Stable protein production process using bacterial host cells

Abstract

The invention relates to the field of industrial fermentation and protein production. Specifically, the present invention relates to a method for producing a protein of interest in a fermentation medium, the method comprising the steps of: a) inoculating the fermentation medium with a bacillus host cell comprising a gene encoding the protein of interest under the control of a promoter; b) culturing the Bacillus host cell in the fermentation medium under conditions that facilitate growth of the Bacillus host cell and expression of the protein of interest, c) adding sulfate to the fermentation medium to achieve a concentration of sulfate of at least 20 mM in the fermentation medium; and d) precipitating and / or crystallizing the protein of interest during culture; wherein the fermentation medium comprises an amino acid derivative in an amount of from 0 g / l to 30 g / l of the fermentation medium. Further contemplated is the use of a combination of a sulfate and an amino acid derivative for the production of a protein of interest in a Bacillus host cell in a fermentation medium and a crystalline protein of interest obtained or obtainable by the method of the invention.

Description

[0001] The present invention relates to the field of industrial fermentation and protein production. In particular, the present invention relates to a method for producing a protein of interest in a fermentation medium, the method comprising the following steps: a) inoculating the fermentation medium with a Bacillus host cell comprising a gene encoding a protein of interest under the control of a promoter; b) culturing the Bacillus host cell in the fermentation medium under conditions that are favorable for the growth of the Bacillus host cell and the expression of the protein of interest, c) adding sulfate to the fermentation medium to reach a concentration of at least 20 mM sulfate in the fermentation medium; and d) allowing the protein of interest to precipitate and / or crystallize during the culturing; wherein the fermentation medium comprises an amino acid derivative in an amount of 0 g / L-30 g / L of the fermentation medium, preferably in an amount of 0.1 g-30 g / L of the fermentation medium.

[0002] Further contemplated is the use of a combination of sulfate and an amino acid derivative for the production of a protein of interest in a Bacillus host cell in a fermentation medium and a crystallized protein of interest obtained or obtainable by the method of the invention.

[0003] Microorganisms are widely used as industrial workhorses for the production of proteins of interest, particularly enzymes. The biotechnological production of proteins of interest is carried out via a fermentation process involving the cultivation of microorganisms and subsequent purification of the product.

[0004] Microorganisms, such as Bacillus species, are able to secrete significant amounts of proteins of interest into the fermentation medium. This allows for simpler purification of the protein of interest compared to intracellular production and explains the success of Bacillus host cells in industrial applications. In addition, high titers of the protein of interest can be obtained and are also desirable. Recent advances in strains and fermentation methods have led to increased titers of the protein of interest.

[0005] However, high titers of the protein of interest may also have disadvantages. For example, high titers of the protein of interest may lead to undesirable protein instability and other undesirable effects on the protein of interest and / or the host organism, such as irreversible aggregation, inactivation and / or (auto)proteolytic processes, which may reduce the health of the host or may reduce the yield of the protein of interest already during cultivation and subsequent purification processes.

[0006] Different methods have been reported to increase the yield of proteins of interest produced by Bacillus cells and to stabilize the proteins of interest during the culture in large-scale bioreactors. These methods involve, for example, changes in the composition of the culture medium. Other methods involve lowering the temperature, especially reducing the likelihood of inclusion body formation (Hashemi 2012, Food Bioprocess Technol 5: 1093-1099; Wenzel 2011, Applied and Environmental Microbiology 77: 6419-6425).

[0007] WO93 / 13125 describes a protein production method for proteins that are prone to inactivation and degradation during fermentation. The method is intended to produce a protease variant carrying a specific amino acid replacement in a Bacillus subtilis strain by continuously and reversibly protecting the protease from the inactivation or degradation during the production phase. This protection is largely achieved by genetic engineering to reduce the solubility of the protein of interest, namely by introducing the specific amino acid replacement in the protease sequence. The protease is precipitated in the fermentation medium, then separated and purified. However, this protein production method does not provide a solution for preparing an enzyme that is easy to (automatically) hydrolyze and does not carry the replacement that causes protective precipitation.

[0008] WO 2018 / 185048 discloses a recovery method for recovering a protein of interest that is partially precipitated during fermentation. The recovery method involves several steps, including a first separation step, followed by a second solubilization step.

[0009] Similarly, US6316240 relates to the recovery of glycosidases or peptidases in an insoluble state from a culture medium. A method is disclosed which comprises suspending the solid fraction of a fermentation medium and adjusting the pH to allow solubilization and recovery of the protein of interest. Another method for recovering a protein of interest from an insoluble state is disclosed in EP 2125865 which describes a method for solubilizing protease crystals or protease precipitates in a fermentation broth.

[0010] Although several methods for recovering protein crystals and precipitates have been described, there is still a further need to achieve simple and stable production of native or wild-type enzymes that have not been engineered for precipitation or crystal formation, but are susceptible to (auto) hydrolysis or are otherwise unstable. Precipitation needs to be carried out under fermentation conditions, preferably under conditions that are conducive to the growth of the bacillus host cell and the expression of the protein of interest, usually under conditions where the viability of the bacillus host cell is not affected. Most preferably, precipitation is carried out under the optimal or near-optimal growth conditions of the bacillus host. The relevant parameters for optimal growth are, for example, pH, salt or temperature, and are known to those skilled in the art.

[0011] Therefore, means to further improve the yield and stabilize the protein of interest during large-scale industrial fermentation processes are still highly desirable.

[0012] The technical problem on which the present invention is based can be regarded as providing means and methods for meeting the above-mentioned needs. This technical problem is solved by the embodiments characterized in the claims and hereinafter.

[0013] Therefore, the present invention relates to a method for producing a protein of interest in a fermentation medium, the method comprising the steps of: a) inoculating the fermentation medium with a Bacillus host cell comprising a gene encoding the protein of interest under the control of a promoter; b) culturing the Bacillus host cell in the fermentation medium under conditions favorable for growth of the Bacillus host cell and expression of the protein of interest, c) adding sulfate to the fermentation medium to reach a concentration of sulfate of at least 20 mM in the fermentation medium; and d) precipitating and / or crystallizing the protein of interest during the culturing; wherein the fermentation medium comprises the amino acid derivative in an amount of 0 g / l-30 g / l of the fermentation medium.

[0014] It should be understood that the present invention is not limited to the specific methods, protocols, reagents, etc. described herein, as these may vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art.

[0015] It should be understood that "a" or "an" as used in the specification and claims may mean one or more, depending on the context in which it is used. Thus, for example, reference to a "host cell" may mean that at least one host cell can be utilized.

[0016] As used below, the terms "having", "comprising" or "including" or any of their arbitrary grammatical variants are used in a non-exclusive manner. Therefore, these terms can refer to the situation in which there are no additional features in the entity described in this context except the features introduced by these terms, and can also refer to the situation in which there are one or more additional features. For example, the expressions "A has B", "A contains B" and "A includes B" can all refer to the situation in which there are no other elements in A except B (i.e., the situation in which A is composed of B alone and exclusively) and the situation in which there are one or more other elements (such as element C, element C and D or even other elements) in entity A except B. The term "comprising" as used herein should not be understood as restrictive. The term instead indicates that there may be more than the actual items referred to, for example, if the term refers to a method including certain steps, the presence of additional steps should not be excluded. However, the term also covers embodiments in which only the items referred to are present, i.e., the term only has the restrictive meaning of "consisting of...".

[0017] In addition, as used below, the terms "particularly", "more particularly", "generally" and "more generally" or similar terms are used in conjunction with additional or alternative features without limiting the alternative possibilities. Therefore, the features introduced by these terms are additional or alternative features and are not intended to limit the scope of the claims in any way. As will be appreciated by those skilled in the art, the present invention can be implemented by using alternative features. Similarly, the features introduced by "in an embodiment of the present invention" or similar expressions are intended to be additional or alternative features, without any restrictions on alternative embodiments of the present invention, without any restrictions on the scope of the present invention, and without any restrictions on the possibility of combining the features introduced in such a manner with other additional or alternative or non-additional or alternative features of the present invention. Not mandatory, but preferred features are also characterized by the terms "preferably", "more preferably" or "most preferably".

[0018] In addition, it should be understood that the term "at least" means that the item or parameter referred to by the term is limited in one direction, but is open in one or more other directions. In addition, it should be understood that the term "at least one" used herein means that one or more items mentioned after the term can be used according to the present invention. For example, if the term indicates that at least one feed solution should be used, this can be understood as one feed solution or more than one feed solution, i.e., two, three, four, five or any other number of feed solutions. Depending on the item referred to by the term, the technician understands the upper limit (if any) that the term may refer to.

[0019] The term "about" as used herein means that with respect to any number cited after the term, there is an interval accuracy in which a technical effect can be achieved. Therefore, about as referred to herein preferably refers to an exact numerical value or a range around the exact numerical value of ±20%, preferably ±15%, more preferably ±10%, or even more preferably ±5%.

[0020] Therefore, the present invention provides a method that can be used for culturing Bacillus host cells in both laboratory and industrial scale fermentation processes. "Industrial fermentation" mentioned according to the present invention refers to a cultivation method in which at least 100 g of carbon source will be added per kg of initial fermentation medium, preferably at least 200 g of carbon source will be added per kg of initial fermentation medium.

[0021] Such large-scale fermentation methods generally include growing inoculated bacillus host cells under conditions that allow the growth and expression of the protein of interest to be produced. Typically, host cells of suitable microorganisms (such as bacillus host cells) are grown in complex or defined fermentation media, and a carbon source is supplied with a constant or varying amount during the incubation period. More typically, according to the present invention, bacillus host cells are grown in a chemically defined culture medium as described elsewhere herein.

[0022] The method according to the present invention comprises at least the following steps: a) inoculating a fermentation medium with a Bacillus host cell comprising a gene encoding a protein of interest under the control of a promoter; b) culturing the Bacillus host cell in the fermentation medium under conditions conducive to the growth of the Bacillus host cell and the expression of the protein of interest, c) adding sulfate to the fermentation medium to reach a concentration of at least 20 mM sulfate in the fermentation medium; and d) allowing the protein of interest to precipitate and / or crystallize during the culturing. In addition, the method may comprise the step of adding an amino acid derivative to the fermentation medium. Typically, the method comprises the step of adding an amino acid derivative to the fermentation medium in an amount of 0.1 g / L to 30 g / L of the fermentation medium.

[0023] The steps as outlined above may generally be performed in the order given or in any other possible order or in parallel.

[0024] It should be understood that, in particular, steps b), c), d) and the optional step of adding amino acid derivatives can be performed sequentially in a given order or in any other possible order, or can be performed in parallel. Typically, in the case where the initial fermentation medium already contains sulfate and / or has already added amino acid derivatives, the steps can be performed in parallel. Alternatively, step b) can be combined with the step of adding amino acid derivatives, and the step of sulfate addition can be performed thereafter. Also alternatively, step c) and the step of adding amino acid derivatives can be performed in parallel. In the method according to the present invention, it is preferred to maintain the desired high concentration of sulfate throughout the fermentation process. This can be achieved specifically by adding sulfate to the fermentation medium together with the feed to reach and / or maintain a concentration of at least 20 mM sulfate in the fermentation medium. Higher concentrations of sulfate may be required and are defined elsewhere herein.

[0025] The method according to the invention may also include additional steps. These additional steps may include terminating the culture and / or obtaining a product, such as a protein of interest, from the Bacillus host cell culture by appropriate purification techniques. Preferably, the method of the invention further includes the step of obtaining the protein of interest from the Bacillus host cell culture obtained after the culture (usually at the end of the fermentation process).

[0026] In the method of the present invention, generally as a first step, ie at the beginning of the fermentation process, the fermentation medium is inoculated with Bacillus host cells comprising a gene encoding a protein of interest under the control of a promoter.

[0027] The term "Bacillus host cell" refers to a Bacillus cell that serves as a host for an expression construct of a gene encoding a protein of interest. The expression construct may be a naturally occurring expression construct, a recombinantly introduced expression construct, or a naturally occurring expression construct that has been genetically modified in a Bacillus cell. The Bacillus host cell may be a host cell from any member of the bacterial genus Bacillus, preferably Bacillus licheniformis, Bacillus subtilis, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus jautus, Bacillus jautus, Bacillus megaterium, Bacillus pumilus, Geobacillus stearothermophilus, Bacillus thuringiensis or Bacillus velezii. velezensis). More preferably, the bacillus host cell is a cell of Bacillus amyloliquefaciens, Bacillus clausii, Bacillus halodurans, Bacillus lentus, Bacillus licheniformis, Bacillus pumilus, Bacillus subtilis or Bacillus velezensis. Still more preferably, the bacillus host cell is a Bacillus licheniformis, Bacillus pumilus or Bacillus subtilis host cell, even more preferably a Bacillus licheniformis or Bacillus subtilis host cell, most preferably a Bacillus licheniformis host cell. Particularly preferably, the Bacillus licheniformis is selected from the group consisting of Bacillus licheniformis deposited under the American Type Culture Collection numbers ATCC 14580, ATCC 31972, ATCC 53926, ATCC 53757, ATCC 55768 and under the DSMZ numbers (German Collection of Microorganisms and Cell Cultures GmbH) DSM 13, DSM 394, DSM 641, DSM 1913, DSM 11259 and DSM 26543.

[0028] Typically, the host cell belongs to a Bacillus licheniformis species, such as a host cell of Bacillus licheniformis strain ATCC 14580 (which is identical to DSM 13, see Veith et al., "The complete genome sequence of Bacilluslicheniformis DSM 13, an organism with great industrial potential." J. Mol. Microbiol. Biotechnol. (2004) 7: 204-211). Alternatively, the host cell may be a host cell of Bacillus licheniformis strain ATCC 53926. Alternatively, the host cell may be a host cell of Bacillus licheniformis strain ATCC 31972. Alternatively, the host cell may be a host cell of Bacillus licheniformis strain ATCC 53757. Alternatively, the host cell may be a host cell of Bacillus licheniformis strain ATCC 53926. Alternatively, the host cell may be a host cell of Bacillus licheniformis strain ATCC 55768. Alternatively, the host cell may be a host cell of Bacillus licheniformis strain DSM 394. Alternatively, the host cell may be a host cell of Bacillus licheniformis strain DSM 641. Alternatively, the host cell may be a host cell of Bacillus licheniformis strain DSM 1913. Alternatively, the host cell may be a host cell of Bacillus licheniformis strain DSM 11259. Alternatively, the host cell may be a host cell of Bacillus licheniformis strain DSM 26543.

[0029] The bacillus host cell used in the method of the present invention should contain an expression construct of a gene encoding a protein of interest to be expressed by the host cell. As used herein, the term "expression construct" refers to a polynucleotide comprising a nucleic acid sequence (e.g., a gene encoding a protein of interest) operably linked to an expression control sequence (e.g., a promoter). Typically, an expression construct used in the method according to the present invention may at least comprise a nucleic acid sequence encoding a protein of interest operably linked to a promoter.

[0030] Promoter as referred to herein is the nucleotide sequence located upstream of a gene, and the gene is located on the same chain as the gene that can transcribe the gene. The activity of the promoter (also referred to as promoter activity) is understood herein as the ability of the promoter to enable the gene to transcribe and initiate the transcription of the gene, in other words, it is understood as the ability of the promoter to drive gene expression. The promoter is followed by the transcription start site of the gene. The promoter is usually recognized by RNA polymerase together with the transcription factor required for initiating transcription. The functional fragment or functional variant of the promoter is a nucleotide sequence that can be recognized by RNA polymerase and can initiate transcription. The functional fragment or functional variant of the promoter is also included in the promoter in the sense of the present invention.

[0031] The promoter may be an inducer-dependent promoter, whose activity depends on the presence of an activation signal molecule, i.e., an inducer molecule, or may be an inducer-independent promoter, i.e., independent of the presence of an inducer molecule added to the fermentation medium, and is constitutively active or a promoter whose activity can be increased regardless of the presence or absence of an inducer molecule added to the fermentation medium. Preferably, the promoter is an inducer-independent promoter.

[0032] Preferably, the promoter is selected from the group consisting of the promoter sequences of the following items: aprE promoter (the natural promoter from the gene encoding Bacillus subtilis Carlsberg protease), amyQ promoter from Bacillus amyloliquefaciens, amyL promoter from Bacillus licheniformis and variants thereof (preferably as described in US5698415), bacteriophage SPO1 promoter (such as promoter PE4, PE5 or P15) (preferably as described in WO2015118126 or Stewart, CR, Gaslightwala, I., Hinata, K., Krolikowski, KA, Needleman, DS, Peng, AS, Peterman, MA, Tobias, A. and Wei, P. 1998, Genes and regulatory sites of the "host-takeover module" in the terminal redundancy of Bacillus subtilis bacteriophage SPO1. Virology 246 (2), 329-340), cryIIIA promoter from Bacillus thuringiensis (preferably as described in WO9425612 or Agaisse, H. and Lereclus, D. 1994. Structural and functional analysis of the promoter region involved in full expression of the cryIIIA toxin gene of Bacillus thuringiensis. Mol. Microbiol. 13 (1). 97-107.), and combinations thereof, as well as active fragments or variants thereof.

[0033] Preferably, as described herein, the promoter sequence can be combined with a 5'-UTR sequence that is native or heterologous to the host cell. Preferably, the promoter is an inducing agent-independent promoter. More preferably, the promoter is selected from the group consisting of: veg promoter, lepA promoter, serA promoter, ymdA promoter, fba promoter, aprE promoter, amyQ promoter, amyL promoter, phage SPO1 promoter, cryIIIA promoter, combinations thereof, and active fragments or variants thereof. Even more preferably, the promoter sequence is selected from the group consisting of: aprE promoter, amyL promoter, veg promoter, phage SPO1 promoter, and cryIIIA promoter, combinations thereof, or active fragments or variants thereof. Still even more preferably, the promoter is selected from the group consisting of an aprE promoter, an SPO1 promoter (such as PE4, PE5 or P15) (preferably as described in WO15118126), a tandem promoter comprising the promoter sequences amyl and amyQ (preferably as described in WO9943835) and a triple promoter comprising the promoter sequences amyL, amyQ and cryIIIa (preferably as described in WO2005098016). Most preferably, the promoter is an aprE promoter, preferably an aprE promoter from Bacillus amyloliquefaciens, Bacillus clausii, Bacillus halodurans, Bacillus lentus, Bacillus licheniformis, Bacillus pumilus, Bacillus subtilis or Bacillus velez, more preferably an aprE promoter from Bacillus licheniformis, Bacillus pumilus or Bacillus subtilis, most preferably an aprE promoter from Bacillus licheniformis.

[0034] The use of a non-inducer-dependent promoter as specified above can be advantageous because it allows the gene of interest to be expressed continuously throughout the fermentation, resulting in continuous and stable protein production without the need for an inducer molecule. Therefore, the use of a non-inducer-dependent promoter can help to increase the yield of the protein of interest. In addition, the use of a non-inducer-dependent promoter as specified above can be advantageous because no additional feed tube for inducer addition is required, thus providing a simpler and more robust technical setup for the production line.

[0035] It should be understood that the activity of the promoter used according to the method of the present invention is preferably independent of heat-inducible elements. Therefore, the promoter used as an expression control sequence according to the present invention may preferably be a temperature-insensitive promoter and / or lack a heat-inducible element.

[0036] In contrast, an "inducer-dependent promoter" is understood herein as a promoter whose activity increases when an "inducer molecule" is added to a fermentation medium to achieve transcription of a gene operably connected to the promoter. Therefore, for an inducer-dependent promoter, the presence of an inducer molecule triggers an increase in gene expression operably connected to the promoter via signal transduction. Gene expression need not be absent before being activated by the inducer molecule, but can exist at a low level of baseline gene expression, which will increase after the addition of the inducer molecule. An "inducer molecule" is a molecule whose presence in a fermentation medium can affect the increase in gene expression by increasing the activity of an inducer-dependent promoter operably connected to a gene. Inducer molecules known in the art include carbohydrates or their analogs, which can also serve as secondary carbon sources in addition to primary carbon sources (such as glucose). Typically, bacillus host cells are not genetically modified in their ability to take in or metabolize inducer molecules.

[0037] As referred to herein, "protein of interest" refers to any protein, peptide or fragment thereof intended to be produced in a Bacillus host cell. Therefore, protein encompasses polypeptides, peptides, fragments thereof, fusion proteins, and the like.

[0038] Preferably, the protein of interest is an enzyme. In particular, an enzyme that is susceptible to proteolytic degradation during fermentation or has autoproteolytic activity, such as any enzyme that catalyzes a proteolytic reaction. More specifically, the enzyme is classified as an oxidoreductase (EC 1), a transferase (EC 2), a hydrolase (EC 3), a lyase (EC 4), an isomerase (EC 5) or a ligase (EC 6) (EC numbering according to enzyme nomenclature, including the recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (1992) including its supplements published from 1993 to 1999). Preferably, the protein of interest is an enzyme suitable for use in detergents.

[0039] Typically, the protein of interest is a protein that precipitates at a concentration of at least 50 mM sulfate or a similar precipitant in the fermentation medium, more typically, the protein of interest is precipitated at a concentration of at least 100 mM, at least 150 mM, at least 200 mM, at least 250 mM, at least 300 mM, at least 350 mM, or at least 400 mM sulfate or a similar precipitant. More typically, the protein of interest is an enzyme as described elsewhere herein, which precipitates as described above; even more typically, the protein of interest is a protease that precipitates at a concentration of at least 50 mM sulfate or a similar precipitant, more typically, the protein of interest is precipitated at a concentration of at least 100 mM, at least 150 mM, or at least 200 mM sulfate or a similar precipitant in the fermentation medium.

[0040] Typically, the protein of interest is precipitated in the fermentation medium at a concentration starting at 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 12.5 g / L, 15 g / L, 17.5 g / L, 20 g / L, 22.5 g / L, 25 g / L, 27.5 g / L, 30 g / L, 32.5 g / L, 35 g / L, 37.5 g / L, 40 g / L or 45 g / L of protein based on measurement of the protein of interest, typically activity of an enzyme of interest within the fermentation medium.

[0041] Typically, the pH of the fermentation medium is pH 5-9, more preferably 6 to 8.5, and most preferably 6.8 to 7.8. These pH ranges are advantageous because they contribute to good growth of the bacillus host cells and secretion of the protein of interest. Therefore, it is also expected that precipitation of the protein of interest may occur under these conditions. In other words, in the fermentation medium, precipitation of the protein of interest typically occurs at a pH value within the pH range of pH 5-9, more preferably pH 6 to pH 8.5, and most preferably pH 6.8 to pH 7.8.

[0042] More preferably, the enzyme is a hydrolase (EC 3), preferably a glycosidase (EC 3.2) or a peptidase (EC 3.4). Particularly preferred enzymes are enzymes selected from the group consisting of amylases (particularly α-amylases (EC 3.2.1.1)), cellulases (EC 3.2.1.4), lactases (EC 3.2.1.108), mannanases (EC 3.2.1.25), lipases (EC 3.1.1.3), phytases (EC 3.1.3.8), nucleases (EC 3.1.11 to EC 3.1.31) and proteases (EC 3.4); in particular, enzymes selected from the group consisting of amylases, proteases, lipases, mannanases, phytases, xylanases, phosphatases, glucoamylases, nucleases and cellulases, preferably amylases or proteases, preferably proteases. Still more preferably, the enzyme is a serine protease (EC 3.4.21), preferably a subtilisin (EC 3.4.21.62).

[0043] Typically, the protein of interest is a protease selected from a protease having an amino acid sequence with at least 80% sequence identity, such as at least 85% sequence identity, such as at least 95% sequence identity, such as at least 96% sequence identity, such as at least 97% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity with any one of SEQ ID NOs: 1-13. More typically, the protein of interest is a protease selected from a protease having an amino acid sequence as specified by any one of SEQ ID NOs: 1-13 or a variant thereof. Alternatively, the protein of interest is an amylase selected from an amylase having an amino acid sequence with at least 80% sequence identity, such as at least 85% sequence identity, such as at least 95% sequence identity, such as at least 96% sequence identity, such as at least 97% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity with any one of SEQ ID NOs: 14-18. More typically, the protein of interest is an amylase selected from an amylase having an amino acid sequence as specified in any one of SEQ ID NOs: 14-18, or a variant thereof.

[0044] However, it should be understood that variants of those enzymes can exist naturally, i.e., as allelic variants between different strains or from other strains, or artificially generated. Typically, those variants comprise an amino acid sequence that differs from the sequence shown in any one of SEQ ID NOs: 1-18 by at least one amino acid substitution, addition and / or deletion, wherein the enzyme variant comprising the sequence substantially retains the same biological activity as the enzyme comprising the amino acid sequence shown in any one of SEQ ID NOs: 1-18. More typically, the variant comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 1-18, still retaining the desired activity. Preferably, such variants comprise an amino acid sequence having m% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs 1-18, still retaining the desired activity, wherein m is an integer from 80 to 100 or an integer from 90 to 100. Even more preferably, such variants comprise an amino acid sequence having 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 at least 99% sequence identity to the amino acid sequence shown in any one of SEQ ID NOs 1-18, while still retaining the desired activity.

[0045] Particularly preferred proteins of interest are proteases comprising an amino acid sequence having 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 at least 99% sequence identity to SEQ ID NO: 1, 2, 7 or 8 and comprising at least two additional negative charges in the loop region of amino acid residues 98 to 104 according to the numbering of SEQ ID NO: 3 compared to SEQ ID NO: 1.

[0046] The term "at least two negative charges are introduced into a specific amino acid sequence" refers to that the net charge of the specific amino acid sequence is increased by at least two negative charges. The net charge of the specific amino acid sequence is increased by at least two negative charges by changing the amino acid sequence, and can be changed by one or more amino acid sequences selected from the group consisting of substitution, deletion and insertion, preferably by one or more amino acid substitutions. The net charge of the specific amino acid sequence is increased by at least two negative charges by removing the positive charge or by introducing a negative charge or by a combination thereof. The four amino acids aspartic acid (Asp, D), glutamic acid (Glu, E), lysine (Lys, K) and arginine (Arg, R) have side chains that can be charged at neutral pH. At pH 7.0, two amino acids are negatively charged: aspartic acid (Asp, D) and glutamic acid (Glu, E) (acidic side chains), and two amino acids are positively charged: lysine (Lys, K) and arginine (Arg, R) (basic side chains). Thus, the introduction of at least two negative charges in the amino acid sequence can be achieved, for example, by replacing arginine with glutamic acid, replacing two uncharged leucine residues with two glutamic acid residues, by inserting two aspartic acid residues or by deleting two lysine residues. The introduction of at least two negative charges by modifying the amino acid sequence is preferably evaluated under conditions that typically occur in a washing step, preferably at pH 6-11, preferably at pH 7-9, more preferably at pH 7.5-8.5, further preferably at pH 7.0-8.0, most preferably at pH 7.0 or pH 8.0.

[0047] Preferably, the at least two additional negative charges in the loop region of amino acid residues 98 to 104 according to the numbering of SEQ ID NO: 3 are obtained by one or more amino acid changes selected from the group consisting of substitution, deletion and insertion, preferably by substitution and / or insertion, most preferably by substitution.

[0048] In a preferred protease, the at least two additional negative charges in the loop region of residues 98 to 104 compared to SEQ ID NO: 1 can be caused by one or more amino acid substitutions at amino acid positions selected from the group consisting of 98, 99, 100, 101, 102, 103 and 104 according to the numbering of SEQ ID NO: 3, preferably at position 101.

[0049] Also preferably, at least two additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 3 are obtained by one or more amino acid changes selected from the group consisting of X99E, X101D and X101E, preferably D99E, R101D and R101E, most preferably R101E. X generally refers to any amino acid residue at a given position.

[0050] More preferably, the loop sequence 98 to 104 of the preferred protease has two additional negative charges compared to SEQ ID NO: 1, having the following sequence ADGEGAI, ADGDGAI, ADGDGSV, ADGEGSV, AADGSGSV, AADGEGSV or ASEGEGSV, wherein the longer sequence has an insertion in the loop sequence.

[0051] Particularly preferred are proteases comprising an amino acid sequence having 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 at least 99% sequence identity to SEQ ID NO: 1, 2, 7 or 8 and comprising glutamic acid (E) at position 101 according to the numbering of SEQ ID NO: 3.

[0052] More preferred is a protease having at least 90% sequence identity to SEQ ID NO: 1 and, compared to SEQ ID NO: 1, comprising the amino acid substitution R101E or R101D, preferably R101E, according to the numbering of SEQ ID NO: 3.

[0053] Also preferred are proteases having at least 90% sequence identity to SEQ ID NO: 1 and comprising, compared to SEQ ID NO: 1, the amino acid substitutions R101E or R101D (preferably R101E) and the amino acid substitutions S3T, V4I and V205I according to the numbering of SEQ ID NO: 3.

[0054] Also preferred are proteases having at least 90% sequence identity to SEQ ID NO: 1 and comprising, compared to SEQ ID NO: 1, the amino acid substitutions R101E or R101D, preferably R101E, and one, preferably two, substitutions selected from the group consisting of S156D and L262E according to the numbering of SEQ ID NO: 3.

[0055] Most preferred is a protease according to any one of SEQ ID NO:2, SEQ ID NO:7 or SEQ ID NO:8.

[0056] Sequence identity is usually provided in the form of "sequence identity %". In order to determine the sequence identity percentage between two amino acid sequences in the first step, a paired sequence alignment is generated between the two sequences, wherein the two sequences are compared (i.e., paired global alignment) over their full length. Preferably, this alignment is generated by a program implementing Needleman and Wunsch algorithm (J.Mol.Biol. (1979) 48, pp. 443-453), preferably by using the program "NEEDLE" (European Molecular Biology Open Software Suite (EMBOSS)) with program default parameters (gap opening = 10.0, gap extension = 0.5 and matrix = EBLOSUM62). For purposes of the present invention, a preferred alignment is an alignment from which the highest sequence identity can be determined. After aligning the two sequences, in the second step, the identity value should be determined according to the alignment. Therefore, according to the present invention, the following calculation of identity percentage is applicable: x%-identity=(same residue / length of the alignment region showing the corresponding sequence of the present invention over its full length)*100. Therefore, the sequence identity associated with the comparison of two amino acid sequences according to this embodiment is calculated by dividing the number of identical residues by the length of the alignment region showing the corresponding sequences of the invention over its entire length. This value is multiplied by 100 to obtain "sequence identity %".

[0057] In particular, enzyme variants comprising an amino acid sequence having at least one amino acid substitution, addition and / or deletion may also comprise conservative mutations which appear to have minimal effect on protein folding, resulting in certain enzyme properties being substantially maintained when compared to those of the parent enzyme. For the determination of % similarity according to the invention, the following applies, which also conforms to the BLOSUM62 matrix, which is one of the most commonly used amino acid similarity matrices for database searching and sequence alignment: amino acid A is similar to amino acid S; amino acid D is similar to amino acid E; N; amino acid E is similar to amino acid D; K; Q; amino acid F is similar to amino acid W; Y; amino acid H is similar to amino acid N; Y; amino acid I is similar to amino acid L; M; V; amino acid K is similar to amino acid E; Q; R; amino acid L is similar to amino acid I; M; V; amino acid M is similar to amino acid I; L; V; amino acid N is similar to amino acid D; H; S; amino acid Q is similar to amino acid E; K; R; amino acid R is similar to amino acid K; Q; amino acid S is similar to amino acid A; N; T; amino acid T is similar to amino acid S; amino acid V is similar to amino acid I; L; M; amino acid W is similar to amino acid F; Y; amino acid Y is similar to amino acid F; H; W.

[0058] Conservative amino acid substitutions can occur in the full-length sequence of the polypeptide sequence of a functional protein (such as an enzyme). Therefore, according to the present invention, the following percentage similarity calculation is applicable: m%-similarity=[(same residue+similar residue) / the length of the comparison region showing the corresponding sequence of the present invention in its full length]*100. Therefore, the sequence similarity related to the comparison of two amino acid sequences herein is calculated by adding the number of identical residues to the number of similar residues divided by the length of the comparison region showing the corresponding sequence of the present invention in its full length. This value is multiplied by 100 to obtain "similarity %". In particular, compared with the full-length polypeptide sequence, a variant enzyme comprising a conservative mutation having at least m% similarity to the corresponding parent sequence is expected to have substantially unchanged enzyme properties, wherein m is 70 to 100, 80 to 100 or 90 to 100, preferably 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 integers. Even more preferably, such variants comprise an amino acid sequence having 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 at least 99% sequence similarity to the amino acid sequence shown in any one of SEQ ID NOs 1-18.

[0059] Preferably, the protein of interest is usually secreted into the fermentation medium by the bacillus host cell. In other words, preferably, the method according to the present invention utilizes a secretory expression system. It is advantageous to secrete the protein of interest into the fermentation medium because it makes the protein of interest easy to separate from the fermentation medium. In order to secrete the protein of interest into the fermentation medium, the nucleic acid construct can usually include a polynucleotide encoding a signal peptide, which instructs the secretion of the protein of interest into the fermentation medium. Various signal peptides are known in the art. These signal peptides are also referred to as "secretion signals" in this article. Preferred signal peptides are selected from the group consisting of the signal peptide of the AprE protein from Bacillus subtilis or the signal peptide of the YvcE protein from Bacillus subtilis.

[0060] The signal peptide from the AprE protein of Bacillus subtilis or the signal peptide from the YvcE protein of Bacillus subtilis is particularly suitable for secreting enzymes (such as amylases) from the Bacillus cells into the fermentation medium. Since the YvcE signal peptide is suitable for secreting a variety of different enzymes, including amylases and proteases, this signal peptide can be used, preferably in combination with the production method according to the present invention as described herein.

[0061] It should be understood that each of the expression control sequence, the nucleic acid sequence encoding the protein of interest and / or the aforementioned additional elements can be from a Bacillus host cell or can be from another species, i.e., a species that is heterologous to the Bacillus host cell. In particular, the protein of interest can be heterologously expressed, i.e., the protein of interest can be heterologous to the Bacillus host cell.

[0062] Generally, the bacillus host cell comprises an expression construct, which is at least included in the gene encoding the protein of interest under the control of a promoter. More generally, the expression construct can be an arrangement of an interested gene and an expression control sequence and / or other elements, and the other elements include, for example, a secretion signal as described elsewhere herein. In particular, the expression construct can include a secretion signal as described elsewhere herein, which can specifically cause the protein of interest to be secreted into the fermentation medium. The interested gene, expression control sequence and / or other elements or their parts can be natural, i.e., endogenous and present in the genome of the bacillus host cell. In addition, the term "expression construct" can also be understood to include a natural expression construct that has been genetically manipulated, for example, by genome editing and / or mutagenesis techniques.

[0063] The expression construct can also be an exogenously introduced expression construct. In the exogenously introduced expression construct, the expression control sequence, the gene encoding the protein of interest and / or other elements can be natural relative to the host cell, or can be derived from other strains, i.e., be heterologous relative to the bacillus host cell. According to the present invention, the expression construct can be introduced into the bacillus host cell by any method known in the art, and the method especially includes well-known conversion, transfection, transduction and conjugation techniques etc. Preferably, the exogenously introduced expression construct is contained in a vector, preferably contained in an expression vector. The expression vector can preferably be located outside the chromosomal DNA of the bacillus host cell, i.e., exist free with one or more copies. However, the expression vector can also preferably be integrated into the chromosomal DNA of the bacillus cell with one or more copies. The expression vector can be linear or circular. Preferably, the expression vector is a viral vector or a plasmid.

[0064] For autonomous replication, the expression vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. Bacterial origins of replication include, but are not limited to, plasmids pUB110, pC194, pTB19, pAMβ1, and pTA1060 that permit replication in Bacillus (Janniere, L., Bruand, C., and Ehrlich, SD (1990). Structurally stable Bacillus subtilis cloning vectors. Gene 87, 53-6; Ehrlich, SD, Bruand, C., Sozhamannan, S., Dabert, P., Gros, MF, Janniere, L., and Gruss, A. (1991). Plasmid replication and structural stability in Bacillus subtilis. Res. Microbiol. 142, 869-873) and pE194 (Dempsey, LA, and Dubnau, DA (1989). Localization of the replication origin of plasmid pE194.J.Bacteriol.171,2866-2869) replication origin. The replication origin can be a replication origin with a mutation that makes its function temperature-sensitive in the host cell (see, for example, Ehrlich, 1978, Proceedings of the National Academy of Sciences USA 75:1433-1436). However, the expression vector preferably contains one or more selective markers that allow the bacillus host cell of transformation to be easily selected. Selective markers are genes encoding products that provide biocide resistance, resistance to heavy metals, prototrophy of auxotrophic types, etc. Bacterial selective markers include but are not limited to dal genes from bacillus subtilis or bacillus licheniformis, or markers that confer antibiotic resistance (such as ampicillin, kanamycin, erythromycin, chloramphenicol or tetracycline resistance). In addition, selection can be completed by co-transformation, for example, as described in WO9109129, wherein the selective marker is on a separate carrier.

[0065] The method according to the present invention comprises the step of inoculating the fermentation medium with a bacillus host cell, which is included in a gene encoding a protein of interest under the control of a promoter, which will be described in more detail below. As used herein, the term "inoculation" refers to introducing a bacillus host cell into a fermentation medium for cultivation. Inoculating the fermentation medium with a bacillus host cell can be achieved by introducing the bacillus host cell of a preculture (starter culture) into an initial fermentation medium. Preferably, the fermentation medium or the initial fermentation medium is inoculated with a preculture grown under conditions known to those skilled in the art. The preculture can be obtained by culturing cells in a preculture, which can be a preculture or a composite preculture determined by a chemical composition. The preculture can be the same or different from the fermentation medium used to cultivate in the method of the present invention. The composite preculture can contain a composite nitrogen source and / or a composite carbon source. Preferably, the preculture used for inoculation is obtained by using a composite culture medium. The preculture can be added in whole or in part to the fermentation medium of the main fermentation, usually to the initial fermentation medium. Preferably, the Bacillus host cells in the preculture are actively growing cells, i.e. they are in a phase of increasing cell number. Typically, the cells in the preculture are in a lag phase when the preculture is inoculated and transition to an exponential growth phase over time. Preferably, the volume ratio between the preculture used for inoculation and the fermentation medium used for the main fermentation is 0.1% to 30% (v / v), more preferably, the volume ratio between the preculture used for inoculation and the initial fermentation medium is 0.1% to 30% (v / v).

[0066] The method according to the present invention also includes the step of cultivating bacillus host cells in a fermentation medium under conditions conducive to the growth of bacillus host cells and the expression of the protein of interest. As used herein, the term "cultivating / cultivation" refers to keeping the bacillus host cells contained in the fermentation medium alive and / or proliferating for at least a predetermined time. The term can encompass exponential cell growth phases as well as stable growth phases or other growth phases, such as controlled growth. Typically, the term "cultivating" refers to the proliferation of bacillus host cells in a fermentation medium described elsewhere herein.

[0067] As used herein, the term "fermentation medium" refers to a water-based solution containing one or more compounds that can support cell growth. Preferably, the fermentation medium according to the present invention is a composite fermentation medium or a chemically determined fermentation medium. More preferably, the fermentation medium according to the present invention is a chemically determined fermentation medium. The term "initial fermentation medium" generally refers to the fermentation medium initially used when fermentation is started by inoculating the medium with bacillus host cells, such as bacillus cells from pre-cultures described elsewhere herein. The term "addition to the process" or "addition to the fermentation process" should be understood as adding to the initial fermentation medium or adding a suitable feed solution during the fermentation process. The components in the fermentation medium generally relate to components or compounds (such as nutrients or other culture medium components) that are initially present (i.e., present in the "initial fermentation medium"), and the sum of those components or compounds that are added to the fermentation medium (e.g., in the feed solution and / or pH adjustment solution) and / or due to the metabolic activity of the cells during the fermentation process.

[0068] As used herein, composite fermentation medium refers to a fermentation medium containing a composite nutrient source of 0.5% to 30% (w / v) of the fermentation medium. Composite nutrient sources are nutrient sources composed of compounds with unclear chemical composition, i.e., compounds with unknown chemical formula, preferably comprising unclear organic nitrogen-containing and / or carbon-containing compounds. In contrast, "chemically determined nutrient sources" (e.g., "chemically determined carbon sources" or "chemically determined nitrogen sources") are understood to be nutrient sources composed of compounds with determined chemical composition. Chemically determined components are components known by their chemical formulas. Composite nitrogen sources are nutrient sources composed of one or more nitrogen-containing compounds with unclear chemical composition (i.e., nitrogen-containing compounds with unknown chemical formulas), preferably comprising organic nitrogen-containing compounds, such as proteins and / or amino acids with unknown composition. Composite carbon sources are carbon sources composed of one or more carbon-containing compounds with unclear chemical composition (i.e., carbon-containing compounds with unknown chemical formulas), preferably comprising organic carbon-containing compounds, such as carbohydrates with unknown composition. It is clear to the technician that composite nutrient sources can be mixtures of different composite nutrient sources. Thus, a complex nitrogen source may comprise a complex carbon source, and vice versa, and a complex nitrogen source may be metabolized by cells in such a way that it acts as a carbon source, and vice versa.

[0069] The composite nutrient source can be a composite nitrogen source. Composite nitrogen sources include, but are not limited to, protein-containing substances, such as extracts from microorganisms, animals or plant cells, for example, vegetable protein products, soy flour, corn flour, pea flour, corn gluten, cottonseed meal, peanut meal, potato flour, meat, casein, gelatin, whey, fish meal, yeast protein, yeast extract, tryptone, peptone, bacterial tryptone, bacterial peptone, waste from microbial cells, plants, meat or animal body processing, and combinations thereof. In one embodiment, the composite nitrogen source is selected from the group consisting of vegetable protein (preferably potato protein, soy protein, corn protein, peanut protein, cotton protein and / or pea protein), casein, tryptone, peptone and yeast extract and combinations thereof.

[0070] Preferably, the fermentation medium can also include definite medium components. Preferably, the fermentation medium also includes a definite nitrogen source. Examples of inorganic nitrogen sources are ammonium, nitrates and nitrites and combinations thereof. In a preferred embodiment, the fermentation medium includes a nitrogen source, wherein the nitrogen source is a complex or a definite nitrogen source or a combination thereof. In one embodiment, the definite nitrogen source is selected from the group consisting of: ammonia, ammonium, ammonium salts (e.g., ammonium chloride, ammonium nitrate, ammonium phosphate, ammonium sulfate, ammonium acetate), urea, nitrates, nitrates, nitrites and amino acids, preferably glutamic acid, and combinations thereof.

[0071] Preferably, the amount of the complex nutrient source is 2% to 15% (v / w) of the fermentation medium. In another embodiment, the amount of the complex nutrient source is 3% to 10% (v / w) of the fermentation medium.

[0072] Also preferably, the composite fermentation medium may also include a carbon source. The carbon source is preferably a composite or a determined carbon source or a combination thereof. Preferably, the composite nutrient source includes a carbohydrate source. Various sugars and sugar-containing substances are suitable carbon sources, and sugars may be present at different stages of polymerization. The preferred composite carbon source for the present invention is selected from the group consisting of molasses, corn syrup, sucrose, dextrin, starch, starch hydrolysate and cellulose hydrolysate and a combination thereof. The preferred determined carbon source is selected from the group consisting of carbohydrates, organic acids and alcohols, preferably glucose, fructose, galactose, xylose, arabinose, sucrose, maltose, lactose, acetic acid, propionic acid, lactic acid, formic acid, malic acid, citric acid, fumaric acid, glycerol, inositol, mannitol and sorbitol, and a combination thereof. Preferably, the determined carbon source is provided in the form of syrup, which may contain up to 20%, preferably up to 10%, more preferably up to 5% impurities. In one embodiment, the carbon source is beet syrup, cane syrup, corn syrup, preferably high fructose corn syrup. In another embodiment, the complex carbon source is selected from the group consisting of molasses, corn syrup, dextrin and starch, or a combination thereof, and wherein the determined carbon source is selected from the group consisting of glucose, fructose, galactose, xylose, arabinose, sucrose, maltose, dextrin, lactose, or a combination thereof.

[0073] Preferably, the fermentation medium is a complex medium comprising a complex nitrogen source and a complex carbon source. More preferably, the fermentation medium is a complex medium comprising a complex nitrogen source and a carbon source, wherein the complex nitrogen source can be partially hydrolyzed as described in WO 2004 / 003216.

[0074] However, as further described elsewhere herein, the fermentation medium typically also may include a source of hydrogen, oxygen, sulfur, phosphorus, magnesium, sodium, potassium, trace elements, and vitamins.

[0075] Usually, the fermentation medium used in the method according to the present invention is a fermentation medium with a chemical composition. A fermentation medium with a chemical composition is a fermentation medium that is basically composed of components determined by the chemical composition of known concentrations. The components determined by the chemical composition are components known by their chemical formulas. The fermentation medium that is basically composed of components determined by the chemical composition includes a culture medium that does not contain a complex nutrient source, particularly does not contain a complex carbon source and / or a complex nitrogen source, i.e. does not contain a complex raw material with a component with an uncertain chemical composition. The fermentation medium that is basically composed of components determined by the chemical composition can also include such a culture medium, which contains a basic small amount of complex nutrient source, such as a complex nitrogen source and / or a carbon source, whose amount is defined as follows, and which is usually not enough to maintain the growth of the bacillus host cell and / or to ensure the formation of a sufficient amount of biomass.

[0076] In this regard, composite raw materials have a composition with unclear chemical composition, because, for example, these raw materials contain many different compounds, including complex heteropolymeric compounds, and have variable compositions due to seasonal variations and differences in geographical origin. Typical examples of composite raw materials used as composite carbon sources and / or nitrogen sources in fermentation are soybean meal, cottonseed meal, corn steep liquor, yeast extract, casein hydrolyzate, molasses, etc. Basically, a small amount of composite carbon source and / or nitrogen source can be present in a fermentation medium determined by the chemical composition of the present invention, for example, as a legacy of an inoculum for main fermentation. The inoculum for main fermentation is not necessarily obtained by fermentation on a medium determined by chemical composition. Most commonly, a small amount of composite nitrogen source is present in a fermentation medium determined by the chemical composition of the main fermentation, and a legacy from the inoculum can be detected. A small amount of composite culture medium components, such as composite carbon and / or nitrogen source, can also be introduced into the fermentation medium by adding a small amount of these composite components to the fermentation medium. It may be advantageous to use composite carbon and / or nitrogen source in the fermentation process of the inoculum for main fermentation, for example, to accelerate the formation of biomass, i.e., to increase the growth rate of microorganisms, and / or to promote internal pH control. For the same reason, it may be advantageous to add a substantially small amount of a composite carbon source and / or a nitrogen source (e.g., yeast extract) to the initial stage of the main fermentation, especially in order to accelerate biomass formation in the early stages of the fermentation process. A substantially small amount of a composite nutrient source that can be added to a chemically determined fermentation medium in the fermentation method according to the present invention is defined as an amount of at most 10% of the total amount of the corresponding nutrients added in the fermentation method. In particular, a substantially small amount of a composite carbon source and / or a nitrogen source that can be added to a chemically determined fermentation medium is defined as an amount of at most 10% of the composite carbon source of the total carbon amount and / or an amount of at most 10% of the composite nitrogen source of the total nitrogen amount added in the fermentation process, preferably an amount of at most 5% of the composite carbon source of the total carbon amount and / or an amount of at most 5% of the composite nitrogen source of the total nitrogen amount, more preferably an amount of at most 1% of the composite carbon source of the total carbon amount and / or an amount of at most 1% of the composite nitrogen source of the total nitrogen amount added in the fermentation process. Preferably, at most 10% of the total amount of carbon and / or at most 10% of the total amount of nitrogen added during the fermentation, preferably at most 5% of the total amount of carbon and / or at most 5% of the total amount of nitrogen, more preferably at most 1% of the total amount of carbon and / or at most 1% of the total amount of nitrogen added via carryover from the inoculum. Most preferably, no complex carbon source and / or complex nitrogen source is added to the fermentation medium during the fermentation.

[0077] A chemically defined nutrient source (eg a chemically defined carbon source or a chemically defined nitrogen source) as referred to herein is to be understood as a nutrient source consisting of chemically defined compounds.

[0078] Cultivating microorganisms in a chemically defined fermentation medium requires culturing cells in a medium containing a nutrient source determined by various chemical compositions, wherein the nutrient source is selected from the group consisting of a hydrogen source determined by the chemical composition, an oxygen source determined by the chemical composition, a carbon source determined by the chemical composition, a nitrogen source determined by the chemical composition, a sulfur source determined by the chemical composition, a phosphorus source determined by the chemical composition, a magnesium source determined by the chemical composition, a sodium source determined by the chemical composition, a potassium source determined by the chemical composition, a trace element source determined by the chemical composition, and a vitamin source determined by the chemical composition. Preferably, the carbon source determined by the chemical composition is selected from the group consisting of carbohydrates, organic acids, hydrocarbons, alcohols, and mixtures thereof. Preferred carbohydrates are selected from the group consisting of glucose, fructose, galactose, xylose, arabinose, sucrose, maltose, maltotriose, lactose, dextrin, maltodextrin, starch, and inulin, and mixtures thereof. Preferred alcohols are selected from the group consisting of glycerol, methanol and ethanol, inositol, mannitol, and sorbitol, and mixtures thereof. Preferred organic acid is selected from the group consisting of acetic acid, propionic acid, lactic acid, formic acid, malic acid, citric acid, fumaric acid and higher alkanoic acid and their mixture.Preferably, the carbon source determined by the chemical composition comprises glucose or sucrose.More preferably, the carbon source determined by the chemical composition comprises glucose, and even more preferably, the major amount of the carbon source determined by the chemical composition provides as glucose.

[0079] Most preferably, the chemically defined carbon source is glucose. It should be understood that the chemically defined carbon source can be provided in the form of syrup, preferably as glucose syrup. As understood herein, glucose as referred to herein should include glucose syrup. Glucose syrup is a viscous sugar solution with a high sugar concentration. The sugar in the glucose syrup is mainly glucose, with a small amount of maltose and maltotriose, and its concentration varies according to the quality grade of the syrup. Preferably, in addition to glucose, maltose and maltotriose, the syrup can contain up to 10%, preferably up to 5%, more preferably up to 3% impurities. Preferably, the glucose syrup is from corn.

[0080] The nitrogen source determined by the chemical composition is preferably selected from the group consisting of the following items: urea, ammonia, nitrate, nitrate, nitrite, ammonium salt (such as ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate and ammonium nitrate) and amino acid (such as glutamate or lysine) and their combination. More preferably, the nitrogen source determined by the chemical composition is selected from the group consisting of ammonia, ammonium sulfate and ammonium phosphate. Most preferably, the nitrogen source determined by the chemical composition is ammonia. The advantage of using ammonia as the nitrogen source determined by the chemical composition is that ammonia can be used as a pH control agent in addition.

[0081] Additional compounds may be added to the complex and chemically defined fermentation medium as described below.

[0082] Typically during cell culture, oxygen is provided by aerating the fermentation medium by aeration and / or aeration. Hydrogen is typically provided by the presence of water in the aqueous fermentation medium. However, hydrogen and oxygen are also contained in the carbon source and / or nitrogen source and may be provided in this manner.

[0083] Magnesium may be provided to the fermentation medium via one or more magnesium salts, preferably selected from the group consisting of magnesium chloride, magnesium sulfate, magnesium nitrate, magnesium phosphate and combinations thereof, or via magnesium hydroxide, or via a combination of one or more magnesium salts and magnesium hydroxide.

[0084] Sodium may be added to the fermentation medium via one or more sodium salts, preferably selected from the group consisting of sodium chloride, sodium nitrate, sodium sulfate, sodium phosphate, sodium hydroxide, and combinations thereof.

[0085] Calcium may be added to the fermentation medium via one or more calcium salts, preferably selected from the group consisting of calcium sulfate, calcium chloride, calcium nitrate, calcium phosphate, calcium hydroxide, and combinations thereof.

[0086] Potassium may be added to the fermentation medium in a chemically defined form by one or more potassium salts, preferably selected from the group consisting of potassium chloride, potassium nitrate, potassium sulfate, potassium phosphate, potassium hydroxide and combinations thereof.

[0087] Phosphorus may be added to the fermentation medium via one or more phosphorus-containing salts, preferably selected from the group consisting of potassium phosphate, sodium phosphate, magnesium phosphate, phosphoric acid and combinations thereof. Preferably, at least 1 g of phosphorus is added per liter of initial fermentation medium.

[0088] Sulfur is added to the fermentation medium via one or more sulfur-containing salts, preferably selected from the group consisting of ammonium sulfate, potassium sulfate, sodium sulfate, magnesium sulfate, sulfuric acid, and combinations thereof.

[0089] In the method according to the invention, sulfate is added to the fermentation medium to reach a concentration of at least 20 mM sulfate in the fermentation medium. Preferably, sulfate is added with the feed to reach or maintain the desired amount. Alternatively, sulfate is already present in the initial fermentation medium in the desired amount, or is added thereto at the beginning of the fermentation, or is added to the fermentation medium at least at the beginning of the production phase of the protein of interest. Even more preferably, sulfate is present in high amounts in the initial fermentation medium and is added with the feed during the fermentation process so that the desired sulfate concentration is maintained or reached.

[0090] "The beginning of the production phase of a protein of interest" refers to the time point at which the protein of interest can be first detected during the main fermentation, in other words, the time point at which protein production is started. The beginning of the production of a protein of interest can be detected by detecting the protein of interest. The detection of a protein of interest can usually be carried out by using standardized tests known to those skilled in the art and determining the presence of protein activity according to the type of the protein of interest. Generally, if the protein of interest is an enzyme, these are tests for determining enzyme activity. In the case where the protein of interest is a protease, the test is for determining proteolytic activity, such as a test for determining substrate cleavage that results in a detectable product (such as a colorimetric or fluorescent product). Such determinations are generally known to those skilled in the art, and are usually even commercially available. For example, protease activity can be determined by using commercially available protease substrate succinyl-Ala-Ala-Pro-Phe-p-nitroaniline (Suc-AAPF-pNA, referred to as AAPF; see, for example, DelMar et al., (1979), Analytical Biochem 99, 316-320). pNA is cleaved from the substrate molecule by protease activity, in particular by proteolytic cleavage in TRIS (2-amino-2-(hydroxymethyl)propane-1,3-diol) buffer at 30°C and pH 8.6. Protease activity results in the release of free pNA from the substrate. Free pNA is yellow and can be quantified by measuring OD405.

[0091] In particular, the amount of sulfate added to the initial fermentation medium reaches a concentration of at least 25mM, at least 50mM, at least 100mM, at least 150mM, at least 200mM, at least 250mM, at least 300mM or at least 400mM sulfate. More particularly, the amount of sulfate added to the fermentation medium reaches a concentration of at least 25mM, at least 50mM, at least 100mM, at least 150mM, at least 200mM, at least 250mM or at least 300mM, at least 400mM or at least 500mM sulfate in the fermentation medium. These concentrations reflect the amount of sulfate or other precipitation agent required according to the present invention. In other words, these concentrations reflect the required amount. Typically, the concentration in the fermentation medium does not exceed 1M sulfate. Preferred concentrations of sulfate are in the range of 100 mM to 750 mM, more preferably 150 mM to 700 mM, still more preferably 200 mM to 600 mM; 300 mM to 550 mM.

[0092] More particularly, sulfate can be added as a feed solution to the fermentation medium during the fermentation process. Even more particularly, the sulfate concentration can reach its maximum value during the main fermentation. At the end of the fermentation process, for example before the step of obtaining the protein of interest, the sulfate concentration is preferably below the maximum value. This sulfate feeding approach may be advantageous because it avoids undesirable sulfate carryover into subsequent process steps, such as the step of purifying the protein of interest.

[0093] Typically and advantageously, the amount of sulfate added to the fermentation medium is sufficient to effect precipitation and / or crystallization of the protein of interest.

[0094] As an alternative to the sulfates mentioned herein or in addition to the sulfates mentioned herein, other precipitants or crystallization aids can be used. Typical precipitants include alkali metal salts and alkaline earth metal salts and their corresponding ammonium salts, preferably precipitants include phosphates and citrates, halide salts and acetates of the above-mentioned alkali metals, alkaline earth metals and ammonium. Typical crystallization aids include organic solvents, such as methanol, ethanol, isopropanol, acetone, dimethylformamide, etc. Other typical precipitants include polyethylene glycols of different molecular weights, such as PEG400 or PEG800 or PEG 4000. It should be understood that precipitation can also be performed by using a mixture of different precipitants.

[0095] According to the present invention, sulfate can be added to the fermentation medium in the form of any suitable sulfate-containing compound (including sulfate-containing salts or acids). The sulfate compound can be added in liquid or solid form. Typically, sulfate is selected from the group of sulfate-containing compounds consisting of (NH4)2SO4, MgSO4, Na2SO4, sulfuric acid and combinations thereof, preferably sulfuric acid or Na2SO4. Typically, the addition of sulfate-containing compounds can include a base and a conjugate acid, such as adding sodium hydroxide and sulfuric acid.

[0096] Preferably, sulfate is added to the fermentation medium as a feed solution and / or is present in the initial fermentation medium during the culture period. More preferably, sulfate is usually present in the initial fermentation medium in an amount of at least 20mM, more usually in an amount of at least 25mM, at least 50mM, at least 100mM, at least 150mM, at least 200mM, at least 250mM or at least 300mM sulfate. Particularly preferably, in the initial fermentation medium, the amount of sulfate is in the range of 150mM to 300mM. Additionally or alternatively, sulfate can be added to the fermentation medium as a feed solution, as described elsewhere herein. Most preferably, sulfate is present in the initial fermentation medium and is added to the fermentation medium as a feed solution during the culture period (e.g., during the fermentation period). Even more preferably, sulfate is added to the fermentation medium during fermentation, e.g. added together with the feed, to reach a desired concentration of sulfate in the fermentation medium of at least 100 mM, typically at least 150 mM, at least 200 mM, at least 250 mM, at least 300 mM, at least 400 mM or at least 500 mM. In particular, once the desired concentration is reached, this concentration is maintained throughout the main fermentation. Preferably, in the step of adding sulfate to the fermentation medium, sulfate is added to reach a concentration of sulfate in the fermentation medium in the range of 100 mM to 750 mM, more preferably in the range of 200 mM to 600 mM, even more preferably in the range of 300 mM to 550 mM.

[0097] Advantageously, the inventors have found that the high concentration of sulfate claimed in combination with amino acid derivatives in the fermentation medium (particularly at the beginning of the fermentation process) has a favorable effect on the production of the protein of interest, such as the increase in the productivity and / or output of the protein of interest. Without being bound by theory, the inventors believe that the combination results in a large amount of effective precipitation and / or crystallization of the protein of interest. This is even applicable to the protein of interest with a strong tendency to self-proteolysis and or the protein of interest that does not form crystals according to common production methods, particularly according to common production methods without the required amount of sulfate and the required amount of amino acid derivatives, such as methods with less than the required amount of the compound in the fermentation medium. Therefore, the autoproteolytic activity of the protein of interest is maintained at a minimum level. In addition, the proteolysis or hydrolysis damage to the protein of interest caused by the host cell enzyme or the unfavorable conditions in the fermentation medium is maintained at a minimum. Therefore, the precipitation and / or crystallization of the protein of interest combined with the presence of amino acid derivatives is considered to stabilize the protein of interest during the fermentation process and lead to an increase in productivity and output.

[0098] The claimed method is particularly advantageous for producing proteins of interest that are susceptible to proteolysis by host proteases (i.e., enzymes such as amylases, proteases, lipases, mannanases, phytases, xylanases, phosphatases, glucoamylases, nucleases and celluloses); or for producing proteins of interest that are susceptible to autoproteolysis, such as proteases. More particularly, the method according to the invention is advantageous for proteins of interest that have not been genetically engineered to reduce solubility, for example by enhancing their precipitation and / or crystallization capabilities.

[0099] Preferably, the fermentation medium and / or the initial fermentation medium comprises one or more selected from the group consisting of:

[0100] 0.1g to 50g nitrogen per liter of fermentation medium;

[0101] 1g to 6g phosphorus per liter of fermentation medium;

[0102] 0.15g to 2g sulfur per liter of fermentation medium;

[0103] 0.4g to 8g potassium per liter of fermentation medium;

[0104] 0.01g to 2g sodium per liter of fermentation medium;

[0105] 0.01 g to 3 g calcium per liter of fermentation medium; and

[0106] 0.1g to 10g magnesium per liter of fermentation medium.

[0107] According to the invention, the fermentation medium comprises the amino acid derivative in an amount of 0 g / l - 30 g / l of the fermentation medium.

[0108] Preferably, the fermentation medium contains the amino acid derivative in an amount of 0.005 g / L to 30 g / L of the fermentation medium, more preferably in an amount of 0.005 g / L to 12 g / L of the fermentation medium. Also preferred is a fermentation medium in which the amino acid derivative is in an amount of 0.1 g / L to 30 g / L, more preferably in which the amino acid derivative is in an amount of 0.5 g / L to 5 g / L of the fermentation medium.

[0109] The amino acid derivative may alternatively or additionally be present in the feed solution in an amount of 0.0125 g / L-30 g / L or in an amount of 0.1 g / L-30 g / L of the feed solution. Preferably, the amino acid derivative is present in the initial fermentation medium, and more preferably, it is not present in the feed.

[0110] Typically, the amino acid derivative may be present in the fermentation medium in an amount of at least 5 mg / L; more typically, the amino acid derivative is present in the fermentation medium in an amount of 0.005 g / L-30 g / L, and even more typically, the amino acid derivative is present in the fermentation medium in an amount of 0.005 g / L-12 g / L. Even more typically, the amino acid derivative is present in an amount of 0.05 g / L to 10 g / L, and even more typically, in an amount of 0.5 g / L to 5 g / L. It is particularly preferred that the amount of the amino acid derivative (such as betaine) is in the range of 0.1 g / L to 20 g / L, more preferably in the range of 0.5 g / L to 10 g / L, and even more preferably in the range of 0.75 g / L to 5 g / L of the fermentation medium. The inventors have found that the addition of sulfate and amino acid derivatives (particularly betaine) in the amounts described herein has a favorable effect on productivity in Bacillus host cells.

[0111] The amount of amino acid derivatives present in the fermentation medium may generally depend on the Bacillus host cell used or may be independent of the cell. In the case where the Bacillus host cell is a Bacillus licheniformis cell, the amino acid derivatives may not be added to the fermentation medium, and thus the amount of amino acid derivatives in the fermentation medium may generally be 0 mg / L fermentation medium. In other words, in the case where the Bacillus host cell is a Bacillus licheniformis cell, the amino acid derivatives may not be present in the fermentation medium and the feed solution, and are not present in the fermentation medium.

[0112] In the case where the bacillus host cell is a Bacillus licheniformis cell, the amount of the amino acid derivative added to the process can be 0 mg / l of the fermentation medium. Thus, for the Bacillus licheniformis host cell, the fermentation medium may not contain any significant amount of the amino acid derivative. However, even in the case where the bacillus host cell is a Bacillus licheniformis cell, typically the amount of the amino acid derivative can be at least 0.5 mg / L of the fermentation medium, or at least 2 mg / L of the fermentation medium, or the amount of the amino acid derivative can be in the range of 0.75 g / L to 5 g / L of the fermentation medium.

[0113] Alternatively, when the Bacillus host cell is a Bacillus subtilis cell, the amount of the amino acid derivative may be at least 10 mg / L of fermentation medium.

[0114] "Amino acid derivatives" according to the present invention refer to amino acid compounds or compounds related thereto. These compounds may generally be referred to as osmoprotectants. Preferably, the amino acid compound is selected from the group consisting of betaine, choline, proline and ectoine, preferably betaine. The term betaine is preferably understood to mean trimethylglycine.

[0115] Alternatively, the fermentation medium may comprise a quaternary ammonium compound, such as a compound selected from betaine and choline.

[0116] Typically, the method according to the invention comprises the step of adding the amino acid derivative to the fermentation medium. More typically, the amino acid derivative is added to the fermentation medium as a feed solution during the culture period and / or is present in the initial fermentation medium. However, the amino acid derivative may also be added to the fermentation medium as a feed solution during the culture period or be present in the initial fermentation medium. Preferably, the amino acid derivative is present in the initial fermentation medium.

[0117] Adding a specific amount of an amino acid derivative to a fermentation medium containing a desired amount of sulfate may be advantageous because it may help to further enhance the growth of the Bacillus host cell under the conditions designed to limit autoproteolytic processes and / or avoid instability or reduced activity of the protein of interest. Without wishing to be bound by theory, the inventors of the present invention assume that the specific combination of the addition of the amino acid derivative and the appropriate amount of sulfate results in a significant increase in the production of the protein of interest. This is also shown in the examples given below.

[0118] Typically, the feed solution differs from the fermentation medium and / or the initial fermentation medium by one or more of the compounds of the group listed above. Even more typically, the feed solution differs from the fermentation medium and / or the initial fermentation medium by the amount of one or more of the compounds of the group listed above.

[0119] One or more trace element ions can be added to the fermentation medium, preferably each with an amount lower than the initial fermentation medium of 10mmol / L. These trace element ions are selected from the group consisting of iron, copper, manganese, zinc, cobalt, nickel, molybdenum, selenium and boron and their combinations. Preferably, trace element ions iron, copper, manganese, zinc, cobalt, nickel and molybdenum are added to the fermentation medium. Preferably, one or more trace element ions are added to the fermentation medium in an amount selected from the group consisting of the following items: 50 μmol to 5mmol of iron per liter of initial culture medium, 40 μmol to 4mmol of copper per liter of initial culture medium, 30 μmol to 3mmol of manganese per liter of initial culture medium, 20 μmol to 2mmol of zinc per liter of initial culture medium, 1 μmol to 100 μmol of cobalt per liter of initial culture medium, 2 μmol to 200 μmol of nickel per liter of initial culture medium and 0.3 μmol to 30 μmol of molybdenum per liter of initial culture medium, and their combinations. In order to add each trace element, one or more selected from the group consisting of chloride, phosphate, sulfate, nitrate, citrate, and acetate may be preferably used.

[0120] Compounds that may optionally be included in the fermentation medium are chelating agents such as citric acid, MGDA, NTA or GLDA, and buffers such as monopotassium phosphate and dipotassium phosphate, calcium carbonate, etc. When processing a process without external pH control, it is preferred to add a buffer. In addition, a defoamer may be added before and / or during the fermentation process.

[0121] Vitamins refer to a group of structurally unrelated organic compounds that are necessary for the normal metabolism of cells. The ability of known cells to synthesize the vitamins they need varies greatly. Vitamins should be added to the fermentation medium of Bacillus cells that cannot synthesize the vitamins. Vitamins can be selected from the group consisting of thiamine, riboflavin, pyridoxal, nicotinic acid or nicotinamide, pantothenic acid, cyanocobalamin, folic acid, biotin, lipoic acid, purines, pyrimidines, inositol and hemin.

[0122] Preferably, the fermentation medium also contains a selection agent, for example an antibiotic such as ampicillin, tetracycline, kanamycin, hygromycin, bleomycin, chloramphenicol, streptomycin or phleomycin, to which the selectable marker of the cells confers resistance.

[0123] The amount of essential compounds added to the culture medium will depend primarily on the amount of biomass formed during the fermentation process. The amount of biomass formed can vary widely, typically the amount of biomass is from about 10 grams to about 150 grams of dry cell mass per liter of fermentation medium. Typically, fermentations that produce an amount of biomass below about 10 g of dry cell mass per liter of fermentation medium are not considered industrially relevant for protein production.

[0124] The optimum amount of each component of the culture medium determined, and which compounds are essential and which are non-essential, will depend on the type of bacillus cells fermented in the culture medium, the amount of biomass, and the product to be formed. Typically, the amount of the culture medium components necessary for the growth of microbial cells can be determined relative to the amount of the carbon source used in the fermentation, typically relative to the main carbon source, since the amount of biomass formed will be primarily determined by the amount of the carbon source used. Particularly preferred fermentation media are also described in the examples below.

[0125] Preferably, the fermentation medium is sterilized before use to prevent or reduce the growth of microorganisms different from the inoculated microbial cells during the fermentation process. Sterilization can be performed using methods known in the art, such as, but not limited to, autoclaving or sterile filtration. Some or all of the medium components can be sterilized separately from other medium components to avoid interaction of the medium components during the sterilization process or to avoid decomposition of the medium components under sterilizing conditions.

[0126] In the method according to the invention, the Bacillus host cells are cultured in a fermentation medium under conditions that are favorable for the growth of the Bacillus host cells and the expression of the protein of interest. The phrase "conditions that are favorable for the growth of the Bacillus host cells and the expression of the protein of interest" means conditions other than the temperature or fermentation medium used for the culture. Such conditions include pH during the culture, physical movement of the culture by shaking or stirring, and / or atmospheric conditions applied to the culture.

[0127] The pH of the fermentation medium during the culture period can be adjusted or maintained. Preferably, the pH of the medium is adjusted before inoculation. It is envisioned that the preferred pH value for the fermentation medium is in the range of about pH 6.6 to about pH 9, preferably in the range of about pH 6.6 to about pH 8.5, more preferably in the range of about pH 6.8 to about pH 8.5, and most preferably in the range of about pH 6.8 to about pH 8.0. For example, for a bacillus cell host cell culture, the pH is preferably adjusted to or above about pH 6.8, about pH 7.0, about pH 7.2, about pH 7.4 or about pH 7.6. Preferably, during the culture period of the bacillus host cell culture, the pH of the fermentation medium is adjusted to about pH 6.8 to about pH 9, preferably about pH 6.8 to about pH 8.5, more preferably about pH 7.0 to about pH 8.5, and most preferably in the range of about pH 7.0 to about pH 8.0.

[0128] Physical motion can be applied by stirring and / or shaking the fermentation medium. Suitable techniques for achieving physical motion during cultivation of bacillus host cells in the fermentation medium are well known in the art and can be applied by the technician without further effort. In addition to stirring, oxygen or other gases can also be applied to the culture by regulating suitable atmospheric conditions. Preferably, oxygen is supplied with 0 bar to 3 bar air or oxygen.

[0129] Furthermore, additional conditions including the selection of appropriate bioreactors or vessels for culturing Bacillus host cells are well known in the art and can be performed by the skilled artisan without further elaboration.

[0130] As used herein, the term "feed solution" refers to a solution added to the fermentation medium after the initial fermentation medium is inoculated with Bacillus host cells. The initial fermentation medium generally refers to the fermentation medium present in the fermentor when inoculated with Bacillus host cells. The feed solution comprises compounds that support the growth of the cells. The feed solution may be enriched in one or more compounds compared to the fermentation medium.

[0131] For example, when the culture is operated in a fed-batch mode, the feed medium or feed solution used can be any of the above-mentioned medium components or a combination thereof. It should be understood herein that at least a portion of the compound provided as a feed solution can already be present in the fermentation medium to a certain extent before feeding the compound. Preferably, the feed solution generally provides a major carbon source comprising at least one carbohydrate during the culture phase. Alternatively, the feed solution provides a major carbon source comprising at least one carbohydrate in the first culture phase and / or in one or more subsequent culture phases. More preferably, the carbohydrates contained in the feed solution represent the major carbon source consumed or metabolized by the host cell. Still more preferably, the feed solution comprises a chemically determined carbon source, even more preferably glucose. Even more preferably, the feed solution comprises 40% to 75% glucose, preferably 42% to 70% glucose, more preferably 45% to 67% glucose, even more preferably 50% to 65% glucose. Even more preferably, glucose is the major carbon source present in the feed solution and / or fermentation medium. Typically, the same feed solution can be used for seed fermenters and production bioreactors operated in a fed-batch mode. The feed solution for the seed fermenter operated in fed-batch mode may be different from the feed solution used in the production bioreactor. However, the feed solution for the seed fermenter operated in fed-batch mode and the feed solution used in the production bioreactor may have the same concentration of glucose, but the feed solution used in the production bioreactor contains salts that are not present in the feed solution for the seed fermenter operated in fed-batch mode.

[0132] During the fermentation process, the feed solution can be added continuously or discontinuously. The discontinuous addition of the feed solution can be performed once in a rapid perfusion mode during the fermentation process, or several times with different or identical volumes. The continuous addition of the feed solution can be performed at the same or different rates (i.e., volume / time) during the fermentation process. A combination of continuous and discontinuous feeding profiles can also be applied during the fermentation process. The components of the fermentation medium provided as the feed solution can be added to one feed solution or added as different feed solutions. In the case of applying more than one feed solution, the feed solution can have the same or different feeding modes as described above. Particularly preferred feed solutions are also described in the following embodiments. According to the present invention, the feed solution can include an amino acid derivative in an amount of 0.0125g / l–30g / l feed solution.

[0133] Typically, the major carbon source can be provided to the fermentation medium in a feed solution different from a feed solution comprising additional nutrients. In other words, typically at least two different feed solutions can be added to the fermentation medium during the fermentation process, more typically at least two feed solutions of different composition can be added to the fermentation medium during the fermentation process. Even more typically, one feed solution comprises the major carbon source and another feed solution comprises additional nutrients, such as salt and / or sulfate.

[0134] As used herein, the phrase "precipitation and / or crystallization of a protein of interest" refers to the formation of any type of insoluble particles of a protein of interest or a protein of interest in a fermentation medium. These particles can be in the form of crystals, amorphous precipitates, or aggregates, or mixtures thereof. Precipitation and / or crystallization of a protein of interest can generally be achieved by adding sulfate to the fermentation medium, as described elsewhere herein. More generally, the protein of interest is secreted into the fermentation medium outside the bacillus host cell, and after secretion into the fermentation medium, precipitates, crystallizes, or aggregates. Alternatively, the protein of interest is present in the bacillus host cell in the form of insoluble particles before being released into the fermentation medium. Precipitation and / or crystallization of a protein of interest generally occurs at a concentration of at least 20 mM sulfate in the fermentation medium or a precipitant as defined elsewhere herein. More typically, precipitation and / or crystallization of the protein of interest occurs at a concentration of sulfate or a similar precipitant of at least 50 mM, at least 100 mM, at least 150 mM, at least 200 mM, at least 250 mM, at least 300 mM, at least 350 mM, or at least 400 mM in the fermentation medium. Although precipitation and / or crystallization of the protein of interest can occur at the above sulfate concentrations, it is beneficial to use a higher sulfate concentration within the desired range that exceeds the concentration at which precipitation and / or crystallization of the protein of interest occurs because it can increase the amount and / or speed of the precipitation and / or crystallization process.

[0135] In particular, the method according to the invention has the step of adding sulfate to the fermentation medium to reach a sulfate concentration in the fermentation medium sufficient to allow or induce precipitation and / or crystallization of the protein of interest during cultivation.

[0136] The precipitation and / or crystallization of the protein of interest in the fermentation medium can be further enhanced by adding a flocculant. Typical flocculants are known in the art and include cationic, anionic and / or nonionic polyacrylamide, polyethyleneimine (PEI), poly(diallyldimethylammonium chloride) (pDADMAC), polyamines, natural polymers from microorganisms (e.g., alginate, chitosan, gelatin, etc.), and chemical flocculants, such as soluble Fe or Al compounds disclosed in WO 96 / 38469. The type and amount of flocculant is selected based on the characteristics of the fermentation medium of the specific selection, and will generally be determined using simple routine experiments. This is known in the art, and the type and amount of one or more flocculants are selected to promote separation fully within the skill of skilled professionals. For example, the method disclosed in WO 2004 / 001054 can be used according to the present invention.

[0137] The step of "precipitating and / or crystallizing the protein of interest during the culture" refers to a step in which precipitation and / or crystallization of the protein of interest occurs. This is generally achieved by adding a sufficient amount or generally the desired amount of sulfate, and may further depend on the protein of interest. Thus, the addition of the desired amount of sulfate specifically induces precipitation and / or crystallization of the protein of interest. In addition, the pH of the fermentation medium may be adjusted to enhance precipitation and / or crystallization of the protein of interest. Still further, protein seeds may be added in an amount of up to about 10% by weight, based on the weight of the solution, to additionally promote the crystallization process.

[0138] Preferably, the method according to the present invention comprises the step of obtaining the protein of interest. As used herein, the phrase "obtaining the protein of interest" generally refers to obtaining a product (such as the protein of interest) from a Bacillus host cell culture by an appropriate purification technique (including techniques known in the art and including techniques described elsewhere herein). More preferably, the step of obtaining the protein of interest comprises the step of purifying the protein of interest, preferably by separating a liquid fraction and a solid fraction of the fermentation medium, thereby obtaining the protein of interest at least in part in the solid fraction.

[0139] Solid / liquid separation techniques are known in the art and include techniques such as centrifugation, filtration or sedimentation followed by decantation. These can generally be used in the method according to the invention, in particular in the step of separating the liquid fraction and the solid fraction of the fermentation medium.

[0140] In particular, the solid / liquid separation technique produces a fraction comprising at least the liquid portion of the fermentation medium (usually referred to as the "liquid fraction") and a solid fraction comprising at least the solid portion present in the fermentation medium. Preferably, the solid fraction of the fermentation medium comprises at least the precipitate and / or crystals of the protein of interest. However, the liquid fraction may comprise at least a portion of the protein of interest, in particular the portion solubilized in the fermentation medium.

[0141] The term "solid fraction of the fermentation medium" denotes the fraction of the fermentation medium obtained after solid / liquid separation, comprising cells, insoluble substrates and insoluble fermentation products, such as precipitated or crystallized protein of interest.

[0142] More generally, the steps for purifying the protein of interest include

[0143] (i) separating the fermentation medium in a first phase and a second phase, wherein the first phase comprises a liquid fraction, the protein of interest in soluble form and optionally cells and cell debris, and the second phase (solid fraction) comprises the protein of interest, cells and cell debris in precipitated form; and

[0144] (ii) optionally dissolving the protein of interest in precipitated or crystallized form in the second phase (solid fraction).

[0145] In order to purify the protein of interest, step (i) may be performed before step (ii). Alternatively, an additional step of solubilizing or dissolving the protein of interest in precipitated or crystalline form present in the fermentation medium may be performed before step (i).

[0146] Therefore, after the step of separating the solid and liquid fractions, or before the step of separating the solid and liquid fractions, the method according to the present invention may comprise dissolving or solubilizing the protein of interest present in a crystalline or precipitated form. In particular, the protein of interest may be dissolved or solubilized by at least one of the following steps:

[0147] - dissolving the solid fraction in a suitable solvent;

[0148] - adding compounds that promote solubilization, such as divalent soluble salts of calcium, magnesium, iron, zinc; and / or

[0149] - Adjust pH.

[0150] However, it may be advantageous to carry out a step of separating the solid and liquid fractions, e.g. in step (i), before solubilizing or dissolving the precipitated or crystallized form of the protein of interest, e.g. in step (ii), since it requires only small amounts of suitable solvents, compounds promoting solubilization and / or pH adjusting agents.

[0151] The divalent salt used to promote solubilization may preferably be a phosphate, sulfate, acetate, nitrate or chloride salt.

[0152] The pH can be adjusted to dissolve the protein of interest depending on the type of protein of interest. For example, where the protein of interest is an amylase, the pH is typically adjusted to a high value, such as a value within the range of 9.0-12.0. Where the protein of interest is a protease, the pH is typically adjusted to a low value, such as a range of 1.5 to 6.0, more typically adjusted to a value within the range of 3.0 to 5.5. Even more typically, the pH can be adjusted to a pH value below 7.0, 6.0, below 5.5, below 5.0, or below 4.5 but above 3.0.

[0153] Methods for dissolving or solubilizing proteins in solid form, particularly precipitated and / or crystallized form, are known in the art and are disclosed in, for example, EP 2125865; US 3,316,240 and WO 93 / 13125. These methods are also suitable for use according to the present invention. The terms "solubilize" and "dissolve" are used interchangeably herein.

[0154] In the method according to the invention, the solubilization step can be carried out by diluting the solid fraction with a suitable solvent (e.g., with water or an aqueous medium). Further optionally, a compound that promotes solubilization is added and the pH is adjusted. Typically, based on the amount of the solid fraction and the concentration of the usual precipitant, the solubilization can be carried out by diluting the second fraction containing the protein of interest in solid form by 100%-2000% (w / w), preferably 100%-1000% (w / w).

[0155] The solvent can typically be water or an aqueous medium. The pH adjustment can be performed before, simultaneously with, or after the addition of the divalent salt. Additional steps may be included, such as mixing may be performed. The mixing time will typically depend on the selected temperature and the crystal morphology and / or structure of the protein of interest in question. According to the present invention, more than 80% of the crystals and / or precipitates of the protein of interest and / or the protein of interest associated with cell agglomerates and / or insolubles can be solubilized; preferably more than 85%; more preferably more than 90%, in particular more than 95% of the crystals and / or precipitates of the protein of interest and / or the desired product and / or insolubles associated with cell agglomerates can be solubilized.

[0156] Furthermore, the solubilization of the protein of interest can be carried out by using polyols, such as low molecular weight polyethylene glycol and C2 to C8 alcohols having at least two OH groups, preferably having only two OH groups, particularly preferred are polyols in which the two OH groups are present on adjacent carbon atoms in the chain, and the C2-C8 alcohols are aliphatic and have a linear carbon chain.

[0157] Preferred polyols include ethylene glycol, propylene glycol, monopropylene glycol, glycerol, low molecular weight (about 900 or less) polyethylene glycols, and mixtures thereof.

[0158] Furthermore, the method of the invention may further comprise a second separation step of removing cells and cell debris from the mixture comprising the solubilized protein of interest. This second separation may generally be a further solid / liquid separation as described elsewhere herein.

[0159] The method according to the present invention may also include one or more of the following additional purification steps, for example, for recovering the protein of interest: ultracentrifugation, evaporation, centrifugation, spray drying, pelleting, freeze drying, stabilization, etc. These purification steps and methods are known in the art and can be adapted to the present invention by means of routine experiments.

[0160] In addition, the method according to the present invention may include the step of adding one or more stabilizers and / or adding one or more protease inhibitors. These additives are known in the art and can be used to stabilize the protein of interest. In addition, the method according to the present invention may include the step of preparing a preparation containing the protein of interest. "Protein preparation" means any non-composite preparation containing a small amount of ingredients, wherein the ingredients are used to stabilize the protein (particularly the protein of interest) contained in the protein preparation and / or the purpose of stabilizing the protein preparation itself. The term "protein stability" relates to the retention rate of protein activity over time during storage or operation. The term "protein preparation stability" relates to maintaining the physical appearance of the protein preparation during storage or operation and avoiding bacterial contamination during storage or operation.

[0161] The protein preparation can be solid or liquid. The protein preparation can be obtained by using technology known in the art. For example, but not limited to this, solid enzyme preparations can be obtained by extrusion or granulation. Suitable extrusion and granulation techniques are known in the art, and are described in, for example, WO 94 / 19444A1 and WO 97 / 43482A1. The protein of interest obtained in the method according to the present invention can optionally be concentrated by partially removing water. Water can be removed by methods known to the technician, such as evaporation, spray drying or spray granulation.

[0162] In addition, the step of purifying the protein of interest may optionally include one or more pretreatment steps, usually before solid / liquid separation, which may include such as dilution, adjusting the pH and / or temperature in the fermentation medium, adding stabilizers that can prevent further growth of the bacillus host cells to the fermentation medium, adding inhibitors that can reduce protease activity and thus limit degradation due to proteolytic activity. Pretreatment may also include a lysis step, such as treating the liquid culture medium with a chemical lysis agent (such as one or more cell wall degrading enzymes, such as lysozyme). Preferred lysis methods include adding lysozyme and / or hydrolases to solubilize suspended matter as cells or residual fermentation feedstock.

[0163] The present invention further contemplates the use of high sulfate concentrations in fermentation media for producing proteins of interest in bacillus host cells. The terms "high sulfate concentration" and "required sulfate concentration" are used interchangeably herein. Typically, high sulfate concentration refers to the amount of at least 20 mM sulfate in the fermentation medium, preferably at least 25 mM, at least 50 mM, at least 100 mM, at least 150 mM, at least 200 mM, at least 250 mM, at least 300 mM sulfate, at least 400 mM or at least 500 mM sulfate. Typically, in the fermentation medium, the sulfate concentration is no more than a concentration of at most 10 M, at most 5 M, at most 2 M, at most 1000 mM, at most 800 mM or at most 600 mM.

[0164] Furthermore, the present invention contemplates the use of a combination of sulfate and amino acid derivatives for producing a protein of interest in a Bacillus host cell in a fermentation medium.

[0165] Furthermore, the present invention relates to a precipitated or crystallized protein of interest obtained or obtainable by a method according to the present invention.

[0166] Advantageously, it has been found that the method according to the present invention provides an improved way of producing proteins of interest that may be unstable or undergo self-proteolysis under conventional industrial fermentation processes. The claimed method particularly provides a means for producing stable proteins of interest with high productivity and high yield, thereby reducing production costs to an attractive level. The method achieves high productivity because the total amount of the protein of interest produced per reactor volume per hour of processing time is increased, which is superior to prior art processes. The method is suitable for large-scale industrial fermentation processes and is cost-effective because no specialized equipment or expensive specialty chemicals are required. In addition, the method according to the present invention is applicable to various proteins of interest, particularly proteases, and to various bacillus host cell strains.

[0167] The descriptions and explanations of the terms given above in this specification apply to all embodiments characterized herein.The following embodiments are particularly preferred embodiments according to the present invention.

[0168] Implementation plan:

[0169] 1. A method for producing a protein of interest in a fermentation medium, the method comprising the following steps:

[0170] a) inoculating a fermentation medium with a Bacillus host cell comprising a gene encoding a protein of interest under the control of a promoter;

[0171] b) culturing the Bacillus host cells in the fermentation medium under conditions favorable for growth of the Bacillus host cells and expression of the protein of interest,

[0172] c) adding sulfate to the fermentation medium to achieve a concentration of at least 20 mM sulfate in the fermentation medium; and

[0173] d) allowing the protein of interest to precipitate and / or crystallize during the incubation period;

[0174] The fermentation medium comprises the amino acid derivative in an amount of 0 g / L-30 g / L of the fermentation medium, preferably in an amount of 0.1 mg / L-30 mg / L of the fermentation medium.

[0175] 2. The method according to embodiment 1, wherein the Bacillus host cell is a cell of Bacillus amyloliquefaciens, Bacillus clausii, Bacillus halodurans, Bacillus lentus, Bacillus licheniformis, Bacillus pumilus, Bacillus subtilis or Bacillus velez, preferably Bacillus licheniformis.

[0176] 3. The method according to any one of the preceding embodiments, wherein the protein of interest is secreted into the fermentation medium.

[0177] 4. The method according to any one of the preceding embodiments, comprising the following steps:

[0178] e) obtaining the protein of interest.

[0179] 5. The method according to the aforementioned embodiment 4, wherein the method further comprises the step of purifying the protein of interest, preferably by separating the liquid fraction and the solid fraction of the fermentation medium, thereby obtaining at least part of the protein of interest in the solid fraction.

[0180] 6. The method according to any one of the preceding embodiments, wherein the fermentation medium is a chemically defined fermentation medium.

[0181] 7. The method according to any one of the preceding embodiments, wherein the protein of interest is heterologously expressed, preferably wherein the protein of interest is an enzyme, more preferably wherein the protein of interest is selected from the group consisting of amylases, proteases, lipases, mannanases, phytases, xylanases, phosphatases, glucoamylases, nucleases and cellulases, more preferably selected from amylases, proteases and mannanases; even more preferably, the protein of interest is a protease.

[0182] 8. The method according to any one of the preceding embodiments, wherein the protein of interest is a protease selected from a protease having at least 80% sequence identity, such as at least 85% sequence identity, such as at least 95% sequence identity, such as at least 96% sequence identity, with any one of SEQ ID NOs: 1-13.

[0183] The protein of interest is a protease having an amino acid sequence having at least 80% sequence identity, such as at least 85% sequence identity, such as at least 95% sequence identity, such as at least 96% sequence identity, with any one of SEQ ID NOs: 14-18.

[0184] Amylases having an amino acid sequence with at least 97% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity.

[0185] 9. The method according to any one of the preceding embodiments, wherein the sulfate is present in the initial fermentation medium.

[0186] 10. The method according to any one of the preceding embodiments, wherein the initial fermentation medium comprises at least 20 mM sulfate, more typically at least 25 mM, at least 50 mM, at least 100 mM, at least 150 mM, at least 200 mM, at least 250 mM or at least 300 mM sulfate.

[0187] 11. The method according to any one of the preceding embodiments, wherein the sulfate is added to the fermentation medium as a feed solution during cultivation.

[0188] 12. The method according to any one of the preceding embodiments, wherein the sulfate is selected from the group of sulfate-containing compounds consisting of (NH4)2SO4, MgSO4, Na2SO4, sulfuric acid and combinations thereof, preferably sulfuric acid or Na2SO4.

[0189] 13. The method according to any one of the preceding embodiments, wherein the amino acid derivative is provided as a feed solution to the fermentation medium during cultivation and / or is present in the initial fermentation medium.

[0190] 14. The method according to any one of the preceding embodiments, wherein the amino acid derivative is selected from the group consisting of betaine, choline, proline and ectoine, preferably betaine, more preferably trimethylglycine.

[0191] 15. The method according to any one of the preceding embodiments, wherein the fermentation medium comprises the amino acid derivative in an amount of 0.005 g / l - 12 g / l fermentation medium.

[0192] 16. The method according to any one of the preceding embodiments, wherein the amino acid derivative is present in the feed solution in an amount of 0.0125 g / l - 30 g / l of the feed solution.

[0193] 17. The method according to any one of the preceding embodiments, wherein the Bacillus host cell comprises an expression construct comprising at least the gene encoding the protein of interest under the control of a promoter.

[0194] 18. The method according to the preceding embodiment, wherein the expression construct comprises a secretion signal.

[0195] 19. The method according to the preceding embodiment 5, wherein the step of separating the liquid fraction and the solid fraction of the fermentation medium comprises centrifugal filtration, or decantation after sedimentation.

[0196] 20. The method according to embodiment 5, wherein the step of purifying the protein of interest comprises:

[0197] (i) separating the fermentation medium in a first phase and a second phase, wherein the first phase comprises the liquid fraction, the protein of interest in soluble form and optionally cells and cell debris, and the second phase, the solid fraction comprises the protein of interest, cells and cell debris in precipitated form; and

[0198] (ii) solubilizing the protein of interest in precipitated form in the second phase (solid fraction).

[0199] 21. The method according to the preceding embodiment, wherein the protein of interest contained in the solid fraction is solubilized, preferably by at least one of the following steps:

[0200] - dissolving the solid fraction in a suitable solvent;

[0201] - adding compounds that promote solubilization, such as divalent soluble salts of magnesium, iron, zinc; and / or

[0202] - Adjust pH.

[0203] 22. The method according to embodiment 21, wherein the solubilization is carried out by using a polyol, such as a low molecular weight polyethylene glycol and a polyol having at least two OH groups, preferably having only two OH groups.

[0204] The C2 to C8 alcohols of the group are particularly preferably polyols in which the two OH groups are present on adjacent carbon atoms in the chain, and the C2-C8 alcohols are aliphatic and have a linear carbon chain.

[0205] 23. The method of embodiment 21, wherein the divalent salt is a phosphate, sulfate, acetate, nitrate or chloride salt.

[0206] 24. The method according to the preceding embodiments 21 to 23, wherein the pH is adjusted to a pH value in the range of 1.5 to 6.0.

[0207] 25. The method according to any one of the preceding embodiments 21 to 24, wherein the pH is adjusted to a pH value in the range of 3.0 to 5.5.

[0208] 26. The method according to any one of the preceding embodiments, further comprising one or more of the following additional purification steps: ultracentrifugation, evaporation, centrifugation, spray drying, granulation, freeze drying, stabilization, etc.

[0209] 27. The method according to any one of the preceding embodiments, further comprising the step of preparing a preparation containing the protein of interest.

[0210] 28. The method according to any one of the preceding embodiments, further comprising the step of adding one or more stabilizers and / or adding one or more protease inhibitors.

[0211] 29. The method according to any one of the preceding embodiments, wherein when the Bacillus host cell is a Bacillus licheniformis cell, the amount of the amino acid derivative added to the method is 0 mg / L fermentation medium.

[0212] 30. The method according to any one of the preceding embodiments, wherein when the Bacillus host cell is a Bacillus subtilis cell, the amount of the amino acid derivative is at least 10 mg / 1 of the fermentation medium.

[0213] 31. The method according to any one of the preceding embodiments, wherein the protein of interest is a protease selected from any one of SEQ ID NO: 2, 7 or 8.

[0214] 32. The method of any one of the preceding embodiments, wherein the Bacillus host cell is a Bacillus licheniformis cell, the amount of the amino acid derivative is at least 0.5 mg / L of the fermentation medium, and the protein of interest is a protease selected from any one of SEQ ID NO: 2, 7 or 8.

[0215] 33. Use of high sulfate concentration in a fermentation medium for producing a protein of interest in a Bacillus host cell.

[0216] 34. The use according to the preceding embodiments, wherein high sulfate concentration refers to an amount of sulfate in the fermentation medium of at least 20 mM, preferably at least 25 mM, at least 50 mM, at least 100 mM, at least 150 mM, at least 200 mM, at least 250 mM, at least 300 mM, at least 300 mM sulfate, at least 400 mM or at least 500 mM sulfate.

[0217] 36. Use of a combination of sulfate and an amino acid derivative for producing a protein of interest in a Bacillus host cell in a fermentation medium.

[0218] 37. A crystallized protein of interest obtained or obtainable by the method according to any one of the preceding embodiments.

[0219] The entire contents of all references as well as the disclosures specifically mentioned in this specification are incorporated herein by reference.

[0220] sequence

[0221] SEQ ID NOs: 1-13 correspond to protease sequences.

[0222] SEQ ID NOs: 14-18 correspond to amylase sequences.

[0223] SEQ ID NOs: 19-25 correspond to protease loop sequences.

[0224] SEQ ID NO: 26 corresponds to the sequence of a protease substrate peptide.

[0225] Attached photos

[0226] Figure 1 Normalized productivity is shown for control fermentations without added sulfate and betaine (open circles) and fermentations with added SO4 and betaine (closed circles).

[0227] Figure 2 Normalized productivity is shown for control fermentations without sulfate and betaine addition (open circles), fermentations with sulfate addition (diamonds), and fermentations with sulfate and betaine addition (triangles).

[0228] Figure 3 Normalized productivity is shown for control fermentations without added betaine (open circles) and fermentations with added betaine (diamonds). Example

[0229] The examples will merely illustrate the present invention. They should in no way be construed as limiting the scope.

[0230] Example 1: Improving the productivity of alkaline protease 1 by adding sulfate and betaine

[0231] A Bacillus licheniformis strain comprising a gene expressing an alkaline protease as shown in SEQ ID NO: 2 and described in WO 95 / 23221 was cultured in a fermentation process using a chemically defined fermentation medium providing the components listed in Tables 1 and 2:

[0232] Table 1: Macro and trace elements provided to the control fermentation and the fermentation according to the invention during the fermentation process Solution

[0233]

[0234] Table 2: Sulfate and betaine added to the fermentation medium during fermentation

[0235] Compound concentration [g / L initial volume]

[0236] Sodium sulfate Na2SO4 85

[0237] Betaine C5H Monohydrate 11 NO2xH2O 3

[0238] In the method according to the present invention, the sulfate concentration in the initial fermentation medium is 27 g / L sodium sulfate and 3 g / L betaine monohydrate.

[0239] Table 3: Trace element composition of trace element solution containing 40 g / L citric acid

[0240]

[0241] Fermentation was started with a medium containing 8 g / L glucose. Two conditions were tested: a control fermentation without the addition of sodium sulfate and betaine and an experimental fermentation with the addition of 85 g / L sodium sulfate and 3 g / L betaine monohydrate. A solution containing 50% glucose was used as feed solution. In Table 1, the total amount of elements related to the initial fermentation volume, totaled together in the fermentation medium (i.e. in the initial fermentation medium and in the feed), is given. The amount of sulfate and betaine added to the fermentation medium in the experimental fermentation is specified in Table 2. In both experiments, the total amount of glucose added was higher than 200 g per liter of initial medium, as required by industrially relevant fermentation methods. Ammonia was used to control the pH to above 7 during the fermentation. The culture temperature was controlled at 30°C. The culture time was 84 hours.

[0242] Measurement of protease titer

[0243] The titer of the protease produced during the fermentation process was determined at different time points. Proteolytic activity was determined by using succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Suc-AAPF-pNA, referred to as AAPF; see, e.g., DelMar et al., (1979), Analytical Biochem 99, 316-320) as a substrate. pNA was cleaved from the substrate molecule by proteolytic cleavage in 30°C, pH 8.6 TRIS buffer, resulting in the release of yellow free pNA, which was quantified by measuring OD405.

[0244] result

[0245] The results are depicted in Figure 1 In the figure, the normalized productivity development over time is shown. The productivity is defined as the total amount of protease produced per reactor volume per hour of treatment time. The final productivity of the control fermentation was used as a normalized 100% value. In both fermentations, the productivity increased with treatment time and reached a stable level in the later stage of the treatment. The fermentation with the addition of sulfate and betaine (fermentation according to the present invention) outperformed the control fermentation without the addition of sulfate and betaine, with a productivity increase of 15%. Therefore, the addition of sulfate and betaine to the process increases the amount of the protein of interest produced by the Bacillus host cells.

[0246] Example 2: Improving the productivity of alkaline protease 1 by adding sulfate and betaine

[0247] A Bacillus licheniformis strain comprising a gene expressing an alkaline protease as shown in SEQ ID NO: 2 and described in WO 95 / 23221 was cultured in a fermentation process using a chemically defined fermentation medium providing the components listed in Table 1 and Table 4:

[0248] Table 4: Sulfate and betaine added to the fermentation medium in the experimental fermentations

[0249] Compound concentration [g / L / initial volume]

[0250] Sodium sulfate Na2SO4 95

[0251] Betaine C5H Monohydrate 11 NO2xH2O 3*

[0252] *Added only in the second experimental fermentation

[0253] During the second experimental fermentation, the sulfate concentration in the initial fermentation medium was 38 g / L sodium sulfate and 3 g / L betaine monohydrate.

[0254] The fermentation was started with a medium containing 8 g / L glucose. Three conditions were tested: a control fermentation without addition of sodium sulfate and without addition of betaine, an experimental fermentation with addition of 95 g / L sodium sulfate per initial volume and a second experimental fermentation with addition of 95 g / L sodium sulfate per initial volume and 3 g / L betaine monohydrate per initial volume. A solution containing 50% glucose was used as feed solution. In Table 1, the total amounts of elements in the fermentation medium (i.e. in the initial fermentation medium and in the feed) totaled together with the initial fermentation volume are given in Table 4.

[0255] In both experiments, the total amount of glucose added was higher than 200 g per liter of initial culture medium, as required by industrially relevant fermentation methods. Ammonia was used to control the pH above 7 during fermentation. The culture temperature was controlled at 30° C. The culture time was 96 hours.

[0256] Measurement of protease titer

[0257] The titer of the protease produced during the fermentation process was determined at different time points. Proteolytic activity was determined by using succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Suc-AAPF-pNA, referred to as AAPF; see, e.g., DelMar et al., (1979), Analytical Biochem 99, 316-320) as a substrate. pNA was cleaved from the substrate molecule by proteolytic cleavage in 30°C, pH 8.6 TRIS buffer, resulting in the release of yellow free pNA, which was quantified by measuring OD405.

[0258] result

[0259] The results are depicted in Figure 2In the figure, the development of normalized productivity over time is shown. Productivity is defined as the total amount of protease produced per reactor volume per hour of treatment time. The final productivity of the control fermentation was used as a normalized 100% value. In all fermentations, productivity increased with treatment time and reached a stable level in the later stage of treatment. The fermentation with the addition of sulfate but not betaine was superior to the control fermentation without the addition of sulfate and betaine, with a productivity increase of 22%. The fermentation with the addition of sulfate and betaine (fermentation according to the present invention) was superior to the control fermentation, with a productivity increase of 35%. Therefore, the addition of sulfate and betaine to the process increased the amount of the protein of interest produced by the Bacillus host cells compared to the control fermentation and compared to the fermentation with only sulfate added.

[0260] Example 3: Addition of betaine alone does not increase the productivity of alkaline protease 1

[0261] The Bacillus strain from Example 1 was cultured according to the control fermentation described in Example 1. In the second fermentation, 3 g / L betaine monohydrate was added per initial volume.

[0262] Measurement of protease titer

[0263] The measurement of protease titer was performed as described in Example 1.

[0264] result

[0265] The results are depicted in Figure 3 In , the development of the normalized productivity over time is shown. The productivity is defined as the total amount of protease produced per reactor volume per hour of treatment time. The final productivity of the control fermentation was used as a normalized 100% value. In both fermentations, the productivity increased with treatment time and reached a stable level in the later stage of the treatment. The fermentation with added betaine did not show any increase in productivity compared to the control fermentation. Therefore, the addition of betaine to the process did not increase the amount of the protein of interest produced by the Bacillus host cells compared to the control fermentation.

Claims

1. A method for producing a protein of interest in a fermentation medium, the method comprising the following steps: a) inoculating a fermentation medium with a Bacillus host cell comprising a gene encoding a protein of interest under the control of a promoter; b) culturing the Bacillus host cells in the fermentation medium under conditions favorable for growth of the Bacillus host cells and expression of the protein of interest, c) adding sulfate to the fermentation medium to achieve a concentration of at least 20 mM sulfate in the fermentation medium; and d) allowing the protein of interest to precipitate and / or crystallize during the incubation period; Wherein the fermentation medium comprises the amino acid derivative in an amount of 0.1 g / l - 30 g / l of the fermentation medium.

2. The method according to any one of the preceding claims, wherein the Bacillus host cell is a cell of Bacillus amyloliquefaciens, Bacillus clausii, Bacillus halodurans, Bacillus lentus, Bacillus licheniformis, Bacillus pumilus, Bacillus subtilis or Bacillus velez, preferably Bacillus licheniformis.

3. The method according to any one of the preceding claims, wherein the protein of interest is secreted into the fermentation medium.

4. The method according to any one of the preceding claims, comprising the steps of: e) obtaining the protein of interest.

5. The method according to any one of the preceding claims, wherein the method further comprises the step of purifying the protein of interest, preferably by separating a liquid fraction and a solid fraction of the fermentation medium, thereby obtaining at least part of the protein of interest in the solid fraction.

6. The method according to any one of the preceding claims, wherein the protein of interest is heterologously expressed, preferably wherein the protein of interest is an enzyme, more preferably wherein the protein of interest is selected from the group consisting of amylases, proteases, lipases, mannanases, phytases, xylanases, phosphatases, glucoamylases, nucleases and cellulases, more preferably selected from amylases, proteases and mannanases; even more preferably, the protein of interest is a protease.

7. The method according to any one of the preceding claims, wherein the protein of interest is a protease selected from proteases having an amino acid sequence having at least 80% sequence identity, such as at least 85% sequence identity, such as at least 95% sequence identity, such as at least 96% sequence identity, such as at least 97% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity to any one of SEQ ID NOs: 1-13, or wherein the protein of interest is an amylase selected from amylases having an amino acid sequence having at least 80% sequence identity, such as at least 85% sequence identity, such as at least 95% sequence identity, such as at least 96% sequence identity, such as at least 97% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity to any one of SEQ ID NOs: 14-18.

8. The method according to any of the preceding claims, wherein the sulfate is selected from the group of sulfate-containing compounds consisting of (NH4)2SO4, MgSO4, Na2SO4, sulfuric acid and combinations thereof, preferably sulfuric acid or Na2SO4.

9. The method according to any one of the preceding claims, wherein the amino acid derivative is selected from the group consisting of betaine, choline, proline and ectoine, preferably betaine, more preferably trimethylglycine.

10. The process according to any one of the preceding claims, wherein the fermentation medium comprises the amino acid derivative in an amount of 0.005 g / l - 12 g / l of the fermentation medium; and / or wherein the feed solution comprises the amino acid derivative in an amount of 0.0125 g / l - 30 g / l of the feed solution.

11. The method according to any one of the preceding claims, wherein the Bacillus host cell comprises an expression construct comprising at least the gene encoding the protein of interest under the control of a promoter.

12. The method of claim 11, wherein the expression construct comprises a secretion signal.

13. The method of claim 5, wherein the step of separating the liquid fraction and the solid fraction of the fermentation medium comprises centrifugal filtration, or sedimentation followed by decantation.

14. The method according to claim 5 or 13, wherein the protein of interest contained in the solid fraction is solubilized, preferably by at least one of the following steps: - decomposing the solid fraction in a suitable solvent; - adding compounds that promote solubilization, such as divalent soluble salts of magnesium, iron, zinc; and / or - Adjust pH.

15. Use of a combination of sulfate and an amino acid derivative for producing a protein of interest in a Bacillus host cell in a fermentation medium.

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