Proregion mutations to enhance protein production in gram-positive bacterial cells

Abstract

The present disclosure generally relates to recombinant polynucleotides comprising novel proregion DNA sequences. Certain aspects of the present disclosure relate to recombinant Gram-positive bacterial strains comprising one or more introduced polynucleotides comprising a novel proregion DNA sequence operably linked to a DNA sequence encoding a protein of interest.

Description

Technical Field

[0001] The present disclosure generally relates to the fields of microbial host cells, molecular biology, protein engineering, fermentation, protein production, etc. Certain aspects of the present disclosure relate to novel pro-region nucleic acid (DNA) sequences, recombinant polynucleotides comprising novel pro-region DNA sequences, genetically modified (recombinant) Gram-positive bacterial strains comprising one or more introduced polynucleotides, etc., wherein the one or more introduced polynucleotides comprise novel pro-region DNA sequences operably linked to nucleic acid (DNA) sequences encoding target proteins.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application No. 63 / 326,615, filed on April 1, 2022, which is hereby incorporated by reference in its entirety.

[0004] References to sequence listings

[0005] The electronic submission of a text file sequence listing named "NB41862WOPCT_SequenceListing.xml" was created on March 29, 2023 and is 51 KB in size and is hereby incorporated by reference in its entirety. Background Art

[0006] Gram-positive microorganisms are commonly used in large-scale industrial fermentations because of their ability to secrete their fermentation products into their culture medium. The secreted protein is exported through the cell membrane and cell wall and then subsequently released into the external medium. For example, large-scale industrial fermentation and secretion of heterologous polypeptides are widely used technologies in industry, wherein microbial cells are transformed with nucleic acids encoding heterologous polypeptides to be expressed. Although there are multiple advances in protein production methods, there is still a need in the art to provide more efficient protein expression methods, which are intended to enhance the production of target proteins for a variety of industrial applications. Summary of the invention

[0007] As generally described herein, the present disclosure provides, inter alia, compositions and methods for producing target proteins in Gram-positive bacterial (host) cells. Certain embodiments relate to novel pro-region nucleic acid (DNA) sequences, recombinant polynucleotides (e.g., vectors, plasmids, expression cassettes, etc.) comprising novel pro-region DNA sequences, recombinant polynucleotides comprising novel pro-region sequences operably linked to downstream (3') gene coding sequences, recombinant polynucleotides comprising novel pro-region sequences operably linked to upstream (5') DNA sequences encoding proprotein signal (secretion) sequences, etc.

[0008] In certain aspects, the disclosure provides recombinant Gram-positive bacterial strains that express one or more introduced polynucleotides encoding target proteins. In specific embodiments, one or more introduced polynucleotides include a novel pro-region DNA sequence operably connected to a DNA sequence encoding a target protein (the DNA sequence encoding the target protein may include an upstream (5') protein signal sequence operably connected thereto). In certain other aspects, the disclosure provides compositions and methods for designing / constructing recombinant Gram-positive bacterial strains expressing one or more introduced novel polynucleotide constructs encoding a target protein, compositions and methods for culturing recombinant strains expressing a target protein, compositions and methods for enhancing the production of a target protein, etc.

[0009] For example, in one or more embodiments, the present disclosure provides novel variant proregion sequences as shown in one or more amino acid sequences of SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32 and SEQ ID NO:33 and / or combinations thereof. In other one or more embodiments, the present disclosure provides novel variant proregion sequences as shown in one or more amino acid sequences of SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32 and SEQ ID NO:33 and / or combinations thereof. Figure 1 , Figure 2 ,or Figure 3 In yet one or more other embodiments, the present disclosure provides novel variant pre-region sequences shown or described in one of Tables 1-5 and / or a combination thereof.

[0010] Therefore, certain one or more embodiments relate to variant (mutant) proregion sequences comprising one or more amino acid substitutions, one or more amino acid insertions, etc. In certain embodiments, the variant proregion sequence comprises an amino acid substitution at position 30, wherein the amino acid positions of the variant proregion are numbered according to SEQ ID NO: 15. In other embodiments, the variant proregion sequence comprises an amino acid substitution at position 30 and one or more positions selected from 1, 2, 3, 4, 6, 14, 16, 19, 20, 23, 36, 37, 38, 39, 42, 43, 44, 49, 50, 64, 65, 67, 68, 71, 79, 83 and 84, wherein the amino acid positions of the variant proregion are numbered according to SEQ ID NO: 15. In one or more other embodiments, the variant proregion sequence is derived from a parent or reference polypeptide having at least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to positions 1-84 of SEQ ID NO: 15. In certain other embodiments, the variant proregion sequence comprises an amino acid insertion of at least glycine (G) at position 2 and lysine (K) at position 3, wherein the amino acid positions of the variant proregion are numbered according to SEQ ID NO: 14. In other one or more embodiments, the variant proregion sequence comprises an amino acid insertion of at least glycine (G) at position 2 and lysine (K) at position 3, and an amino acid substitution at one or more positions selected from 1, 32, 38, 46, 66, 67, 70, and 73, wherein the amino acid positions of the variant proregion are numbered according to SEQ ID NO: 14. In some other embodiments, the variant proregion is derived from a parent or reference polypeptide having at least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to positions 1-86 of SEQ ID NO: 14. In yet other embodiments, the variant proregion sequence comprises an amino acid insertion of at least glycine (G) at position 2, lysine (K) at position 3, and alanine (A) at position 4, wherein the amino acid positions of the variant proregion are numbered according to SEQ ID NO: 30. In certain aspects, the variant proregion is derived from a parent or reference polypeptide having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to positions 1-87 of SEQ ID NO: 30.In certain other one or more embodiments, the variant pre-region sequence comprises an amino acid insertion of at least glycine (G) at position 2, lysine (K) at position 3, and serine (S) at position 4, wherein the amino acid positions of the variant pre-region are numbered according to SEQ ID NO: 29. In some other embodiments, the variant pre-region is derived from a parent polypeptide or a reference polypeptide having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to positions 1-87 of SEQ ID NO: 29. In other one or more embodiments, the variant pre-region sequence comprises an amino acid insertion of at least glycine (G) at position 2, lysine (K) at position 3, alanine (A) at position 4, and alanine (A) at position 5, wherein the amino acid positions of the variant pre-region are numbered according to SEQ ID NO: 31. In certain one or more embodiments or aspects, the variant proregion is derived from a parent or reference polypeptide having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to positions 1-88 of SEQ ID NO: 31. In other embodiments, the variant proregion sequence comprises a glutamic acid (E) to glycine (G) substitution at position 30 (E30G), a leucine (L) to lysine (K) substitution at position 68 (L68K), and an isoleucine (I) to valine (V) substitution at position 73 (I72V), wherein the amino acid positions of the variant proregion are numbered according to SEQ ID NO: 32. In other one or more aspects or embodiments, the variant pro-region sequence comprises a glutamic acid (E) to glycine (G) substitution at position 30 (E30G), a leucine (L) to lysine (K) substitution at position 68 (L68K), and a glutamic acid (E) to isoleucine (I) substitution at position 80 (E80I), wherein the amino acid positions of the variant pro-region are numbered according to SEQ ID NO: 33. In certain embodiments, the variant pro-region is derived from a parent or reference polypeptide having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to positions 1-84 of SEQ ID NO: 15, SEQ ID NO: 32, and / or SEQ ID NO: 33. In other aspects, the variant front region of the present disclosure comprises Table 1-5. Figure 1 , Figure 2 , Figure 3 , SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:33, and any combination thereof. In one or more embodiments, the amino acid modifications shown in Tables 1-5, Figure 1 , Figure 2 , Figure 3 , SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:33, and combinations thereof, are derived from a parent or reference polypeptide having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:15.

[0011] Therefore, some other one or more embodiments provide recombinant nucleic acids encoding novel variant pre-region sequences of the present invention. Therefore, some embodiments relate to polynucleotides comprising variant pre-region nucleic acids, etc. In one or more embodiments, the present disclosure provides polynucleotides comprising upstream (5') nucleic acids (sequences) encoding variant pre-regions of the present invention, and the upstream nucleic acids (sequences) are operably connected to downstream (3') nucleic acid sequences encoding heterologous target proteins (POI). In some other aspects, the present disclosure provides polynucleotides, which include upstream (5') nucleic acids encoding preprotein signal (secretion) sequences, which are operably connected to downstream (3') nucleic acid sequences encoding variant pre-regions of the present invention, and the downstream nucleic acid sequences encoding variant pre-regions of the present invention are operably connected to downstream nucleic acid sequences encoding target proteins (POI). Some other embodiments relate to expression cassettes, which include upstream (5') promoter regions sequences operably connected to downstream (3') polynucleotides of the present disclosure.

[0012] In certain other one or more embodiments, the present disclosure relates to recombinant (modified) Gram-positive bacterial cells / strains comprising one or more introduced polynucleotides or expression cassettes of the present disclosure. Therefore, other embodiments of the present disclosure relate to methods / processes for producing heterologous target proteins in recombinant Gram-positive cells as described and described herein.

[0013] In some one or more embodiments, the present disclosure provides methods for producing a heterologous POI in a Gram-positive bacterial cell, the methods comprising introducing an expression cassette into a Gram-positive cell, the expression cassette comprising an upstream promoter region operably linked to a downstream nucleic acid sequence encoding a variant pro-region, the variant pro-region comprising an amino acid substitution at position 30 and one or more positions selected from 1, 2, 3, 4, 6, 14, 16, 19, 20, 23, 36, 37, 38, 39, 42, 43, 44, 49, 50, 64, 65, 67, 68, 71, 79, 83 and 84, wherein the variant pro-region nucleic acid (sequence) is operably linked to the downstream nucleic acid sequence encoding the POI, wherein the amino acid positions of the variant pro-region are numbered according to SEQ ID NO: 15; and growing / culturing / fermenting the modified cells under conditions for producing the POI. In certain other embodiments, the present disclosure provides methods for producing heterologous POIs in Gram-positive bacterial cells, the methods comprising introducing an expression cassette into a Gram-positive cell, the expression cassette comprising an upstream promoter region sequence operably linked to a downstream nucleic acid (sequence) encoding a variant pro-region, the variant pro-region comprising an amino acid insertion of glycine (G) at position 2 and lysine (K) at position 3, and an amino acid substitution at one or more positions selected from 1, 32, 38, 46, 66, 67, 70 and 73, wherein the variant pro-region nucleic acid (sequence) is operably linked to a downstream nucleic acid sequence encoding a POI, wherein the amino acid positions of the variant pro-region are numbered according to SEQ ID NO: 14; and growing / culturing / fermenting the modified cells under conditions for producing the POI. In yet other embodiments, the present disclosure relates to methods for producing heterologous POIs in Gram-positive bacterial cells, the methods comprising introducing an expression cassette of the present disclosure into a Gram-positive cell, and growing / culturing / fermenting the modified cells under conditions for producing the POI. In some one or more embodiments of these methods, the box includes a nucleic acid (sequence) encoding a preprotein signal (secretion) sequence, which is operably connected and is located between a promoter and a variant pre-region sequence. In some other embodiments of these methods, relative to a control gram-positive cell, the modified cell produces an increased amount of POI. For example, as shown in the following control gram-positive cells, it includes an expression cassette introduced, the expression cassette includes an identical upstream promoter sequence (i.e., identical to a modified cell) operably connected to a downstream nucleic acid encoding a pre-region sequence comprising SEQ ID NO: 15, and the downstream nucleic acid encoding the pre-region sequence is operably connected to a downstream nucleic acid sequence encoding the same POI (i.e., identical to a modified cell). In some embodiments of these methods, the box introduced is integrated into the genome of the cell. In some embodiments of these methods, at least two boxes are introduced into the cell.Certain other embodiment(s) of the present disclosure are described herein and set forth below. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The amino acid sequence of the wild-type (reference) Bacillus lentus proregion and certain SEL variant proregion sequences disclosed herein are shown. Figure 1 As shown, the reference Bacillus lentus proregion sequence comprises eighty-four (84) amino acid (residue) positions as shown in SEQ ID NO: 15 (wherein the glutamic acid at position 30 ( E ) residues are underlined), variant pro-region sequence A comprises the eighty-four (84) amino acid positions set forth in SEQ ID NO:9 (wherein the histidine (H) residue at position 30 is in bold), variant pro-region sequence B comprises the eighty-four (84) amino acid positions set forth in SEQ ID NO:11 (wherein the glycine (G) residue at position 30 is in bold), variant pro-region sequence C comprises the eighty-six (86) amino acid positions set forth in SEQ ID NO:14 (wherein the glycine (G) and lysine (K) residues inserted at positions 2 and 3, respectively, are shown in bold (GK), and the E residues are underlined), variant pro-region sequence D comprises the eighty-seven (87) amino acid positions set forth in SEQ ID NO:29 (wherein the glycine (G), lysine (K), and serine (S) residues inserted at positions 2-4, respectively, are shown in bold (GKS)), variant pro-region sequence E comprises the eighty-seven (87) amino acid positions set forth in SEQ ID NO:30 (wherein the glycine (G), lysine (K), and alanine (A) residues inserted at positions 2-4, respectively, are shown in bold (GKA)), variant pro-region sequence F comprises the eighty-eight (88) amino acid positions set forth in SEQ ID NO:31 (wherein the glycine (G), lysine (K), alanine (A), and alanine (A) residues inserted at positions 2-5, respectively, are shown in bold (GKAA)), variant pro-region sequence G comprises SEQ ID NO: The eighty-four (84) amino acid positions shown in NO:32 (wherein the glycine (G) residue at position 30 is shown in bold, and the L68K and I72V substitutions are underlined) and the variant proregion sequence H comprises the eighty-four (84) amino acid positions shown in SEQ ID NO:33 (wherein the glycine (G) residue at position 30 is shown in bold, and the L68K and E80I substitutions are underlined).

[0015] Figure 2The amino acid sequences of variant proregion sequence A (SEQ 09), variant proregion sequence B (SEQ 11), variant proregion sequence C (SEQ 14), variant proregion sequence D (SEQ 29), variant proregion sequence E (SEQ 30), variant proregion sequence F (SEQ 31), variant proregion sequence G (SEQ 32), and variant proregion sequence H (SEQ 33) aligned with the wild-type (WT) proregion sequence (SEQ 15) are shown. Figure 2 As shown, the WT proregion comprises a glutamic acid (E) residue at position 30 (E; SEQ 15), the variant proregion sequence A comprises a histidine (H) residue at position 30 (H; SEQ 09), the variant proregion sequence B comprises a glycine (G) residue at position 30 (G; SEQ 11), the variant proregion sequence C comprises two (2) amino acid insertions of glycine (G) and lysine (K) (GK insertion; SEQ 14), the variant proregion sequence D comprises three (3) amino acid insertions of glycine (G), lysine (K) and serine (S) (GKS insertion; SEQ 29), the variant proregion sequence E comprises three (3) amino acid insertions of glycine (G), lysine (K) and alanine (A) (GKA insertion; SEQ 30), and the variant proregion sequence F comprises four (4) amino acid insertions of glycine (G), lysine (K), alanine (A), and alanine (A) (GKAA insertion; SEQ 31), the variant pro-region sequence G comprises a glycine (G) residue at position 30, a lysine (K) residue at position 68, and a valine (V) residue at position 72, and the variant pro-region sequence H comprises a glycine (G) residue at position 30, a lysine (K) residue at position 68, and an isoleucine (I) residue at position 80. For example, as shown in the alignment of the WT pro-region sequence (SEQ 15) and the variant pro-region sequence C (SEQ 14), two (2) amino acid residues GK are inserted between the alanine (A) at position 1 and the glutamic acid (E) at position 2 of the WT pro-region (SEQ ID NO: 15), resulting in a variant pro-region sequence C of 86 amino acids of SEQ ID NO: 14. Figure 2 As shown, the two hyphens (--) shown in SEQ 15, SEQ 09, SEQ 11, SEQ 32 and SEQ 33 indicate two (2) gaps in amino acid positions relative to the variant proregion sequence C shown in SEQ 14.

[0016] Figure 3 The following amino acid (residue) position numbers are shown: Reference (wild type) Bacillus lentus proregion amino acid sequence (E 30 , SEQ ID NO: 15), variant pre-region sequence A (H 30 ; SEQ ID NO: 09), variant proregion sequence B (G 30; SEQ ID NO:11), variant proregion sequence C (G2K3 insertion, SEQ ID NO:14), variant proregion sequence D (G2K3S4 insertion, SEQ ID NO:29), variant proregion sequence E (G2K3A4 insertion, SEQ ID NO:30), variant proregion sequence F (G2K3A4A5 insertion, SEQ ID NO:31), variant proregion sequence G (with E30G, L68K and I72V substitutions; SEQ ID NO:32) and variant proregion sequence F (with E30G, L68K and E80I substitutions; SEQ ID NO:33), wherein the amino acid position is indicated by subscript numbers from the 5' (N-terminus) to the 3' (C-terminus) direction, for example, alanine (A1) at position 1 of SEQ ID NO:15, glutamic acid (E2) at position 2 of SEQ ID NO:15, etc. For example, the mutant front region sequence disclosed in the present invention can refer to the wild-type (reference) front region sequence (numbering SEQ ID NO: 15, E30), variant (reference) front region sequence A (numbering SEQ ID NO: 9, H30), variant (reference) front region sequence B (numbering SEQ ID NO: 11, G30), variant (reference) front region sequence C (numbering SEQ ID NO: 14, GK insertion), variant (reference) front region sequence D (numbering SEQ ID NO: 29, GKS insertion), variant (reference) front region sequence E (numbering SEQ ID NO: 20, GKA insertion), variant (reference) front region sequence F (numbering SEQ ID NO: 31, GKAA insertion), variant (reference) front region sequence G (numbering SEQ ID NO: 32, L68K / I72V substitution), variant (reference) front region sequence H (numbering SEQ ID NO: 33, L68K / E80I substitution) or a combination thereof, such as Figure 3 shown.

[0017] Biological sequence description

[0018] SEQ ID NO: 1 is a nucleic acid (DNA) sequence comprising the upstream (5') aprE gene flanking region, the Bacillus subtilis rrnI-P2 promoter variant and the 5'-aprE UTR region.

[0019] SEQ ID NO: 2 is the amino acid sequence of a wild-type Bacillus gibsonii subtilisin designated "BG46".

[0020] SEQ ID NO: 3 is the amino acid sequence of a variant B. gibsonii BG46 subtilisin designated "BG46_Variant 1".

[0021] SEQ ID NO: 4 is a DNA sequence encoding the signal sequence of the wild-type Bacillus subtilis AprE protein.

[0022] SEQ ID NO:5 is a DNA sequence encoding the variant proregion sequence A (30H, SEQ ID NO:9).

[0023] SEQ ID NO: 6 is the DNA sequence of the wild-type Bacillus amyloliquefaciens BPN' terminator.

[0024] SEQ ID NO: 7 is the DNA sequence of the kanamycin (kan) gene expression cassette.

[0025] SEQ ID NO: 8 is the amino acid sequence of a variant Bacillus gibsonii BG46 subtilisin designated "BG46_variant 2".

[0026] SEQ ID NO:9 is the amino acid sequence encoded by the variant Proregion A DNA sequence (SEQ ID NO:5).

[0027] SEQ ID NO: 10 is a DNA sequence encoding variant proregion sequence B (30G, SEQ ID NO: 11).

[0028] SEQ ID NO: 11 is the amino acid sequence encoded by the variant Proregion B DNA sequence (SEQ ID NO: 10).

[0029] SEQ ID NO: 12 is the amino acid sequence of the Bacillus amyloliquefaciens BPN' pro-region sequence.

[0030] SEQ ID NO: 13 is a DNA sequence encoding a variant proregion sequence C (GK insertion, SEQ ID NO: 14).

[0031] SEQ ID NO: 14 is the amino acid sequence encoded by the variant Proregion C DNA sequence (SEQ ID NO: 13).

[0032] SEQ ID NO: 15 is the amino acid sequence of the wild-type (WT) B. lentus proregion sequence.

[0033] SEQ ID NO: 16 is the DNA sequence of the 5'FR of the serA gene of B. licheniformis.

[0034] SEQ ID NO: 17 is a DNA sequence comprising the rrnI-p3 promoter region.

[0035] SEQ ID NO: 18 is the DNA sequence of Bacillus subtilis aprE 5'-UTR.

[0036] SEQ ID NO: 19 is the DNA sequence of the Bacillus licheniformis amyL terminator.

[0037] SEQ ID NO: 20 is the DNA sequence of the 3'FR of the serA gene of Bacillus licheniformis.

[0038] SEQ ID NO: 21 is the DNA sequence of the 5'FR of the lysA gene of Bacillus licheniformis.

[0039] SEQ ID NO: 22 is the DNA sequence of the 3'FR of the lysA gene of Bacillus licheniformis.

[0040] SEQ ID NO:23 is the DNA sequence encoding the variant proregion sequence C (GKA insertion, SEQ ID NO:14).

[0041] SEQ ID NO:24 is the amino acid sequence encoded by the variant Proregion C DNA sequence (SEQ ID NO:23).

[0042] SEQ ID NO:25 is the DNA sequence encoding the variant proregion sequence C (GKAA insertion, SEQ ID NO:14).

[0043] SEQ ID NO:26 is the amino acid sequence encoded by the variant Proregion C DNA sequence (SEQ ID NO:25).

[0044] SEQ ID NO:27 is the DNA sequence encoding the variant proregion sequence C (GKAS insertion, SEQ ID NO:14).

[0045] SEQ ID NO:28 is the amino acid sequence encoded by the variant Proregion C DNA sequence (SEQ ID NO:27).

[0046] SEQ ID NO: 29 is the amino acid sequence of variant proregion sequence D.

[0047] SEQ ID NO:30 is the amino acid sequence of variant proregion sequence E.

[0048] SEQ ID NO:31 is the amino acid sequence of variant proregion sequence F.

[0049] SEQ ID NO:32 is the amino acid sequence of variant proregion sequence G.

[0050] SEQ ID NO: 33 is the amino acid sequence of variant proregion sequence H. DETAILED DESCRIPTION

[0051] As briefly described above and described in detail below, the present disclosure provides, among other things, novel pro-region nucleic acid (DNA) sequences, recombinant polynucleotides (e.g., vectors, plasmids, expression cassettes, etc.) comprising novel pro-region DNA sequences, recombinant polynucleotides comprising novel pro-region sequences operably linked to downstream (3') gene coding sequences, recombinant polynucleotides comprising novel pro-region sequences operably linked to upstream (5') DNA sequences encoding protein signal (secretion) sequences, and the like. In certain aspects, the present disclosure provides recombinant Gram-positive bacterial strains that express one or more introduced polynucleotides encoding target proteins. In a specific embodiment, one or more introduced polynucleotides comprise novel pro-region DNA sequences operably linked to a DNA sequence encoding a target protein (the DNA sequence encoding the target protein may comprise an upstream (5') protein signal sequence operably linked thereto). Therefore, certain other aspects of the present disclosure provide, among other things, compositions and methods for designing / constructing recombinant Gram-positive bacterial strains expressing one or more introduced novel polynucleotide constructs encoding a target protein, compositions and methods for culturing recombinant strains expressing a target protein, compositions and methods for enhancing the production of a target protein, and the like.

[0052] I. Definitions

[0053] In view of the disclosed recombinant (modified) strains and methods thereof described herein, the following terms and phrases are defined.Terms not defined herein shall be given their ordinary meaning as used in the art.

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the compositions and methods of the present invention are applied. Although any methods and materials similar or equivalent to the methods and materials described herein may also be used in the practice or testing of the compositions and methods of the present invention, representative illustrative methods and materials are now described. All publications and patents cited herein are incorporated by reference in their entirety.

[0055] It is further noted that the claims may be drafted to exclude any optional element. Therefore, this statement is intended to serve as antecedent basis (or conditional thereto) for use of exclusive terminology such as "solely," "only," "exclusively," "not including," and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0056] As will be apparent to those skilled in the art after reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features that can be readily separated or combined with the features of any of the other several embodiments without departing from the scope or spirit of the inventive compositions and methods described herein. Any recited method may be performed in the order of events recited or in any other order that is logically possible.

[0057] As used herein, the phrases "Gram-positive bacteria," "Gram-positive cells," "Gram-positive bacterial strains," and / or "Gram-positive bacterial cells" have the same meaning as used in the art. For example, Gram-positive bacterial cells include all strains of the Actinobacteria and Firmicutes. In certain embodiments, such Gram-positive bacteria belong to the classes Bacillus, Clostridia, and Mollicutes.

[0058] As used herein, "Bacillus" includes all species within the genus "Bacillus" as known to those of skill in the art, including, but not limited to, B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, and B. thuringiensis. It will be recognized that the genus Bacillus is continually undergoing taxonomic reorganization. Thus, the genus is intended to include species that have been reclassified, including, but not limited to, organisms such as Bacillus stearothermophilus (which is now named "Geobacillus stearothermophilus").

[0059] As used herein, the term "recombinant" or "non-natural" refers to an organism, microorganism, cell, nucleic acid molecule or vector that has at least one engineered genetic change or has been modified by the introduction of a heterologous nucleic acid molecule, or refers to a cell (e.g., a microbial cell) that has been altered so that the expression of a heterologous or endogenous nucleic acid molecule or gene can be controlled. Recombination also refers to a cell derived from a non-natural cell, or a progeny of a non-natural cell with one or more such modifications. Genetic changes include, for example, the introduction of modifications of expressible nucleic acid molecules encoding proteins, or the addition, deletion, substitution of other nucleic acid molecules, or other functional changes in the genetic material of the cell. For example, a recombinant cell can express genes or other nucleic acid molecules (e.g., fusion proteins or chimeric proteins) that are not found in natural (wild-type) cells in the same or homologous form, or can provide an altered endogenous gene expression pattern, such as overexpression, underexpression, minimal expression, or no expression at all. "Recombination" or the generation of a "recombined" nucleic acid is typically an assembly of two or more nucleic acid fragments, wherein the assembly produces a chimeric gene.

[0060] The term "derived" encompasses the terms "originating from," "obtained," "obtainable," and "produced," and generally indicates that a specified material or composition finds its origin in another specified material or composition, or has characteristics that can be described with reference to another specified material or composition. For example, the recombinant Gram-positive bacterial cells of the present disclosure can be derived from / obtained from any known Gram-positive bacterial strain.

[0061] As used herein, "nucleic acid" refers to nucleotide or polynucleotide sequences and fragments or portions thereof, as well as DNA, cDNA and RNA of genomic or synthetic origin, which may be double-stranded or single-stranded, whether representing a sense strand or an antisense strand. It should be understood that due to the degeneracy of the genetic code, a variety of nucleotide sequences may encode a given protein.

[0062] It should be understood that the polynucleotides (or nucleic acid molecules) described herein include "genes", "vectors" and "plasmids".

[0063] Accordingly, the term "gene" refers to a polynucleotide encoding a specific sequence of amino acids, which contains all or part of a protein coding sequence, and may include regulatory (non-transcribed) DNA sequences, such as a promoter sequence, which determines, for example, the conditions under which a gene is expressed. The transcribed region of a gene may include an untranslated region (UTR) (including a 5'-untranslated region (UTR) and a 3'-UTR) and a coding sequence.

[0064] As used herein, "endogenous gene" refers to a gene in its natural location in the genome of an organism.

[0065] As used herein, a "heterologous" gene, "non-endogenous" gene, or "foreign" gene refers to a gene not normally found in the host organism, but introduced into the host organism by gene transfer. The term "foreign" gene(s) encompasses native genes inserted into a non-native organism and / or chimeric genes inserted into a native or non-native organism.

[0066] As used herein, "heterologous control sequences" refer to gene expression control sequences (e.g., promoters, enhancers, terminators, etc.) that do not function in nature to regulate (control) the expression of a gene of interest. Typically, heterologous nucleic acids are not endogenous (natural) to the cell or part of the genome in which they are present, and have been added to the cell by infection, transfection, transduction, transformation, microinjection, electroporation, etc. "Heterologous" nucleic acid constructs may contain control sequence / DNA coding (ORF) sequence combinations that are the same or different from those found in the natural host cell.

[0067] As used herein, the term "expression" refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid molecules of the present disclosure. Expression can also refer to the translation of mRNA into polypeptides. Therefore, the term "expression" includes any steps involved in the production of polypeptides, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, secretion, etc.

[0068] As used herein, the term "coding sequence" refers to a nucleotide sequence that directly specifies the amino acid sequence of its (encoded) protein product. The boundaries of a coding sequence are usually determined by an open reading frame (hereinafter, "ORF") that usually begins with the ATG start codon. Coding sequences typically include DNA, cDNA, and recombinant nucleotide sequences.

[0069] As used herein, the terms "promoter", "promoter element", "promoter sequence" and the like refer to nucleic acid (DNA) sequences that can control the transcription of a gene coding sequence (CDS) into messenger RNA (mRNA), when the promoter region sequence is placed upstream (5') and operably connected to the downstream (3') gene CDS. As commonly understood by those skilled in the art, a promoter typically provides a site for specific binding and transcription initiation by RNA polymerase. In some aspects, the term "promoter" refers to the minimum portion of the promoter nucleic acid sequence required to initiate transcription (i.e., comprising an RNA polymerase binding site). For example, a promoter typically comprises a "-10" (consensus sequence) element and a "-35" (consensus sequence) element, which are located upstream (5') and are relative to the +1 transcription start site (TSS) of the gene CDS to be transcribed. The core promoter -10 and -35 elements are commonly referred to in the art as "TATAAT" (Pribnow box) consensus region and "TTGACA" consensus region, respectively. The core promoter (10 and -35) regions are typically separated (spaced) by about fifteen to twenty (15-20) intervening base pairs (nucleotides).

[0070] Promoters can all be derived from natural genes, or be composed of different elements derived from different promoters found in nature, or even contain synthetic nucleic acid segments. It will be appreciated by those skilled in the art that different promoters can direct genes to be expressed in different cell types, or at different developmental stages, or in response to different environments or physiological conditions. Promoters can be constitutive promoters, inducible promoters, adjustable promoters, hybrid promoters, synthetic promoters, tandem promoters, etc. Promoters that make genes expressed in most cell types at most times are generally referred to as "constitutive promoters". It should be further recognized that, since in most cases, the exact boundaries of regulatory sequences have not yet been fully defined, DNA fragments of different lengths can have the same promoter activity. In certain aspects, the upstream (5') promoter sequence (pro) is operably linked to a downstream DNA sequence encoding a protein signal sequence (SS), which is operably linked to a downstream DNA sequence encoding a pre-region sequence (PRO), which is operably linked to a downstream (3') DNA sequence (ORF) encoding a mature protein of interest, which can be schematically presented as 5'-[pro]-[SS]-[PRO]-[ORF]-3'.

[0071] As used herein, "a functional promoter sequence that controls the expression of a target gene linked to a protein coding sequence of a target gene" refers to a promoter sequence that controls the transcription and translation of a coding sequence in a desired Gram-positive host cell. For example, in certain embodiments, the present disclosure provides a polynucleotide comprising an upstream (5') promoter (or a 5' promoter region, or a tandem 5' promoter, etc.) that is functional in a Gram-positive cell, wherein the functional promoter region is operably linked to a nucleic acid sequence encoding a target protein.

[0072] As used herein, the term "precursor protein" refers to an inactive form of a protein. In some aspects, a full-length protein is synthesized as a precursor in the form of a presequence and a mature protein (abbreviated as "pre-protein"). In other aspects, a full-length protein is synthesized as a precursor in the form of a signal peptide sequence, a presequence and a mature protein (abbreviated as "pre-pro-protein"). For example, a presequence is usually used as a signal peptide for transport, and a presequence is typically essential for the correct folding of a related (mature) protein.

[0073] As used herein, the term "mature protein" refers to the active form of a protein as opposed to the inactive precursor (full-length) protein.

[0074] As used herein, the terms "signal sequence", "secretion signal" and "signal peptide" can be used interchangeably and refer to a sequence of amino acid residues that can participate in the secretion or directional transport of a precursor protein. Typically, the signal (pre) sequence is cleaved from the precursor protein by a signal peptidase during translocation. The signal (pre) sequence is typically located at the N-terminus of the mature protein sequence, or at the N-terminus of the pre-region (pro) sequence, where the signal (pre) sequence and the pre-region (pro) sequence are used in an operable combination and are located upstream (5') of the mature POI sequence.

[0075] As used herein, the terms "pro sequence", "pro-sequence" and "pro-region sequence" are used interchangeably and are abbreviated as "PRO" sequence, "Pro" sequence, "pro" sequence, etc. The term pro sequence as used herein has the same meaning as understood in the art. For example, the Bacillus subtilis alkaline serine protease "subtilisin" is first produced as pre-pro-subtilisin, which consists of a signal (pre) sequence for protein secretion, followed by a pre-region (pro) sequence of seventy-seven (77) amino acids, followed by an amino acid sequence encoding a mature subtilisin (e.g., pre-pro-subtilisin). The pro sequence acts as an intramolecular chaperone (e.g., directly catalyzes a protein folding reaction) and is generally essential for the correct folding of the associated (mature) protein. Similarly, both the folding and intracellular transport (or secretion) of the mature target protein may require a pro sequence, indicating that these two functions are closely related. In certain aspects, the pro-region sequence of the present disclosure comprises an amino acid sequence derived from the wild-type (WT, reference) Bacillus lentus pro-region sequence of SEQ ID NO: 15. In certain embodiments, the pro-region sequence is derived from an amino acid sequence comprising homology to SEQ ID NO: 15, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32 and / or SEQ ID NO: 33. Thus, in certain aspects, the amino acid modifications of one or more pro-region variants described herein are numbered by reference to the pro-region amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 14.

[0076] For example, the amino acid sequence of one or more pre-region variants described herein can be compared with the amino acid sequence of SEQ ID NO: 15 using an alignment algorithm, and each amino acid residue in a given amino acid sequence aligned (preferably optimally aligned) with the amino acid residues of SEQ ID NO: 15 is conveniently numbered by reference to the numerical position of the corresponding amino acid residue. When compared with a query sequence (also sometimes referred to as a "reference sequence"), a sequence alignment algorithm will identify one or more positions at which insertion or deletion occurs in the subject sequence. Sequence alignments with other pre-region amino acid sequences can be determined using an amino acid alignment algorithm.

[0077] In certain aspects, references to the proregion amino acid sequence "positions" may be presented as single-letter amino acids (residues) followed by the position number (e.g., SEQ ID NO: 15, the alanine at position 1 is presented as "A1", the glutamic acid at position 2 is presented as "E2", etc.). In related embodiments, references to variant (mutant) proregion amino acid sequences may be presented as single-letter amino acids (residues), wherein the amino acid position of the parent (reference) sequence is numbered, followed by the mutant amino acid (residue) at the same position (e.g., SEQ ID NO: 15, the alanine (A) at position 1 is substituted with glycine (G) as "A1G", the glutamic acid (E) at position 30 is substituted with histidine (H) as "E30H", etc.). Multiple amino acid residues can also be substituted at the same position in the pro-region amino acid sequence, where the amino acid position of the parent (reference) sequence is numbered, followed by one or more mutant amino acid residues at the same position separated by a slash (e.g., SEQ ID NO: 15, the replacement of alanine (A) at position 1 with glycine (G), glutamic acid (E) or lysine (K) can be presented as "A1G / E / K", etc.).

[0078] Certain ranges are provided herein where a numerical value is preceded by the term "about". The term "about" is used herein to provide textual support for the exact number that follows it and for numbers that are close to or approximate to the number that follows the term. When determining whether a number is close to or approximate to a specifically stated number, the close or approximate unstated number can be a number that provides a substantial equivalent of the specifically stated number in the context in which it is presented. For example, with respect to a numerical value, the term "about" refers to a range of -10% to +10% of the numerical value, unless the term is otherwise specifically defined in the context.

[0079] As used herein, polynucleotides encoding a "PRO" sequence, polynucleotides encoding a "pro-region" sequence, and polynucleotides encoding a "pro-sequence" can be used interchangeably and refer to a DNA sequence located immediately upstream (5') and operably linked to a downstream (3') gene coding sequence (CDS) encoding a mature protein of interest (POI). In certain aspects, a polynucleotide encoding a pro-region sequence refers to a DNA sequence located and operably linked to an upstream (5') protein signal sequence (SS) and a downstream (3') gene CDS encoding a mature POI. For example, a pro-region DNA sequence (PRO) is operably linked to an upstream (5') DNA sequence (SS) encoding a protein signal sequence and is operably linked to a downstream (3') DNA gene CDS encoding a mature POI (e.g., 5'-[SS]-[PRO]-[ORF]-3'; N-[pre]-[pro]-[protein]-C).

[0080] As used herein, the "wild-type (WT) Bacillus lentus pro-region" sequence comprises the amino acid sequence set forth in SEQ ID NO: 15. In certain aspects, the WT (reference) pro-region sequence (SEQ ID NO: 15) may be referred to as the "E30 pro-sequence" or the "E 30 pre-sequence".

[0081] As used herein, the mutant proregion sequence designated as "variant proregion A" comprises the amino acid sequence shown in SEQ ID NO: 9. In certain embodiments, the variant proregion A sequence (SEQ ID NO: 9) may be referred to as "H30 prosequence" or "H30 prosequence". 30 pre-sequence".

[0082] As used herein, the mutant proregion sequence designated as "variant proregion B" comprises the amino acid sequence shown in SEQ ID NO: 11. In certain embodiments, the variant proregion B sequence (SEQ ID NO: 11) may be referred to as "G30 prosequence" or "G 30 pre-sequence".

[0083] As used herein, the mutant proregion sequence designated as "variant proregion C" comprises the amino acid sequence shown in SEQ ID NO: 14. In certain embodiments, the variant proregion C sequence (SEQ ID NO: 14) may be referred to as a "GK preinsertion sequence" or a "G2K3 preinsertion sequence".

[0084] As used herein, the mutant proregion sequence designated as "variant proregion D" comprises the amino acid sequence shown in SEQ ID NO: 29. In certain embodiments, the variant proregion D sequence (SEQ ID NO: 29) may be referred to as "GKS preinsertion sequence" or "G2K3S4 preinsertion sequence".

[0085] As used herein, the mutant proregion sequence designated as "variant proregion E" comprises the amino acid sequence shown in SEQ ID NO: 30. In certain embodiments, the variant proregion E sequence (SEQ ID NO: 30) may be referred to as "GKA preinsertion sequence" or "G2K3A4 preinsertion sequence".

[0086] As used herein, the mutant proregion sequence designated as "variant proregion F" comprises the amino acid sequence shown in SEQ ID NO: 31. In certain embodiments, the variant proregion F sequence (SEQ ID NO: 31) may be referred to as "GKAA preinsertion sequence" or "G2K3A4A5 preinsertion sequence".

[0087] As used herein, the mutant proregion sequence designated as "variant proregion G" comprises the amino acid sequence set forth in SEQ ID NO: 32. In certain embodiments, the variant proregion G sequence (SEQ ID NO: 32) may be referred to as the L68K / I72V prosequence.

[0088] As used herein, the mutant proregion sequence designated as "variant proregion H" comprises the amino acid sequence set forth in SEQ ID NO: 32. In certain embodiments, the variant proregion H sequence (SEQ ID NO: 33) may be referred to as the L68K / E80I prosequence.

[0089] like Figure 3 As shown in the figure, the amino acid (residue) position numbering can refer to the WT proregion amino acid sequence (E30, SEQ ID NO: 15), the variant proregion sequence A (H30; SEQ ID NO: 09), the variant proregion sequence B (G 30 ; SEQ ID NO:11), variant proregion sequence C (GK insertion, SEQ ID NO:14), variant proregion sequence D (GKS insertion, SEQ ID NO:29), variant proregion sequence E (GKA insertion, SEQ ID NO:30), variant proregion sequence F (GKAA insertion, SEQ ID NO:31), variant proregion sequence G (L68K / I72V, SEQ ID NO:32) and / or variant proregion sequence H (L68K / E80I, SEQ ID NO:33).

[0090] As used herein, the phrase "polynucleotide encoding a full-length protein" refers to a DNA sequence encoding a "precursor" protein; and the phrase "polynucleotide encoding a mature protein" refers to a DNA sequence encoding a "mature" protein as defined herein. In certain aspects, the polynucleotide encoding the precursor protein comprises an upstream (5') DNA sequence encoding at least a pre-region amino acid sequence, which is operably linked to a downstream (3') DNA sequence (e.g., an open reading frame, ORF) encoding an amino acid sequence of a mature protein of interest (POI). In other aspects, the polynucleotide encoding the precursor protein comprises an upstream (5') DNA sequence encoding at least a protein signal sequence, which is operably linked to a downstream (3') DNA sequence encoding a pre-region amino acid sequence, which is operably linked to a downstream (3') ORF encoding an amino acid sequence of a mature POI.

[0091] As used herein, the term "untranslated region" may be abbreviated as "UTR".

[0092] As used herein, the phrases "5' untranslated region," "5' untranslated region," and / or "5' transcription leader sequence" are used interchangeably and abbreviated as "5'-UTR." As is generally understood in the art, the 5'-UTR is known to be the region of the messenger RNA (mRNA) that is located directly upstream (5') of the start codon.

[0093] When a nucleic acid is in a functional relationship with another nucleic acid sequence, the nucleic acid is "operably linked". For example, if the DNA encoding a secretory leader sequence (i.e., a signal sequence) is expressed as a preprotein that participates in the secretion of a polypeptide, the DNA encoding the secretory leader sequence is operably linked to the DNA encoding the polypeptide; if a promoter or enhancer affects the transcription of a coding sequence, the promoter or enhancer is operably linked to the sequence (CDS, ORF); or if a ribosome binding site is positioned to promote translation, the ribosome binding site is operably linked to a coding sequence. Generally, "operably linked" means that the DNA sequence being linked is continuous, and in the case of a secretory leader sequence, it is continuous and in the reading phase. However, enhancers do not have to be continuous. Connection is achieved by connecting at a convenient restriction site. If such a site does not exist, a synthetic oligonucleotide adapter or joint is used according to conventional practice. Therefore, the term operably linked generally refers to the association (juxtaposition) of nucleic acid sequences on a single nucleic acid fragment, so that the function of one nucleic acid fragment is affected by another. For example, if a promoter (pro) controls the transcription of a gene coding sequence (gene CDS), then the promoter is operably linked to the gene CDS (eg, 5'-[pro]-[gene CDS] 3').

[0094] As used herein, the phrase "variant rrnI-P2 promoter and 5'-UTR region" (abbreviated as "P2 promoter / 5'-UTR region") refers to the variant Bacillus subtilis rrnI-P2 promoter / 5'-aprE UTR region DNA sequence shown in SEQ ID NO:1.

[0095] As used herein, the DNA encoding the “wild-type Bacillus subtilis aprE signal peptide sequence” may be abbreviated as “aprESS” and comprises the nucleotide sequence of SEQ ID NO:4.

[0096] As used herein, “wild-type Bacillus amyloliquefaciens BPN′ terminator (BPN′term)” may be abbreviated as “term” and comprises the nucleotide sequence of SEQ ID NO:6.

[0097] As used herein, the variant Bacillus gibsonii (BG46) subtilisin comprising the amino acid sequence of SEQ ID NO: 3 is abbreviated as "BG46_variant 1".

[0098] As used herein, the variant Bacillus gibsonii (BG46) subtilisin comprising the amino acid sequence of SEQ ID NO: 8 is abbreviated as "BG46_Variant 2", which BG46_Variant 2 is derived from the wild-type Bacillus gibsonii (BG46) subtilisin reporter protein (SEQ ID NO: 2).

[0099] As used herein, "suitable regulatory sequence" refers to a nucleotide sequence that is located upstream (5' non-coding sequence), internal or downstream (3' non-coding sequence) of a coding sequence and affects transcription, RNA processing or stability or translation of the relevant coding sequence. Regulatory sequences can include promoters, transcription leader sequences, RNA processing sites, effector binding sites and stem-loop structures.

[0100] As used herein, "host cell" refers to a cell with the ability to serve as a host or expression vehicle for a newly introduced DNA sequence. Thus, in certain embodiments of the present disclosure, the host cell is a Gram-positive cell (e.g., Bacillus species) and / or a Gram-negative cell (e.g., Escherichia coli).

[0101] As used herein, a "modified cell" refers to a recombinant cell that comprises at least one genetic modification that is not present in a parent cell, reference cell, or control cell from which the modified cell is derived.

[0102] As used herein, when the expression and / or production of a protein of interest (POI) in a recombinant (modified) cell is compared to the expression and / or production of the same POI in an unmodified (control) cell, it is to be understood that the modified and unmodified cells are grown / cultured / fermented under the same conditions (e.g., the same conditions such as culture medium, temperature, pH, etc.).

[0103] As used herein, when used in phrases such as "the recombinant cells 'express / produce' an increased amount of a protein of interest relative to unmodified (control) cells", "increased amount" specifically refers to an "increased amount" of a protein of interest (POI) expressed / produced in the recombinant cells, which "increased amount" is always relative to unmodified (control) cells expressing / producing the same POI, wherein the modified and unmodified cells are grown / cultured / fermented under the same conditions.

[0104] As used herein, "increasing" protein production or "increased" protein production means that the amount of protein (e.g., target protein) produced is increased. The protein can be produced in the host cell, or secreted (or transported) into the culture medium. In certain embodiments, the target protein is produced (secreted) into the culture medium. Increased protein production can be detected, for example, as a higher maximum level of protein or enzyme activity (such as, for example, amylase activity) compared to the parent host cell, or the total extracellular protein produced.

[0105] As used herein, the terms "modification" and "genetic modification" are used interchangeably and include: (a) introduction, substitution or removal of one or more nucleotides in a gene (or its ORF), or introduction, substitution or removal of one or more nucleotides in a regulatory element required for transcription or translation of a gene or its ORF, (b) gene disruption, (c) gene conversion, (d) gene deletion, (e) gene down-regulation, (f) specific mutagenesis of any one or more genes disclosed herein and / or (g) random mutagenesis.

[0106] In some aspects, genetic modification refers in particular to the introduction, substitution or removal of one or more nucleotides in a nucleic acid (DNA) sequence encoding a proregion (amino acid) sequence of the present disclosure. For example, in certain embodiments, a DNA sequence encoding a natural proregion amino acid sequence shown in SEQ ID NO: 15 is genetically modified as described herein.

[0107] As used herein, the term "introduced", as used in phrases such as "introducing 'gene', 'polynucleotide', 'open reading frame (ORF)', 'gene coding sequence', 'vector', 'expression cassette' into Gram-positive bacterial cells", etc., includes methods known in the art for introducing polynucleotides (DNA) into cells, including but not limited to protoplast fusion, natural or artificial transformation (e.g., calcium chloride, electroporation), transduction, transfection, conjugation, etc.

[0108] As used herein, "transformed" or "transformation" means transforming a cell by using recombinant DNA techniques. Transformation typically occurs by inserting one or more nucleotide sequences (e.g., polynucleotides, ORFs, or genes) into a cell. The inserted nucleotide sequence may be a heterologous nucleotide sequence (i.e., a sequence that does not naturally occur in the cell to be transformed). Thus, transformation generally refers to the introduction of exogenous DNA into a host cell such that the DNA remains as a chromosomal integrant or a self-replicating extrachromosomal vector.

[0109] As used herein, "transforming DNA", "transforming sequence" and "DNA construct" refer to the DNA for introducing a sequence into a host cell or an organism. Transforming DNA is a DNA for introducing a sequence into a host cell or an organism. DNA can be produced in vitro by PCR or any other suitable technology. In certain embodiments, the transforming DNA comprises an input sequence, and in other embodiments, it further comprises an input sequence flanked by homology boxes. In yet other embodiments, the transforming DNA comprises other non-homologous sequences (i.e., stuffing sequences or flanking) added to the end. The end can be closed so that the transforming DNA forms a closed loop, such as, for example, as inserted into a vector.

[0110] As used herein, "disruption of a gene" or "gene disruption" can be used interchangeably and broadly refers to any genetic modification that substantially prevents a host cell from producing a functional gene product (e.g., protein). Therefore, as used herein, gene disruption includes, but is not limited to, frameshift mutations, premature termination codons (i.e., so that no functional protein is produced), substitutions of internal deletions of proteins that eliminate or reduce activity (so that no functional protein is produced), insertions that destroy coding sequences, mutations that remove the operable connection between the natural promoter required for transcription and the open reading frame, etc.

[0111] As used herein, "input sequence" refers to a DNA sequence introduced into the bacterial cell chromosome. In certain embodiments, the input sequence is a part of a DNA construct. In other embodiments, the input sequence encodes one or more target proteins. In certain embodiments, the input sequence comprises a sequence that may or may not be present in the genome of the cell to be transformed (that is, it may be a homologous or heterologous sequence). In certain embodiments, the input sequence encodes one or more target proteins, genes and / or mutations or modified genes. In alternative embodiments, the input sequence encodes functional wild-type genes or operons, functional mutant genes or operons or non-functional genes or operons. In certain embodiments, non-functional sequences can be inserted into genes to destroy the function of genes. In another embodiment, the input sequence includes a selective marker. In a further embodiment, the input sequence includes two homology boxes.

[0112] As used herein, "homology box" refers to a nucleic acid sequence homologous to a sequence in a bacterial cell chromosome. More particularly, according to the present invention, a homology box is an upstream or downstream region that has a sequence identity between about 80% and 100%, a sequence identity between about 90% and 100%, or a sequence identity between about 95% and 100% with a gene or a part of a gene to be deleted, destroyed, inactivated, downregulated, etc. The direct flanking coding region. These sequences guide the integration position of the DNA construct in the bacterial cell chromosome and guide which part of the chromosome is replaced by the input sequence. Although it is not intended to limit the present disclosure, the homology box can include between about 1 base pair (bp) and 200 kilobases (kb). Preferably, the homology box includes between about 1bp and 10.0kb; between 1bp and 5.0kb; between 1bp and 2.5kb; between 1bp and 1.0kb; and between 0.25kb and 2.5kb. Homology boxes can also include about 10.0 kb, 5.0 kb, 2.5 kb, 2.0 kb, 1.5 kb, 1.0 kb, 0.5 kb, 0.25 kb, and 0.1 kb. In some embodiments, the 5' and 3' ends of the selectable marker are flanked by homology boxes, wherein the homology boxes contain nucleic acid sequences that closely flank the coding region of the gene.

[0113] As used herein, a host cell "genome", a bacterial (host) cell "genome", or a Bacillus sp. (host) cell "genome" includes chromosomal genes and extrachromosomal genes.

[0114] As used herein, the terms "plasmid", "vector" and "cassette" refer to an extrachromosomal element that usually carries genes that are typically not part of the central metabolism of the cell and is usually in the form of a circular double-stranded DNA molecule. Such an element may be a linear or circular autonomously replicating sequence derived from single-stranded or double-stranded DNA or RNA from any source, a genome integrating sequence, a bacteriophage, or a nucleotide sequence in which multiple nucleotide sequences have been linked or recombined into a single construct that is capable of introducing a promoter fragment and a DNA sequence for a selected gene product, along with appropriate 3' non-translated sequences, into a cell.

[0115] As used herein, the term "plasmid" refers to a circular double-stranded (ds) DNA construct used as a cloning vector and forming an extrachromosomal self-replicating genetic element in many bacteria and some eukaryotic organisms. In some embodiments, the plasmid is incorporated into the genome of the host cell. In some embodiments, the plasmid is present in the parent cell and is lost in the daughter cell.

[0116] As used herein, "transformation cassette" refers to a specific vector that contains a gene (or its ORF) and has elements in addition to the foreign gene that facilitate transformation of a specific host cell.

[0117] As used herein, the term "vector" refers to any nucleic acid that can replicate (propagate) in a cell and can carry a new gene or DNA segment to a cell. Therefore, the term refers to a nucleic acid construct designed to be transferred between different host cells. Vectors include viruses, phages, proviruses, plasmids, phagemids, transposons, and artificial chromosomes such as YAC (yeast artificial chromosome), BAC (bacterial artificial chromosome), PLAC (plant artificial chromosome), etc. that are "episomes" (i.e., they replicate autonomously or can be integrated into the chromosome of a host organism).

[0118] "Expression vector" refers to a vector that has the ability to be incorporated into a cell and express heterologous DNA in the cell. Many prokaryotic and eukaryotic expression vectors are commercially available and known to those skilled in the art. The selection of an appropriate expression vector is within the knowledge of those skilled in the art.

[0119] As used herein, the terms "expression cassette" and "expression vector" refer to a nucleic acid construct having a series of specified nucleic acid elements (i.e., these are vectors or vector elements, as described above) that allow a specific nucleic acid to be transcribed in a target cell, which is recombinant or synthetically produced. The recombinant expression cassette can be incorporated into a plasmid, a chromosome, a mitochondrial DNA, a plastid DNA, a virus, or a nucleic acid fragment. Typically, the recombinant expression cassette portion of the expression vector includes (in addition to other sequences) a nucleic acid sequence to be transcribed and a promoter. In certain embodiments, the DNA construct also includes a series of specified nucleic acid elements that allow a specific nucleic acid to be transcribed in a target cell. In certain embodiments, the DNA construct of the present disclosure includes a selectable marker and an inactivated chromosome, or a gene, or a DNA segment as defined herein.

[0120] As used herein, "targeting vector" is a vector comprising a polynucleotide sequence that is homologous to the region in the chromosome of the host cell to which the targeting vector is transformed and that can drive homologous recombination at the region. For example, the targeting vector can be used for introducing mutations into the chromosome of the host cell by homologous recombination. In certain embodiments, the targeting vector comprises other non-homologous sequences (i.e., stuffing sequences or flanking sequences) that are for example added to the end. The end can be closed so that the targeting vector forms a closed loop, for example, as inserted into a vector. For example, in certain embodiments, the cell is modified (for example, transformed) by introducing one or more "targeting vectors" in a parental Bacillus licheniformis (host) cell.

[0121] As used herein, the term "protein of interest" or "POI" refers to a polypeptide of interest that is desired to be expressed in a modified (recombinant) Gram-positive host cell, wherein the POI is preferably expressed at an increased level (i.e., relative to "unmodified" (parent or control) cells). Thus, as used herein, a POI may be an enzyme, a substrate binding protein, a surfactant protein, a structural protein, a receptor protein, and the like. In certain embodiments, the modified cells of the present disclosure produce an increased amount of a heterologous protein of interest relative to a control cell. In specific embodiments, the increased amount of the protein of interest produced by the modified cells of the present disclosure is at least 0.5% increase, at least 1.0% increase, at least 5.0% increase, or more than 5.0% increase relative to a control cell.

[0122] Similarly, as defined herein, "target gene" or "GOI" refers to a nucleic acid sequence (eg, polynucleotide, gene, or ORF) encoding a POI. A "target gene" encoding a "target protein" may be a naturally occurring gene, a mutated gene, or a synthetic gene.

[0123] As used herein, the terms "polypeptide" and "protein" are used interchangeably and refer to a polymer of any length comprising amino acid residues linked by peptide bonds. Conventional one (1) letter or three (3) letter codes for amino acid residues are used herein. A polypeptide may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The term polypeptide also encompasses amino acid polymers that have been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more amino acid analogs (including, for example, non-natural amino acids, etc.), as well as other modifications known in the art.

[0124] In certain embodiments, the genes of the present disclosure encode commercially relevant proteins of industrial interest, such as enzymes (e.g., acetylesterase, aminopeptidase, amylase, arabinanase, arabinofuranosidase, carbonic anhydrase, carboxypeptidase, catalase, cellulase, chitinase, chymosin, cutinase, deoxyribonuclease, epimerase, esterase, α-galactosidase, β-galactosidase, α-glucanase, glucan lyase, endo-β-glucanase, glucoamylase, glucose oxidase, α-glucosidase, β-glucosidase, glucuronidase, glycosylation ... enzymes, such as glycosyl hydrolases, hemicellulases, hexose oxidases, hydrolases, invertases, isomerases, laccases, lipases, lyases, mannosidases, oxidases, oxidoreductases, pectate lyases, pectin acetylesterases, pectin depolymerases, pectin methylesterases, pectinolytic enzymes, perhydrolases, polyol oxidases, peroxidases, phenoloxidases, phytases, polygalacturonases, proteases, peptidases, rhamnogalacturonases, ribonucleases, transferases, transporters, transglutaminases, xylanases, hexose oxidases, and combinations thereof).

[0125] As used herein, a "variant" polypeptide refers to a polypeptide derived from a parent (or reference) polypeptide, typically by recombinant DNA technology, by substitution, addition, or deletion of one or more amino acids. Variant polypeptides may differ from parent polypeptides by a small number of amino acid residues and can be defined by their level of primary amino acid sequence homology / identity with the parent (reference) polypeptide.

[0126] In certain one or more embodiments, the variant polypeptide has at least about 40% to about 70%, at least 75%, at least 80%, at least 85%, 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%, or even at least 99% amino acid sequence identity to the parent (reference) polypeptide sequence.

[0127] As used herein, "variant" polynucleotide refers to a polynucleotide having a specified degree of sequence homology / identity with a parent (or reference) polynucleotide, or a polynucleotide that hybridizes with a parent polynucleotide (or its complementary sequence) under stringent hybridization conditions. In certain one or more embodiments, the variant polynucleotide of the present disclosure has at least about 40% to at least about 100% nucleotide sequence identity with a parent (reference) polynucleotide sequence. In certain other embodiments, the variant polynucleotide has at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 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%, or at least 99% nucleotide sequence identity with a parent (reference) polynucleotide sequence.

[0128] As used herein, "mutation" refers to any change or alteration in a nucleic acid sequence. There are several types of mutations, including point mutations, deletion mutations, silent mutations, frameshift mutations, splicing mutations, etc. Mutations can be made specifically (e.g., via site-directed mutagenesis) or randomly (e.g., via chemical reagents, by repair minus bacterial strain passage).

[0129] As used herein, the term "substitution" in the context of a polypeptide or sequence thereof means that one amino acid is replaced (ie, substituted) by another amino acid.

[0130] As used herein, the term "homology" relates to homologous polynucleotides or polypeptides. If two or more polynucleotides or two or more polypeptides are homologous, this means that the homologous polynucleotides or polypeptides have at least 60%, more preferably at least 70%, even more preferably at least 85%, still more preferably at least 90%, more preferably at least 95%, and most preferably at least 98% "degree of identity". Whether two polynucleotides or polypeptide sequences have a sufficiently high degree of identity as defined herein, two sequences can be suitably studied by using a computer program known in the art to compare, such as " GAP " provided in the GCG program package (Program Manual for the Wisconsin Package, 8th edition, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, 53711, U.S.) (Needleman and Wunsch, (1970)). DNA sequence comparisons were performed using GAP with the following settings: GAP creation penalty 5.0 and GAP extension penalty 0.3.

[0131] As used herein, the term "percent identity (%)" refers to the level of nucleic acid or amino acid sequence identity between nucleic acid sequences encoding polypeptides or amino acid sequences of polypeptides when aligned using sequence alignment programs.

[0132] As used herein, "specific productivity" is the total amount of protein produced per cell per time in a given period of time.

[0133] As used herein, the terms "purified," "isolated," or "enriched" mean that a biomolecule (e.g., a polypeptide or polynucleotide) is altered from its native state by separating it from some or all of the naturally occurring components with which it is associated in nature. Such separation or purification can be accomplished by art-recognized separation techniques such as ion exchange chromatography, affinity chromatography, hydrophobic separation, dialysis, protease treatment, ammonium sulfate precipitation or other protein salt precipitation, centrifugation, size exclusion chromatography, filtration, microfiltration, gel electrophoresis, or gradient separation to remove whole cells, cell debris, impurities, foreign proteins, or enzymes that are not desired in the final composition. Components that provide additional benefits, such as activators, anti-inhibitors, desired ions, compounds that control pH, or other enzymes or chemicals, can then be further added to the purified or isolated biomolecule composition.

[0134] II. Mutated PRO sequences for enhanced protein production

[0135] As described herein generally and set forth in the examples below, the applicant has designed and constructed a site evaluation library (SEL) to test / screen certain genetic modifications (mutations) to improve the productivity of recombinant proteins. In particular, as set forth in the examples, recombinant proteins (enzymes) are utilized as reporter proteins to monitor protein expression in the recombinant Bacillus strains of the present disclosure. Certain proregion sequences suitable for expression / production of heterologous (recombination) mature proteins have been described. For example, PCT Publication No. WO 2008 / 112258 and PCT Publication No. 2010 / 123754 describe the prosequences of Bacillus Clausii alkaline precursor protease and Bacillus lentus subtilisin precursor protease, wherein certain mutation presequences promote the productivity enhancement of mature protease. PCT Publication No. WO 1993 / 20214 describes certain AprE signals (pre) and proregion sequences for expressing Gram-negative lipase in Bacillus subtilis.

[0136] More particularly, as described in the Examples below, the pro-region of the expression construct encoding the exemplary (subtilisin) reporter protein was genetically modified (SEL). In particular, as generally described in the Examples (see Figure 1-Figure 3) A proregion sequence derived from the wild-type (reference) prosequence (SEQ ID NO: 15) was subjected to SEL to screen for mutations that enhance the productivity of the mature subtilisin reporter protein.

[0137] As described in Example 1, the variant pre-region sequence A (30H, SEQ ID NO: 9; Figure 1 ) was used as a template to establish a site evaluation library (SEL) and developed into a 4.4 kb fragment, wherein the linear DNA of the expression cassette was used to transform competent Bacillus cells. As described in Example 1, sequence analysis was performed to determine unique pre-region sequence A variants and / or pre-region sequence B variants in the SEL, wherein each of the 84 amino acid positions of the variant pre-region sequence A (SEQ ID NO: 9) was changed (substituted) to all other nineteen (19) naturally occurring amino acid residues. In particular, Table 1 (Example 1) shows the results of the reporter protein productivity (performance index (PI) value) compared to the reference construct of the reporter protein expressed using the WT PRO sequence (SEQ ID NO: 15) after seventy-two (72) hours. For example, as shown in Table 1, the PI of the pre-region sequence A (E30H, SEQ ID NO: 9) increased by about 50% relative to the PI of the WT PRO sequence (30E; SEQ ID NO: 15). Likewise, as shown in Table 1, mutations in any of amino acid positions 1, 2, 3, 4, 6, 14, 19, 20, 23, 36, 37, 38, 39, 42, 43, 44, 49, 50, 64, 65, 67, 68, 71, 83 or 84 combined with position 30 showed enhanced reporter protein productivity at 72 hours relative to the reference pro-region sequence (SEQ ID NO: 15). In addition, approximately 80% of the selected mutant combinations had additional positive charges (see Table 1, Δ charge).

[0138] As further described in Example 3, the proregion of the expression construct encoding the exemplary (subtilisin) reporter protein (BG46_variant 2) was genetically modified (SEL). More specifically, the variant proregion sequence B (30G, SEQ ID NO: 11; Figure 1 ) as a template to develop a linear DNA expression cassette comprising one or more pro-regions of the present disclosure. In particular, the 5' sequence of the seventy-eight (78) amino acid wild-type AprE propeptide sequence (SEQ ID NO: 12) was used to exchange the first three (3) amino acid residues (positions 1-3, "AGK"; SEQ ID NO: 12) with the first amino acid residue being alanine (A), resulting in a variant pro-region sequence C (SEQ ID NO: 14; GK insertion, Figure 2). For example, as shown in Table 1 (Example 1), a mutant of the proregion variant A sequence (SEQ ID NO: 9; Table 1, mutant "E30G") showed high expression of the reporter protein (PI 2.3) after 72 hours of growth compared to the wild-type proregion (Table 1, PI 1.0) and the proregion variant A sequence (SEQ ID NO: 11; Table 1, mutant "E30H", PI 1.5).

[0139] Example 4 of the present disclosure further describes the preparation of a combinatorial library based on a reference proregion sequence (SEQ ID NO: 15), wherein amino acids glycine (G) and lysine (K) ("GK") are inserted. More specifically, as described in Example 3, amino acid residue GK is inserted into the N-terminus of the wild-type proregion sequence (SEQ ID NO: 15), thereby generating a proregion variant C amino acid sequence as shown in SEQ ID NO: 14, which comprises 86 amino acid positions, such as Figure 2 As shown in the comparison. For example, Table 2 (Example 4) presents the combination of pre-region mutations showing that BG46_variant 2 reporter protein productivity (PI) increases, wherein the change in charge (Δ charge) is generally between +1 and +4 (Table 2). In some aspects, the applicant has noted that such a positive charge is suitable as a combination in the loop region of the pre-region sequence (e.g., as reference SEQ ID NO:15 residue positions 66-73).

[0140] As shown in Table 2, the PI values ​​of the SEL proregion variant C sequence (derived from SEQ ID NO: 14) were compared to the PI values ​​of the wild-type (WT) proregion sequence (SEQ ID NO: 15), wherein the amino acid position numbers (Table 2, column 1) were compared to the WT proregion sequence (i.e., without the "AGK" insertion). Thus, as described in Example 4, the proregion variant C sequence shown in Table 2 showed high expression of the reporter protein (PI values ​​between about 1.9 and 2.3) after 72 hours of growth compared to the wild-type proregion (Table 2, PI 1.0) and the proregion variant B sequence (mutant "E30G", PI 1.7).

[0141] As generally described in Example 5, BG46_Variant 2 subtilisin was used as a reporter protein to monitor expression in Bacillus licheniformis strains. More specifically, DNA encoding a variant pro-region sequence of the present disclosure was operably linked to a DNA sequence encoding a mature BG46_Variant 2 protein, wherein the strain construction was generally described in Example 5. For example, Table 3 shows the PI values ​​of Bacillus licheniformis strains comprising a variant pro-region sequence relative to a control pro-region sequence B (SEQ ID NO: 11) comprising an E30G mutation, wherein the variant pro-region sequence exhibited high expression of the reporter protein compared to the control pro-region sequence (E30G) after 72 hours of growth.

[0142] As further described in Example 6, the variant proregion sequence C (SEQ ID NO: 14) is subjected to additional N-terminal modifications. For example, the variant proregion sequence C (having 86 amino acid positions) is further modified so that an additional amino acid, such as alanine (A) or serine (S), is introduced (inserted) after the lysine (K) in position three (3). In particular, as described in Example 6 (and as Figure 1-Figure 3 As shown in Table 4, variant proregion sequences D (SEQ ID NO: 29, GKS insertion), E (SEQ ID NO: 30, GKA insertion) and F (GKAA insertion; SEQ ID NO: 31) were constructed, wherein the performance index (PI) values ​​(Table 4) of the proregion variant sequences D, E and F were compared with the variant proregion sequence C (SEQ ID NO: 14, "GK" insertion). As presented in Table 4, after 72 hours of growth, the PI index of the three N-terminal proregion mutants (GKA, GKS, GKAA) showed increased expression of the reporter protein BG46_variant 2 subtilisin (SEQ ID NO: 8) compared to the proregion sequence C (GK; SEQ ID NO: 14).

[0143] Likewise, certain C-terminal modifications were made to the pro-region sequences as described in Example 7. In particular, variant pro-region sequence B (SEQ ID NO: 11) was further engineered to replace the leucine (L) residue at position sixty-eight (68) with lysine (K), and the isoleucine (I) at position seventy-two (72) with valine (V), thereby generating variant pro-region sequence G (SEQ ID NO: 32), or was engineered to replace the leucine (L) at position sixty-eight (68) with lysine (K), and the glutamic acid (E) at position seventy-two (80) with isoleucine (I), thereby generating variant pro-region sequence F (SEQ ID NO: 33), as described in Example 7. Figure 1-Figure 3 As presented in Table 5, the PI values ​​of the variant proregion sequences G (SEQ ID NO: 32) and H (SEQ ID NO: 33) were increased compared to the reference (control) variant proregion B sequence (SEQ ID NO: 11).

[0144] Therefore, certain embodiments of the present disclosure provide, inter alia, recombinant polynucleotides encoding novel pro-region sequences, expression cassettes comprising DNA sequences encoding novel pro-region sequences operably linked to DNA sequences encoding mature target proteins, modified Gram-positive bacterial cells / strains expressing polynucleotides encoding precursor proteins comprising novel pro-region sequences, etc. In certain aspects, the novel variant (mutant) pro-region sequences of the present disclosure comprise an amino acid sequence derived from the wild-type Bacillus lentus pro-sequence of SEQ ID NO: 15.

[0145] As used herein, the phrase "variant proregion" refers to a polypeptide sequence derived from a reference proregion sequence, typically by recombinant DNA technology, by substitution, addition, or deletion of one or more amino acids. The variant proregion (amino acid) sequence may differ from the reference (parent) proregion sequence by a small amount of amino acid residues, and can be defined by their primary amino acid sequence homology / identity level with the reference (parent) proregion sequence.

[0146] As described above, in the context of two polynucleotides or polypeptide sequences, the term "identity" refers to the nucleotides or amino acids in the two sequences that are identical when the maximum correspondence is aligned, as measured using a sequence comparison or analysis algorithm described below and known in the art. The phrase "percent identity (%)" (abbreviated as "PID") refers to polynucleotide (nucleic acid) or polypeptide (amino acid) sequence identity. The percent identity can be determined using standard techniques known in the art. In some aspects, the percent amino acid identity shared by the target sequence can be determined by comparing the sequence information directly, such as by using alignment programs / algorithms such as BLAST, MUSCLE, or CLUSTAL. For example, Altschul et al. (1990) and Karlin et al. (1993) describe the BLAST algorithm. In particular, the amino acid sequence identity percentage (%) value is determined by the number of matching identical residues divided by the total number of residues of the "reference" sequence (including any gaps created by the program for the best / maximum alignment). The BLAST algorithm refers to the "reference" sequence as the "query" sequence.

[0147] As used herein, "homologous proregion" refers to a proregion with different similarities in primary, secondary and / or tertiary structures. When comparing proteins, protein homology can refer to the similarity of linear amino acid sequences. Homology can be determined by, for example, comparing the amino acid sequences using programs such as BLAST, MUSCLE or CLUSTAL. Homology searches for protein sequences can be performed using BLASTP and PSI-BLAST from NCBI BLAST using a threshold value (E value cutoff) of 0.001 (e.g., see Altschul et al., 1997).

[0148] The BLAST program uses several search parameters, most of which are set to default values. The NCBI BLAST algorithm finds the most related sequences in terms of biological similarity, but is not recommended for query sequences of less than 20 residues (Altschul et al., 1997 and Schaffer et al., 2001). Exemplary default BLAST parameters for nucleic acid sequence searches include: Neighborhood Word Length Threshold = 11; E-value Cutoff = 10; Scoring Matrix = NUC.3.1 (match = 1, mismatch = -3); Gap Open = 5; and Gap Extension = 2. Exemplary default BLAST parameters for amino acid sequence searches include: Word Length = 3; E-value Cutoff = 10; Scoring Matrix = BLOSUM62; Gap Open = 11; and Gap Extension = 1. Using this information, protein sequences can be grouped and / or phylogenetic trees can be constructed therefrom. Amino acid sequences can be entered in programs such as the Vector NTI Advance suite, and a guide tree can be created using the Neighbor Joining (NJ) method (Saitou and Nei, 1987). The tree structure can be calculated using the Kimura correction for sequence distances and ignoring positions with gaps. Programs such as AlignX can display the calculated distance values ​​in brackets after the molecule name displayed on the phylogenetic tree.

[0149] Understanding homology between molecules can reveal information about the evolutionary history of the molecules as well as their functions; if a newly sequenced protein is homologous to an already characterized protein, there is a strong indication of the biochemical function of the new protein. If two molecules are derived from a common ancestor, they are said to be homologous. Homologous molecules or homologs can be divided into two categories: paralogs and orthologs. Paralogs are homologs that exist within one species. Paralogs often differ in their detailed biochemical functions. Orthologs are homologs that exist in different species and have very similar or identical functions. Protein superfamily is the largest grouping (clade) of proteins for which a common ancestor can be inferred. Usually this common ancestor is based on sequence alignments and mechanistic similarities.

[0150] The CLUSTAL W algorithm is another example of a sequence alignment algorithm (Thompson et al., 1994). The default parameters of the CLUSTAL W algorithm include: Gap Open Penalty = 10.0; Gap Extension Penalty = 0.05; Protein Weight Matrix = BLOSUM Series; DNA Weight Matrix = IUB; Delayed Divergent Sequence % = 40; Gap Separation Distance = 8; DNA Transition Weight = 0.50; List Hydrophilic Residues = GPSNDQEKR; Use Negative Matrix = Off; Switch Special Residue Penalty = On; Switch Hydrophilic Penalty = On; and Switch End Gap Separation Penalty = Off. In the CLUSTAL algorithm, deletions occurring at either end are included. For example, a variant having five amino acid deletions at either end of a 500 amino acid polypeptide (or within a polypeptide) will have a sequence identity percentage of 99% (495 / 500 identical residues × 100) relative to a "reference" polypeptide. Such a variant will be covered by a variant having "at least 99% sequence identity" with the polypeptide.

[0151] In certain embodiments, the variant pre-region sequence has at least about 40% to about 70%, at least 75%, at least 80%, at least 85%, 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%, or even at least 99% amino acid sequence identity with a reference (parent) pre-region sequence of the present disclosure. In certain embodiments, the pre-region sequence is derived from an amino acid sequence comprising homology with SEQ ID NO: 15, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32 and / or SEQ ID NO: 33. Thus, in certain aspects, the amino acid modifications of one or more pro-region variants described herein are numbered by reference to the pro-region amino acid sequence of SEQ ID NO:15, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 and / or SEQ ID NO:33.

[0152] In certain aspects, the novel (variant) proregion sequence is derived from a parent (reference) proregion sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 15. In certain embodiments, the variant proregion sequence comprises a mutation at amino acid (residue) position 30 and one or more amino acid residue positions selected from the group consisting of the following positions, according to the SEQ ID NO: 15 amino acid (position) numbering: 1, 2, 3, 4, 6, 14, 16, 19, 20, 23, 36, 37, 38, 39, 42, 43, 44, 49, 50, 64, 65, 67, 68, 71, 79, 83 and 84.

[0153] In other embodiments, the variant proregion sequences of the present disclosure comprise amino acid insertions of glycine (G) at position 2 and lysine (K) at position 3, and amino acid substitutions at one or more positions selected from positions 1, 32, 38, 46, 66, 67, 70, and 73, wherein the amino acid positions are numbered according to SEQ ID NO: 14. In certain embodiments, the novel (variant) proregion sequence is derived from a reference proregion sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 14.

[0154] In other embodiments, the variant proregion sequences of the present disclosure comprise an amino acid insertion of glycine (G) at position 2, lysine (K) at position 3, and serine (S) at position 4, wherein the amino acid positions are numbered according to SEQ ID NO: 29. In certain embodiments, the novel (variant) proregion sequence is derived from a reference proregion sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 29.

[0155] In other embodiments, the variant proregion sequence of the present disclosure comprises an amino acid insertion of glycine (G) at position 2, lysine (K) at position 3, and alanine (A) at position 4, wherein the amino acid positions are numbered according to SEQ ID NO: 30. In certain embodiments, the variant proregion sequence is derived from a reference proregion sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 30.

[0156] In other embodiments, the variant proregion sequence of the present disclosure comprises an amino acid insertion of glycine (G) at position 2, lysine (K) at position 3, alanine (A) at position 4, and alanine (A) at position 5, wherein the amino acid positions are numbered according to SEQ ID NO: 31. In certain embodiments, the variant proregion sequence is derived from a reference proregion sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 31.

[0157] In certain other embodiments, the variant pro-region sequence is derived from a parent (reference) pro-region sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with the amino acid sequence of SEQ ID NO:11 (G30; pro-region B sequence).

[0158] In certain embodiments, the variant proregion sequence comprises at least one amino acid substitution at a position selected from position 68, position 72, and position 80, wherein the amino acid positions are numbered according to SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:32, or SEQ ID NO:33.

[0159] In certain other embodiments, the variant proregion sequence comprises a mutation at amino acid (residue) position 30 and one or more amino acid residue positions selected from the group consisting of: 1, 2, 3, 4, 6, 14, 16, 19, 20, 23, 36, 37, 38, 39, 42, 43, 44, 49, 50, 64, 65, 67, 68, 71, 79, 83 and 84, wherein the amino acid (position) numbering is based on SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:32, or SEQ ID NO:33.

[0160] In certain embodiments, the variant pre-region sequence is designed, engineered, constructed, etc., such that the net charge of the pre-region sequence is positive (e.g., +1 net charge) relative to a reference (parent) pre-region sequence (e.g., SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33). In certain embodiments, the variant pre-region sequence of the present disclosure is designed, engineered, constructed, etc., such that the net charge of the pre-region sequence is at least positive 1 (+1) to about positive 4 (+4). In one or more other embodiments, the variant pre-region sequence of the present disclosure comprises one or more amino acid modifications shown in Table 1, Table 2, Table 3, Table 4, and / or Table 5.

[0161] III. Recombinant Polynucleotides and Molecular Biology

[0162] As generally described above, some embodiments of the present disclosure relate to novel mutation front region sequences. In related aspects, the present disclosure provides recombinant polynucleotides, which include one or more mutation front region nucleic acid (DNA) sequences. Therefore, some embodiments relate to recombinant polynucleotides (e.g., vectors, plasmids, expression cassettes, etc.), recombinant gram-positive bacterial cells / strains expressing target proteins, etc. In some aspects, the present disclosure provides polynucleotide constructs suitable for introducing into recombinant gram-positive bacterial cells (strains) for enhancing target protein production. In some aspects, the polynucleotide constructs of the present disclosure are referred to as expression cassettes, wherein the cassette is in the 5' to 3' direction and includes at least an upstream (5') front region DNA sequence in an operable combination, and the front region DNA sequence is connected to the downstream (3') gene CDS encoding a mature target protein (POI).

[0163] For example, one or more nucleotide sequences described herein can be produced by using any suitable synthesis, operation and / or separation technology or its combination.For example, one or more polynucleotides described herein can be produced using standard nucleic acid synthesis techniques well known to those skilled in the art such as solid phase synthesis techniques.In such technology, typically synthesize up to fifty (50) or more fragments of nucleotide bases, then connect (for example, by enzyme or chemical connection method) to form any desired continuous nucleic acid sequence basically.It is also possible to promote the synthesis of one or more polynucleotides described herein by any suitable method known in the art, including but not limited to the chemical synthesis using the following method: classical phosphoramidite method (for example, Beaucage and Caruthers, 1981), or as typically in the method described by Matthes et al. (1984) practiced in automatic synthesis method.One or more polynucleotides described herein can also be produced by using automatic DNA synthesizer. Custom nucleic acids can be ordered from various commercial sources (e.g., ATUM (DNA 2.0), Newark, CA, USA; Life Tech (GeneArt), Carlsbad, CA, USA; GenScript, Ontario, Canada; Base Clear BV, Leiden, Netherlands; Integrated DNA Technologies, Skokie, IL, USA; Ginkgo Bioworks (Gen9), Boston, MA, USA; and Twist Bioscience, San Francisco, CA, USA). Other techniques and related principles for synthesizing nucleic acids are described and known in the art.

[0164] Recombinant DNA techniques for modifying nucleic acids are well known in the art, such as restriction endonuclease digestion, ligation, reverse transcription and cDNA production and polymerase chain reaction (e.g., PCR). One or more polynucleotides described herein can also be obtained by screening a cDNA library using one or more oligonucleotide probes, which can be hybridized with a polynucleotide encoding one or more variants described herein or amplified by PCR. The procedures for screening and separating cDNA clones and PCR amplification procedures are well known to those skilled in the art and are described in standard references known to those skilled in the art. One or more polynucleotides described herein can be obtained by changing the naturally occurring polynucleotide backbone (e.g., encoding one or more variant pre-region sequences described herein) by, for example, known mutagenesis procedures (e.g., site-directed mutagenesis, site-saturation mutagenesis, and in vitro recombination). A variety of methods suitable for generating modified polynucleotides described herein encoding one or more variants described herein are known in the art, including, but not limited to, site-saturation mutagenesis, scanning mutagenesis, insertion mutagenesis, deletion mutagenesis, random mutagenesis, site-directed mutagenesis and directed evolution, and various other recombination methods.

[0165] As described generally above and further described in the examples below, certain embodiments of the present disclosure relate to recombinant (modified) Gram-positive cells capable of producing an increased amount of heterologous target protein. Therefore, certain embodiments relate to methods for constructing such recombinant Gram-positive cells with increased protein production capacity. In certain embodiments, one or more expression cassettes encoding the target protein are introduced into the Gram-positive cells of the present disclosure. In an exemplary embodiment, these cassettes are integrated into the genome of the cell. Therefore, certain embodiments relate to nucleic acid molecules, polynucleotides (e.g., vectors, plasmids, expression cassettes), regulatory elements, etc., suitable for constructing recombinant (modified) Gram-positive host cells.

[0166] Thus, as shown in the examples and generally described herein, recombinant cells of the present disclosure can be constructed by those skilled in the art using standard and conventional recombinant DNA and molecular cloning techniques well known in the art. Methods for genetic modification include, but are not limited to, (a) introducing, replacing, or removing one or more nucleotides in a gene, or introducing, replacing, or removing one or more nucleotides in a regulatory element required for transcription or translation of a gene, (b) gene disruption, (c) gene conversion, (d) gene deletion, (e) gene downregulation, (f) site-specific mutagenesis, and / or (g) random mutagenesis.

[0167] In certain embodiments, the modified cells of the present disclosure can be constructed by reducing or eliminating the expression of a gene using methods well known in the art (e.g., insertion, disruption, substitution or deletion). The portion of the gene to be modified or inactivated can be, for example, a coding region or a regulatory element required for expression of the coding region.

[0168] Examples of such regulatory or control sequences may be promoter sequences or functional portions thereof (i.e., portions sufficient to affect the expression of the nucleic acid sequence). Other control sequences for modification include, but are not limited to, leader sequences, propeptide sequences, signal sequences, transcription terminators, transcription activators, and the like.

[0169] In certain other embodiments, modified cells are constructed by gene deletion to eliminate or reduce the expression of genes.Gene deletion technology enables partial or complete removal of one or more genes, thereby eliminating their expression, or expressing non-functional (or reduced activity) protein products.In such methods, the deletion of one or more genes can be completed by homologous recombination using a plasmid constructed to continuously contain 5' and 3' districts flanking the gene.Continuous 5' and 3' districts can be introduced into cells, for example, on a temperature-sensitive plasmid, combined with a second selectable marker at a permissive temperature to allow the plasmid to be established in the cell.Then the cell is transferred to a non-permissive temperature to select cells that integrate the plasmid into one of the homologous flanking regions of the chromosome.The selection of plasmid integration is affected by selecting a second selectable marker.After integration, the recombination event at the second homologous flanking region is stimulated by moving the cell to a permissive temperature for several generations without selecting.Cells are plated to obtain single colonies, and it is checked whether the colony loses two selectable markers.Therefore, those skilled in the art can easily identify the nucleotide region (suitable for complete or partial deletion) in the coding sequence of the gene and / or the non-coding sequence of the gene.

[0170] In other embodiments, modified cells are constructed by introducing, replacing or removing one or more nucleotides in a gene or a regulatory element required for its transcription or translation. For example, nucleotides can be inserted or removed to cause the introduction of a stop codon, the removal of a start codon or a frameshift of an open reading frame. Such modifications can be accomplished by site-directed mutagenesis or PCR-generated mutagenesis according to methods known in the art. Therefore, in certain embodiments, a gene of the present disclosure is inactivated by complete or partial deletion.

[0171] In another embodiment, modified cells are constructed by gene conversion processes. For example, in a gene conversion method, a nucleic acid sequence corresponding to one or more genes is induced in vitro to produce a defective nucleic acid sequence, which is then transformed into a parental cell to produce a defective gene. By homologous recombination, the defective nucleic acid sequence replaces the endogenous gene. It may be desirable that the defective gene or gene fragment also encodes a marker that can be used to select a transformant containing the defective gene. For example, the defective gene can be associated with a selectable marker and introduced into a non-replicating or temperature-sensitive plasmid. The selection of plasmid integration is affected by selecting a marker under conditions that do not allow plasmid replication. The selection of a second recombination event that causes gene replacement is affected by checking whether the bacterium colony loses the selectable marker and whether the mutated gene is obtained. Alternatively, the defective nucleic acid sequence can contain the insertion, substitution or deletion of one or more nucleotides of a gene, as described below.

[0172] In other embodiments, modified cells are constructed using nucleotide sequences complementary to the nucleotide sequence of the gene by the antisense technology established. More particularly, the expression of the gene of Gram-positive cells can be reduced (downregulated) or eliminated by introducing a nucleotide sequence complementary to the nucleotide sequence of the gene, which can be transcribed in the cell and can hybridize with the mRNA produced in the cell. Under conditions where the complementary antisense nucleotide sequence is allowed to hybridize with the mRNA, the amount of the translated protein is therefore reduced or eliminated. Such antisense methods include, but are not limited to, RNA interference (RNAi), small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotides, etc., all of which are well known to skilled artisans.

[0173] In other embodiments, modified cells are produced / constructed via CRISPR-Cas9 editing.For example, the gene encoding the target protein can be edited or destroyed (or missing or downregulated) by the endonuclease guided by nucleic acid, which is combined by guiding RNA (e.g., Cas9) and Cpf1 or guiding DNA (e.g., NgAgo) to find its target DNA, which recruits the endonuclease to the target sequence on DNA, wherein the endonuclease can produce single-strand or double-strand breaks in DNA. This targeted DNA break becomes a substrate for DNA repair and can be recombined with the editing template provided to destroy or delete the gene. For example, a gene encoding a nucleic acid-guided endonuclease (for this purpose, Cas9 from Streptococcus pyogenes) or a codon-optimized gene encoding a Cas9 nuclease is operably connected to a promoter active in Gram-positive cells and a terminator active in Gram-positive cells, thereby producing a Gram-positive cell Cas9 expression cassette. Similarly, one skilled in the art easily identifies one or more target sites unique to the target gene. For example, to construct a DNA construct encoding a gRNA-directed to a target site within a gene of interest, a variable targeting domain (VT) will contain nucleotides of the target site, which are located 5' of the protospacer sequence adjacent to the motif (TGG) of the (PAM) protospacer sequence, and these nucleotides are fused to the DNA encoding the Cas9 endonuclease recognition domain (CER) of Streptococcus pyogenes Cas9. The DNA encoding the VT domain is combined with the DNA encoding the CER domain to produce the DNA encoding the gRNA. Therefore, the Gram-positive expression cassette of the gRNA is produced by operably linking the DNA encoding the gRNA to a promoter active in a Gram-positive cell and a terminator active in a Gram-positive cell.

[0174] In certain embodiments, DNA breaks induced by endonucleases are repaired / replaced with input sequences. For example, in order to accurately repair DNA breaks generated by the above-mentioned Cas9 expression cassette and gRNA expression cassette, a nucleotide editing template is provided so that the cell's DNA repair machinery can utilize the editing template. For example, about 500 bp 5' of the target gene can be fused to about 500 bp 3' of the target gene to generate an editing template that is used by the Gram-positive host machinery to repair DNA breaks generated by the RGEN.

[0175] Cas9 expression cassette, gRNA expression cassette and editing template can be delivered to filamentous fungal cells using many different methods (e.g., protoplast fusion, electroporation, natural competence or induced competence). By amplifying the locus with forward and reverse primers, the transformed cells are screened by amplifying the locus of the target gene by PCR. These primers can amplify the wild-type locus or the modified locus edited by RGEN. These fragments are then sequenced using sequencing primers to identify edited bacterium colonies.

[0176] In other embodiments, modified cells are constructed by random or specific mutagenesis using methods well known in the art (including but not limited to chemical mutagenesis and transposition). Gene modification can be performed by subjecting the parental cell to mutagenesis and screening for mutant cells whose gene expression has been reduced or eliminated. Mutagenesis that can be specific or random can be performed, for example, by using a suitable physical or chemical mutagen, using a suitable oligonucleotide, or subjecting the DNA sequence to mutagenesis produced by PCR. In addition, mutagenesis can be performed using any combination of these mutagenesis methods.

[0177] Examples of physical or chemical mutagens suitable for the purposes of the present invention include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-methyl-N'-nitrosoguanidine (NTG), O-methylhydroxylamine, nitrous acid, ethyl methanesulfonate (EMS), sodium bisulfite, formic acid, and nucleotide analogs. When such agents are used, mutagenesis is typically performed by incubating the parental cells to be mutagenized under appropriate conditions in the presence of the selected mutagen, and selecting mutant cells that exhibit reduced or no expression of the gene.

[0178] PCT Publication No. WO 2003 / 083125 discloses methods for modifying Gram-positive (Bacillus) cells, such as using PCR fusion to generate Bacillus deletion strains and DNA constructs to bypass E. coli. PCT Publication No. WO 2002 / 14490 discloses methods for modifying Bacillus cells, which methods include (1) construction and transformation of an integrating plasmid (pComK), (2) random mutagenesis of coding sequences, signal sequences, and propeptide sequences, (3) homologous recombination, (4) improving transformation efficiency by adding non-homologous flanks to the transforming DNA, (5) optimizing double crossover integration, (6) site-directed mutagenesis, and (7) markerless deletion.

[0179] Suitable methods for introducing polynucleotide sequences into bacterial cells (e.g., Gram-negative cells, Gram-positive cells) are well known to those skilled in the art. In fact, methods such as transformation, including protoplast transformation and mid-plate collection, transduction, and protoplast fusion, are known and suitable for use in the present disclosure. Transformation methods are particularly preferably used to introduce the DNA construct of the present disclosure into host cells.

[0180] In addition to common methods, in some embodiments, host cells are directly transformed (i.e., before introducing the host cells, no intermediate cells are used to amplify the DNA construct or otherwise process the DNA construct). The introduction of the DNA construct into the host cell includes those physical and chemical methods known in the art for introducing DNA into the host cell without inserting into a plasmid or a vector. Such methods include but are not limited to calcium chloride precipitation, electroporation, naked DNA, liposomes, etc. In other embodiments, the DNA construct is co-transformed with a plasmid without inserting into the plasmid. In other embodiments, by methods known in the art, a selective marker is deleted or substantially excised from a modified Bacillus strain. In certain embodiments, the carrier is decomposed from the host chromosome, and the flanking region is left on the chromosome, while the intrinsic chromosome region is removed.

[0181] Promoters and promoter sequence regions for expressing genes, their coding sequences (CDS), open reading frames (ORFs) and / or variant sequences in Gram-positive cells are generally known to those skilled in the art. Promoter sequences of the present disclosure are generally selected so that they function in Gram-positive cells. For example, promoters that can be used to drive gene expression in Bacillus cells include, but are not limited to, the subtilis alkaline protease (aprE) promoter, the alpha-amylase promoter (amyE) of Bacillus subtilis, the alpha-amylase promoter (amyL) of Bacillus licheniformis, the alpha-amylase promoter of Bacillus amyloliquefaciens, the neutral protease (nprE) promoter from Bacillus subtilis, the mutant aprE promoter, or any other promoter from Bacillus licheniformis or other related Bacillus. Methods for screening and generating promoter libraries with a range of activities (promoter strengths) in Bacillus cells are described in Publication No. WO 2002 / 14490.

[0182] IV. Fermentation of Gram-positive cells for protein production

[0183] As generally described above, certain embodiments relate to compositions and methods for constructing and obtaining Gram-positive cells with increased protein production phenotypes. Therefore, certain embodiments relate to methods for producing target proteins in Gram-positive cells by fermenting cells in suitable culture media. Fermentation methods well known in the art can be used to ferment Gram-positive cells of the present disclosure.

[0184] In certain embodiments, cells are cultured under batch or continuous fermentation conditions. Classical batch fermentation is a closed system, wherein the composition of the culture medium is set at the beginning of fermentation, and the composition does not change during fermentation. At the beginning of fermentation, one or more desired organisms are inoculated into the culture medium. In this method, fermentation is allowed to occur without adding any component to the system. Typically, batch fermentation meets the qualification of "batch" about adding a carbon source, and control factors (such as pH and oxygen concentration) are often attempted. The metabolites and biomass composition of the batch system are constantly changing until the fermentation stops. In typical batch culture, cells can progress to the high growth logarithmic phase through a static lag phase, and finally enter a stationary phase where the growth rate decreases or stops. If not treated, the cells in the stationary phase eventually die. Usually, the cells in the logarithmic phase are responsible for the mass production of products.

[0185] A suitable variation of a standard batch system is a "fed-batch" fermentation system. In this variation of a typical batch system, substrate is added in increments as the fermentation progresses. Fed-batch systems are useful when catabolite repression may inhibit the metabolism of the cell and when it is desired to have a limited amount of substrate in the culture medium. The measurement of actual substrate concentration in a fed-batch system is difficult and is therefore estimated based on the variation of measurable factors such as pH, dissolved oxygen and the partial pressure of waste gases such as CO2. Batch and fed-batch fermentation are commonly used and known in the art.

[0186] Continuous fermentation is an open system, wherein the fermentation medium of limitation is continuously added to the bioreactor, and an equal amount of conditioned medium is removed simultaneously for processing. Continuous fermentation usually maintains the culture at a constant high density, wherein the cell is mainly in the logarithmic phase growth. Continuous fermentation allows one or more factors affecting cell growth and / or product concentration to be regulated. For example, in one embodiment, limiting nutrients (such as carbon source or nitrogen source) are maintained at a fixed rate, and all other parameters are allowed to be regulated. In other systems, many factors affecting growth can be constantly changed, and the cell concentration measured by the turbidity of the culture medium remains unchanged. Continuous systems strive to maintain steady-state growth conditions. Therefore, the cell loss caused by extracting the culture medium should be balanced with the cell growth rate in the fermentation. The method for adjusting the nutrients and growth factors used for continuous fermentation technology and the technology for maximizing the product formation rate are well known in the field of industrial microbiology.

[0187] In certain embodiments, the target protein expressed / produced by the Gram-positive cells of the present disclosure can be recovered from the culture medium by conventional procedures, including separation of host cells from the culture medium by centrifugation or filtration, or, if necessary, destruction of the cells and removal of the supernatant from the cell parts and debris. Typically, after clarification, the protein component of the supernatant or filtrate is precipitated with the aid of a salt (e.g., ammonium sulfate). The precipitated protein is then dissolved and can be purified by a variety of chromatographic procedures (e.g., ion exchange chromatography, gel filtration).

[0188] In certain embodiments, cells are cultured under batch or continuous fermentation conditions. Classical batch fermentation is a closed system, wherein the composition of the culture medium is set at the beginning of fermentation, and the composition does not change during fermentation. At the beginning of fermentation, one or more desired organisms are inoculated into the culture medium. In this method, fermentation is allowed to occur without adding any component to the system. Typically, batch fermentation meets the qualification of "batch" about adding a carbon source, and control factors (such as pH and oxygen concentration) are often attempted. The metabolites and biomass composition of the batch system are constantly changing until the fermentation stops. In typical batch culture, cells can progress to the high growth logarithmic phase through a static lag phase, and finally enter a stationary phase where the growth rate decreases or stops. If not treated, the cells in the stationary phase eventually die. Usually, the cells in the logarithmic phase are responsible for the mass production of products.

[0189] A suitable variation of a standard batch system is a "fed-batch" fermentation system. In this variation of a typical batch system, substrate is added in increments as the fermentation progresses. Fed-batch systems are useful when catabolite repression may inhibit the metabolism of the cell and when it is desired to have a limited amount of substrate in the culture medium. The measurement of actual substrate concentration in a fed-batch system is difficult and is therefore estimated based on the variation of measurable factors such as pH, dissolved oxygen and the partial pressure of waste gases such as CO2. Batch and fed-batch fermentation are commonly used and known in the art.

[0190] Continuous fermentation is an open system, wherein the fermentation medium of limitation is continuously added to the bioreactor, and an equal amount of conditioned medium is removed simultaneously for processing. Continuous fermentation usually maintains the culture at a constant high density, wherein the cell is mainly in the logarithmic phase growth. Continuous fermentation allows one or more factors affecting cell growth and / or product concentration to be regulated. For example, in one embodiment, limiting nutrients (such as carbon source or nitrogen source) are maintained at a fixed rate, and all other parameters are allowed to be regulated. In other systems, many factors affecting growth can be constantly changed, and the cell concentration measured by the turbidity of the culture medium remains unchanged. Continuous systems strive to maintain steady-state growth conditions. Therefore, the cell loss caused by extracting the culture medium should be balanced with the cell growth rate in the fermentation. The method for adjusting the nutrients and growth factors used for continuous fermentation technology and the technology for maximizing the product formation rate are well known in the field of industrial microbiology.

[0191] In certain embodiments, the target protein expressed / produced by the Gram-positive cells of the present disclosure can be recovered from the culture medium by conventional procedures, including separation of host cells from the culture medium by centrifugation or filtration, or, if necessary, destruction of the cells and removal of the supernatant from the cell parts and debris. Typically, after clarification, the protein component of the supernatant or filtrate is precipitated with the aid of a salt (e.g., ammonium sulfate). The precipitated protein is then dissolved and can be purified by a variety of chromatographic procedures (e.g., ion exchange chromatography, gel filtration).

[0192] V. Target protein

[0193] The protein of interest (POI) of the present disclosure may be any endogenous or heterologous protein, and it may be a variant of such a POI. The protein may contain one or more disulfide bridges, or may be a protein whose functional form is a monomer or a multimer, i.e., the protein has a quaternary structure and is composed of a plurality of identical (homologous) or non-identical (heterologous) subunits, wherein the POI or its variant POI is preferably a POI having a desired property.

[0194] For example, in certain embodiments, the modified Gram-positive cells of the present disclosure produce at least about 0.1% more, at least about 0.5% more, at least about 1% more, at least about 5% more, at least about 6% more, at least about 7% more, at least about 8% more, at least about 9% more, or at least about 10% more, or more, POI relative to their unmodified (reference or control) cells.

[0195] In certain embodiments, the modified Gram-positive cells of the present disclosure exhibit an increase in the specific productivity (Qp) of the POI relative to the control cells. For example, the detection of specific productivity (Qp) is a suitable method for evaluating protein production. The specific productivity (Qp) can be determined using the following equation:

[0196] “Qp=gP / gDCW·hr”

[0197] Wherein "gP" is grams of protein produced in the tank; "gDCW" is grams of dry cell weight (DCW) in the tank; and "hr" is the fermentation time in hours from the inoculation time, which includes production time as well as growth time.

[0198] Thus, in certain other embodiments, the modified Gram-positive cells of the present disclosure comprise an increase in specific productivity (Qp) of at least about 0.1%, at least about 1%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, or at least about 10% or more relative to unmodified (parent) cells.

[0199] In certain embodiments, the POI or its variant POI is selected from the group consisting of: acetylesterase, aminopeptidase, amylase, arabinanase, arabinofuranosidase, carbonic anhydrase, carboxypeptidase, catalase, cellulase, chitinase, rennetinase, cutinase, deoxyribonuclease, epimerase, esterase, α-galactosidase, β-galactosidase, α-glucanase, glucan lyase, endo-β-glucanase, glucoamylase, glucose oxidase, α-glucosidase, β-glucosidase, glucuronidase, glycosyl Hydrolases, hemicellulases, hexose oxidases, hydrolases, invertases, isomerases, laccases, ligases, lipases, lyases, mannosidases, oxidases, oxidoreductases, pectate lyases, pectin acetylesterases, pectin depolymerases, pectin methylesterases, pectinolytic enzymes, perhydrolases, polyol oxidases, peroxidases, phenoloxidases, phytases, polygalacturonases, proteases, peptidases, rhamnogalacturonases, ribonucleases, transferases, transporters, transglutaminases, xylanases, hexose oxidases, and combinations thereof.

[0200] Thus, in certain embodiments, the POI or variant POI thereof is an enzyme selected from the group consisting of Enzyme Commission (EC) numbers EC 1, EC 2, EC 3, EC 4, EC 5, or EC 6.

[0201] There are various assays known to those of ordinary skill in the art for detecting and measuring the activity of proteins expressed intracellularly and extracellularly.

[0202] VI. Exemplary Embodiments

[0203] Non-limiting examples of the compositions and methods disclosed herein are as follows:

[0204] 1. A variant proregion sequence comprising an amino acid substitution at position 30 and one or more positions selected from 1, 2, 3, 4, 6, 14, 16, 19, 20, 23, 36, 37, 38, 39, 42, 43, 44, 49, 50, 64, 65, 67, 68, 71, 79, 83 and 84, wherein the amino acid positions of the variant proregion are numbered according to SEQ ID NO: 15.

[0205] 2. A variant proregion as described in Example 1, which is derived from a parent polypeptide or reference polypeptide having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with positions 1-84 of SEQ ID NO:15.

[0206] 3. A variant proregion sequence comprising an amino acid insertion of glycine (G) at position 2 and lysine (K) at position 3, and an amino acid substitution at one or more positions selected from 1, 32, 38, 46, 66, 67, 70 and 73, wherein the amino acid positions of the variant proregion are numbered according to SEQ ID NO:14.

[0207] 4. A variant proregion as described in Example 3, which is derived from a parent polypeptide or reference polypeptide having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with positions 1-86 of SEQ ID NO:14.

[0208] 5. A variant proregion sequence comprising amino acid insertions of glycine (G) at position 2, lysine (K) at position 3, and alanine (A) at position 4, wherein the amino acid positions of the variant proregion are numbered according to SEQ ID NO: 30.

[0209] 6. A variant proregion as described in Example 5, which is derived from a parent polypeptide or reference polypeptide having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with positions 1-87 of SEQ ID NO:30.

[0210] 7. A variant proregion sequence comprising amino acid insertions of glycine (G) at position 2, lysine (K) at position 3, and serine (S) at position 4, wherein the amino acid positions of the variant proregion are numbered according to SEQ ID NO: 29.

[0211] 8. A variant proregion as described in Example 7, which is derived from a parent polypeptide or reference polypeptide having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with positions 1-87 of SEQ ID NO:29.

[0212] 9. A variant proregion sequence comprising amino acid insertions of glycine (G) at position 2, lysine (K) at position 3, alanine (A) at position 4, and alanine (A) at position 5, wherein the amino acid positions of the variant proregion are numbered according to SEQ ID NO:31.

[0213] 10. The variant proregion of Example 9, which is derived from a parent polypeptide or reference polypeptide having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with positions 1-88 of SEQ ID NO:31.

[0214] 11. A variant pro-region sequence comprising a glutamic acid (E) to glycine (G) substitution at position 30 (E30G), a leucine (L) to lysine (K) substitution at position 68 (L68K), and an isoleucine (I) to valine (V) substitution at position 73 (I72V), wherein the amino acid positions of the variant pro-region are numbered according to SEQ ID NO:32.

[0215] 12. A variant proregion as described in Example 11, which is derived from a parent polypeptide or reference polypeptide having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with positions 1-84 of SEQ ID NO:15 or SEQ ID NO:32.

[0216] 13. A variant pro-region sequence comprising a glutamic acid (E) to glycine (G) substitution at position 30 (E30G), a leucine (L) to lysine (K) substitution at position 68 (L68K), and a glutamic acid (E) to isoleucine (I) substitution at position 80 (E80I), wherein the amino acid positions of the variant pro-region are numbered according to SEQ ID NO:33.

[0217] 14. A variant proregion as described in Example 13, which is derived from a parent polypeptide or reference polypeptide having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with positions 1-84 of SEQ ID NO:15 or SEQ ID NO:33.

[0218] 15. A nucleic acid encoding the variant proregion of any one of embodiments 1-14.

[0219] 16. A polynucleotide comprising the variant pro-region nucleic acid as described in Example 15.

[0220] 17. A polynucleotide comprising an upstream (5') nucleic acid encoding the variant proregion of any one of embodiments 1-14, wherein the upstream nucleic acid is operably linked to a downstream (3') nucleic acid sequence encoding a protein of interest (POI).

[0221] 18. A polynucleotide comprising an upstream (5′) nucleic acid encoding a protein signal (secretion) sequence, the upstream nucleic acid being operably linked to a downstream (3′) nucleic acid sequence encoding a variant pro-region as described in any one of Examples 1-14, the downstream nucleic acid sequence encoding a variant pro-region as described in any one of Examples 1-14 being operably linked to a downstream nucleic acid sequence encoding a target protein (POI).

[0222] 19. An expression cassette comprising an upstream (5') promoter region sequence operably linked to a downstream (3') polynucleotide as described in any one of embodiments 16-18.

[0223] 20. A Gram-positive host cell comprising an introduced polynucleotide of any one of embodiments 16-18 or an introduced cassette of embodiment 19.

[0224] 21. The variant proregion of embodiment 1, wherein the one or more amino acid substitutions increase the net charge of the variant proregion sequence relative to the reference proregion of SEQ ID NO: 15.

[0225] 22. The variant proregion of embodiment 3, wherein the one or more amino acid substitutions increase the net charge of the variant proregion sequence relative to the reference proregion of SEQ ID NO: 14.

[0226] 23. The variant proregion of Embodiment 21 or Embodiment 22, wherein the increase in net charge is at least 1 (+1) to 4 (+4).

[0227] 24. The variant proregion as described in Example 1, comprising the amino acid modifications shown in Table 1.

[0228] 25. The variant proregion of embodiment 1, wherein the amino acid substitution at position 30 is glycine (G), histidine (H) or asparagine (N).

[0229] 26. The variant proregion of embodiment 1, wherein the amino acid substitution at position 1 is threonine (T).

[0230] 27. The variant proregion of embodiment 1, wherein the amino acid substitution at position 2 is alanine (A) or proline (P).

[0231] 28. The variant proregion of embodiment 1, wherein the amino acid substitution at position 3 is alanine (A), cysteine ​​(C), glycine (G), arginine (R), serine (S) or valine (V).

[0232] 29. The variant proregion of embodiment 1, wherein the amino acid substitution at position 4 is glutamine (Q).

[0233] 30. The variant proregion of embodiment 1, wherein the amino acid substitution at position 6 is arginine (R).

[0234] 31. The variant proregion of embodiment 1, wherein the amino acid substitution at position 14 is threonine (T).

[0235] 32. The variant proregion of embodiment 1, wherein the amino acid substitution at position 19 is arginine (R).

[0236] 33. The variant proregion of embodiment 1, wherein the amino acid substitution at position 20 is lysine (K).

[0237] 34. The variant proregion of embodiment 1, wherein the amino acid substitution at position 23 is lysine (K), asparagine (N) or glutamine (Q).

[0238] 35. The variant proregion of Example 1, wherein the amino acid substitution at position 36 is glycine (G).

[0239] 36. The variant proregion of embodiment 1, wherein the amino acid substitution at position 37 is threonine (T).

[0240] 37. The variant proregion of embodiment 1, wherein the amino acid substitution at position 38 is histidine (H).

[0241] 38. The variant proregion of embodiment 1, wherein the amino acid substitution at position 39 is lysine (K) or glutamine (Q).

[0242] 39. The variant proregion of embodiment 1, wherein the amino acid substitution at position 44 is isoleucine (I).

[0243] 40. The variant proregion of embodiment 1, wherein the amino acid substitution at position 49 is threonine (T) or glutamine (Q).

[0244] 41. The variant proregion of embodiment 1, wherein the amino acid substitution at position 50 is arginine (R).

[0245] 42. The variant proregion of embodiment 1, wherein the amino acid substitution at position 64 is lysine (K).

[0246] 43. The variant proregion of embodiment 1, wherein the amino acid substitution at position 65 is lysine (K).

[0247] 44. The variant proregion of embodiment 1, wherein the amino acid substitution at position 67 is serine (S).

[0248] 45. The variant proregion of embodiment 1, wherein the amino acid substitution at position 68 is lysine (K) or methionine (M).

[0249] 46. ​​The variant proregion of embodiment 1, wherein the amino acid substitution at position 79 is glutamine (Q).

[0250] 47. The variant proregion of embodiment 1, wherein the amino acid substitution at position 83 is isoleucine (I).

[0251] 48. The variant proregion of embodiment 1, wherein the amino acid substitution at position 84 is leucine (L).

[0252] 49. The variant proregion of Example 3, comprising the amino acid modifications shown in Table 2, Table 3, Table 4, SEQ ID NO: 14, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 and combinations thereof.

[0253] 50. The variant proregion of embodiment 3, wherein the amino acid substitution at position 1 is threonine (T).

[0254] 51. The variant proregion of Example 3, wherein the amino acid substitution at position 32 is glycine (G).

[0255] 52. The variant proregion of Example 3, wherein the amino acid substitution at position 38 is glycine (G).

[0256] 53. The variant proregion of embodiment 3, wherein the amino acid substitution at position 46 is isoleucine (I).

[0257] 54. The variant proregion of embodiment 3, wherein the amino acid substitution at position 66 is lysine (K).

[0258] 55. The variant proregion of embodiment 3, wherein the amino acid substitution at position 67 is lysine (K).

[0259] 56. The variant proregion of embodiment 3, wherein the amino acid substitution at position 70 is lysine (K).

[0260] 57. The variant proregion of Example 3, wherein the amino acid substitution at position 73 is arginine (R).

[0261] 58. A variant front region comprising Table 1-5, Figure 1 , Figure 2 , Figure 3 , the amino acid modifications shown in any one of SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, and SEQ ID NO:33, and combinations thereof.

[0262] 59. The variant proregion of Example 58, which is derived from a parent polypeptide or reference polypeptide having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with positions 1-84 of SEQ ID NO:15.

[0263] 60. A method for producing a heterologous protein of interest (POI) in a Gram-positive bacterial cell, the method comprising (a) introducing an expression cassette into a Gram-positive cell, the expression cassette comprising an upstream (5′) promoter operably linked to a downstream (3′) nucleic acid encoding a variant pro-region sequence, the variant pro-region sequence comprising an amino acid substitution at position 30 and one or more positions selected from 1, 2, 3, 4, 6, 14, 16, 19, 20, 23, 36, 37, 38, 39, 42, 43, 44, 49, 50, 64, 65, 67, 68, 71, 79, 83 and 84, the downstream nucleic acid encoding the variant pro-region sequence being operably linked to a downstream (3′) nucleic acid sequence encoding the POI, wherein the amino acid positions of the variant pro-region are numbered according to SEQ ID NO: 15; and (b) growing / culturing / fermenting the modified cells under suitable conditions for producing the POI.

[0264] 61. A method for producing a heterologous protein of interest (POI) in a Gram-positive bacterial cell, the method comprising (a) introducing an expression cassette into a Gram-positive cell, the expression cassette comprising an upstream (5′) promoter sequence operably linked to a downstream (3′) nucleic acid encoding a variant pro-region sequence, the variant pro-region sequence comprising an amino acid insertion of glycine (G) at position 2, lysine (K) at position 3, and an amino acid substitution at one or more positions selected from 1, 32, 38, 46, 66, 67, 70, and 73, the downstream nucleic acid encoding the variant pro-region sequence being operably linked to a downstream (3′) nucleic acid sequence encoding the POI, wherein the amino acid positions of the variant pro-region are numbered according to SEQ ID NO: 14; and (b) growing / culturing / fermenting the modified cells under suitable conditions for producing the POI.

[0265] 62. A method for producing a heterologous protein of interest (POI) in a Gram-positive bacterial cell, the method comprising (a) introducing into the Gram-positive cell an expression cassette as described in Example 19, and (b) growing / culturing / fermenting the modified cell under suitable conditions for producing the POI.

[0266] 63. A method as described in any of embodiments 60-62, wherein the box further comprises a nucleic acid encoding a preprotein signal (secretion) sequence, which is operably linked and located between the promoter and the variant pre-region sequence.

[0267] 64. The method of any one of embodiments 60-62, wherein the modified cell produces an increased amount of the POI relative to a control Gram-positive cell having an introduced expression cassette comprising the same upstream (5′) promoter sequence operably linked to a downstream (3′) nucleic acid encoding a proregion sequence of SEQ ID NO: 15, wherein the downstream nucleic acid encoding the proregion sequence is operably linked to a downstream nucleic acid sequence encoding the same POI.

[0268] 65. A method as described in Example 64, wherein the increase in the POI relative to control cells is at least about 0.1% more, at least about 0.5% more, at least about 1% more, at least about 5% more, at least about 6% more, at least about 7% more, at least about 8% more, at least about 9% more, or at least about 10% more or more POI.

[0269] 66. The method of any one of embodiments 60-63, wherein the modified cell has a performance index (PI) value of at least about 1.1 relative to a control Gram-positive cell having an introduced expression cassette comprising the same upstream promoter sequence operably linked to a downstream nucleic acid encoding a proregion sequence of SEQ ID NO: 15, the downstream nucleic acid encoding the proregion sequence operably linked to a downstream nucleic acid sequence encoding the same POI as the modified cell, wherein the modified cell and control cells are cultured under the same conditions.

[0270] 67. The method of any one of embodiments 60-63, wherein the introduced cassette is integrated into the genome of the cell.

[0271] 68. The method of any one of embodiments 60-63, comprising at least two introduced boxes.

[0272] 69. The method of any one of embodiments 60-63, wherein the POI is selected from the group consisting of an enzyme, an antibody, a receptor, and a regulatory protein.

[0273] 70. The method of any one of embodiments 60-63, wherein the POI is selected from the group consisting of acetylesterase, aminopeptidase, amylase, arabinanase, arabinofuranosidase, carbonic anhydrase, carboxypeptidase, catalase, cellulase, chitinase, rennetinase, cutinase, deoxyribonuclease, epimerase, esterase, α-galactosidase, β-galactosidase, α-glucanase, glucan lyase, endo-β-glucanase, glucoamylase, glucose oxidase, α-glucosidase, β-glucosidase, glucuronidase, enzymes, such as amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids,

[0274] 71. The method of claim 70, wherein the protease is a native or variant subtilisin.

[0275] 72. The method of any one of claims 60-71, wherein the Gram-positive bacterial cell is a Bacillus sp. cell, optionally wherein the Bacillus sp. cell is selected from the group consisting of Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus clausii, Bacillus halodurans, Bacillus megaterium, Bacillus coagulans, Bacillus circulans, Bacillus radiata, Bacillus thuringiensis, and Geobacillus stearothermophilus.

[0276] Examples

[0277] Certain aspects of the present disclosure may be further understood from the following examples, which should not be construed as limiting. Modifications to materials and methods will be apparent to those skilled in the art. Standard recombinant DNA and molecular cloning techniques used herein are well known in the art (Ausubel et al., 1987; Sambrook et al., 1989).

[0278] Example 1

[0279] Modification of the GG36 PRO sequence by site evaluation library

[0280] In this example, a variant subtilisin protein (BG46_Variant 1; SEQ ID NO: 2) was used as a reporter protein to monitor protein expression as described herein. More specifically, a DNA fragment comprising the upstream (5') aprE gene flanking region is operably linked to a polynucleotide construct (e.g., an expression cassette) comprising a variant Bacillus subtilis rrnI-P2 promoter / 5'-aprE UTR region DNA sequence, thereby generating SEQ ID NO: 1, which is operably linked to a DNA sequence encoding an AprE signal sequence (SEQ ID NO: 4), which is operably linked to a DNA sequence (SEQ ID NO: 5) encoding a variant pre-region sequence A (SEQ ID NO: 9), which is operably linked to a DNA sequence encoding a mature BG46_variant 1 (SEQ ID NO: 2), which is operably linked to a DNA sequence encoding a mature BG46_variant 1 (SEQ ID NO: 6), and the polynucleotide construct is operably linked to a downstream (3') aprE gene flanking region sequence comprising a kanamycin (kan) gene expression cassette (SEQ ID NO: 7). More specifically, the DNA fragments are assembled using standard molecular biology techniques and used as templates to develop linear DNA expression cassettes comprising one or more of the proregion modifications (mutations) described herein.

[0281] Site Evaluation Library (SEL) of Pre-Sequence A

[0282] The amino acid sequence encoding the variant proregion sequence A (30H, SEQ ID NO: 9) was used as a template to create a site evaluation library (SEL) and has been developed as a 4.4 kb fragment by Twist Biosciences headquarters (South San Francisco). The linear DNA of the expression cassette was used to transform competent Bacillus subtilis cells, where the transformation mixture was plated onto LA plates containing 1.8 ppm kanamycin and incubated overnight at 37° C. Single colonies were picked and grown in Luria broth at 37° C. under antibiotic selection.

[0283] Sequence analysis was performed to identify unique Pro-Sequence A variants, which were carefully selected into a 96-well microtiter plate (MTP). For example, the first (1 st ) amino acid position is alanine ("Ala" or "A"), which can be changed (substituted) with all other nineteen (19) naturally occurring amino acid residues, and the second (2 nd) amino acid position is glutamic acid ("Glu" or "E"), it can be changed (substituted) to all other nineteen (19) naturally occurring amino acid residues, and so on, until all eighty-four (84) positions of the proregion sequence A (SEQ ID NO:9) have been evaluated (e.g., see Figure 1 ).

[0284] In the reporter protein expression experiment, the transformed cells were placed in a 96-well MTP, grown in culture medium (enriched semi-defined medium based on MOP buffer) at 32°C in a shaking incubator at 300 rpm and 80% humidity for 3 days, and centrifuged and filtered. The clarified culture supernatant was used to measure (assay) the reporter protease activity to determine the productivity level, wherein the sample was collected after seventy-two (72) hours. The reporter protease activity assay is further described in Example 2 below.

[0285] Location Affects Productivity

[0286] As described in the table below, Table 1 shows the results of relative reporter protein productivity compared to a reference construct in which the reporter protein gene is expressed with the native GG36 pro-region sequence (SEQ ID NO: 15), wherein expression is measured by the activity assay described in Example 2 below. For example, as shown in Table 1, the performance index (PI) values ​​for samples collected after seventy-two (72) hours are given (PI calculation is as described in Example 2), wherein the amino acid (residue) positions that affect productivity in conjunction with position 30 are 1, 2, 3, 4, 6, 14, 19, 20, 23, 36, 37, 38, 39, 42, 43, 44, 49, 50, 64, 65, 67, 68, 71, 83, 84. Changes in the 5' amino acid, as well as the 3' amino acid modification affected productivity, and in addition 80% of the selected combinations had an additional positive charge.

[0287] Table 1 Comparison of performance indexes between the pro-region variant A sequences and WT PRO

[0288]

[0289]

[0290] Example 2

[0291] Protease activity assay

[0292] The protease activity of BG46_variant 1 subtilisin was determined by measuring the hydrolysis of a synthetic suc-AAPF-pNA peptide substrate. For the AAPF assay, the reagent solution used was: 100 mM Tris pH 8.6, 10 mM CalCl2, 0.005% -80 (Tris / Ca buffer) and 160mM suc-AAPF-pNA in DMSO (suc-AAPF-pNA stock solution; Sigma: S-7388). To prepare the working solution, one (1) mL of suc-AAPF-pNA stock solution was added to 100 mL of Tris / Ca buffer and mixed. The enzyme sample was added to a microtiter plate (MTP) containing one (1) mg / mL suc-AAPF-pNA working solution, and the activity was determined kinetically at 405 nm at room temperature over three to five (3-5) minutes using a SpectraMax plate reader. The protease activity was expressed as mOD / minute. The activity of each constructed variant was measured and compared to a reference construct grown in the same plate. The reference sample value was divided by the variant sample value, i.e., the performance index (PI).

[0293] Example 3

[0294] Modification of 5' GG36 PRO sequence

[0295] In this example, BG46_variant 2 subtilisin (SEQ ID NO: 8) was used as a reporter protein to monitor expression as described herein. More specifically, a DNA fragment comprising the upstream (5') aprE gene flanking region is operably linked to a polynucleotide construct comprising a variant (5') Bacillus subtilis rrnI-P2 promoter / 5'-UTR aprE region DNA sequence, thereby generating SEQ ID NO: 1, which is operably linked to a DNA sequence encoding an AprE signal peptide sequence (SEQ ID NO: 4), which is operably linked to a DNA sequence encoding a variant proregion sequence B (SEQ ID NO: 9, 30H) (SEQ ID NO: 10), which is operably linked to a DNA sequence encoding a mature BG46_variant 2 subtilisin (SEQ ID NO: 8), which is operably linked to a DNA sequence encoding a mature BG46_variant 2 subtilisin (SEQ ID NO: 8), which is operably linked to a Bacillus amyloliquefaciens BPN' terminator DNA sequence (SEQ ID NO: 6), and the polynucleotide construct is operably linked to a kanamycin (kan) gene expression cassette (SEQ ID NO: 10). More specifically, the DNA fragments are assembled using standard molecular biology techniques and used as templates to develop linear DNA expression cassettes comprising one or more promoter region modifications described herein.

[0296] Introduction of 5' pro-region modification

[0297] The 5' (N-terminal) sequence of the seventy-eight (78) amino acid AprE proregion sequence (SEQ ID NO: 12) was used to exchange the first three (3) amino acid residues with the first amino acid residue (A) of the variant proregion sequence C (SEQ ID NO: 14), resulting in SEQ ID NO: 13. As shown in Table 1 above, mutants of the proregion variant A sequence (i.e., Table 1, "E030G"; SEQ ID NO: 11) showed high expression of the reporter protein. Expression constructs were prepared using standard molecular biology techniques and compared to the reference proregion. The variants (mutants) were developed as 4.4 kb linear DNA fragments and used to transform competent Bacillus subtilis cells, where the transformation mixture was plated onto LA plates containing 1.8 ppm kanamycin and incubated overnight at 37°C. Single colonies were picked and grown in Luria broth at 37°C under antibiotic selection.

[0298] In the reporter protein expression experiment, the transformed cells were placed in a 96-well MTP, grown in a shaking incubator at 300 rpm, 80% humidity at 32 ° C in a medium (enriched semi-defined medium based on MOP buffer) for 3 days, and centrifuged and filtered. The clarified culture supernatant was used to measure (measure) the reporter protease activity to determine the productivity level, wherein the sample was collected after 72 hours. The reporter protease activity assay was performed as described in Example 2 above, wherein the protein productivity of the variant front region C was about 1.1 times that of E030G-GG36-Pro (SEQ ID NO: 11).

[0299] Example 4

[0300] Further modification of the GG36 PRO sequence

[0301] In this example, BG46_variant 2 subtilisin (SEQ ID NO: 8) was used as a reporter protein to monitor expression as described herein. Construction was performed as described in Example 3 above. More specifically, a combinatorial library was prepared on a reference proregion sequence (SEQ ID NO: 15) in which the amino acids glycine (G) and lysine (K) were inserted at the 5' (N-terminal) sequence ("GK") (e.g., see Figure 2 , SEQ ID NO:14).

[0302] Combinatorial library of variants in the proregion C

[0303] Select from table 1 data to combine front region mutations, wherein use standard molecular biology techniques to prepare expression constructs and compare the constructs with reference front regions.Linear DNA expression cassettes are used to transform competent Bacillus subtilis cells, wherein the transformation mixture is plated on LA plates containing 1.8ppm kanamycin and incubated overnight at 37°C.Pick single colonies and make them grow in Luria broth at 37°C under antibiotic selection.In reporter protein expression experiments, the transformed cells are placed in 96-well MTPs, grown in a shaking incubator at 32°C with 300rpm, 80% humidity for 3 days in a culture medium (enriched semi-defined culture medium based on MOP buffer), and centrifuged and filtered.Use clarified culture supernatant to measure (measure) reporter protease activity to determine productivity levels, wherein samples are collected after 72 hours.Report protease activity assay as described in above-mentioned example 2.

[0304] Front-end and mid-end combination to improve productivity

[0305] As presented below, Table 2 discloses the sequence results of the pre-region mutation combinations showing increased productivity of the expressed reporter protein (SEQ ID NO: 8), wherein the delta (Δ) charge varies between +1 and +4. More particularly, the positive charge is combined as in the loop region (i.e., residue positions 66-73). The performance index (PI) values ​​of the samples collected after 72 hours are given, which are calculated as described above in Example 2. For example, the PI values ​​of these pre-region variants are compared with the WT pre-region sequence (Table 2, SEQ ID NO: 15), wherein the amino acid position number (Table 2, column 1) is compared with SEQ ID NO: 14, without the "GK" insertion. As Figure 3 As shown, variant proregion sequences can be numbered according to the position numbering of the reference variant proregion sequence C (SEQ ID NO: 14; comprising 86 amino acid positions) or the wild-type (reference) proregion sequence (SEQ ID NO: 15; comprising 84 amino acid positions).

[0306] Table 2

[0307] The sequence of the proregion variant C (SEQ ID NO: 14) was compared with the WT PRO without GK insertion (SEQ ID

[0308] NO:15) Performance index comparison

[0309]

[0310]

[0311] Example 5

[0312] Modification of the 5'GG36 PRO sequence of Bacillus licheniformis

[0313] In this example, BG46_variant 2 subtilisin (SEQ ID NO: 8) was used as a reporter protein to monitor expression as described herein. The construction was generally performed as follows. A first DNA fragment comprising a (5′) serA gene flanking region (5′ serA gene FR) including a Bacillus licheniformis serA selectable marker expression cassette (SEQ ID NO: 16) is operably linked to a polynucleotide construct comprising a Bacillus subtilis rrnI-p3 promoter region DNA sequence (SEQ ID NO: 17) upstream (5′) of a DNA sequence operably linked to a Bacillus subtilis aprE 5′ untranslated region (5′-UTR; SEQ ID NO: 18), the untranslated region DNA sequence being operably linked to a DNA encoding a Bacillus subtilis AprE signal sequence (SEQ ID NO: 4), which is operably linked to a DNA encoding a variant proregion C sequence (SEQ ID NO: 14), which is operably linked to a DNA encoding a mature BG46_variant 2 subtilisin protease (SEQ ID NO: 19). IDNO:8), the DNA sequence encoding the mature BG46_variant 2 subtilisin is operably linked to the Bacillus licheniformis amyL terminator (SEQ ID NO:19), which in turn is operably linked to the (3′) serA gene flanking region (3′ serA gene FR) (SEQ ID NO:20).

[0314] A second DNA fragment comprising a (5′) lysA gene flanking region (5′ lysA gene FR) including a Bacillus licheniformis lysA selectable marker expression cassette (SEQ ID NO:21) is operably linked to a polynucleotide construct comprising a DNA sequence of the Bacillus subtilis rrnI-p3 promoter region upstream (5′) (SEQ ID NO:17) operably linked to a DNA sequence of the Bacillus subtilis aprE 5′-UTR (SEQ ID NO:18), the DNA sequence of the untranslated region being operably linked to a DNA encoding the Bacillus subtilis AprE signal sequence (SEQ ID NO:4), which is operably linked to a DNA encoding a variant proregion C sequence (SEQ ID NO:14), which is operably linked to a DNA encoding a mature BG46_variant 2 subtilisin protease (SEQ ID NO:19). NO:8), the DNA sequence encoding the mature BG46_variant 2 subtilisin is operably linked to the Bacillus licheniformis amyL terminator (SEQ ID NO:10), which in turn is operably linked to the (3′) lysA gene flanking region (3′ lysA gene FR; SEQ ID NO:22).

[0315] More particularly, these DNA fragments are assembled using standard molecular biology techniques and used as templates to develop linear DNA expression cassettes comprising one or more pro-region sequence modifications described herein. For example, a Bacillus licheniformis strain comprising a variant pro-region sequence was constructed by integrating a first and a second DNA fragment (as described above) into the genome, wherein the first and second fragments contain the pro-region sequence variants shown in Table 3 below. As shown in Table 3, the production of the reporter protein was determined as previously described using standard methods (e.g., see WO 2019 / 055261) and normalized relative to a control pro-region sequence containing the E30G variant.

[0316] Table 3

[0317] Sequence of the proregion variant C (GK, SEQ: 14) in Bacillus licheniformis and the control proregion sequence B

[0318] Comparison of performance index of (E32G; SEQ:11)

[0319] Variant relative to WT PI Δ Charge E32G 1.0 1 E32G-L70K 1.1 2 E32G-A73R 1.3 2 E32G-L70K-A73R 1.2 3

[0320] Example 6

[0321] Additional modification of the 5' GG36 PRO sequence

[0322] In this example, BG46_variant 2 subtilisin (SEQ ID NO: 8) was used as a reporter protein to monitor expression as described herein. In particular, the 5 (N-terminal) sequence of the mutated GG36 PRO sequence consisting of 86 amino acids (SEQ ID NO: 14; variant pro-region sequence C) was further modified to introduce an additional amino acid, such as alanine (A) or serine (S), after the lysine at position three (3) (e.g., to potentially promote the release of the mature protease into the culture medium). For example, Figure 1-3 These N-terminal modifications are shown to produce three (3) variant proregion sequences DF (SEQ ID NOs: 29-31).

[0323] These N-terminally modified GG36 PRO sequences (SEQ ID NO:29, SEQ ID NO:30 and SEQ ID NO:31) were assembled in an expression cassette comprising an upstream (5') aprE gene flanking region operably linked to a polynucleotide construct comprising a variant (5') Bacillus subtilis rrnI-P2 promoter / 5'-UTR aprE region DNA sequence, thereby generating SEQ ID NO:1, which was operably linked to a DNA sequence encoding an AprE signal peptide sequence (SEQ ID NO:4), which was operably linked to a DNA sequence encoding a variant proregion sequence, which was operably linked to a DNA sequence encoding a mature BG46_variant 2 subtilisin (SEQ ID NO:8), which was operably linked to a Bacillus amyloliquefaciens BPN' terminator DNA sequence (SEQ ID NO:9). NO:6), which polynucleotide construct is operably linked to the downstream (3') aprE gene flanking region (3' aprE gene FR) containing a downstream kanamycin (kan) gene expression cassette (SEQ ID NO:7). The cassette containing the mutant / variant PRO sequence is transformed into Bacillus subtilis cells using standard molecular biology techniques.

[0324] The transformed cells were placed in a 96-well MTP, grown in a medium (enriched semi-defined medium based on MOP buffer) at 32°C in a shaking incubator at 300 rpm and 80% humidity for 3 days, and centrifuged and filtered. The clarified culture supernatant was used to measure (assay) the reporter protease activity to determine the productivity level, wherein the samples were collected after seventy-two (72) hours. In particular, Table 4 shows the results of the reporter protein productivity (performance index (PI) value) after 72 hours compared to the reference construct expressing the reporter protein using the WT PRO sequence (SEQ ID NO: 14).

[0325] Table 4

[0326] Comparison of performance indexes of the proregion variants DF (SEQ ID NO: 29-31) and the variant C proregion sequence (SEQ ID NO: 14)

[0327] name Variants PI SEQ ID Variant C GK 1.00 14 Variant E GKA 1.3 30 Variant D GKS 1.2 29 Variant F GKA 1.4 31

[0328] As shown in Table 4 above, the PI values ​​of the proregion variants were compared to the proregion sequence variant proregion sequence C (SEQ ID NO: 14, "G2K3"), where the amino acid position numbering is compared to SEQ ID NO: 14 (i.e., with a "GK" insertion). After 72 hours of growth, the PI index of the three (3) N-terminal proregion mutants (AGKA, AGKS, AGKAA) showed high expression of the reporter protein BG46_variant 2 subtilisin (SEQ ID NO: 8) compared to variant C (AGK; SEQ ID NO: 14).

[0329] Example 7

[0330] C-terminal modification of the GG36 PRO sequence

[0331] In this example, the GG36 variant proregion sequence B (SEQ ID NO: 11) was further engineered to replace the leucine (L) at position sixty-eight (68) with lysine (K) and the isoleucine (I) at position seventy-two (72) with valine (V), or to replace the glutamic acid (E) at position eighty (80) with isoleucine (I), resulting in two (2) engineered proregion sequence variant sequences G (SEQ ID NO: 32) and H (SEQ ID NO: 33), e.g., see Figure 1-Figure 3 In particular, transformed cells were grown as described above and reporter protease activity was measured (assayed) in clarified culture supernatants to determine productivity levels, with samples collected after 72 hours.

[0332] For example, Table 5 below shows the results of reporter protein productivity (PI value) after 72 hours compared to a reference construct expressing a reporter protein using a variant B proregion sequence (SEQ ID NO: 11). More particularly, as shown in Table 5, the PI values ​​of variant proregion sequences G and H were increased compared to the reference / control variant B proregion sequence, wherein variant proregion sequence G (comprising L68K and I72V mutations) produced a PI of 1.08 relative to proregion sequence B (E30G), and variant proregion sequence H (comprising L68K and E80I mutations) produced a PI of 1.18 relative to proregion sequence B (E30G).

[0333] Table 5 Comparison of performance indexes of front region variants G and H with front region variant C

[0334] name Variants PI Δ Charge relative to WT Variant B E30G 1.0 1 Variant G E30G L68K I72V 1.4 2 Variant F E30G L68K E80I 1.5 3

[0335] References

[0336] PCT Publication No. WO1993 / 20214

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[0339] PCT Publication No. WO2008 / 112258

[0340] PCT Publication No. WO2010 / 123754

[0341] PCT Publication No. WO2016 / 205710

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Claims

1. A variant proregion sequence comprising an amino acid substitution at position 30 and one or more positions selected from 1, 2, 3, 4, 6, 14, 16, 19, 20, 23, 36, 37, 38, 39, 42, 43, 44, 49, 50, 64, 65, 67, 68, 71, 79, 83 and 84, wherein the amino acid positions of the variant proregion are numbered according to SEQ ID NO:

15.

2. A variant proregion as described in claim 1, which is derived from a parent polypeptide or reference polypeptide having at least about 70%, about 75%, about 80%, about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with SEQ ID NO:

15.

3. A variant proregion sequence comprising an amino acid insertion of glycine (G) at position 2 and lysine (K) at position 3, and an amino acid substitution at one or more positions selected from 1, 32, 38, 46, 66, 67, 70 and 73, wherein the amino acid positions of the variant proregion are numbered according to SEQ ID NO:

14.

4. A variant proregion as described in claim 3, which is derived from a parent polypeptide or reference polypeptide having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with SEQ ID NO:

14.

5. A variant pro-region sequence comprising a glutamic acid (E) to glycine (G) substitution at position 30 (E30G), a leucine (L) to lysine (K) substitution at position 68 (L68K), and an isoleucine (I) to valine (V) substitution at position 73 (I72V), wherein the amino acid positions of the variant pro-region are numbered according to SEQ ID NO: 15; or a variant pro-region sequence comprising a glutamic acid (E) to glycine (G) substitution at position 30 (E30G), a leucine (L) to lysine (K) substitution at position 68 (L68K), and a glutamic acid (E) to isoleucine (I) substitution at position 80 (E80I), wherein the amino acid positions of the variant pro-region are numbered according to SEQ ID NO:

15.

6. A variant proregion as described in claim 5, which is derived from a parent polypeptide or reference polypeptide having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with SEQ ID NO:

15.

7. A polynucleotide comprising the variant proregion nucleic acid of any one of claims 1-6.

8. A polynucleotide comprising an upstream nucleic acid encoding the variant proregion according to any one of claims 1 to 6, wherein the upstream nucleic acid is operably linked to a downstream nucleic acid sequence encoding a protein of interest (POI).

9. A polynucleotide comprising an upstream nucleic acid encoding a signal sequence, wherein the upstream nucleic acid is operably linked to a downstream nucleic acid sequence encoding a variant front region as described in any one of claims 1-6, and the downstream nucleic acid sequence encoding the variant front region as described in any one of claims 1-6 is operably linked to a downstream nucleic acid sequence encoding a target protein (POI).

10. An expression cassette comprising an upstream promoter operably linked to a downstream polynucleotide as claimed in any one of claims 7 to 9.

11. A Gram-positive host cell comprising the cassette of claim 10 introduced therein.

12. A method for producing a protein of interest (POI) in a Gram-positive bacterial cell, the method comprising: (a) introducing an expression cassette into a Gram-positive cell, the expression cassette comprising an upstream promoter operably linked to a downstream nucleic acid encoding a variant pro-region sequence, the variant pro-region sequence comprising an amino acid substitution at position 30 and one or more positions selected from 1, 2, 3, 4, 6, 14, 16, 19, 20, 23, 36, 37, 38, 39, 42, 43, 44, 49, 50, 64, 65, 67, 68, 71, 79, 83 and 84, the downstream nucleic acid encoding the variant pro-region sequence being operably linked to a downstream nucleic acid encoding the POI, wherein the amino acid positions of the variant pro-region are numbered according to SEQ ID NO: 15, and (b) culturing the modified cells under conditions for producing the POI.

13. A method for producing a protein of interest (POI) in a Gram-positive bacterial cell, the method comprising: (a) introducing an expression cassette into a Gram-positive cell, the expression cassette comprising an upstream promoter sequence operably linked to a downstream nucleic acid encoding a variant pro-region sequence, the variant pro-region sequence comprising an amino acid insertion of glycine (G) at position 2, lysine (K) at position 3, and an amino acid substitution at one or more positions selected from 1, 32, 38, 46, 66, 67, 70, and 73, the downstream nucleic acid encoding the variant pro-region sequence being operably linked to a downstream nucleic acid encoding the POI, wherein the amino acid positions of the variant pro-region are numbered according to SEQ ID NO: 14, and (b) culturing the modified cells under conditions for producing the POI.

14. The method of claim 12 or claim 13, wherein the cassette further comprises a nucleic acid encoding a signal sequence operably linked and positioned between the upstream promoter and the downstream variant proregion.

15. The method of claim 12 or claim 13, wherein the modified cells produce an increased amount of the POI relative to a control Gram-positive cell having an introduced expression cassette comprising the same upstream promoter sequence operably linked to a downstream nucleic acid encoding a proregion sequence of SEQ ID NO: 15, wherein the downstream nucleic acid encoding the proregion sequence is operably linked to a downstream nucleic acid sequence encoding the same POI.

16. The method of claim 12 or claim 13, wherein the increased amount of POI is at least about 0.1% relative to the control cells.

17. The method of claim 12 or claim 13, wherein the POI is selected from the group consisting of acetylesterase, aminopeptidase, amylase, arabinanase, arabinofuranosidase, carbonic anhydrase, carboxypeptidase, catalase, cellulase, chitinase, rennetinase, cutinase, deoxyribonuclease, epimerase, esterase, α-galactosidase, β-galactosidase, α-glucanase, glucan lyase, endo-β-glucanase, glucoamylase, glucose oxidase, α-glucosidase, β-glucosidase, glucose lyase, Aldolases, glycosyl hydrolases, hemicellulases, hexose oxidases, hydrolases, invertases, isomerases, laccases, ligases, lipases, lyases, mannosidases, oxidases, oxidoreductases, pectate lyases, pectin acetylesterases, pectin depolymerases, pectin methylesterases, pectinolytic enzymes, perhydrolases, polyol oxidases, peroxidases, phenoloxidases, phytases, polygalacturonases, proteases, peptidases, rhamnogalacturonases, ribonucleases, transferases, transporters, transglutaminases, xylanases, hexose oxidases, and combinations thereof.

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