Fermentation method

Abstract

The invention provides an improved recombinant fermentation process, where the fermentation medium is supplemented with a plant protein and incubated with a proteolytic enzyme prior to inoculation with a microorganism.

Description

[0001] FERMENTATION METHOD

[0002] Reference to a Sequence Listing

[0003] This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

[0004] FIELD OF THE INVENTION

[0005] The present invention relates to an improved recombinant fermentation process, comprising a plant protein degradation step.

[0006] BACKGROUND

[0007] Fermentation of microorganisms may require a substrate containing a source of amino acids. In small scale laboratory fermentation / cultivation it is common to use peptone, but this is too expensive for industrial scale fermentation, where various proteins can be used as a cheap (and crude) indirect source of amino acids.

[0008] While such crude proteins need to be degraded to become a source of amino acids, microbial production organisms used to produce proteinaceous products are often recombinantly modified to reduce the expression level of proteases. Even when proteases are produced by the production organism, the amount of protease will be insufficient to avoid growth rate limitation at the outset of the fermentation.

[0009] In a typical fermentation, formation of a protein of interest (product) will lag behind formation of biomass (cell growth), and therefore, even when the product is a protease this will not solve the problem of growth rate limitation.

[0010] Thus, the object of the present invention is an improved fermentation process, where a plant protein source is used to provide a fermentation substrate rich in amino acids.

[0011] SUMMARY OF THE INVENTION

[0012] The present invention provides, in a first aspect, a method for producing a protein of interest, comprising

[0013] (a) providing a fermentation medium comprising a plant protein,

[0014] (b) incubating the fermentation medium with a proteolytic enzyme, and

[0015] (c) cultivating a microorganism capable of secreting the protein of interest in the fermentation medium.

[0016] Other aspects and embodiments of the invention are apparent from the description and examples.

[0017] Unless otherwise indicated, or if it is apparent from the context that something else is meant, all percentages are percentage by weight (% w / w). As used herein, the term "consists essentially of" (and grammatical variants thereof), as applied to the compositions and methods of the invention, means that the compositions / methods may contain additional components so long as the additional components do not materially alter the composition / method.

[0018] As used herein, the term "essentially free of' (and grammatical variants thereof), as applied to the compositions and methods of the invention, means that the compositions / methods may contain minor amounts of the specified component so long as the amount of the component does not materially alter, or provide any material effect on, the composition / method. In an embodiment, "essentially free of" means 0% w / w.

[0019] Sequences

[0020] SEQ ID NO: 1: amino acid sequence of a protease from Bacillus lentus.

[0021] SEQ ID NO: 2: amino acid sequence of a protease from Bacillus licheniformis.

[0022] SEQ ID NO: 3: amino acid sequence of a protease from Bacillus amyloliquefaciens.

[0023] SEQ ID NO: 4: amino acid sequence of a protease from Bacillus gibsonii.

[0024] SEQ ID NO: 5: amino acid sequence of a protease from Bacillus gibsonii.

[0025] DETAILED DESCRIPTION

[0026] We have found that it is possible to improve industrial fermentation processes by introducing a pre-inoculation step, where a fermentation medium is supplemented with a plant protein and incubated with a proteolytic enzyme to (partially) hydrolyze the plant protein. The fermentation medium may be incubated with the proteolytic enzyme at a different pH than the pH used subsequently for cultivating the microorganism producing the protein of interest.

[0027] Thus, the invention provides a method for producing a protein of interest, comprising:

[0028] (a) providing a fermentation medium comprising a plant protein,

[0029] (b) incubating the fermentation medium with a proteolytic enzyme,

[0030] (c) cultivating a microorganism capable of secreting the protein of interest in the fermentation medium.

[0031] In an embodiment, the incubation in step (b) is carried out for at least 10 minutes. There is no upper limit, but for practical reasons it would rarely exceed 12 or 24 hours. The higher temperature, the shorter incubation time is needed. Suitable incubation temperatures could be in the range of 20-70°C, but it depends on the thermostability of the proteolytic enzyme. It is generally desirable to use a high incubation temperature for a short time to reduce capex of the overall fermentation process.

[0032] In an embodiment, the fermentation medium in step (a) comprises the plant protein in an amount of 0.1-50% w / w, preferably in an amount of 0.1-30% w / w, in an amount of 0.1-25% w / w, or in an amount of 0.1-20% w / w. In an embodiment, the fermentation medium in step (b) comprises the proteolytic enzyme in an amount of 0.0001-1 % w / w of active enzyme protein; preferably in an amount of 0.0005- 0.5% w / w of active enzyme protein. If a high amount of proteolytic enzyme is used in step (b), a shorter incubation time (or lower temperature) is needed. Likewise, a lower amount of proteolytic enzyme can be used if the incubation time is extended.

[0033] In an embodiment, the fermentation medium is incubated with the proteolytic enzyme at a pH in the range of pH 6 to pH 9.

[0034] In an embodiment, the proteolytic enzyme is inactivated by heat treatment after incubation with the fermentation medium in step (b) and before cultivating the microorganism in step (c). Preferably, the heat treatment includes heating to at least 90°C, such as at least 100°C, at least 110°C, or at least 120°C. The heat treatment may be a separate step, or it may be carried out as part of a heat sterilization of the fermentation medium, prior to inoculation.

[0035] The fermentation medium may be diluted with water and / or additional nutrients may be added before cultivating the microorganism. Additional fermentation medium may be added during cultivation of the microorganism (fed-batch) to supplement with amino acids from the hydrolyzed plant protein.

[0036] In an embodiment, the microorganism is cultivated at a pH in the range of pH 3 to pH 8. The pH may be controlled / adjusted by addition of NaOH.

[0037] In an embodiment, the fermentation medium in step (b) is transferred to a fermentation tank before cultivating the microorganism in step (c). Preferably, the fermentation tank has a volume of at least 1 m3(1000 L), at least 5 m3(5000 L), at least 10 m3(10000 L), at least 20 m3(20000 L), at least 50 m3(50000 L), or at least 100 m3(100000 L). Preferably, the production bioreactor may have a volume of 1-5 m3, 5-10 m3, 10-20 m3, 20-50 m3, 50-100 m3, or 100-150 m3. The fermentation medium may be sterilized in the fermentation tank before cultivating the microorganism in step (c).

[0038] Fermentation

[0039] The (submerged) fermentation of the invention is a process where one or more microorganisms are grown in a liquid medium / substrate comprising the necessary nutrients, minerals, vitamins and other components necessary for the growth of the microorganism. Fermentation processes may be aerobic, where air, oxygen or a mixture is added to the fermenter, or it may be anaerobic where no oxygen is added. Typically, fermentation processes comprise stirring or agitation of the fermentation medium to secure uniform conditions in all parts of the fermentation tank (“fermenter” or “bioreactor”). In aerobic fermentation processes, stirring / agitation is usually required to secure a good distribution of (small) air bubbles in the fermentation medium, which is required for a good oxygen transfer from the gas phase into the liquid phase.

[0040] Industrial fermentation processes are typically carried out in large fermentation tanks or bioreactors having a size of more than 10 m3An industrial production bioreactor may have a volume of at least 1 m3, at least 5 m3, at least 10 m3, at least 20 m3, at least 50 m3, or at least 100 m3. Preferably, the production bioreactor may have a volume of 1-5 m3, 5-10 m3, 10-20 m3, 20-50 m3, 50-100 m3, or 100-150 m3.

[0041] The fermentation process may be a batch process, where all ingredients are added to the fermentation tank that is inoculated / seeded with the microorganism from a pre-culture, and the fermentation proceeds until completion. The inoculant or seed is produced in one or more small fermenter(s) in batch or fed-batch operation. Such progressively larger fermenters is often referred to as a seed train. The volume of the inoculum for inoculation of the main bioreactor can range from 0.1 to 15% (v / v) of the volume of the initial volume of the production bioreactor.

[0042] In the case of an aerobic fermentation process, oxygen is delivered throughout the fermentation process. The fermentation process may be a fed-batch process, starting as a batch or fed-batch fermentation, but a given time, typically when the cell density has reached a certain predetermined level, a nutrient solution (feed) is supplied (fed) to the fermenter until the end of the fermentation process. Such fermentation processes are known in the art and the present invention is not limited to any particular fermentation process.

[0043] The fermentation process may be a process where a single microorganism is grown in the fermenter or it may be a co-cultivation process, where two or more microorganisms are inoculated and grown simultaneously during the fermentation process.

[0044] The fermentation medium is according to the invention intended to mean the nutrient composition wherein the one or more microorganisms are grown in the fermentation process. The fermentation medium is generally an aqueous solution comprising a mixture of nutrients, vitamins, minerals and other components necessary for the growth of the particular selected microorganisms for the particular fermentation process. As examples of suitable nutrients can be mentioned carbohydrates, such as mono-, di-, oligo-or polysaccharides e.g. glucose, maltose, lactose, xylose, arabinose, dextrins, maltodextrin and starches; amino acids, di- oligo- and polypeptides; lipids e.g. mono-, di- or triglycerides, milk, juices and fractions thereof. Other examples of suitable substrates included hydrolysates of naturally occurring materials, e.g. hydrolysed starch, cellulose or lignocellulosic materials. Further examples of suitable nutrients include stream from industrial processes, such as molasses, sugar been pulp, cereal fractions, corn steep liquor etc. As example of suitable minerals can be mentioned sodium, potassium, calcium, magnesium, iron and ammonium salts with suitable anions such as chloride, carbonate, sulphate, phosphate, and nitrates, and further micronutrients, i.e. components required in small amounts for growth such a cupper, iron, molybdenum, cobalt, zinc and iodine. The fermentation medium may be a defined medium, i.e. a medium composed of clearly defined components or it may be a complex medium comprising one or more complex nutrient that cannot be clearly defined, such as (hydrolysed) materials, e.g. hydrolysed starch, hydrolysed lignocellulosic material, corn step liquor or molasses.

[0045] When the microorganism is inoculated into the fermentation medium the microorganism grows and convert the nutrients into cell material (biomass), products, metabolites and waste material forming a fermentation broth. Thus, the term fermentation broth in intended to mean an aqueous composition comprising nutrients, minerals, cells, cellular materials, products, metabolites, waste products etc; provided by the growth of the one or more microorganism(s) in the fermentation medium.

[0046] The microorganism(s) may in principle be selected among any prokaryotic or eukaryotic organisms, which organisms are capable of growing in a (submerged) fermentation.

[0047] In a preferred embodiment, the microorganism is bacterial; preferably prokaryotic; more preferably Gram positive or Gram negative; even more preferably the microorganism is selected among Gram-positive bacteria, including, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces', or selected among Gram-negative bacteria, inducing, Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, llyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma', most preferably, the one or more microorganism comprises a Bacillus species, preferably selected among Bacillus alkalophilus, Bacillus altitudinis, Bacillus amyloliquefaciens, B. amyloliquefaciens subsp. plantarum, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus methylotrophicus, Bacillus pumilus, Bacillus safensis, Bacillus stearothermophilus, Bacillus subtilis and Bacillus thuringiensis.

[0048] In another preferred embodiment, the microorganism is a Lactobacillus species, preferably selected among Lactobacillus reuteri, Lactobacillus casei, Lactobacillus paracasei, L. paracasei subsp. paracasei, L. paracasei subsp. tolerans, Lactobacillus rhamnosus, Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus crispatus and Lactobacillus delbrueckii.

[0049] The cell may be a fungal cell. “Fungi” as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as well as the Oomycota and all mitosporic fungi (as defined by Hawksworth et al., In, Ainsworth and Bisby’s Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK).

[0050] The fungal cell may be a yeast cell. “Yeast” as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfect! (Blastomycetes). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, Passmore, and Davenport, editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

[0051] The fungal cell may be a filamentous fungal cell. “Filamentous fungi” include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). The filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative.

[0052] In still another preferred embodiment, the microorganism is eukaryotic, more preferably the one or more microorganism is a fungus, even more preferably a filamentous fungus, including Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thiel avia, Tolypocladium, Trametes, or Trichoderma or a yeast, including Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, Yarrowia\, Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica', most preferably the one or more microorganism is an Aspergillus, Penicillium or Trichoderma species, preferably selected from Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Penicillium purpurogenum, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei and Trichoderma viride.

[0053] In an embodiment, the microorganism has a growth optimum at a pH below pH 8, or below pH 7; more preferably at pH 3-8 or at pH 3-7; more preferably at pH 4-8 or at pH 4-7.

[0054] In a particular embodiment, the microorganism is a strain of Bacillus, Aspergillus, or Trichoderma.

[0055] Recovery

[0056] The protein of interest may be recovered from the fermentation medium / broth using standard downstream processing operations.

[0057] The relevant downstream processing technology to be applied depends on the nature of the protein of interest. Typically, recovery of the protein of interest from the fermentation medium / broth includes downstream processing steps selected from the group consisting of pretreatment of broth (such as flocculation), removal of cells and other solid material from broth (primary separation), filtration, concentration (for example by membrane separation, precipitation, or evaporation), stabilization, and standardization.

[0058] Many other recovery procedures and steps may be applied, e.g., pH-adjustments, variation in temperature, crystallization, treatment of the solution comprising the protein of interest with active carbon, use of chromatography, and use of various adsorbents.

[0059] Plant proteins

[0060] The plant protein used in the method of the invention may be any plant derived protein useful as a source of amino acids in a fermentation medium. Plant proteins are often isolated from whole-plant (meal) compositions, but some plants are so protein rich that they can be used almost directly as a plant protein source in a fermentation medium. Such whole-plant compositions may also serve as a source of other substrates, such as carbohydrates, lipids, and / or other metabolites.

[0061] Examples of plant proteins include, but are not limited to, soybean protein, potato protein, corn (gluten) protein, wheat (gluten) protein, pea protein, rice bran protein, alfalfa protein, sunflower protein, cottonseed protein, and rapeseed protein. A preferred plant protein is potato protein.

[0062] Proteolytic enzymes

[0063] Suitable proteolytic enzymes (proteases) may be of any origin, but are preferably of bacterial or fungal origin, optionally in the form of protein engineered or chemically modified mutants. The protease may be an alkaline protease, such as a serine protease or a metalloprotease. A serine protease may for example be of the S1 family, such as trypsin, or the S8 family, such as a subtilisin. A metalloprotease may for example be a thermolysin, e.g. from the M4 family, or another metalloprotease such as those from the M5, M7 or M8 families.

[0064] In an embodiment, the proteolytic enzyme (protease) is a serine protease, preferably a subtilisin. Subtilisins advantageously have little activity at acidic pH, such as below pH 6 or below pH 5, which can be used as a partial (reversible) inactivation of the proteolytic activity.

[0065] The proteolytic enzyme may advantageously be thermostable. For example, it may retain at least 10% residual activity, preferably 20%, 30%, 40%, or 50% residual activity, after incubation at 50°C for 30 minutes.

[0066] The term "subtilases" refers to a sub-group of serine proteases according to Siezen et al., Protein Eng. 4 (1991) 719-737 and Siezen et al., Protein Sci. 6 (1997) 501-523. Serine proteases are a subgroup of proteases characterized by having a serine in the active site, which forms a covalent adduct with the substrate. The subtilases may be divided into six subdivisions, the Subtilisin family, the Thermitase family, the Proteinase K family, the Lantibiotic peptidase family, the Kexin family and the Pyrolysin family. Although proteases suitable for use in the method of the invention may be obtained from a variety of organisms, including fungi such as Aspergillus, particularly useful proteases have generally been obtained from bacteria and in particular from Bacillus.

[0067] In a particular embodiment, the proteolytic enzyme is a subtilisin derived from a Bacillus sp. Examples of Bacillus species from which subtilisins have been derived include Bacillus lentus, Bacillus alkalophilus, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus pumilus and Bacillus gibsonii. Particular subtilisins include subtilisin lentus, subtilisin Novo, subtilisin Carlsberg, subtilisin BPN’, subtilisin 309, subtilisin 147 and subtilisin 168 and e.g. protease PD138 (described in WO 93 / 18140).

[0068] In an embodiment of the invention, the amino acid sequence of the proteolytic enzyme (such as a serine protease or a subtilisin) has at least 80% sequence identity, preferably 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 of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0069] Amino acid alterations, as described above, may be of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding module.

[0070] Essential amino acids in a polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant molecules are tested for protease activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899- 904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acids can also be inferred from an alignment with a related polypeptide, and / or be inferred from sequence homology and conserved catalytic machinery with a related polypeptide or within a polypeptide or protein family with polypeptides / proteins descending from a common ancestor, typically having similar three-dimensional structures, functions, and significant sequence similarity. Additionally or alternatively, protein structure prediction tools can be used for protein structure modelling to identify essential amino acids and / or active sites of polypeptides. See, for example, Jumper et al., 2021 , “Highly accurate protein structure prediction with AlphaFold”, Nature 596: 583-589.

[0071] Single or multiple amino acid substitutions, deletions, and / or insertions can be made and tested using known methods of mutagenesis, recombination, and / or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241 : 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95 / 17413; or WO 95 / 22625. Other methods that can be used include error-prone PCR, CRISPR gene editing, phage display (e.g., Lowman et al., 1991 , Biochemistry 30: 10832-10837; US 5,223,409; WO 92 / 06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).

[0072] For purposes of the present invention, the sequence identity between two amino acid sequences is determined as the output of “longest identity” using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. In order for the Needle program to report the longest identity, the -nobrief option must be specified in the command line. The output of Needle labeled “longest identity” is calculated as follows:

[0073] (Identical Residues x 100) / (Length of Alignment - Total Number of Gaps in Alignment)

[0074] Suitable commercially available proteolytic enzymes include those sold under the trade names Alcalase, Duralase, Durazym, Relase, Relase Ultra, Savinase, Savinase Ultra, Primase, Polarzyme, Kannase, Liquanase, Liquanase Ultra, Ovozyme, Coronase, Coronase Ultra, Blaze, Neutrase, Everlase, Esperase, Progress Uno, Progress Key and Progress Excel (Novozymes), those sold under the tradename Maxatase, Maxacai, Maxapem, Purafect, Purafect Prime, Purafect MA, Purafect Ox, Purafect OxP, Puramax, Properase, FN2, FN3, FN4, Excellase, Eraser, Opticlean, Optimase, Preferenz P200, and Preferenz P300 (DuPont / IFF), BLAP (sequence shown in Figure 29 of US 5352604) and variants hereof (Henkel AG), KAP (Bacillus alkalophilus subtilisin from Kao), and Lavergy Pro (BASF).

[0075] Protein of interest

[0076] The protein of interest may be any useful protein produced (secreted) by cultivating a (recombinant) microbial organism in a fermentation medium.

[0077] Proteins may be small (peptides; <50 amino acids) or large (polypeptides; >50 amino acids) biomolecules that perform a vast array of functions within living organisms, including catalyzing reactions, DNA replication, responding to stimuli, providing structure to cells and organisms, and transporting molecules from one location to another. Proteins are composed of chains of polymerized amino acids, which are folded in a very specific three-dimensional structure. The three-dimensional structure is critical for maintaining the function of the protein. Some chemicals can change the folding, or even unfold (denaturing) the three-dimensional structure, which will result in loss of function, such as loss of enzymatic activity.

[0078] In an embodiment, the protein of interest is a polypeptide.

[0079] Proteins fall into at least three distinct groups, namely enzymes, cell signaling and ligand binding proteins, and structural proteins.

[0080] Enzymes are described below. Cell signaling and ligand binding proteins include many pharmaceutical proteins such as, for example, agonists, antagonists, receptors, membrane proteins, ion channels, antibodies (e.g. single-domain antibodies) and hormones; while structural proteins provide stiffness and rigidity to otherwise-fluid biological components.

[0081] Preferably, the proteins of interest are enzymes or cell signaling and ligand binding proteins; more preferably the proteins are enzymes.

[0082] Enzymes

[0083] The enzymes used as a protein of interest are catalytic proteins, and the term “active enzyme protein” is defined herein as the amount of catalytic protein(s), which exhibits enzymatic activity. This can be determined using an activity based analytical enzyme assay. In such assays, the enzyme typically catalyzes a reaction generating a colored compound. The amount of the colored compound can be measured and correlated to the concentration of the active enzyme protein. This technique is well-known in the art.

[0084] The enzyme(s) may be selected from the group consisting of protease, lipase, cutinase, amylase, carbohydrase, cellulase, pectinase, mannanase, arabinase, galactanase, xylanase, muramidase, nuclease (DNase, RNase), dispersin, catalase, perhydrolase, and oxidase (such as laccase and / or peroxidase).

[0085] The protease may be a serine protease, for example a subtilisin. The amylase may be an alpha-amylase or a glucoamylase. The cellulase may be an endo-1 ,4-beta-glucanase, also referred to as endoglucanase.

[0086] The enzyme may be a naturally occurring enzyme of bacterial or fungal origin, or it may be a variant derived from one or more naturally occurring enzymes by gene shuffling and / or by substituting, deleting or inserting one or more amino acids. Chemically modified or protein engineered mutants are included.

[0087] In an embodiment, the protein of interest is not a protease.

[0088] Further embodiments of the invention include:

[0089] Embodiment 1. A method for producing a protein of interest, comprising

[0090] (a) providing a fermentation medium comprising a plant protein, (b) incubating the fermentation medium with a proteolytic enzyme, and

[0091] (c) cultivating a microorganism capable of secreting the protein of interest in the fermentation medium.

[0092] Embodiment 2. The method of the preceding embodiment, wherein the protein of interest is secreted into the fermentation medium.

[0093] Embodiment 3. The method of any of the preceding embodiments, which further comprises recovering the protein of interest from the fermentation medium.

[0094] Embodiment 4. The method of any of the preceding embodiments, wherein the protein of interest is a cell signaling or ligand binding protein.

[0095] Embodiment 5. The method of any of the preceding embodiments, wherein the protein of interest is an enzyme.

[0096] Embodiment 6. The method of any of the preceding embodiments, wherein the protein of interest is not a protease.

[0097] Embodiment 7. The method of any of the preceding embodiments, wherein the protein of interest is an enzyme selected from the group consisting of protease, lipase, cutinase, amylase, carbohydrase, cellulase, pectinase, mannanase, arabinase, galactanase, xylanase, muramidase, nuclease, dispersin, catalase, perhydrolase, oxidase, laccase, and peroxidase.

[0098] Embodiment 8. The method of any of the preceding embodiments, wherein the protein of interest is an enzyme selected from the group consisting of lipase, cutinase, amylase, carbohydrase, cellulase, pectinase, mannanase, arabinase, galactanase, xylanase, nuclease, dispersin, catalase, perhydrolase, oxidase, laccase, and peroxidase.

[0099] Embodiment 9. The method of any of the preceding embodiments, wherein the plant protein is selected from the group consisting of soybean protein, potato protein, corn (gluten) protein, wheat (gluten) protein, pea protein, rice bran protein, alfalfa protein, sunflower protein, cottonseed protein, and rapeseed protein.

[0100] Embodiment 10. The method of any of the preceding embodiments, wherein the plant protein is potato or soybean protein.

[0101] Embodiment 11. The method of any of the preceding embodiments, wherein the fermentation medium in step (a) comprises the plant protein in an amount of 0.1-50% w / w.

[0102] Embodiment 12. The method of any of the preceding embodiments, wherein the fermentation medium in step (a) comprises the plant protein in an amount of 0.1-30% w / w.

[0103] Embodiment 13. The method of any of the preceding embodiments, wherein the fermentation medium in step (a) comprises the plant protein in an amount of 0.1-25% w / w.

[0104] Embodiment 14. The method of any of the preceding embodiments, wherein the fermentation medium in step (a) comprises the plant protein in an amount of 0.1-20% w / w. Embodiment 15. The method of any of the preceding embodiments, wherein the amount of proteolytic enzyme in step (b) is 0.0001-1% w / w of active enzyme protein; preferably 0.0001- 0.5% w / w, or 0.0001-0.1% w / w of active enzyme protein.

[0105] Embodiment 16. The method of any of the preceding embodiments, wherein the amount of proteolytic enzyme in step (b) is 0.0005-1% w / w of active enzyme protein; preferably 0.0005- 0.5% w / w, or 0.0005-0.1% w / w of active enzyme protein.

[0106] Embodiment 17. The method of any of the preceding embodiments, wherein the amount of proteolytic enzyme in step (b) is 0.001-1% w / w of active enzyme protein; preferably 0.001- 0.5% w / w, or 0.001-0.1% w / w of active enzyme protein.

[0107] Embodiment 18. The method of any of the preceding embodiments, wherein the proteolytic enzyme is a serine protease.

[0108] Embodiment 19. The method of any of the preceding embodiments, wherein the proteolytic enzyme is a subtilisin.

[0109] Embodiment 20. The method of any of the preceding embodiments, wherein the proteolytic enzyme retains at least 10% residual activity after incubation at 50°C for 30 minutes.

[0110] Embodiment 21. The method of any of the preceding embodiments, wherein the proteolytic enzyme has at least 80% amino acid sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0111] Embodiment 22. The method of any of the preceding embodiments, wherein the proteolytic enzyme has at least 85% amino acid sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0112] Embodiment 23. The method of any of the preceding embodiments, wherein the proteolytic enzyme has at least 90% amino acid sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0113] Embodiment 24. The method of any of the preceding embodiments, wherein the proteolytic enzyme has at least 95% amino acid sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0114] Embodiment 25. The method of any of the preceding embodiments, wherein the proteolytic enzyme has at least 96% amino acid sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0115] Embodiment 26. The method of any of the preceding embodiments, wherein the proteolytic enzyme has at least 97% amino acid sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0116] Embodiment 27. The method of any of the preceding embodiments, wherein the proteolytic enzyme has at least 98% amino acid sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. Embodiment 28. The method of any of the preceding embodiments, wherein the proteolytic enzyme has at least 99% amino acid sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0117] Embodiment 29. The method of any of embodiments 13-20, wherein the amino acid changes are substitutions.

[0118] Embodiment 30. The method of any of the preceding embodiments, wherein the proteolytic enzyme is inactivated by heat treatment before cultivating the microorganism; preferably the heat treatment includes heating to at least 90°C, such as at least 100°C, at least 110°C, or at least 120°C.

[0119] Embodiment 31. The method of any of the preceding embodiments, wherein the proteolytic enzyme is inactivated by heat treatment after incubation with the fermentation medium in step (b) and before cultivating the microorganism in step (c); preferably the heat treatment includes heating to at least 90°C, such as at least 100°C, at least 110°C, or at least 120°C.

[0120] Embodiment 32. The method of any of the preceding embodiments, wherein the fermentation is incubated with the proteolytic enzyme for at least 10 minutes.

[0121] Embodiment 33. The method of any of the preceding embodiments, wherein the fermentation is incubated with the proteolytic enzyme for up to 24 hours; preferably up to 12 hours.

[0122] Embodiment 34. The method of any of the preceding embodiments, wherein the fermentation medium is incubated with the proteolytic enzyme at a temperature in the range of 20-70°C.

[0123] Embodiment 35. The method of any of the preceding embodiments, wherein the fermentation medium is incubated with the proteolytic enzyme at a pH in the range of pH 6 to pH 9.

[0124] Embodiment 36. The method of any of the preceding embodiments, wherein the fermentation medium is supplemented with additional nutrients after incubation with the proteolytic enzyme.

[0125] Embodiment 37. The method of any of the preceding embodiments, wherein the fermentation medium is supplemented with a sugar source after incubation with the proteolytic enzyme.

[0126] Embodiment 38. The method of any of the preceding embodiments, wherein the microorganism is cultivated in the fermentation medium at a pH in the range of pH 3 to pH 8; preferably the pH is controlled by addition of NaOH.

[0127] Embodiment 39. The method of any of the preceding embodiments, wherein the microorganism is a recombinant microorganism. Embodiment 40. The method of any of the preceding embodiments, wherein the microorganism is selected from the group consisting of Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces.

[0128] Embodiment 41. The method of any of the preceding embodiments, wherein the microorganism is a strain selected from the group consisting of Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, Bacillus thuringiensis,

[0129] Embodiment 42. The method of any of the preceding embodiments, wherein the microorganism is a strain selected from the group consisting of Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans.

[0130] Embodiment 43. The method of any of the preceding embodiments, wherein the microorganism is selected from the group consisting of Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, llyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0131] Embodiment 44. The method of any of the preceding embodiments, wherein the microorganism is a filamentous fungus.

[0132] Embodiment 45. The method of any of the preceding embodiments, wherein the microorganism is selected from the group consisting of Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, and Trichoderma.

[0133] Embodiment 46. The method of any of the preceding embodiments, wherein the microorganism is a strain selected from the group consisting of Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Talaromyces emersonii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, and Trichoderma viride.

[0134] Embodiment 47. The method of any of the preceding embodiments, wherein the microorganism is selected from the group consisting of Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, and Yarrowia.

[0135] Embodiment 48. The method of any of the preceding embodiments, wherein the microorganism is a strain selected from the group consisting of Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, and Yarrowia lipolytica.

[0136] Embodiment 49. The method of any of the preceding embodiments, wherein the microorganism is a strain of Bacillus, Aspergillus, or Trichoderma.

[0137] Embodiment 50. The method of any of the preceding embodiments, wherein the microorganism is a strain of Bacillus or Trichoderma.

[0138] Embodiment 51. The method of any of the preceding embodiments, wherein the microorganism has a reduced protease expression level.

[0139] Embodiment 52. The method of any of the preceding embodiments, wherein step (b) is carried out at a different pH than step (c).

[0140] Embodiment 53. The method of any of the preceding embodiments, wherein the fermentation medium in step (b) is transferred to a fermentation tank before cultivating the microorganism in step (c).

[0141] Embodiment 54. The method of the preceding embodiment, wherein the fermentation tank has a volume of at least 1 m3(1000 L), at least 5 m3(5000 L), at least 10 m3(10000 L), at least 20 m3(20000 L), at least 50 m3(50000 L), or at least 100 m3(100000 L).

[0142] Embodiment 55. The method of the preceding embodiment, wherein the fermentation tank has a volume of 1-5 m3, 5-10 m3, 10-20 m3, 20-50 m3, 50-100 m3, or 100-150 m3.

[0143] Embodiment 56. The method of any of embodiments 53-55, wherein the fermentation medium is sterilized in the fermentation tank before cultivating the microorganism in step (c).

[0144] EXAMPLES

[0145] Chemicals were commercial products of at least reagent grade. Alcalase is a subtilisin available from Novozymes.

[0146] EXAMPLE 1

[0147] A fermentation medium was prepared by mixing 2200 kg potato protein with 220 kg Alcalase 2.4L (Novozymes) and various nutrient salts in a total volume of 47000 kg.

[0148] The fermentation medium was incubated at 55°C for 120 minutes at pH 7. The pH was controlled with NaOH. After incubation, the fermentation medium was heated to 90°C to inactivate the proteolytic enzyme, transferred to a fermentation tank, and sterilized at 123°C.

[0149] The fermentation medium was supplemented with glucose and used to cultivate a recombinant strain of Bacillus licheniformis which secreted a protease into the fermentation medium. The protease was subsequently recovered from the fermentation medium.

[0150] EXAMPLE 2

[0151] A fermentation medium was prepared by mixing 8250 kg potato protein with 129 kg Alcalase 2.4L (Novozymes) and various nutrient salts in a total volume of 52000 kg.

[0152] The fermentation medium was incubated at 55°C for 30 minutes at pH 7. After incubation, the fermentation medium was heated to 90°C to inactivate the proteolytic enzyme, transferred to a fermentation tank, and sterilized at 123°C.

[0153] The fermentation medium was supplemented with glucose and used to cultivate a recombinant strain of Bacillus subtilis which secreted an amylase into the fermentation medium. The amylase was subsequently recovered from the fermentation medium.

[0154] EXAMPLE 3

[0155] A fermentation medium was prepared by mixing 2000 kg potato protein with 189 kg Alcalase 2.4L (Novozymes) and various nutrient salts in a total volume of 50000 kg.

[0156] The fermentation medium was incubated at 55°C for 120 minutes at pH 7. The pH was controlled with NaOH. After incubation, the fermentation medium was heated to 90°C to inactivate the proteolytic enzyme, transferred to a fermentation tank, and sterilized at 123°C.

[0157] The fermentation medium was supplemented with glucose and used to cultivate a recombinant strain of Bacillus licheniformis which secreted an amylase into the fermentation medium. The amylase was subsequently recovered from the fermentation medium.

[0158] EXAMPLE 4

[0159] A fermentation medium was prepared by mixing 800 kg potato protein with 84 kg Alcalase 2.4L (Novozymes) and various nutrient salts in a total volume of 43000 kg. The fermentation medium was incubated at 55°C for 120 minutes at pH 7. After incubation, the fermentation medium was heated to 90°C to inactivate the proteolytic enzyme, transferred to a fermentation tank, and sterilized at 123°C.

[0160] The fermentation medium was supplemented with glucose and used to cultivate a recombinant strain of Trichoderma reesei which secreted a cellulase into the fermentation medium. The cellulase was subsequently recovered from the fermentation medium.

[0161] EXAMPLE 5

[0162] A fermentation medium was prepared by mixing 1356 kg potato protein with 135.6 kg Alcalase 2.4L (Novozymes) and various nutrient salts in a total volume of 47000 kg.

[0163] The fermentation medium was incubated at 55°C for 120 minutes at pH 7. After incubation, the fermentation medium was heated to 90°C to inactivate the proteolytic enzyme, transferred to a fermentation tank, and sterilized at 123°C.

[0164] The fermentation medium was supplemented with glucose and used to cultivate a recombinant strain of Trichoderma reesei which secreted a muramidase into the fermentation medium. The muramidase was subsequently recovered from the fermentation medium.

[0165] EXAMPLE 6

[0166] A fermentation medium was prepared by mixing 4700 kg potato protein with 108.75 kg Alcalase 2.4L (Novozymes) and various nutrient salts in a total volume of 25000 kg.

[0167] The fermentation medium was incubated at 53°C for 60 minutes at pH 7. The pH was controlled with NaOH. After incubation, the fermentation medium was heated to 90°C to inactivate the proteolytic enzyme, transferred to a fermentation tank, and sterilized at 123°C.

[0168] The fermentation medium was supplemented with glucose and used to cultivate a recombinant strain of Bacillus halodurans which secreted a protease into the fermentation medium. The protease was subsequently recovered from the fermentation medium.

Claims

CLAIMS1. A method for producing a protein of interest, comprising(a) providing a fermentation medium comprising a plant protein,(b) incubating the fermentation medium with a proteolytic enzyme, and(c) cultivating a microorganism capable of secreting the protein of interest in the fermentation medium.

2. The method of the preceding claim, wherein the protein of interest is secreted into the fermentation medium, and subsequently recovered from the fermentation medium.

3. The method of any of the preceding claims, wherein the protein of interest is not a protease.

4. The method of any of the preceding claims, wherein the protein of interest is an enzyme.

5. The method of any of the preceding claims, wherein the microorganism is a recombinant microorganism.

6. The method of any of the preceding claims, wherein the microorganism is a strain of Bacillus, Aspergillus, or Trichoderma.

7. The method of any of the preceding claims, wherein the plant protein is selected from the group consisting of soybean protein, potato protein, corn (gluten) protein, wheat (gluten) protein, pea protein, rice bran protein, alfalfa protein, sunflower protein, cottonseed protein, and rapeseed protein; preferably the plant protein is potato or soybean protein.

8. The method of any of the preceding claims, wherein the fermentation medium in step (a) comprises the plant protein in an amount of 0.1-50% w / w, preferably in an amount of 0.1-30% w / w, in an amount of 0.1-25% w / w, or in an amount of 0.1-20% w / w.

9. The method of any of the preceding claims, wherein the proteolytic enzyme is a serine protease, preferably a subtilisin.

10. The method of any of the preceding claims, wherein step (b) is carried out at a pH in the range of pH 6 to pH 9.

11. The method of any of the preceding claims, wherein step (b) is carried out at a temperature in the range of 20-70°C for at least 10 minutes.

12. The method of any of the preceding claims, wherein the proteolytic enzyme is inactivated by heat treatment after step (b) and before step (c); preferably the heat treatment includes heating to at least 90°C, such as at least 100°C, at least 110°C, or at least 120°C.

13. The method of any of the preceding claims, wherein the fermentation medium is supplemented with additional nutrients after incubation with the proteolytic enzyme.

14. The method of any of the preceding claims, wherein the microorganism is cultivated in a batch or fed-batch fermentation.

15. The method of any of the preceding claims, wherein the microorganism is cultivated at a pH in the range of pH 3 to pH 8; preferably the pH is controlled by addition of NaOH.

16. The method of any of the preceding claims, wherein the fermentation medium in step (b) is transferred to a fermentation tank before cultivating the microorganism in step (c); preferably the fermentation tank has a volume of at least 1 m3, at least 5 m3, at least 10 m3, at least 20 m3, at least 50 m3, or at least 100 m3.