglycoside (EPS). D-maltoheptaglycoside is a closed oligosaccharide that can be cleaved by endoamylase. After cleavage, α-glucosidase releases PNP molecules, which have a yellow color and can therefore be measured by visible spectrophotometry at 405 nm. The kit containing EPS substrate and α-glucosidase is manufactured by Roche Costum Biotech (Cat. No. 10880078103) and is described in Lorentz K. et al. (2000), Clin. Chem., 46 / 5: 644-649. The slope of the time-dependent absorption curve is proportional to the specific activity (activity per mg of enzyme) of the α-amylase under given conditions.

[0297] Each strain was cultured in six replicates, and the enzyme activity values were calculated as the average of the six replicates for each strain. The average enzyme activity of the B. subtilis amylase-expressing strains with the additional prsA gene was normalized to the average activity value of the corresponding B. subtilis amylase-producing "control strain", which was set to 100%.

[0298] Table 8 Amylase expression plasmid pAMY029

[0299] Bacillus subtilis expression strain PrsA gene Relative amylase activity CV BES#190 na 100％ 21％ BES#191 Bacillus pumilus 77％ 15％ BES#192 Bacillus licheniformis 136％ 13％ BES#193 Bacillus lentus 307％ 17％ BES#194 Geobacillus stearothermophilus 220％ 20％

[0300] Table 9: Amylases from pAMY031 expression plasmid

[0301] Bacillus subtilis expression strain PrsA gene Relative amylase activity CV BES#195 na 100％ 11％ BES#196 Bacillus pumilus 128％ 14％ BES#197 Bacillus licheniformis 121％ 7％ BES#198 Bacillus lentus 80％ 8％ BES#199 Geobacillus stearothermophilus 133％ 11％

[0302] Table 10: Amylases from pAMY035 expression plasmids

[0303]

[0304]

[0305] When the prsA genes of Bacillus licheniformis, Bacillus lentus, and Geobacillus stearothermophilus were additionally expressed, the amylase production capacity of pAMY029 increased by 30% to 200%, with the PrsA gene from Bacillus lentus showing the greatest improvement in amylase production (Table 8). Additional expression of the prsA gene from Bacillus pumilus had a negative impact on the amylase production capacity of pAMY029 in Bacillus subtilis.

[0306] In the case of additional expression of the prsA gene of Bacillus licheniformis, Bacillus pumilus and Geobacillus stearothermophilus, the amylase production capacity of the amylase of pAMY031 was increased by 20%-30%, while the additional expression of the prsA gene of Bacillus lentus had a negative impact on the amylase production capacity of pAMY031 in Bacillus subtilis compared with the amylase of pAMY029 (Table 9).

[0307] The amylase production capacity of B. licheniformis amylase AmyL of pAMY035 in B. subtilis was increased by 70%-80% with the additional expression of the prsA gene of all tested species (i.e., B. licheniformis, B. pumilus, B. lentus, and G. stearothermophilus) (Table 10).

[0308] Example 2: Cultivation of Bacillus licheniformis amylase-expressing strains co-expressing prsA genes from various strains

[0309] Bacillus licheniformis amylase expressing strains without the additional prsA gene (control strain) and Bacillus licheniformis amylase expressing strains with the additional psrA gene as listed in Table 6 were cultured under microtiter plate based fed batch process conditions as described for Bacillus subtilis (Example 1).

[0310] At the end of the fermentation process, a culture sample was removed and the supernatant was prepared by centrifugation and sterile filtration through a 0.2 μm filter. Amylase activity was determined as described in Example 1. Enzyme activity was calculated as the average of six replicates for each strain. The average enzyme activity of the B. licheniformis amylase-expressing strain with the additional prsA gene was normalized to the average activity of the corresponding B. licheniformis amylase-expressing "control strain," which was set to 100%.

[0311] Table 11: Amylases from pAMY029 expression plasmids

[0312]

[0313]

[0314] Table 12: Amylases from pAMY031 expression plasmid

[0315] Bacillus licheniformis expression strain PrsA gene Relative amylase activity CV BES#210 na 100％ 7％ BES#211 Bacillus pumilus 333％ 5％ BES#212 Bacillus licheniformis 256％ 3％ BES#213 Bacillus lentus 126％ 4％ BES#214 Geobacillus stearothermophilus 145％ 6％

[0316] Table 13: Amylases from pAMY033 expression plasmids

[0317] Bacillus licheniformis expression strain PrsA gene Relative amylase activity CV BES#215 na 100％ 8％ BES#216 Bacillus pumilus 262％ 5％ BES#217 Bacillus licheniformis 196％ 7％ BES#218 Bacillus lentus 138％ 10％ BES#219 Geobacillus stearothermophilus 146％ 9％

[0318] Table 14: Amylases from pAMY035 expression plasmids

[0319] Bacillus licheniformis expression strain PrsA gene Relative amylase activity CV BES#220 na 100％ 12％ BES#221 Bacillus pumilus 126％ 8％ BES#222 Bacillus licheniformis 166％ 11％ BES#223 Bacillus lentus 159％ 7％ BES#224 Geobacillus stearothermophilus 135％ 10％

[0320] When the prsA genes of Bacillus pumilus, Bacillus licheniformis, Bacillus lentus, and Geobacillus stearothermophilus were additionally expressed, the productivity of pAMY029 amylase in Bacillus licheniformis was increased by 40% to more than 316% (Table 11). Compared with the results with Bacillus subtilis, in which the expression of the prsA gene of Bacillus pumilus (BES#191) had a negative impact on the productivity of pAMY029 amylase, surprisingly, the expression of the prsA gene of Bacillus pumilus in Bacillus licheniformis showed the highest increase in the productivity of pAMY029 amylase.

[0321] Similar to the results with the pAMY029 amylase, expression of the prsA gene of B. pumilus resulted in the highest amylase production capacity of pAMY031 amylase (Table 12) and pAMY033 amylase (Figure 13) in B. licheniformis compared to expression of prsA genes from other species.

[0322] With additional expression of the prsA gene from all tested strains (ie, B. licheniformis, B. pumilus, B. lentus, and G. stearothermophilus), the amylase production capacity of B. licheniformis amylase AmyL from pAMY035 in B. licheniformis was increased by 26%-66% (Table 14).

[0323] Example 3: Cultivation of a Bacillus licheniformis amylase-expressing strain co-expressing the prsA gene of Bacillus pumilus keep

[0324] To further analyze the surprisingly strong effect of additional expression of the B. pumilus prsA gene on the production of heterologous amylases in B. licheniformis, the amylase production capacity of various different amylases was analyzed in B. licheniformis Bli#008 control strain (native prsA gene only) or B. licheniformis Bli#208 strain harboring an additional expression cassette for the B. licheniformis prsA transgene.

[0325] MTP-based fed-batch cultivation was performed as described in Example 2 and enzyme activity was determined.

[0326] Table 15 shows the average enzyme activity values of six replicates of each B. licheniformis amylase expressing strain with the additional prsA gene of B. pumilus (Table 7), which were normalized to the average activity value of the corresponding B. licheniformis amylase expressing "control strain", which was set to 100%.

[0327] Table 15: Increased amylase production by Bacillus licheniformis with additional prsA of Bacillus pumilus

[0328]

[0329]

[0330] The additional expression of the prsA gene of Bacillus pumilus in Bacillus licheniformis surprisingly greatly improved the amylase production capacity of various Bacillus-derived amylases from the amylase expression plasmids pAMY031, pAMY038, pAMY040, pAMY042, pAMY048, pAME050, pAMY053, pAMySQL059 and pAMY061.

Claims

1. A Bacillus licheniformis host cell expressing a) a first polypeptide having peptidyl-prolyl cis-trans isomerase (EC 5.2.1.8) activity, the first polypeptide comprising an amino acid sequence at least 81% identical to SEQ ID NO: 1, and b) a second polypeptide having amylase activity, wherein the second polypeptide is heterologous to the Bacillus licheniformis host cell. 2 . The Bacillus licheniformis host cell of claim 1 , wherein the first polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:

1.

3. The Bacillus licheniformis host cell of claim 1 or 2, wherein the second polypeptide has alpha-amylase activity (EC 3.2.1.1) or maltogenic alpha-amylase activity (EC 3.2.1.133).

4. The Bacillus licheniformis host cell of any one of claims 1 to 3, wherein the second polypeptide comprises an amino acid sequence that is at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%, or 100% identical to SEQ ID NO: 29, 35, 38, 40, 42, 48, 50, 53, 59, or 61.

5. The Bacillus licheniformis host cell of any one of claims 1 to 4, wherein the second polypeptide comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to SEQ ID NO: 29, 35, 38, 40, 42, 48, 50, 53, 59, or 61.

6. The Bacillus licheniformis host cell according to any one of claims 1 to 5, wherein the host cell comprises: a) a first expression cassette for said first polypeptide, said first expression cassette comprising a promoter operably linked to a first polynucleotide encoding said first polypeptide, and optionally a terminator, and b) a second expression cassette for the second polypeptide, the second expression cassette comprising a promoter operably linked to a second polynucleotide encoding the second polypeptide, and optionally a terminator.

7. The Bacillus licheniformis host cell according to claim 6, wherein the promoter of the first expression cassette and / or the promoter of the second expression cassette is an inducer-independent promoter or an inducible promoter.

8. The Bacillus licheniformis host cell according to any one of claims 6 or 7, wherein the first expression cassette and / or the second expression cassette are present on a plasmid in the host cell or stably integrated into the chromosomal DNA of the host cell.

9. The Bacillus licheniformis host cell of any one of claims 1 to 8, wherein the second polypeptide is secreted.

10. The Bacillus licheniformis host cell of any one of claims 1 to 9, wherein the second polypeptide comprises a signal peptide.

11. The Bacillus licheniformis host cell according to any one of claims 1 to 10, wherein the host cell is a host cell from a strain selected from the group consisting of: Bacillus licheniformis strains ATCC 14580, ATCC 31972, ATCC 53757, ATCC 53926, ATCC 55768, DSM 13, DSM 394, DSM 641, DSM 1913, DSM 11259 and DSM 26543.

12. A method for producing a polypeptide having amylase activity, the method comprising a) providing a Bacillus licheniformis host cell according to any one of claims 1 to 11, and b) cultivating the host cell under conditions allowing expression of the polypeptide having amylase activity, and optionally, c) obtaining or purifying the polypeptide having amylase activity.

13. A method for producing a Bacillus licheniformis host cell according to any one of claims 1 to 11, the method comprising a) providing a Bacillus licheniformis host cell, and b) introducing a first polynucleotide encoding the first polypeptide according to claim 1 or 2 and b) a second polynucleotide encoding the second polypeptide according to any one of claims 1, 3, 4 and 5 into the host cell provided in step a).

14. The method according to claim 13, wherein in step b), the first expression cassette and the second expression cassette according to any one of claims 6 to 9 are introduced into the host cell.

15. Use of the first polypeptide having peptidyl-prolyl cis-trans isomerase (EC 5.2.1.8) activity according to claim 1 or 2 and / or a polynucleotide encoding the first polypeptide for increasing the production of the second polypeptide having alpha-amylase activity according to any one of claims 1, 3, 4 and 5 in a Bacillus licheniformis host cell.

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