Fermentation method of recombinant Bacillus spores

Abstract

The present invention provides a method for producing a fermentation product, comprising culturing recombinant exospore-producing Bacillus cells that express a fusion protein of interest in exospores in a medium containing a carbon source and a nitrogen source at a total concentration of 20 g / L or more, to obtain a fermentation broth containing a high titer of recombinant Bacillus spores.

Description

[Technical Field]

[0001] REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 939,560, filed November 22, 2019, which is incorporated herein by reference in its entirety.

[0002] FIELD OF THE INVENTION The present invention relates to the field of bacterial fermentation, and more particularly to improved fermentation media for recombinant Bacillus strains.

[0003] Reference to electronically submitted sequence listings An official copy of the sequence listing will be submitted electronically via EFS-Web as an ASCII sequence listing entitled "BCS199009WO_ST25.txt", created on November 20, 2020, and submitted concurrently with the specification. The sequence listing contained in this ASCII document is a part of the specification and is incorporated herein by reference in its entirety. [Background technology]

[0004] Exosporium-producing Bacillus cells can be engineered to display heterologous proteins on their exosporium using fusion proteins containing a targeting sequence operably linked to the protein of interest. These engineered bacterial systems are useful in a variety of agricultural applications, as they can improve plant growth and / or promote plant health, provide enhanced activity against insects, mites, nematodes, and / or plant pathogens, or provide herbicide resistance. Novel methods for fermenting engineered Bacillus strains for spore display to improve spore titer, sporulation rate, and protein of interest activity are desirable for more efficient and cost-effective production of these cells and the proteins they display. Previous fermentation methods for these engineered bacteria were based on laboratory-scale processes that resulted in relatively low colony-forming unit (CFU) counts or low expression rates of the protein of interest. Novel methods for producing such engineered bacteria would enable more cost-effective use of this technology. Summary of the Invention [Problem to be solved by the invention]

[0005] summary The present invention is directed to a method for fermenting recombinant exosporium-producing Bacillus cells capable of expressing a fusion protein comprising a protein or peptide of interest, and a targeting sequence for displaying the protein or peptide of interest on the exosporium of the recombinant Bacillus cells.

[0006] In one embodiment, the invention includes a method of producing a fermentation product from a recombinant Exosporium-producing Bacillus cell expressing a fusion protein by culturing the recombinant Exosporium-producing Bacillus cell expressing the fusion protein in a medium comprising: i) yeast extract at a concentration of about 2 g / L to about 30 g / L; ii) glucose at a concentration of up to about 35 g / L; iii) Ca 2+Source of ions. In such embodiments, the fusion protein comprises a protein or peptide of interest and a targeting sequence, an exosporium protein or exosporium protein fragment.

[0007] In another embodiment of this method of producing a fermentation product, the medium comprises: i) yeast extract at a concentration of about 3 g / L to about 20 g / L; ii) glucose at a concentration of up to about 35 g / L; iii) soy flour at concentrations up to about 50 g / L; iv) Ca 2+ Source of ions. In one embodiment, the Ca 2+ The source of ions is CaCl2*2H2O. In another embodiment, the above-mentioned medium contains Mg 2+ In a more particular embodiment, the present invention further comprises a source of Mg ions. 2+ The source of the ions is MgSO4*7H2O.

[0008] In another embodiment, the medium comprises cottonseed at a concentration of up to about 10 g / L. The medium can also comprise corn steep at a concentration of up to about 10 g / L.

[0009] In one aspect of these embodiments, a pH of 6-8 is maintained during cultivation in these media. pH maintenance is achieved by the addition of acid or base. Alternatively, or additionally, the disclosed media contain a buffer. In one aspect, the buffer is KHPO and KHPO. In a more specific aspect, KHPO is present at a concentration of at least 1 g / L and KHPO is present at a concentration of at least 0.8 g / L.

[0010] In one embodiment of these methods, the culturing is carried out at 25-35°C. Further, the culturing is carried out for up to 50 hours and / or the culturing is carried out until sporulation of the Bacillus cells is at least 90% complete. In one aspect of this embodiment, the culturing results in a spore titer of at least 1 x 109 spores / mL of fermentation broth is obtained.

[0011] In another embodiment, the disclosed medium comprises one or more carbon sources having a total concentration of at least 20 g / L. In another embodiment, the disclosed medium comprises one or more nitrogen sources having a total concentration of at least 3 g / L. In yet another embodiment, the combined concentration of the one or more carbon sources and one or more nitrogen sources is at least 20 g / L.

[0012] In one embodiment, the present invention provides a method for producing a fermentation product from a recombinant Exosporium-producing Bacillus cell expressing a fusion protein by culturing the recombinant Exosporium-producing Bacillus cell expressing the fusion protein in a medium comprising: a) yeast extract having a concentration of about 3 g / L to about 25 g / L, about 5 g / L to about 15 g / L, or about 10 g / L to about 15 g / L; b) glucose at a concentration of up to about 30 g / L, from about 20 g / L to about 35 g / L, or from about 25 g / L to about 30 g / L; c) soy flour at a concentration of up to about 30 g / L, from about 10 g / L to about 30 g / L, or from about 15 g / L to about 20 g / L; d) a buffer containing KHPO at a concentration of about 1 g / L to about 20 g / L, or about 1 g / L to about 5 g / L, and KHPO at a concentration of about 0.1 g / L to about 5 g / L, or about 0.5 g / L to about 2 g / L, or about 1 g / L to about 3 g / L, or about 0.5 g / L to about 1 g / L; e) CaCl2*2H2O at a concentration of about 0.010 g / L to about 1 g / L, about 0.015 g / L to about 0.80 g / L, or about 0.02 g / L to about 0.4 g / L; and f) MgSO4*7H2O at a concentration of about 0.1 g / L to about 1 g / L, 0.10 g / L to about 0.80 g / L, or about 0.2 g / L to about 0.5 g / L. In such embodiments, the fusion protein comprises a protein or peptide of interest and a targeting sequence, an exosporium protein or exosporium protein fragment.

[0013] In a more particular aspect of this embodiment, the medium further comprises about 0.01 g / L to about 0.1 g / L of ZnSO4*7H2O.

[0014] In one embodiment of the above method, the protein or peptide of interest is a plant growth stimulating protein or peptide, a protein or peptide that protects plants from pathogens, and an insecticidal protein or peptide.

[0015] In another embodiment, the targeting sequence, exosporium protein or exosporium protein fragment comprises: an amino acid sequence having at least about 43% identity with amino acids 20 to 35 of SEQ ID NO: 1, and at least about 54% identity with amino acids 25 to 35; a targeting sequence comprising amino acids 1 to 35 of SEQ ID NO: 1; a targeting sequence comprising amino acids 20 to 35 of SEQ ID NO: 1; a targeting sequence comprising amino acids 22 to 31 of SEQ ID NO: 1; a targeting sequence comprising amino acids 22 to 33 of SEQ ID NO: 1; a targeting sequence comprising amino acids 20 to 31 of SEQ ID NO: 1; a targeting sequence comprising SEQ ID NO: 1; or An Exosporium protein comprising an amino acid sequence having at least 85% identity to SEQ ID NO:2.

[0016] In another embodiment, the Exosporium-producing Bacillus cell is a cell of a member of the Bacillus cereus family. In a more particular embodiment, the Bacillus cereus family member is Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides, Bacillus pseudomycoides, Bacillus samanii, Bacillus gaemokensis, Bacillus weihenstephensis, Bacillus toyoiensis, and combinations thereof.

[0017] In another embodiment, the Bacillus cell producing the recombinant Exoporium is derived from Bacillus thuringiensis BT013A.

[0018] In one embodiment of the above method, the plant growth-promoting protein or peptide is characterized in that it is an enzyme involved in the production or activation of a plant growth-promoting compound, and the compound is selected from the group consisting of acetoin reductase, indole-3-acetamidohydrolase, tryptophan monooxygenase, acetolactate synthetase, α-acetolactate decarboxylase, pyruvate decarboxylase, diacetyl reductase, butanediol dehydrogenase, aminotransferase, tryptophan decarboxylase, amine oxidase, indole-3-pyrazole Vinyl acid decarboxylase, indole-3-acetaldehyde dehydrogenase, tryptophan side chain oxidase, nitrile hydrolase, nitrilase, peptidase, protease, adenosine phosphate isopentenyltransferase, phosphatase, adenosine kinase, adenine phosphoribosyltransferase, CYP735A, 5' ribonucleotide phosphohydrolase, adenosine nucleosidase, zeatin cis-trans isomerase, zeatin O-glucosyltransferase, β-glucosidase, cis-hydroxylase, CK Cis-hydroxylase, CK N-glucosyltransferase, 2,5-ribonucleotide phosphohydrolase, adenosine nucleosidase, purine nucleoside phosphorylase, zeatin reductase, hydroxylamine reductase, 2-oxoglutarate dioxygenase, gibberellin 2B / 3B hydrolase, gibberellin 3-oxidase, gibberellin 20-oxidase, chitosanase, chitinase, β-1,3-glucanase, β-1,4-glucanase, β-1,6-glucanase, aminocyclopropane-1-carboxylic acid deaminase, and enzymes involved in nod factor production.

[0019] In another embodiment, the enzyme degrades or modifies bacterial, fungal, or plant nutritional sources and is selected from the group consisting of cellulase, lipase, lignin oxidase, protease, glycoside hydrolase, phosphatase, nitrogenase, nuclease, amidase, nitrate reductase, nitrite reductase, amylase, ammonia oxidase, ligninase, glucosidase, phospholipase, phytase, pectinase, glucanase, sulfatase, urease, xylanase, and siderophore.

[0020] In another embodiment, the protein or peptide is an insecticidal protein. In a more particular embodiment, the insecticidal protein is a VIP insecticidal protein, an endotoxin, a Cry toxin, a protease inhibitor protein or peptide, a cysteine ​​protease, a serine protease, or a chitinase.

[0021] In another aspect, the protein or peptide is a protein or peptide that protects the plant from pathogens, such as a protease or lactonase.

[0022] In one embodiment of this aspect, the serine protease has an amino acid sequence having at least 95%, at least 98%, or at least 99% identity to any one of SEQ ID NOs:5-7.

[0023] The present invention also includes the fermentation broth or fermentation product produced by the above method.

[0024] In yet another aspect, a fermentation broth is provided that comprises: a) yeast extract at a concentration of about 3 g / L to about 25 g / L; b) glucose at a concentration of up to about 30 g / L; c) soy flour at a concentration of up to about 30 g / L; d) a buffer comprising KHPO at a concentration of about 0.5 g / L to about 5 g / L and KHPO at a concentration of about 0.1 g / L to about 5 g / L; e) CaCl*2H0 at a concentration of about 0.010 g / L to about 1 g / L; and f) MgSO*7H0 at a concentration of about 0.1 g / L to about 1.5 g / L. In certain embodiments, the fermentation broth can further comprise recombinant Exosporium-producing Bacillus cells expressing a fusion protein, wherein the fusion protein comprises a protein or peptide of interest and a targeting sequence, an Exosporium protein, or an Exosporium protein fragment. [Brief explanation of the drawings]

[0025] [Figure 1] Figure 1 shows the scaled performance of tdTomato in conventional yeast extract-based media and in the new media prototypes M0, M2, and M5. [Figure 2] Figure 2 shows the performance of the new medium M2 on several species of Bacillus from the Bacillus cereus family, including Bacillus thuringiensis. [Figure 3] Figure 3 shows tdTomato fluorescence using novel medium M2 in strains #1-4, demonstrating robust protein expression with the use of the novel medium. [Figure 4] FIG. 4 shows spore titers and cargo production when medium M2 is used with bacterial lines displaying tdTomato. [Figure 5] Figure 5 shows the performance of tdTomato at the microreactor scale in basal medium and in the novel medium prototypes M2 and OM3. [Figure 6] FIG. 6 shows the Sep1 protease activity performance in the basal medium and the new medium prototypes M2 and OM3.

[0026] A brief description of arrays SEQ ID NO: 1 is the BclA promoter from B. cereus. SEQ ID NO: 2 is amino acids 1-41 of BdA (B. anthracis Sterne). SEQ ID NO: 3 is the amino acid sequence of the tdTomato fluorescent protein. SEQ ID NO: 4 is full length BdA (B. anthracis Sterne). SEQ ID NO: 5 is the amino acid sequence of serine protease (Sep1) from Bacillus firmus DS-1. SEQ ID NO: 6 is the amino acid sequence of serine protease (Sep1) from Bacillus firmus strain 1. SEQ ID NO: 7 is the amino acid sequence of a serine protease variant having a deletion. SEQ ID NO: 8 is the amino acid sequence of endoglucanase from Bacillus subtilis. SEQ ID NO: 9 is the amino acid sequence of a phospholipase from Bacillus thuringiensis. SEQ ID NO: 10 is the amino acid sequence of chitosinase from Bacillus subtilis.

[0027] Detailed explanation Some Bacillus species produce exosporium as the outermost layer of their endospores. Systems have been developed to engineer recombinant Bacillus cells that display fusion proteins on such exosporium. Examples of such systems are described in U.S. Patent No. 9,133,251 and International Publication Nos. WO 2014 / 145964 and WO 2016 / 044655, each of which is incorporated herein by reference in its entirety. These recombinant exosporium-producing Bacillus cells can express fusion proteins containing a peptide and a targeting sequence, resulting in the peptide being targeted and displayed on the exosporium. Bacillus exosporium display has the potential to deliver peptides or proteins of interest to plants via seed, leaf, or soil treatment. Prior to the present disclosure, the use of media enriched with carbon and nutrient sources for the fermentation of exosporium-producing Bacillus expressing fusion proteins on exosporium was thought to result in detrimental loss of the expressed fusion protein. Previously, very lean media were used, which were based on yeast extract as the primary source of carbon and nitrogen. However, the present disclosure shows that media rich in carbon and nitrogen sources are highly effective, providing high activity of the protein or peptide of interest, both due to higher CFU numbers and higher protein or peptide expression per spore.

[0028] Thus, the present disclosure provides improved methods for fermenting exosporium-producing bacteria engineered to display heterologous proteins on their exosporium using media rich in carbon and nitrogen sources. These novel fermentation methods result in higher sporulation efficiency and improved CFU numbers while retaining high protein activity compared to previously used media. The fermentation media described herein produce high cell densities at low cost at scales ranging from 20 L to over 3000 L. These novel fermentation methods enable improved utilization of genetically engineered Bacillus strains at commercially useful scales.

[0029] I. Fermentation Method of Recombinant Exosporium-Producing Bacillus The present disclosure provides a method for fermenting a Bacillus strain capable of displaying a protein of interest on its exosporium by culturing the strain in the presence of a novel medium provided herein. The novel fermentation medium provided herein results in improved colony forming unit numbers of cultured cells and increased activity of the protein or peptide of interest displayed on the exosporium of the cells. The fermentation medium provided herein is a medium containing yeast extract, soybean flour, glucose, Ca, and / or PEG / PPG-14. 2+ ions, or Mg 2+ The ions may include one or more of the following:

[0030] During fermentation, when nutrients are depleted, cells begin to transition from the growth phase to the sporulation phase, resulting in the end product of fermentation consisting mostly of spores, metabolic products, and residual fermentation medium. In the submerged fermentation culture process described herein, the product of the microbial culture process is called a "fermentation broth." Such a broth can be concentrated as described above. The concentrated fermentation broth can be washed, for example, via a diafiltration process, to remove residual fermentation broth and metabolic products. The term "broth concentrate," as used herein, refers to a fermentation broth that is concentrated by conventional industrial methods but remains in liquid form, as described above. The term "fermentation product," as used herein, refers to the fermentation broth, broth concentrate, and / or dried fermentation broth or broth concentrate, referred to herein as dried fermentation broth.

[0031] Fermentation media disclosed herein can include concentrated sources of amino acids, such as yeast extract, e.g., Yeast Extract for Microbial Growth Media (Sigma, St. Louis, MO, USA), Yeast Extract Bacteriological (Thomas Scientific, Swedesboro, NJ, USA), or Yeast Extract (LabScientific, Highlands, NJ, USA). Other concentrated amino acid sources can also be used, including NZ-AMINE A® (Sigma, St. Louis, MO, USA) or BD Bacto Casamino Acids (BD Biosciences, San Jose, CA, USA). In certain embodiments, yeast extract can be provided at a concentration of up to about 30 g / L, e.g., from about 2 g / L to about 30 g / L, or in some embodiments, from about 5 g / L to about 10 g / L. In certain embodiments, yeast extract may be present in the disclosed media at a concentration of about 3 g / L, about 5 g / L, about 10 g / L, or about 25 g / L.

[0032] The media provided by the present disclosure may further include nutrient sources capable of providing protein, vitamins, minerals, and / or carbohydrates. Nutrient sources for the media used in the disclosed fermentation processes may be selected from the group consisting of soy flour, peptone, nitrate, ammonium chloride, ammonium sulfate, ammonium nitrate, and amino acids. Exemplary sources of these nutrients include soy flour or peptone, such as soy peptone. Nutrient sources for use in the disclosed media include, but are not limited to, soy flour (Sigma, St. Louis, MO, USA) or peptone from GLYCINE MAX® (Sigma, St. Louis, MO, USA). In one embodiment, the total concentration of the nutrient sources may be up to about 50 g / L, up to about 30 g / L, up to about 20 g / L, up to about 15 g / L, up to about 10 g / L, or from about 5 g / L to about 35 g / L, from about 10 g / L to about 30 g / L, or from about 10 g / L to about 25 g / L. In one embodiment, the nutrient source used in the disclosed fermentation process is selected from the group consisting of soy flour and peptone. In certain embodiments, the soy flour may be present at a concentration of up to about 50 g / L, such as from about 5 g / L to about 35 g / L, such as from about 10 g / L to about 30 g / L. In certain embodiments, the soy flour may be present in the disclosed medium at a concentration of about 10 g / L, about 15 g / L, about 20 g / L, or about 30 g / L.

[0033] In further embodiments, the media disclosed herein include one or more carbon sources, including carbohydrates such as glucose. Carbon sources for the media used in the disclosed fermentation processes are selected from the group consisting of fructose, glucose, galactose, sucrose, lactose, mannitol, maltose, trehalose, soluble starch, molasses, sugarcane juice, and beet juice. In one embodiment, the carbon source is selected from the group consisting of fructose, glucose, galactose, sucrose, lactose, mannitol, maltose, and trehalose. In other embodiments, the total concentration of the carbon sources can be up to about 50 g / L, up to about 40 g / L, up to about 35 g / L, up to about 30 g / L, up to about 25 g / L, up to about 20 g / L, or from about 10 g / L to about 50 g / L, from about 20 g / L to about 35 g / L, or from about 25 g / L to about 30 g / L. In certain embodiments, the carbon source may be present in the disclosed media at a concentration of about 25 g / L or about 30 g / L. In exemplary embodiments, glucose may be present at a concentration of up to about 50 g / L, up to about 40 g / L, up to about 35 g / L, up to about 30 g / L, up to about 25 g / L, or up to about 20 g / L, e.g., about 10 g / L to about 50 g / L, or about 15 g / L to about 40 g / L, or up to about 35 g / L, e.g., about 20 g / L to about 35 g / L, e.g., about 25 g / L to about 30 g / L. In certain embodiments, glucose may be present in the disclosed media at a concentration of about 25 g / L or about 30 g / L.

[0034] The novel fermentation medium provided herein may further comprise cottonseed flour at a concentration of up to about 10 g / L, such as about 2 g / L, 5 g / L, or 2 g / L to 7 g / L. Cottonseed flour is a fine yellow powder made from cottonseed germ and is commercially known as PHARMAMEDIA®, available from Archer Daniels Midland. Because cottonseed flour is rich in protein, it is primarily a nitrogen source, but also provides some carbohydrates. The medium of the present disclosure may further comprise corn steep liquor at a concentration of up to about 10 g / L, such as about 2 g / L, 5 g / L, or 2 g / L to 7 g / L. Corn steep liquor is a by-product of corn wet milling and is a viscous concentrate of corn solubles containing amino acids, vitamins, and minerals.

[0035] The fermentation media disclosed herein may further comprise a buffer for adjusting the pH during the fermentation process. Alternatively, the pH of the provided media may be controlled through the addition of an acid or base. Several methods for controlling the pH of a fermentation medium are known in the art, including through buffers known in the art, or through the addition of an acid or base if the fermentation equipment allows this. Buffering of the media provided herein is further described in Example 3 and Table 2.

[0036] The fermentation media disclosed herein may further comprise one or more sources of salts of divalent cations. Sources of salts of divalent cations include Ca 2+ , Mg 2+ and Zn 2+The divalent cation salt source may be selected from the group consisting of chloride, sulfate, hydroxide, carbonate, bicarbonate, and nitrate, of each of the above. In one embodiment, the divalent cation salt source may be selected from the group consisting of calcium chloride or magnesium sulfate. In one embodiment, the divalent cation salt source may be present in the medium at a concentration of about 0.010 g / L to about 2.5 g / L, about 0.02 g / L to about 2 g / L, or about 0.010 g / L to about 1 g / L. In another embodiment, CaCl is present in the medium at a concentration of about 0.010 g / L to about 1 g / L, about 0.015 g / L to about 0.80 g / L, or about 0.02 g / L to about 0.4 g / L; in another embodiment, MgSO is present at a concentration of about 0.1 g / L to about 1 g / L, 0.10 g / L to about 0.80 g / L, or about 0.2 g / L to about 0.5 g / L. In another embodiment, CaCl*2H0 is present in the medium at a concentration of about 0.010 g / L to about 1 g / L, about 0.015 g / L to about 0.80 g / L, or about 0.02 g / L to about 0.4 g / L; in another embodiment, MgSO*7H0 is present at a concentration of about 0.1 g / L to about 1 g / L, 0.10 g / L to about 0.80 g / L, or about 0.2 g / L to about 0.5 g / L. In another embodiment, the salt source further comprises zinc sulfate. In one embodiment, ZnSO is present at a concentration of about 0.010 g / L to about 1 g / L, about 0.015 g / L to about 0.80 g / L, or about 0.02 g / L to about 0.1 g / L. In another embodiment, ZnSO4*7H2O is present at a concentration of about 0.010 g / L to about 1 g / L, about 0.015 g / L to about 0.80 g / L, or about 0.02 g / L to about 0.1 g / L.

[0037] Culturing of the Bacillus strain can be carried out for a time appropriate for the cells to sporulate. For example, culturing can be carried out for about 1 to about 72 hours (hours), about 5 to about 60 hours, about 10 to about 54 hours, or 24 to 48 hours. In one embodiment, culturing can be carried out for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 42, 48, 54, or 60 hours, where any of the values ​​listed can form an upper or lower limit, as appropriate. In another embodiment, the time for culturing may be greater than or equal to about 1, 2, 3, 5, 6, 7, 8, 10, 11, 13, 14, 15, 14, 17, 18, 20, 21, 22, 23, 24, 25, 26, 28, 29, 30, 31, 32, 33, 34, 35, 37, 38, 39, 40, 41, 43, 44, 45, 46, 47, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 hours. In yet another embodiment, the time for culturing can be less than or equal to about 1, 2, 3, 4, 5, 6, 7, 10, 12, 15, 14, 14, 47, 48, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 68, 69, or 70 hours. In yet another aspect, culturing is carried out for about 24 to 50 hours or about 45 to 70 hours.

[0038] The temperature during cultivation can be about 20 to about 55° C., about 25 to about 40° C., or about 28 to about 35° C. In one embodiment, the temperature during cultivation can be about 20 to about 32° C. or about 28 to about 40° C. In another embodiment, cultivation can be carried out at a temperature of about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55° C., where any of the recited values ​​can form an upper or lower limit, as appropriate. In yet another aspect, the culturing can be performed at a temperature greater than or equal to about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55°C. In yet another embodiment, the culture can be performed at a temperature lower than or equal to about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55° C. In one embodiment, the culture can be performed at about 28 to about 35° C. In a further embodiment, the culture can be performed at about 30° C.

[0039] The methods of the present disclosure provide a significant increase in spore titer when compared to laboratory-scale culture medium as described in the Examples section. In certain embodiments, the methods provided herein provide a spore titer of about 1 x 10 when using a basal medium as defined in the Examples section. 8 compared to 1 × 10 9 In certain examples, fermentation of Bacillus strains using the novel media provided herein results in spore titers of at least about 1 x 10 8 At least approximately 1.5 x 10 spores / mL 8 At least approximately 1 x 10 spores / mL 9 spores / mL, or at least approximately 1.5 x 10 9Spore titers of spores / mL may be obtained.

[0040] In embodiments provided herein, fermentation of the recombinant Bacillus strains described herein with the disclosed media resulted in a sporulation rate of at least 95%. For example, fermentation of the recombinant Bacillus strains described herein with the disclosed media can result in a sporulation rate of at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0041] Fermentation of engineered Bacillus using the improved media disclosed herein also results in improved activity of the displayed protein compared to fermentation using a dilute medium, such as a basal medium. In certain embodiments, fermentation of engineered Bacillus using the improved media disclosed herein can result in up to about 2-fold, up to about 5-fold, up to about 10-fold, up to about 20-fold, up to about 30-fold, up to about 50-fold, or up to about 100-fold greater display protein activity and / or expression per mL of fermentation broth than the activity of the displayed protein from the same recombinant strain fermented in a dilute medium, such as a basal medium. Fermentation of engineered Bacillus using the novel media disclosed herein can also result in improved display protein yield per colony-forming unit of spores, which is up to about 2-fold, up to about 5-fold, up to about 10-fold, up to about 20-fold, up to about 30-fold, up to about 50-fold, or up to about 100-fold greater display protein yield per colony-forming unit from the same recombinant strain fermented in a dilute medium, such as a basal medium.

[0042] II. Recombinant Bacillus Strains The novel media and methods disclosed herein can be used to ferment recombinant exosporium-producing Bacillus strains engineered to express a fusion protein containing a targeting sequence and a peptide. Bacillus strains useful in the present invention include strains of any exosporium-producing species of Bacillus, such as strains from the Bacillus cereus family, including Bacillus thuringiensis.

[0043] The recombinant exosporium-producing Bacillus strain can further comprise a fusion protein comprising a targeting sequence and any peptide of interest. The fusion protein comprises a targeting sequence, an exosporium protein, or an exosporium protein fragment that targets the fusion protein to the exosporium of Bacillus cereus, and (a) a plant growth-stimulating protein or peptide; (b) a protein or peptide that protects the plant from pathogens or pests; (c) a protein or peptide that enhances plant stress resistance; (d) a plant-binding protein or peptide; or (e) a plant immune system enhancer protein or peptide. When expressed in Bacillus cereus bacteria, these fusion proteins are targeted to the exosporium layer of the spore, where they are physically oriented so that the protein or peptide is displayed on the outside of the spore.

[0044] This Bacillus exosporium display (BEMD) system can be used to deliver peptides, enzymes, and other proteins to plants (e.g., to plant leaves, fruits, flowers, stems, or roots) or to plant growth media such as soil. Peptides, enzymes, and proteins delivered to soil or other plant growth media in this manner persist and remain active in the soil for extended periods of time. Introducing recombinant exosporium-producing Bacillus cells expressing the fusion proteins described herein into the soil or rhizosphere of plants can beneficially enhance plant growth in many different soil conditions. Using BEMD to produce these enzymes can continue to provide beneficial results to the plant and rhizosphere for several months after the plant's life.

[0045] Targeting sequence Bacillus is a genus of rod-shaped bacteria. Bacteria in the Bacillus cereus family include Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides, Bacillus pseudomycoides, Bacillus samanii, Bacillus gaemokensis, Bacillus toyoiensis, and Bacillus weihenstephensis. Under stressful environmental conditions, Bacillus cereus bacteria undergo sporulation, forming ovoid endospores that can remain dormant for extended periods of time. The outermost layer of the endospore, called the exospore, contains a basal layer surrounded by hair-like projections. The filaments on the hair-like nap are formed primarily by the collagen-like glycoprotein BclA, while the basal layer is composed of many different proteins. Another collagen-related protein, BclB, is also present in the exosporium and is exposed on the endospores of members of the Bacillus cereus family.

[0046] BclA, the major component of the surface nap, has been shown to be attached to the exosporium, with its amino terminus (N-terminus) located in the basal layer and its carboxy terminus (C-terminus) extending outward from the spore.

[0047] It has previously been discovered that certain sequences from the N-terminal regions of BclA and BclB can be used to target peptides or proteins to the exosporium of Bacillus cereus endospores (see U.S. Patent Application Publication Nos. 2010 / 0233124 and 2011 / 0281316, and Thompson et al., "Targeting of the BclA and BclB Proteins to the Bacillus anthraci Spore Surface," Molecular Microbiology, 70(2):421-34 (2008), which are hereby incorporated by reference in their entireties). Additionally, the Bacillus anthracis BetA / BAS3290 protein was found to localize to the exosporium.

[0048] Targeting of a protein of interest (e.g., an enzyme) to an exosporium protein can be achieved using the following motif, which may be present in the targeting sequence, the exosporium protein, or an exosporium protein fragment that targets the fusion protein to the exosporium of the recombinant Bacillus: X1-X2-X3-X4-X5-X6-X7-X8-X9-X 10 -X 11 -X 12 -X 13 -X 14 -X 15 -X 16 Contains, where: X1 is an amino acid or is absent; X2 is phenylalanine (F), leucine (L), isoleucine (I), or methionine (M); X3 is any amino acid; X4 is proline (P) or serine (S); X5 is any amino acid; X6 is leucine (L), asparagine (N), serine (S), or isoleucine (I); X7 is valine (V) or isoleucine (I); X8 is glycine (G); X9 is proline (P); X 10 is threonine (T) or proline (P); X 11 is leucine (L) or phenylalanine (F); X 12 is proline (P); X 13 is any amino acid; X 14 is any amino acid; X 15 is proline (P), glutamine (Q), or threonine (T); X 16 is proline (P), threonine (T) or serine (S).

[0049] In particular, amino acids 20–35 of BclA from Bacillus anthracis Sterne strains have been found to be sufficient for targeting to exosporium.

[0050] Any portion of BclA containing amino acids 20-35 can be used as a targeting sequence. Additionally, full-length exosporium proteins or exosporium protein fragments can be used to target fusion proteins to exosporium. Thus, full-length BclA or fragments of BclA containing amino acids 20-35 can be used to target exosporium.

[0051] The targeting sequence may comprise amino acids 1-35 of SEQ ID NO:2, amino acids 20-35 of SEQ ID NO:2, a methionine linked to amino acids 20-35 of SEQ ID NO:2, SEQ ID NO:2, amino acids 22-31 of SEQ ID NO:2, amino acids 22-33 of SEQ ID NO:2, or amino acids 20-31 of SEQ ID NO:2. Alternatively, the targeting sequence may consist of amino acids 1-35 of SEQ ID NO:2, amino acids 20-35 of SEQ ID NO:2, or SEQ ID NO:2. Alternatively, the targeting sequence may consist of amino acids 22-31 of SEQ ID NO:2, amino acids 22-33 of SEQ ID NO:2, or amino acids 20-31 of SEQ ID NO:2. Alternatively, the exosporium protein may comprise full-length BclA (SEQ ID NO:4), or the exosporium protein fragment may comprise an intermediate-sized fragment of BclA lacking the carboxy terminus, such as amino acids 1-196 of SEQ ID NO:4.

[0052] Fusion proteins The fusion protein may comprise a targeting sequence, an Exosporium protein or protein fragment, and at least one plant growth-stimulating protein or peptide. The plant growth-stimulating protein or peptide may comprise a peptide hormone, a non-hormone peptide, an enzyme involved in the production or activation of a plant growth-stimulating compound, or an enzyme that degrades or modifies a bacterial, fungal, or plant nutrient source. The targeting sequence, Exosporium protein, or Exosporium protein fragment may be any of the aforementioned representative sequences, Exosporium proteins, or Exosporium protein fragments.

[0053] The fusion protein may comprise a targeting sequence, an exosporium protein or exosporium protein fragment, and at least one protein or peptide that protects plants from pathogens. The targeting sequence, exosporium protein, or exosporium protein fragment may be any of the targeting sequences, exosporium proteins, or exosporium protein fragments described above.

[0054] Fusion proteins can be produced using standard cloning and molecular biology methods known in the art. For example, a gene encoding a protein or peptide (e.g., a gene encoding a plant growth-stimulating protein or peptide) can be amplified by polymerase chain reaction (PCR) and ligated to DNA encoding any of the targeting sequences to form a DNA molecule encoding a fusion protein. The DNA molecule encoding the fusion protein can be cloned into any suitable vector, such as a plasmid vector. The vector suitably contains a multiple cloning site into which the DNA molecule encoding the fusion protein can be easily inserted. The vector also suitably contains a selectable marker, such as an antibiotic resistance gene, so that bacteria transformed, transfected, or mated with the vector can be easily identified and isolated. If the vector is a plasmid, the plasmid also suitably contains an origin of replication. The DNA encoding the fusion protein is suitably under the control of a sporulation promoter that will cause expression of the fusion protein in the exosporium of endospores of B. cereus family members (e.g., the native bclA promoter from B. cereus family members). Alternatively, DNA encoding the fusion protein can be integrated into the chromosomal DNA of the B. cereus family member host.

[0055] The fusion protein can also contain an additional polypeptide sequence that is not part of the targeting sequence, an Exosporium protein, an Exosporium protein fragment, or a plant growth-stimulating protein or peptide, a protein or peptide that protects the plant from pathogens, a protein or peptide that enhances stress tolerance in the plant, or a plant-binding protein or peptide. For example, the fusion protein can contain a tag or marker to facilitate purification or visualization of the fusion protein (e.g., a polyhistidine tag or a fluorescent protein such as GFP or YFP) or visualization of recombinant Exosporium-producing Bacillus cell spores expressing the fusion protein.

[0056] Expression of fusion proteins in exosporium using the targeting sequences, exosporium proteins, and exosporium protein fragments described herein is enhanced by the lack of secondary structure at the amino termini of these sequences, allowing the fusion proteins to retain their native folding and activity. Proper folding can be further enhanced by including a short amino acid linker between the targeting sequence, exosporium protein, exosporium protein fragment, and fusion partner protein.

[0057] Plant Growth-Promoting Proteins and Peptides As described above, the fusion protein can include a targeting sequence, an exosporium protein or exosporium protein fragment, and at least one plant growth stimulatory protein or peptide. For example, the plant growth stimulatory protein or peptide can include a peptide hormone, a non-hormonal peptide, an enzyme involved in the production or activation of a plant growth stimulatory compound, or an enzyme that degrades or modifies a bacterial, fungal, or plant nutrient source.

[0058] For example, when the plant growth stimulating protein or peptide comprises a peptide hormone, the peptide hormone can include phytosulfokine (e.g., phytosulfokine-α), clavata 3 (CLV3), systemin, ZmlGF, or SCR / SP11.

[0059] When the plant growth stimulatory protein or peptide comprises a non-hormonal peptide, the non-hormonal peptide can include RKN 16D10, Hg-Syv46, eNOD40 peptide, melittin, mastoparan, Mas7, RHPP, POLARIS, or Kunitztrypsin inhibitor (KTI).

[0060] Plant growth stimulatory proteins or peptides can include enzymes involved in the production or activation of plant growth stimulatory compounds, which can be any enzyme that catalyzes any step in the biological synthesis pathway of a compound that stimulates plant growth or alters plant structure, or any enzyme that catalyzes the conversion of an inactive or less active derivative of a compound that stimulates plant growth or alters plant structure to an active or more active form.

[0061] Plant growth stimulating compounds can include compounds produced by bacteria or fungi in the rhizosphere, such as 2,3-butanediol.

[0062] Alternatively, the plant growth stimulating compound may comprise a plant growth hormone, such as a cytokinin or cytokinin derivative, ethylene, an auxin or an auxin derivative, gibberellic acid or a gibberellic acid derivative, abscisic acid or an abscisic acid derivative, or jasmonic acid or a jasmonic acid derivative.

[0063] When the plant growth stimulating compound comprises a cytokinin or a cytokinin derivative, the cytokinin or cytokinin derivative can comprise kinetin, cis-zeatin, trans-zeatin, 6-benzylaminopurine, dihydroxyzeatin, N6-(D2-isopentenyl)adenine, ribosylzeatin, N6-(D2-isopentenyl)adenosine, 2-methylthio-cis-ribosylzeatin, trans-ribosylzeatin, 2-methylthio-trans-ribosylzeatin, ribosylzeatin-5-monophosphate, N6-dimethylaminopurine, 2'-deoxyzeatin riboside, 4-hydroxy-3-methyltrans-2-butenylaminopurine, ortho-topolin, meta-topolin, benzyladenine, ortho-methyltopolin, meta-methyltopolin, or a combination thereof.

[0064] When the plant growth stimulating compound comprises an auxin or auxin derivative, the auxin or auxin derivative can comprise an active auxin, an inactive auxin, a conjugated auxin, a naturally occurring auxin, or a synthetic auxin, or a combination thereof. For example, the auxin or auxin derivative can comprise indole-3-acetic acid, indole-3-pyruvic acid, indole-3-acetaldoxime, indole-3-acetamide, indole-3-acetonitrile, indole-3-ethanol, indole-3-pyruvic acid, indole-3-acetaldoxime, indole-3-butyric acid, phenylacetic acid, 4-chloroindole-3-acetic acid, glucose-conjugated auxin, or a combination thereof.

[0065] Enzymes involved in the production or activation of plant growth stimulating compounds include acetoin reductase, indole-3-acetamidohydrolase, tryptophan monooxygenase, acetolactate synthetase, α-acetolactate decarboxylase, pyruvate decarboxylase, diacetyl reductase, butanediol dehydrogenase, aminotransferases (e.g., tryptophan aminotransferase), tryptophan decarboxylase, aminooxidase, indole-3-pyruvate decarboxylase, indole-3-acetamidohydrolase, and indole-3-acetamidohydrolase. Cetaldehyde dehydrogenase, tryptophan side chain oxidase, nitrile hydrolase, nitrilase, peptidase, protease, adenosine phosphate isopentenyltransferase, phosphatase, adenosine kinase, adenine phosphoribosyltransferase, CYP735A, 5' ribonucleotide phosphohydrolase, adenosine nucleosidase, zeatin cis-trans isomerase, zeatin O-glucosyltransferase, β-glucosidase, cis-hydroxylase, CK, cis-hydroxylase These can include N-glucosyltransferase, 2,5-ribonucleotide phosphohydrolase, adenosine nucleosidase, purine nucleoside phosphorylase, zeatin reductase, hydroxylamine reductase, 2-oxoglutarate dioxygenase, gibberellin 2B / 3B hydrolase, gibberellin 3-oxidase, gibberellin 20-oxidase, chitosinase, chitinase, β-1,3-glucanase, β-1,4-glucanase, β-1,6-glucanase, aminocyclopropane-1-carboxylic acid deaminase, or enzymes involved in producing nod factors (e.g., nodA, nodB, or nodI).

[0066] When the enzyme comprises a protease or peptidase, the protease or peptidase can be a protease or peptidase that cleaves a protein, peptide, proprotein, or preproprotein to produce a bioactive peptide. The bioactive peptide can be any peptide that exhibits biological activity.

[0067] Examples of bioactive peptides include RKN 16D10 and RHPP.

[0068] Proteases or peptidases that cleave proteins, peptides, proproteins, or preproproteins to produce biologically active peptides can include subtilisin, acid proteases, alkaline proteases, proteinases, endopeptidases, exopeptidases, thermolysin, papain, pepsin, trypsin, pronase, carboxylase, serine proteases, glutamic acid proteases, aspartic acid proteases, cysteine ​​proteases, threonine proteases, or metalloproteases.

[0069] Proteases or peptidases can cleave proteins in protein-rich diets (eg, soybean meal or yeast extract).

[0070] Plant growth stimulating proteins can also include enzymes that degrade or catalyze bacterial, fungal, or plant nutrient sources. Such enzymes include cellulases, lipases, lignin oxidases, proteases, glycoside hydrolases, phosphatases, nitrogenases, nucleases, amidases, nitrate reductases, nitrite reductases, amylases, ammonia oxidases, ligninases, glucosidases, phospholipases, phytases, pectinases, glucanases, sulfatases, ureases, and xylanases. When the enzyme is pectinase, it can also be pectin lyases (also called pectate lyases), pectate lyases, or polygalacturonases (including endopolygalacturonase or exopolygalacturonase). When introduced into a plant's growth medium or applied to a plant, seed, or the area surrounding a plant or plant seed, a fusion protein comprising an enzyme that degrades or modifies bacteria, fungi, or plant nutrient sources can aid in the processing of nutrients in the vicinity of the plant, resulting in enhanced uptake of the nutrient by the plant or enhanced uptake by beneficial bacteria or fungi in the vicinity of the plant. In one embodiment, the phospholipase comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:9.

[0071] Suitable cellulases include endocellulases (e.g., endogluconases such as Bacillus subtilis endoglucanase, Bacillus thuringiensis endoglucanase, Bacillus cereus endoglucanase, or Bacillus clausii endoglucanase), exocellulases (e.g., Trichoderma reesei exocellulase), and β-glucosidases (e.g., Bacillus subtilis β-glucosidase, Bacillus thuringiensis β-glucosidase, Bacillus cereus β-glucosidase, or Bacillus clausii endoglucanase). clausii) β-glucosidase).

[0072] The lipase may include Bacillus subtilis lipase, Bacillus thuringiensis lipase, Bacillus cereus lipase, or Bacillus clausii lipase.

[0073] In one embodiment, the lipase comprises a Bacillus subtilis lipase.

[0074] In another embodiment, the cellulase is a Bacillus subtilis endoglucanase. In one embodiment, the endoglucanase comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:8.

[0075] In yet another embodiment, the fusion protein comprises the E. coli protease PtrB.

[0076] Suitable lignin oxidases include lignin peroxidase, laccase, glyoxal oxidase, ligninase and manganese peroxidase.

[0077] The protease can include subtilisin, acid protease, alkaline protease, proteinase, peptidase, endopeptidase, exopeptidase, thermolysin, papain, pepsin, trypsin, pronase, carboxylase, serine protease, glutamic acid protease, aspartic acid protease, cysteine ​​protease, threonine protease, or metalloprotease.

[0078] The phosphatase can include a phosphate monoester hydrolase, a phosphomonoesterase (e.g., PhoA4), a phosphate diester hydrolase, a phosphodiesterase, a triphosphate monoester hydrolase, a phosphate anhydride hydrolase, a pyrosphatase, a phytase (e.g., Bacillus subtilis EE148 phytase or Bacillus thuringiensis BT013A phytase), a trimetaphosphatase, or a triphosphatase.

[0079] Proteins and peptides that protect plants from pathogens The fusion protein may comprise a targeting sequence, an exosporium protein or exosporium protein fragment, and at least one protein or peptide that protects the plant from a pathogen.

[0080] The protein or peptide may include a protein or peptide that stimulates a plant immune response. For example, the protein or peptide that stimulates a plant immune response may include a plant immune system enhancer protein or peptide. The plant immune system enhancer protein or peptide may be any protein or peptide that has a beneficial effect on the plant's immune system. Suitable plant immune system enhancer proteins and peptides include harpin, α-elastin, β-elastin, systemin, phenylalanine ammonia lyase, elicitin, defensin, cryptogein, flagellin protein, and flagellin peptide (e.g., flg22).

[0081] Alternatively, proteins or peptides that protect plants from pathogens can be proteins or peptides with antibacterial, antifungal, or both antibacterial and antifungal activity. Examples of such proteins and peptides include bacteriocins, lysozyme, lysozyme peptides (e.g., LysM), siderophores, nonribosomally active peptides, conalbumin, albumin, lactoferrin, lactoferrin peptides (e.g., LfcinB), streptavidin, and TasA.

[0082] The protein or peptide that protects plants from pathogens may be a protein or peptide with insecticidal activity, anthelmintic activity, or a combination thereof that inhibits insect or insect predation. For example, the protein or peptide that protects plants from pathogens may include an insecticidal bacterial toxin (e.g., VIP insecticidal protein), an endotoxin, a Cry toxin (e.g., a Cry toxin derived from Bacillus thuringiensis), a protease inhibitor protein or peptide (e.g., a trypsin inhibitor or an arrowhead protease inhibitor), a cysteine ​​protease, or a chitinase. When the Cry toxin is a Cry toxin derived from Bacillus thuringiensis, the Cry toxin may be a Cry5B protein or a Cry21A protein. Cry5B and Cry21A have both insecticidal and nematicidal activity.

[0083] Proteins that protect plants from pathogens can include enzymes. Suitable enzymes include proteases and lactonases. Proteases and lactonases can be specific for bacterial signaling molecules (e.g., bacterial lactone homoserine signaling molecules).

[0084] When the enzyme is a protease, the enzyme can be a serine protease, such as Sep1. Serine proteases are the largest and most widely distributed class of proteases. They cleave peptide bonds at serine residues within specific recognition sites in proteins. These proteases are frequently used by bacteria for nutrient removal from the environment. Serine proteases have also been shown to exhibit nematicidal activity through the digestion of nematode gut tissue. Studies of Bacillus firmus strain DS-1, which exhibits nematicidal activity against Meloidogyne incognita and soybean cyst nematode, revealed that the serine protease produced by the strain possesses serine protease activity and degrades nematode gut tissue. Geng, C., et al., “A Novel Serine Protease, Sep1, from Bacillus firmus DS-1 Has Nematicidal Activity and Degrades Multiple Intestinal-Associated Nematode Proteins”, Scientific Reports, 2016, vol. 6, no. 25012.

[0085] In Table 1, SEQ ID NOS: 5-7 are the amino acid sequences of wild-type and mutant enzymes that exhibit or are predicted to exhibit serine protease activity. Thus, for example, SEQ ID NOS: 5 and 6 provide the amino acid sequences of wild-type serine protease enzymes from two different Bacillus firmus strains, sharing 98% sequence similarity. SEQ ID NOS: 7 provides the amino acid sequence of the same enzyme as SEQ ID NOS: 5, with the exception that amino acids 181-240 of SEQ ID NOS: 5 are deleted, such that SEQ ID NOS: 5 and 7 share 81% sequence similarity. The catalytic residues referenced above in Geng et al., 2016, are maintained in the mutant serine protease amino acid sequence of SEQ ID NOS: 7.

[0086] [Table 1]

[0087] When the enzyme is a lactonase, the lactonase can include 1,4-lactonase, 2-pyrone-4,6-dicarboxylate lactonase, 3-oxoadipate enol-lactonase, actinomycin lactonase, deoxylimonate A-ring-lactonase, gluconolactonase L-rhamnono-1,4-lactonase, limonin-D-ring-lactonase, steroid-lactonase, triacetate-lactonase, or xylono-1,4-lactonase.

[0088] The enzyme can also be an enzyme specific for bacterial or fungal cellular components. For example, the enzyme can include β-1,3-glucanase, β-1,4-glucanase, β-1,6-glucanase, chitosinase, chitinase, chitosinase-like enzyme, lyticase, peptidase, proteinase, protease (e.g., alkaline protease, acid protease, or neutral protease), mutanolysin, stapholysin, or lysozyme. In one embodiment, the chitosinase comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 10.

[0089] Proteins and peptides that enhance plant stress tolerance The fusion protein may comprise a targeting sequence, an exosporium protein or exosporium protein fragment, and at least one protein or peptide that enhances stress tolerance in plants.

[0090] For example, proteins or peptides that enhance stress tolerance in plants include enzymes that degrade stress-related compounds. Stress-related compounds include, but are not limited to, aminocyclopropane-1-carboxylic acid (ACC), reactive oxygen species, nitric oxide, oxylipins, and phenolics. Specific reactive oxygen species include hydroxyl, hydrogen peroxide, oxygen, and superoxide. Enzymes that degrade stress-related compounds may include superoxide dismutase, oxidase, catalase, aminocyclopropane-1-carboxylic acid deaminase, peroxidase, antioxidant enzymes, or antioxidant peptides.

[0091] Proteins or peptides that enhance plant stress resistance can also include proteins or peptides that protect plants from environmental stress.Environmental stress can include, for example, drought, flooding, heat, freezing, salt, heavy metals, low pH, high pH, ​​or a combination thereof.For example, proteins or peptides that protect plants from environmental stress can include ice nucleation protein, prolinase, phenylalanine ammonia lyase, isochorismate synthase, isochorismate pyruvate lyase, or choline dehydrogenase.

[0092] Plant-binding proteins and peptides The fusion protein may comprise a targeting sequence, an exosporium protein or exosporium protein fragment, and at least a plant-binding protein or peptide. The plant-binding protein or peptide may be any protein or peptide capable of specifically or non-specifically binding to any part of a plant (e.g., plant roots, or aerial parts of a plant such as leaves, stems, flowers, or fruits) or plant material. Thus, for example, the plant-binding protein or peptide may be a root-binding protein or peptide, or a leaf-binding protein or peptide.

[0093] Suitable plant-binding proteins and peptides include adhesins (e.g., ricadhesins), flagellins, optins, lectins, expansins, biofilm structural proteins (e.g., TasA or YuaB), fimbrial proteins, pilus proteins, intimins, invasins, agglutinins, and affimbril proteins.

[0094] Recombinant Bacillus expressing the fusion protein The fusion proteins described herein can be expressed by recombinant Exosporium-producing Bacillus cells. The fusion protein can be any of the fusion proteins described above.

[0095] The recombinant Exosporium-producing Bacillus cells can co-express two or more of any of the above fusion proteins. For example, the recombinant Exosporium-producing Bacillus cells can co-express at least one fusion protein comprising a plant-binding protein or peptide with at least one fusion protein comprising a plant growth-stimulating protein or peptide, at least one fusion protein comprising a protein or peptide that protects plants from pathogens, or at least one fusion protein comprising at least one protein or peptide that enhances stress tolerance in plants.

[0096] The recombinant Exosporium-producing Bacillus cells can include Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides, Bacillus pseudomycoides, Bacillus samanii, Bacillus gaemokensis, Bacillus weihenstephensis, Bacillus toyoiensis, or combinations thereof. For example, the recombinant Exosporium-producing Bacillus cells can include Bacillus cereus, Bacillus thuringiensis, Bacillus pseudomycoides, or Bacillus mycoides. In particular, the recombinant Exosporium-producing Bacillus cells can include Bacillus thuringiensis or Bacillus mycoides.

[0097] To generate recombinant Exosporium-producing Bacillus cells expressing a fusion protein, any member of the Bacillus cereus family can be conjugated, transduced, or transformed with a vector encoding the fusion protein using standard methods known in the art (e.g., by electroporation). The bacteria can then be screened to identify transformants by any method known in the art. For example, if the vector contains an antibiotic resistance gene, the bacteria can be screened for antibiotic resistance. Alternatively, DNA encoding the fusion protein can be integrated into the chromosomal DNA of a B. cereus family host. The recombinant Exosporium-producing Bacillus cells can then be exposed to conditions that induce sporulation. Suitable conditions for inducing sporulation are known in the art. For example, recombinant Exosporium-producing Bacillus cells can be plated onto agar plates and incubated at about 30°C for several days (e.g., 3 days).

[0098] Inactivated, non-virulent, or genetically engineered strains of any of the above species can also be used. For example, Bacillus thuringiensis lacking Cry toxins can be used. Alternatively, once recombinant B. cereus spores expressing the fusion protein are generated, they can be inactivated to prevent further germination during use. Any method known in the art for inactivating bacterial spores can be used. Suitable methods include, but are not limited to, heat treatment, gamma irradiation, X-ray irradiation, UV-A irradiation, UV-B irradiation, chemical treatment (e.g., treatment with glutaraldehyde, formaldehyde, hydrogen peroxide, acetic acid, bleach, or any combination thereof), or a combination thereof. Alternatively, spores derived from non-virulent strains or genetically or physically inactivated strains can be used.

[0099] Scalability The novel media of the present invention can be used for fermentation in any suitable vessel, including glass or plastic tubes or microtiter plates, glass or stainless steel flasks, bottles, or carboys, microreactors, or bioreactors. Bioreactors suitable for use with the disclosed media can be up to about 5 L, up to about 20 L, up to about 1000 L, up to about 3000 L, and industrial scale (e.g., up to 30,000 L in volume). [Example]

[0100] Example Example 1: Identification of factors determining yield, sporulation, and cargo protein activity Experiments were conducted to develop a cost-effective fermentation method for recombinant exosporium-producing Bacillus cells expressing a protein or peptide of interest on exosporium. Such a novel fermentation method can obtain high spore titers and protein activity while maintaining high sporulation efficiency. 9 spores / mL to 3.5 x 10 9 Enhanced protein activity compared to basal medium with titer yields in the range of spores / mL, approximately 1 × 10 8 A prototype medium was developed that yielded spores / mL and significantly reduced protein activity. The basal medium was derived from the laboratory-scale medium and used low concentrations of yeast extract as the primary source of carbon and nitrogen ("Base Medium").

[0101] Initial experiments were designed to elucidate the primary factors or combinations of factors driving key responses such as spore titer, sporulation rate, and protein activity. In separate experiments using standard lab media, brain heart infusion broth (BHI) broth + 0.5% glycerol; Lauria-Bertani (LB) broth containing tryptone, yeast extract, and sodium chloride; succinic acid nutrient agar (SNA); and tryptic soy broth (TSB) containing casein digest, soybean meal digest, dextrose, sodium chloride, and dipotassium phosphate did not yield improved results compared to Base Medium. Furthermore, experimental media without yeast extract produced bacterial growth, but sporulation rates varied significantly. These results demonstrate that multiple factors contribute to medium performance.

[0102] Data on fermentation results were compiled, and a machine learning model was trained to predict process yield. Based on several sets of fermentation data, a model was developed to evaluate the relative contribution of several fermentation parameters, including each of the medium components. Several factors (temperature, harvest time, yeast extract, total carbohydrates, total carbon + nitrogen, and total solids) accounted for over 80% of the variability in yield. Further analysis showed that the main factor affecting sporulation efficiency was related to the interaction between the carbon and nitrogen sources.

[0103] Example 2: Identification of novel media candidates Experiments were designed to elucidate the key factors and combinations of factors driving key responses expressed in a model system as fluorescence: spore titer, sporulation rate, and cargo protein activity. Fermentations were performed using a recombinant Bacillus thuringiensis strain, Bt013A, engineered to display the fluorescent protein tdTomato in exosporium, where it could be detected to assess protein activity. Briefly, to construct a Bacillus cereus family member displaying the tdTomato fluorescent protein (the "tdTomato strain"), the pSUPER plasmid was generated by fusing the pUC57 plasmid (containing an ampicillin resistance cassette and ColE1 replication origin) with the pBC16-1 plasmid from Bacillus cereus (containing a tetracycline resistance gene, the repU replication gene, and the oriU replication origin). This 5.8 kb plasmid can replicate in both E. coli and Bacillus and can be selected for by conferring resistance to β-lactam antibiotics in E. coli and tetracycline in Bacillus. The base pSUPER plasmid was modified by insertion of a PCR-generated fragment fusing the BclA promoter (SEQ ID NO: 1), start codon, amino acids 20-35 of BclA (amino acids 20-35 of SEQ ID NO: 2), and an in-frame alanine linker sequence with SEQ ID NO: 3, resulting in a plasmid designated pSUPER-BclA 20-35-SEQ ID NO: 3. This construct was transformed into E. coli and plated onto Lysogeny broth plates supplemented with ampicillin (100 μg / mL) to obtain single colonies. Individual colonies were used to inoculate Lysogeny broth and ampicillin and incubated overnight at 37°C and 300 rpm. Plasmids from the resulting cultures were extracted using a commercially available plasmid purification kit. The DNA concentrations of these plasmid extracts were measured spectrophotometrically, and the resulting plasmids were subjected to analytical digestion with appropriate combinations of restriction enzymes. The resulting digestion patterns were visualized by agarose gel electrophoresis to examine the size of the plasmids and the presence of different plasmid features.The relevant sections of the purified pSUPER derivatives were further examined by Sanger sequencing. The verified pSUPER-BclA 20-35-SEQ ID NO:3 plasmid was introduced into Bacillus thuringiensis BT013A by electroporation. Single transformed colonies were isolated by plating on nutrient broth plates containing tetracycline (10 μg / mL). Individual positive colonies were used to inoculate brain heart infusion broth containing tetracycline (10 μg / mL) and incubated overnight at 30°C and 300 rpm. Genomic DNA from the resulting culture was purified, and the relevant sections of the pSUPER-BclA 20-35-SEQ ID NO:3 plasmid were resequenced to confirm the genetic purity of the cloned sequences. Verified colonies were grown overnight in brain heart infusion broth with 10 μg / mL tetracycline and sporulation was induced by incubation in base medium or experimental medium at 30°C for 48 hours.

[0104] The treatment conditions were as follows: the production medium was inoculated with the seed medium at an optical density of 1.0 Au, and the seed medium was oThe strains were grown at 25°C for 48–64 hours (harvest time dependent on sporulation rate). Microreactor-scale experiments did not control pH due to equipment limitations, but pH was controlled (by acid and base addition) when scaled up to a 5 L bioreactor. These experiments revealed the need for enrichment of the base medium with additional carbon and nitrogen components. Once a selected set of critical parameters was identified, further experiments were designed to find appropriate levels of these components, leading to prototype M0–M5 media. To confirm and compare the results, experiments were conducted to test the performance of the tdTomato strains in three key medium prototypes, M0, M2, and M5, along with the base medium, focusing on spore titer and fluorescence. Whole broths were used to evaluate performance by testing spore titer using a basic hemocytometer and tdTomato fluorescence using a fluorescence microplate reader (Ex: 551 nm, Em: 584 nm). The results are reported in Example 4 below. Table 2 shows the compositions of medium prototypes M0-M5, which provided higher titer yields and enhanced cargo protein activity (fluorescence) compared to Base Medium.

[0105] [Table 2]

[0106] Example 3: Buffering of novel media Experiments were then conducted to test the performance of the tdTomato strain in six pre-existing medium prototypes, M0, M1, M2, M3, M4, and M5, compared to the Base Medium. In Examples 3 and 4, CaCl2*2H2O and MgSO4*7H2O were used for M2 at the lower end of the concentration range shown in Table 2, i.e., 0.025 g / L and 0.02 g / L, respectively. Because there was no pH control in the microreactor in which the experiments were conducted, the pH was observed to drop to very low levels in media M1, M2, M3, M4, and M5. The Base Medium sporulated well, producing the expected spore titer (1 x 108 The spores were visualized as tdTomato fluorescent and were confirmed by plate reader.

[0107] To confirm that reduced pH was indeed the factor resulting in reduced growth and / or poor sporulation in some of the novel media, experiments were performed in a pH-controlled environment in a 5L bioreactor. The strain grew well in several novel media (M0, M2, M5) in a pH-controlled environment, which resulted in higher acid and base consumption than in fermentation runs using the base medium. tdTomato protein production was visualized and measured by fluorescence using a plate reader. Protein concentrations per spore appeared to be higher in the prototype media compared to the base medium.

[0108] To reduce pH fluctuations in non-pH-controlled environments such as microreactors or shake flasks, experiments were designed to test buffers of different strengths in novel media, as shown in Table 3. Buffer optimization appeared to resolve the pH fluctuation problem. 1XB medium exhibited large pH fluctuations, while 2.5XB medium exhibited acceptable fluctuations. 4XB and 6XB media significantly reduced the fluctuations. The terms 1X, 2.5X, 4X, and 6X refer to the calculation of buffer capacity and not to differences in the volumes of buffer components. It should be noted that such buffers are not necessary in fermentors, which allow for monitoring and adjustment of pH during fermentation via the addition of acid or base as needed.

[0109] [Table 3]

[0110] Example 4: Spore titer and cargo protein activity with novel media The effect of a pH-controlled environment was further investigated in a 5-L bioreactor, where the performance of the tdTomato strain was tested in three novel medium prototypes, M0, M2, and M5, as well as in Base Medium. Phosphate levels were as follows: 1 g / L KHPO and 0.8 g / L KHPO. In these experiments, pH was controlled using acid and base addition rather than the buffer system described in Example 3. Spore titers, sporulation rates, and fluorescence are shown in Table 4.

[0111] [Table 4]

[0112] Scaled tdTomato performance in Base Medium and novel medium prototypes M0, M2, and M5 is shown in Figure 1. Fluorescence was higher in the novel media in these experiments. Note that the % sporulation in M5 was not as high as in M0 and M2 due to a malfunctioning pH probe; the pH of that particular M5 batch was suboptimal, resulting in low sporulation. (In other experiments, fermentations in M5 medium typically achieved greater than 95% sporulation.) For M2, the scaled performance showed that the fold increase in fluorescence was greater than the fold increase in cells, indicating that each spore exhibited higher levels of tdTomato protein when fermented with the improved method compared to the method using Base Medium.

[0113] Example 5: Performance of the new medium with other Bacillus strains The above experiments were performed using recombinant exosporium-producing Bacillus thuringiensis cells expressing a protein or peptide of interest in their exosporium via fusion to a targeting sequence. The performance of Medium M2 was also examined using such recombinant exosporium-producing Bacillus cells from several other exosporium-producing Bacillus species. In this Example 5, CaCl2*2H2O and MgSO4*7H2O were used for M2 at the upper end of the concentration range shown in Table 2, 0.375 g / L and 0.45 g / L, respectively.

[0114] As shown in Figure 2, spore titers were 2 x 10 for each strain tested. 9 CFU / mL, which exceeded the spore titer of 1 x 10 observed using Base Medium. 8 This represents a significant increase of over 100%.

[0115] As shown in Figure 3, strong tdTomato fluorescence was observed in strains #1 to #4 using the novel medium M2, indicating robust protein expression with the use of the novel medium.

[0116] As shown in Figure 4, modified medium M2, when used with bacterial lines displaying the tdTomato fluorescent protein, resulted in a substantial increase in spore titer and protein production. Similar to the results in Example 4, the fold increase in fluorescence was higher than the fold increase in spore titer, indicating that each spore had a higher level of the displayed protein than when cells were fermented in Base Medium.

[0117] Example 6: Spore titer and cargo protein activity of tdTomato on novel medium prototypes M2 and OM3 Experiments were conducted to test the performance of the tdTomato strain described in Example 2 with novel media prototypes M2 and OM3 compared to Base Medium. Table 2 shows the compositions of media M2 and OM3, which resulted in higher titer yields and enhanced cargo protein activity (fluorescence) compared to Base Medium. For M2, CaCl2*2H2O and MgSO4*7H2O were used at the lower end of the concentration range shown in Table 2, i.e., 0.025 g / L and 0.02 g / L, respectively.

[0118] The tdTomato strain was fermented at the microreactor scale in Base Medium, Novel Medium M2, and Novel Medium OM3. Spore titer and fluorescence were evaluated as in Example 2, and the performance of the scaled tdTomato is shown in Figure 5. Spore titer and sporulation rate are shown in Table 5.

[0119] [Table 5]

[0120] Example 7: Construction of Bacillus cereus family members displaying serine proteases or serine protease variants Further experiments were performed using a recombinant Bacillus thuringiensis strain, Bt013A, engineered to display on exosporium a serine protease (Sep1 mutant) whose protein activity could be analyzed. Briefly, a Bacillus cereus family member displaying a Sep1 mutant protein and carrying an ExsY knockout ("Sep1 strain") was constructed.

[0121] To construct a Bacillus cereus family member displaying a Sep1 mutant, the pSUPER plasmid was generated by fusing the pUC57 plasmid (containing an ampicillin resistance cassette and ColE1 origin of replication) with the pBC16-1 plasmid from Bacillus cereus (containing a tetracycline resistance gene, the repU replication gene, and the oriU origin of replication). This 5.8 kb plasmid can replicate in both E. coli and Bacillus species and can be selected by conferring resistance to ρ3-lactam antibiotics in E. coli and tetracycline resistance in Bacillus species. The basic pSUPER plasmid was modified by inserting a PCR-generated fragment encoding the Sep1 mutant, fusing the promoter, start codon, targeting sequence, and an in-frame alanine linker according to SEQ ID NO:7. The pSUPER plasmid was obtained. This construct was transformed into E. coli and plated on Lysogeny broth plates containing ampicillin (100 μg / mL) to obtain single colonies. Individual colonies were used to inoculate lysogeny broth and ampicillin and incubated overnight at 37°C and 300 rpm. Plasmids from the resulting cultures were extracted using a commercially available plasmid purification kit. The DNA concentrations of these plasmid extracts were measured spectrophotometrically, and the resulting plasmids were subjected to analytical digestion with the appropriate combination of restriction enzymes. The resulting digestion patterns were visualized by agarose gel electrophoresis to examine the size of the plasmid and the presence of distinct plasmid features. Relevant sections of the purified pSUPER derivatives, such as the Sep1 mutant expression cassette, were further examined by Sanger sequencing.

[0122] The pSUPER plasmid, verified as described above, was introduced into Bacillus thuringiensis BT013A (Accession No. NRL B-50924) by electroporation. Single transformed colonies were isolated by plating on nutrient broth plates containing tetracycline (10 μg / mL). Individual positive colonies were used to inoculate brain heart infusion broth containing tetracycline (10 μg / mL) and incubated overnight at 30°C and 300 rpm. Genomic DNA from the resulting culture was purified, and the relevant section of the pSUPER plasmid was resequenced to confirm the genetic purity of the cloned sequence. Verified colonies were grown overnight in brain heart infusion broth containing 10 μg / mL tetracycline and sporulation was induced by incubation in yeast extract-based medium at 30°C for 48 hours.

[0123] To generate an exsY knockout (KO) mutant of Bacillus thuringiensis BT013A, we constructed a gene shuttle and integration vector containing the pUC57 backbone, which can replicate in E. coli, and the origin of replication and erythromycin resistance cassette from pE194. This construct can replicate in both E. coli and Bacillus species. Constructs were generated containing a 1-kb DNA region corresponding to the upstream region of the exsY gene and a 1-kb region corresponding to the downstream region of the exsY gene, both amplified from Bacillus thuringiensis BT013A. For each construct, the two 1-kb regions were then spliced ​​together using homologous recombination with the overlapping regions and the pKOKI plasmid, respectively. The plasmid constructs were verified by digestion and DNA sequencing. Clones were screened for erythromycin resistance.

[0124] Clones were passaged in brain heart infusion broth at elevated temperatures (40°C). Individual colonies were picked onto LB agar plates containing 5 μg / mL erythromycin, grown at 30°C, and screened for the presence of the chromosomally integrated pKOKI plasmid by colony PCR. Colonies with integration events were subsequently passaged to screen for single colonies that had lost erythromycin resistance (signaling loss of the plasmid and removal of the exsY gene by recombination). The verified deletion was confirmed by PCR amplification and sequencing of the target region on the chromosome. Finally, a PCR-amplified, circularized pBC section of the pSUPER plasmid (described above) was transformed into this exsY mutant strain of BT013A. The resulting strain displays the Sep1 mutant protein and is a Bacillus cereus family member with an exsY knockout ("Sep1 strain").

[0125] For each exsYKO mutant expressing a serine protease variant, an overnight culture was grown in BHI medium at 30°C and 300 rpm in a baffled flask with antibiotic selection. One milliliter of this overnight culture was inoculated into 50 mL of yeast extract-based medium in a baffled flask and grown at 30°C for 2 days. An aliquot of spores was removed and vortexed. Spores were harvested by centrifugation at 8000 × g for 10 minutes, and the supernatant containing exosporium fragments was filtered through a 0.22 μm filter to remove residual spores. No spores were observed in the filtrate.

[0126] Example 8: Spore titer and cargo protein activity of Sep1 strain on novel media prototypes M2 and OM3 Sep1 strain was produced by fermentation in flasks or 20 L fermenters. Briefly, overnight brain heart infusion seed flasks of Sep1 strain containing 10 μg / mL tetracycline were inoculated into 1 L shake flasks or 20 L fermenters of M2 or OM3 medium and grown at 30°C for 48–72 h to produce ≥90% endospores. For M2, CaCl2*2H2O and MgSO4*7H2O were used at the lower end of the concentration range shown in Table 2, i.e., 0.025 g / L and 0.02 g / L, respectively. For this study, the harvested whole cell broth was used as the final product. Exosporium fragments were not harvested prior to the analysis described below.

[0127] Protease activity was measured at the time of fermentation harvest (no downstream processing was performed). Enzyme activity was measured using a synthetic peptide substrate (Ala-Ala-Pro-Phe). The peptide substrate was fused with a C-terminal nitrophenyl and an N-terminal succinyl group. The peptide exhibited an absorbance maximum at 320 nm before protease cleavage and shifted to 390 nm after cleavage. The assay mixture consisted of 2.5 mg / mL peptide substrate in 240 μL of 50 mM Hepes buffer, pH 7.5, containing 5 mM CaCl2. The substrate and buffer were preincubated at room temperature, after which 25 μL of enzyme solution was added. Protease activity of the Sep1 strain in base medium or the new medium prototypes M2 and OM3 is shown in Figure 6.

[0128] Spore titers were determined by hemocytometry in the fermentation whole broth, and the results are shown in Table 6.

[0129] [Table 6]

Claims

1. 1. A method for producing a fermentation product from recombinant Exosporium-producing Bacillus cells expressing a fusion protein, comprising: culturing in a medium a recombinant Exosporium-producing Bacillus cell expressing the fusion protein; The medium is i) yeast extract at a concentration of 3 g / L to 20 g / L; ii) glucose at a concentration of 20-35 g / L; iii) soy flour at a concentration of 10-30 g / L; and iv) Ca ２＋ a source of ions; Including, the recombinant Exosporium-producing Bacillus cell is a cell of a member of the Bacillus cereus family; The method of producing the fusion protein comprises a protein or peptide of interest and an exosporium targeting sequence, an exosporium protein or an exosporium protein fragment.

2. The Ca ２＋ The source of ions is CaCl 2 The method according to claim 1, wherein

3. The medium contains Mg ２＋ The method of claim 1 further comprising a source of ions.

4. The Mg ２＋ The source of ions is MgSO ４ The method according to claim 3, wherein

5. 2. The method of claim 1, wherein the medium further comprises cottonseed flour at a concentration of up to about 10 g / L and / or corn steep liquor at a concentration of up to about 10 g / L.

6. The method according to any one of claims 1 to 5, further comprising maintaining the pH at 6 to 8 during the culturing.

7. 7. The method of claim 6, wherein maintaining the pH is achieved by adding an acid or a base.

8. The method of claim 6 , wherein the medium further comprises a buffer.

9. The buffer is K ２ HPO ４ and K.H. ２ P.O. ４ The method according to claim 8, wherein

10. K ２ HPO ４ is present at a concentration of at least 1 g / L, and KH ２ P.O. ４. The method of claim 9, wherein is present at a concentration of at least 0.8 g / L.

11. 11. The method of claim 1, wherein the culturing is performed at 25°C to 35°C, and / or the culturing is performed for up to 50 hours, and / or the culturing is performed until sporulation of the Bacillus cells is at least 90% complete.

12. The culture yielded at least 1 x 10 ９ The method of any one of claims 1 to 11, wherein a fermentation broth having a spore titer of spores / mL is obtained.

13. The method of any one of claims 1 to 12, wherein the culture medium comprises one or more carbon sources having a total concentration of at least 20 g / L, and optionally one or more nitrogen sources having a total concentration of at least 3 g / L.

14. 14. The method of claim 13, wherein the combined concentration of the one or more carbon sources and the one or more nitrogen sources is at least 15 g / L.

15. The medium is a) yeast extract at a concentration of 5 g / L to 15 g / L; b) glucose at a concentration of 20 g / L to 35 g / L; c) soy flour at a concentration of 10 g / L to 30 g / L; d) K at a concentration of 1 g / L to 5 g / L ２ HPO ４ and KH at a concentration of 0.5 g / L to 2 g / L. ２ P.O. ４ a buffer comprising: e) CaCl at a concentration of 0.015 g / L to 0.80 g / L 2 *2H 2 O; and f) MgSO at a concentration of 0.10 g / L to 0.80 g / L 4 *7H 2 O The method of any one of claims 2 to 14, comprising:

16. The medium is a) yeast extract at a concentration of 10 g / L to 15 g / L; b) glucose at a concentration of 25 g / L to 30 g / L; c) soy flour at a concentration of 15 g / L to 20 g / L; d) K at a concentration of 1 g / L to 3 g / L ２ HPO ４ and KH at a concentration of 0.5 g / L to 1 g / L. ２ P.O. ４ a buffer comprising: e) CaCl at a concentration of 0.02 g / L to 0.4 g / L 2 *2H 2 O; and f) MgSO at a concentration of 0.2 g / L to 0.5 g / L 4 *7H 2 O The method of any one of claims 2 to 15, comprising:

17. The members of the Bacillus cereus family include Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides, Bacillus pseudomycoides, Bacillus samanii, Bacillus gaemokensis, Bacillus weihenstephensis, Bacillus 2. The method of claim 1, wherein the bacterial strain is selected from the group consisting of Bacillus weihenstephensis, Bacillus toyoiensis, and combinations thereof.

18. 18. The method of claim 17, wherein the recombinant Bacillus cell is derived from Bacillus thuringiensis BT013A.

19. A fermentation broth produced by the method according to any one of claims 1 to 18.

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