WO2024133849A1

WO2024133849A1 - Ovalbumin fermentation

Info

Publication number: WO2024133849A1
Application number: PCT/EP2023/087534
Authority: WO
Inventors: Wilhelmus Theodorus Antonius Maria De Laat; Remon VAN DEN BERG
Original assignee: The Protein Brewery B.V.
Priority date: 2022-12-22
Filing date: 2023-12-22
Publication date: 2024-06-27

Abstract

The present invention is in the field of food technology and fermentation technology. The invention relates to the use of a linear polymer as an antifoaming agent in the microbial production of proteins. This allows improved animal-free production of animal-derived proteins for human consumption, by expression of such proteins in microbial cells.

Description

OVALBUMIN FERMENTATION

Field of the invention

Background of the invention

Egg white is used in many food applications for binding, foaming and gelling purposes. With the global population anticipated to rise from 7 billion towards 9 billion in 2050 and 11 billion in 2100, global food production needs to grow drastically. Concurrent to this, due to global warming, there is a reduction in available arable land surface and irrigation water. The process of egg production requires significant amounts of water, via the cultivation of soybean and grains, which are fed to chickens. Additional considerations of this process are the outputs which include nitrogen emissions via manure, the threat to human health by Salmonella and Listeria in egg products, and the increase of antibiotic resistance caused by the use of antibiotics in chicken farms. The health of humans is further threatened by the use of large amounts of pesticides like Fipronil, which is used to fight mites in chicken farms and may accumulate in the eggs themselves. With these concerns in mind, there is an increased demand for plant based food products with good nutritional value and functional properties, especially from vegan consumers (eating no animal derived products).

The concept of expressing egg white proteins like ovalbumin in microbes, and cultivating them in sterile fermenters can be considered as an alternative for obtaining functional ingredients like egg white. To produce egg white proteins by fermentation, preferably the production should be more land- and water-efficient, with less contribution to global warming factors. The production of ovalbumin per hectare was calculated by assuming an average of 7 tonnes of chicken feed per hectare (10 tonnes of corn and 4 tonnes of soybean per HA), then assuming 2 kg of chicken feed per kg egg and 6% egg white protein of the egg (the rest is shell, egg yolk and water). Assuming 54% of ovalbumin in egg white protein, the total production of ovalbumin by the animal value chain system is 7 * 0.06 * 0.54/2 = 1 13 kg of ovalbumin obtained per year.

The production of proteins from sugar, using ammonia as a nitrogen source in a fungal system can be as high as 20% on weight basis. The proteins produced by fungi can be secreted from the cell into the culture medium and then the proteins of interest can be harvest from the fermenter by separating the cells from the secreted proteins by microfiltration, filtration, centrifugation or a combination thereof. The cell free protein solution may then be concentrated by ultrafiltration or vacuum evaporation if necessary, towards the desired protein concentration. Subsequently, the protein may be further purified from fungal background proteins that may be present, such as cellulases (Trichoderma) or amylases (Aspergillus). The proteins can then be dried to powders by methods known in the art, such as freeze drying or spray drying. As fungi can produce up to 100 g/L protein and yeasts only 10 g/L at the same sugar input, there it may be considered that using yeasts will provide only 3 times improvement of the animal system, while with fungi we can reach 30 times higher land use reduction. Many fungi and yeasts have been exploited on an industrial scale for production of enzymes and other proteins. The most utilized yeasts are Saccharomyces cerevisiae, Pichia pastoris, Kluyveromyces lactis and Hansenula polymorpha. While for K. lactis the highest reported protein expression is around few g/L of fermentation broth (van Ooyen et al., 2006), for P. pastoris it is about a factor two higher (Werten M.W.T. et al., 2019). However, a production process, that will be based on working with methylotrophic yeasts (P.pastoris), requires installation of explosion-proof fermentation equipment due to the use of promoters, which rely on methanol as the inducer. Most industrial enzymes are produced by filamentous fungi such as Aspergillus spp., Trichoderma reesei, Myceliophthora thermophila etc. Filamentous fungi are known for their extremely high protein secretion capacity, which is several times higher than that of yeasts. For instance, for A . thermophila, previously known as Chrysosporium lucknowense C1 , which was developed by Dyadic International Inc. (US) for production of industrial and pharma proteins and enzymes, the production titres of secreted proteins higher than 100 g/L have been reported (Visser H. et al., 2011). For A. niger and T. reesei the production titres are believed to be in the range of several dozens of g/L, possibly reaching more than 100 g/L of protein (Owen ward, 2012).

Hen ovalbumin has been expressed in yeast S. cerevisiae (Mercereau-Puijalon O. et al., 1980) and P. pastoris (Ito K. et al., 2005). The total secreted amount of ovalbumin reported by Ito et al. was about 10 mg/L, while ovalbumin was found in the cell-free extract in the amount between 5-20 ng/mg of protein in S. cerevisiae. The P. pastoris secreted ovalbumin was found to be mono- or di-glycosylated and no N-terminal acetylation and no phosphorylation were found when compared to hen ovalbumin (Ito K. et al., 2005, supra). The hen ovalbumin molecule is mainly mono-glycosylated, N-terminal acetylated and phosphorylated at zero, one or two serine residues (Nisbet A. D. et al., 1981). Clara Foods CO., San Francisco, CA (US) describes in their patent application (US 2018355020 A1) cloning and overexpression of several proteins present in a hen egg in P. pastoris, including ovalbumin. The amount of ovalbumin produced is however not mentioned.

There are reports in the literature of heterologous expression of ovalbumin originating from different birds. Kfizkova et al. (1992) isolated cDNA from Japanese quail and expressed it in S. cerevisiae. The protein was detected in cell-free extract. Yang et al. (2009) described the cloning of a Chinese quail ovalbumin gene and overexpression in P. pastoris, with reported yields up to 5.45 g/L of secreted ovalbumin.

WO2021144342 describes the animal-free production of ovalbumin for human consumption by expression of ovalbumin in fungal cells.

There is however, still a need in the art for more efficient microbial production of animal- derived proteins for human consumption, such as meat, dairy and poultry proteins, including egg white proteins such as ovalbumin. There is a need for more stable ovalbumin compositions. There is a need for improved process parameters to allow the more efficient use of reactors such as stirred tank reactors. There is a need for increased production output of proteins of interest.

Summary of the invention

The inventors found that protein production could be improved by using a linear polymeric antifoaming agent. Accordingly, the invention provides a process for producing a protein of interest, the process comprising the steps of: i) providing a host cell capable of expressing the protein of interest; ii) culturing the host cell in a medium in a fermenter under conditions conducive to expression of the protein of interest, wherein the culturing is in the presence of a linear polymeric antifoaming agent; and, iii) optionally, recovering the protein of interest.

Preferably the linear polymeric antifoaming agent is a polyalkylene glycol. Preferably the polyalkylene glycol is polypropylene glycol. The linear polymeric antifoaming agent preferably has a molecular weight of 0.2 to 20 kDa, preferably 0.4 to 10 kDa, more preferably 0.5 to 5 kDa, most preferably 0.6 to 3 kDa. The linear polymeric antifoaming agent is preferably present at a concentration of 0.01 to 10 g/L, more preferably 0.05 to 5 g/L, even more preferably 0.08 to 3 g/L, most preferably 0.1 to 2.5 g/L.

In some embodiments the protein of interest is a food-protein, preferably an animal-derived food-protein. The food-protein is preferably a milk protein, a hemeprotein, or an egg protein, preferably an egg white protein, more preferably ovalbumin.

In the process the host cell preferably comprises an expression cassette, wherein the expression cassette comprises a first nucleotide sequence coding for the protein of interest, wherein the first nucleotide sequence is operably linked to at least one regulatory sequence that is capable of effecting expression of the encoded protein of interest by the host cell. The host cell is preferably a microbial host cell. The microbial host cell can be a yeast or a filamentous fungus, preferably a filamentous fungus such as those belonging to the species Altemaria alternata, Apophysomyces variabilis, Aspergillus spp., Aspergillus fumigatus, Aspergillus flavus, Aspergillus oryzae, Aspergillus niger, Aspergillus awamori, Aspergillus nidulans, Aspergillus terreus, Cladosphialophora spp., Fonsecaea pedrosoi, Fusarium spp., Fusarium oxysporum, Fusarium solani, Lichtheimia spp., Lichtheimia corymbifera, Lichtheimia ramosa, Myceliophthora spp., Myceliophthora thermophila, Rhizopus spp., Rhizopus microsporus, Rhizomucor spp., Rhizomucor pusillus, Rhizomucor miehei, Trichoderma spp., Trichoderma reesei, Trichophyton spp., Trichophyton interdigitale, and Trichophyton rubrum, more preferably the microbial host cell belongs to the species Aspergillus, most preferably the microbial host cell belongs to the species Aspergillus niger.

The fermenter is preferably a stirred tank reactor. In some embodiments of the process, in step b) input of mechanical power into the medium in the fermenter is no more than 2.5, 2.0, 1.8, 1.6, 1.4, 1.0, 0.5, 0.2 or 0.1 kW/m³. Also provided is the use of a linear polymer as an antifoaming agent in the microbial production of an animal-derived food-protein. Also provided is a composition comprising a linear polymer antifoaming agent and a microbial host cell. Also provided is a composition comprising a linear polymer antifoaming agent and an egg protein, preferably an egg white protein, most preferably ovalbumin.

Description of the invention

The invention provides a process for producing a protein of interest, the process comprising the steps of: i) providing a host cell capable of expressing the protein of interest; ii) culturing the host cell in a medium in a fermenter under conditions conducive to expression of the protein of interest, wherein the culturing is in the presence of a linear polymeric antifoaming agent; and, iii) optionally, recovering the protein of interest. Such a process is referred to herein as a process according to the invention.

Also provided is the use of a linear polymer as an antifoaming agent in the microbial production of an animal-derived food-protein. Also provided is the use of a linear polymer for the stabilization of an egg white protein, preferably ovalbumin. Features and definitions of such use are preferably as defined below.

Protein of interest

The present invention is particularly suitable for the animal-free production of proteins that are normally derived from animals and that are commonly used in the preparation of food for human consumption. In principle any protein that is normally obtained from or produced by an animal or part of an animal and that can be used in the preparation of food for human consumption is suitable for being produced in an animal-free manner in accordance with the invention. Thus, in some embodiments of the process according to the invention, the protein of interest is a food-protein, preferably an animal-derived food-protein. In more preferred embodiments of the process according to the invention, the food-protein is a milk protein, a hemeprotein, or an egg protein, preferably an egg white protein, more preferably ovalbumin.

However, more particularly the invention is concerned with the animal-free production of dairy and poultry proteins, more specifically milk proteins and egg proteins. In one embodiment, the animal-derived food-protein of interest is a milk protein, preferably a protein present in the milk of cattle (i.e. bovine or Bos taurus), buffalo (including water buffalo), goats, sheep or camel, or in the milk of other less common milk animals such as yak, horse, reindeer and donkeys. The protein of interest can be a casein or can be a whey protein such as p-lactoglobulin, a-lactalbumin, bovine serum albumin or an immunoglobulin.

In one embodiment, the animal-derived food-protein of interest is a hemeprotein, preferably a hemeprotein from a non-human animal, more preferably a mammal such as a cow, pig, horse, goat or sheep. Preferred animal-derived hemeproteins include hemoglobin and myoglobin. The animal-derived hemeproteins produced in accordance with the invention can be applied as red heme-bound iron protein in meat substitutes.

In one embodiment, the animal-derived food-protein of interest is an egg protein, i.e. a protein that is present in a bird’s egg. The term "bird" as used herein includes both domesticated birds and non-domesticated birds such as wild life birds. Birds e.g. include poultry, fowl, waterfowl, game bird, ratite (e.g., flightless bird), chicken (Gallus gallus domesticus), quail, turkey, duck, ostrich (Struthio camelus), Somali ostrich (Struthio molybdophanes), goose, gull, guinea fowl, pheasant, emu (Dromaius novaehollandiae), American rhea (Rhea americana), Darwin's rhea (Rhea pennata) and kiwi. Preferably, the animal-derived food-protein of interest is an egg white protein. The egg white protein can be an egg white protein selected from the group consisting of ovalbumin, ovotransferrin, ovomucoid, G162M F167A ovomucoid, ovoglobulin G2, ovoglobulin G3, a-ovomucin, p-ovomucin, lysozyme, ovoinhibitor, ovoglycoprotein, flavoprotein, ovomacroglobulin, ovostatin, cystatin, avidin, ovalbumin related protein X and ovalbumin related protein Y (see e.g. US2018/0355020). A particularly preferred egg white protein is ovalbumin.

A particularly preferred animal-derived food-protein of interest to be produced in an animal- free manner in accordance with the invention is the egg white protein ovalbumin.

Providing a host cell

A host cell can be any suitable host cell, preferably it is a microbial host cell such as a fungal host cell. The microbial host cell is preferably a yeast or a filamentous fungus. The host preferably has a genetic modification so as to enable the host cell to produce the protein of interest. A host cell for use in a process of the invention thus preferably comprises an expression cassette, wherein the expression cassette comprises a first nucleotide sequence coding for the protein of interest, wherein the first nucleotide sequence is operably linked to at least one regulatory sequence that is capable of effecting expression of the encoded protein of interest by the host cell. A preferred yeast is Pichia, more preferably Pichia pastoris.

A fungal host is a host cell that belongs to the “fungi”, which are herein defined as eukaryotic microorganisms, which include all species of the subdivision Eumycotina (Alexopoulos, C. J., 1962, In: Introductory Mycology, John Wiley & Sons, Inc., New York). The term fungus thus includes both filamentous fungi and yeast. "Filamentous fungi" are herein defined as eukaryotic microorganisms that include all filamentous forms of the subdivision Eumycotina and Oomycota (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK). The filamentous fungi are 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. A preferred filamentous fungal host cell is a host cell the belongs to a genus selected from the genera including, but are not limited to, Alternaria, Apophysomyces, Aspergillus, Cladosphialophora, Fonsecaea, Fusarium, Lichtheimia, Myceliophthora, Rhizopus, Rhizomucor, Trichoderma and Trichophyton. More preferably, a filamentous fungal host cell belong to a species selected from Alternaria alternata, Apophysomyces variabilis, Aspergillus spp., Aspergillus fumigatus, Aspergillus flavus, Aspergillus oryzae, Aspergillus niger, Aspergillus awamori, Aspergillus nidulans, Aspergillus terreus, Cladosphialophora spp., Fonsecaea pedrosoi, Fusarium spp., Fusarium oxysporum, Fusarium solani, Lichtheimia spp., Lichtheimia corymbifera, Lichtheimia ramosa, Myceliophthora spp., Myceliophthora thermophila, Rhizopus spp., Rhizopus microsporus, Rhizomucor spp., Rhizomucor pusillus, Rhizomucor miehei, Trichoderma spp., Trichoderma reesei Trhichophyton spp., Trichophyton interdigitale, and Trichophyton rubrum. The most preferred filamentous fungal host cell is a strain of the species Aspergillus, most preferably the microbial host cell belongs to the species Aspergillus niger. In other embodiments the host cell belongs to the species Aspergillus, Trichoderma, Pichia, Hansenula, or Myceliophthora, optionally Trichoderma, Pichia, Hansenula, or Myceliophthora. A preferred Pichia is Pichia pastoris.

Several strains of filamentous fungi are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), Chinese Centrum for Industrial Culture Collection (CICC) and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL), including e.g. the strains Aspergillus niger CBS 513.88, CBS124.903, and CICC2462; Aspergillus oryzae ATCC 20423, IFO 4177, ATCC 1011 , CBS205.89, ATCC 9576, ATCC14488-14491 , ATCC 11601 and ATCC12892; P. chrysogenum CBS 455.95, Penicillium citrinum ATCC 38065, Penicillium chrysogenum P2, Talaromyces emersonii CBS 124.902, Acremonium chrysogenum ATCC 36225 or ATCC 48272, Trichoderma reesei ATCC 26921 or ATCC 56765 or ATCC 26921 , Aspergillus sojae ATCC11906 and Chrysosporium lucknowense ATCC44006 and derivatives thereof.

"Yeasts" are herein defined as eukaryotic microorganisms and include all species of the subdivision Eumycotina that predominantly grow in unicellular form. Yeasts may either grow by budding of a unicellular thallus or may grow by fission of the organism. Alternatively, a fungal host cell is a yeast host cell the belongs to a genus selected from the genera including Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia, more preferably a species selected from the species Kluyveromyces lactis, Saccharomyces cerevisiae, Hansenula polymorpha, Yarrowia lipolytica and Pichia pastoris.

The term "fungal", when referring to a protein or nucleic acid molecule thus means a protein or nucleic acid whose amino acid or nucleotide sequence, respectively, naturally occurs in a fungus.

In one particularly preferred embodiment, the filamentous fungal host cell is of a strain that grows, or has the ability to grow with a “yeast like morphology”. The term “yeast like morphology” as used herein for a filamentous fungal host cell, indicates that the filamentous fungus grows with short hyphae and little of branching of the hyphae. A reference strain of Aspergillus niger that grows with a yeast like morphology is strain CICC2462, as obtainable from the China Center of Industrial Culture Collection (CICC, Building 6, No. 24 Yard, Jiuxianqiao Middle Road, Chaoyang District, Beijing, China; www.china-cicc.org). Aspergillus niger CICC2462 is used in the industrial production of glucoamylase and is a morphological mutant strain of A. niger that does not produce spores, has short mycelia, thick hyphae, which results in a low-viscosity fermentation broth, is a strong enzyme producer, has low protease activity, is osmotolerant, and is suitable for high-density submerged liquid fermentation (Zhang et al., Microb Cell Fact. 2016; 15: 68).

A filamentous fungal strain that grows with a yeast like morphology is herein defined as a filamentous fungal strain with at least one of the characteristics of: a) the hyphae of the filamentous fungal strain are on average not more than 5, 10, 20, 50 or 100% longer than the hyphae of the reference strain CICC2462; and b) the hyphae of the filamentous fungal strain show on average not more than 5, 10, 20, 50 or 100% branching than the hyphae of the reference strain CICC2462, whereby preferably the filamentous fungal strain and the reference strain CICC2462 are grown under identical conditions. A preferred filamentous fungal host cell is Aspergillus niger strain CICC2462, or a strain that is a single colony isolate and/or a derivative of strain CICC2462.

The advantage of using as host cell a filamentous fungal strain that grows with a “yeast like morphology” is that the strain can be cultured under condition requiring a minimum input of mechanical power into the medium in the fermenter for aeration, which provides savings on costs for energy. In addition the use of less mechanical power reduces shear forces, thereby reducing destruction of proteins and the cells, which increases yield and facilitates filtration by reducing clogging of filters by cellular debris etc.

In one embodiment, a fungal host cell of the invention comprises a genetic modification that reduces or eliminates the specific activity or amount of at least one enzyme or protein selected from a glucoamylase (e.g. glaA), an amylase, such an acid stable alpha-amylase (e.g. amyA) or a neutral alpha-amylase (e.g. amyB\ and amyBII), an oxalic acid hydrolase (e.g. oahA), a protein involved in mycotoxin biosynthesis and a protease, and a protein encoded by KU70, KU80, hdfA and hdfB or homologues thereof. Preferably in the host the genetic modification reduces the specific activity or amount of the enzyme or protein to no more than 90, 75, 50, 20, 10, 5, 2 or 1 % of the specific activity or amount of the enzyme or protein compared to a corresponding host cell lacking the genetic modification, when cultivated under identical conditions. More preferably, the genetic modification completely eliminates the specific activity or amount of the enzyme or protein in the host cell.

The advantage of reducing or eliminating the expression in the host cell of one or more of glucoamylase (glaA), acid stable alpha-amylase (amyA) and neutral alpha- amylase (amyBI and amyBII) is not only that the cell’s energy and resources are not utilized for these byproducts and/or that downstream processing of the product of interest such as ovalbumin is simplified since there are fewer by-products present, but most importantly, in many of the food applications of ovalbumin the action of these enzymes on starch and starch-derived substrates is preferably avoided.

The advantage of reducing or eliminating the expression in the host cell of oxalic acid hydrolase (oahA) is that the host cell will produce less or no oxalic acid. Oxalic acid is an unwanted by-product in many applications such as food-applications. Furthermore, reducing or eliminating the production of oxalic by the host cell reduces the undesirable lowering of the pH of the culture medium by this acid. Avoiding such low pH requires less buffering, improves product yield and avoids aggregation/precipitation of the ovalbumin produced.

The advantage of reducing or eliminating the expression in the host cell of a protein involved in mycotoxin biosynthesis is that it reduces or avoids the formation of such toxins during the fermentation of product of interest, which is highly undesirable as these toxins present a health hazard to operators, customers and the environment.

The advantage of reducing or eliminating the expression in the host cell of a protease is that it reduces the negative impact of host cell proteases on the yield and quality of the product of interest such as ovalbumin. Preferred genetic modifications or reducing or eliminating the expression in the host cell of one or more proteases include e.g. prfT, which is a transcriptional activator of proteases in eukaryotic cells. Several fungal transcriptional activators of proteases have been recently described in WO 00/20596, WO 01/68864, WO 2006/040312 and WO 2007/062936. These transcriptional activators were isolated from A niger, A. fumigatus, P. chrysogenum and A. oryzae. These transcriptional activators of protease genes can be used to improve a method for producing a polypeptide in a fungal cell, wherein the polypeptide is sensitive for protease degradation. When the host cell is deficient in prfT, the host cell will produce less proteases that are under transcriptional control of prfT. It is therefore advantageous when the host cell according to the invention is deficient in prfT. A host cell of the invention may further be deficient a major protease such as pepA.

For means and method for effecting a genetic modification that reducing or eliminating in the host cell the specific activity or amount of at least one enzyme or protein selected from a glucoamylase (e.g. glaA), an amylase, such an acid stable alpha-amylase (e.g. amyA) or a neutral alpha- amylase (e.g. amyB\ and amyBII), an oxalic acid hydrolase (e.g. oahA), a protein involved in mycotoxin biosynthesis and a protease, reference is made to WO2011/009700.

A further genetic modification that can be applied in a host cell for reducing the background amount of (endogenous) secreted proteins is an inactivation or deletion of the amyR as described by Zhang et al. (2016, supra).

Culturing

An important feature of the invention is that the culturing is in the presence of a linear polymeric antifoaming agent. Such agents are defined in the section below. The culturing, or fermentation, of host cells may be carried out in any suitable way, using a fermenter that is for instance a stirred tank reactor. The reason for this choice by most of the industrial submerged fermentations of fungi lies on the rheology of (filamentous) fungal biomass in submerged cultures, which is affected, amongst others, by the morphology of the growing fungal mycelium. Consequently, in one embodiment the fermentation process of the invention is carried out in a stirred tank reactor. In a preferred embodiment however, the mechanical power into the medium in the fermenter is limited to prevent or reduce precipitation and/or aggregation in the medium of the protein of interest, in particular of an ovalbumin. In one embodiment therefore, the input of mechanical power (during the culturing of the host) into the medium in the fermenter is no more than no more than 2.5, 2.0, 1 .8, 1 .6, 1 .4, 1 .0, 0.5, 0.2 or 0.1 kW/m³.

Alternatively, or in addition to the use of (limited) mechanical power, a bubble column may be employed for the process of the invention. In general, the growth of fungi, in particular the mycelial growth of filamentous fungi, can lead to a highly viscous solution, which can negatively affect the aeration of the medium in the fermenter and thereby the growth and product formation. In order to obtain good oxygen and nutrient transfer of the viscous medium within the reactor, a high-power input for stirring is needed, which can give rise to precipitation and/or aggregation in the medium of the protein of interest to be produced, in particular in the case of an ovalbumin. However, strains of filamentous fungi, which are altered in their morphology, for instance those, which mycelial growth could be described as “yeast like morphology”, e.g. short hyphae and little of branching, could be also fermented in a reactor equipped with a bubble column (see for instance van ‘t Riet and Tramper, 1991 for description of type of bioreactors). Consequently, in one embodiment the fermentation is carried out in a bubble column, preferably without the input of mechanical power into the medium in the fermenter (e.g. for stirring). In this instance, the cultured medium is “stirred” or mixed by the gas (e.g. air or oxygen) bubbles moving upwards through the medium. In one embodiment the fermentation is carried out in a combination of a stirred tank reactor and a bubble column. In a further embodiment, the bubble column makes use of a batch mode, a fed batch mode or a repeated fed batch mode. In one embodiment, a high cell density fermentation is applied, where the operation may be carried out at packed cell volume of more than 10% (cells/volume) and even more then 25%, 30%, 40%, or sometimes even at 45% packed cell volume. In one embodiment, the bubble column is executed with a gas volume of > 0.5 wm (broth volume replacement per minute), even higher than 0.7 wm and most preferably at 1 .0 wm. The air provides good mixing of the fermenter and aeration. Therefore, in one embodiment the bubbles are sparged through holes in a pipe of 4 to 6 mm.

Media for growth of host cells of the invention are generally known in the art. In a preferred embodiment, the medium for culturing a host cell of the invention is a chemically defined medium. Typical composition of the chemically defined media for growth of (filamentous) fungi are e.g. described in US 20140342396 A1. The pH in the fermenter may be controlled using ammonia as titrant.

The process preferably uses a carbon source comprising at least one of glucose, sucrose, isomaltose and maltodextrines. The carbon source may be delivered in the form of thick juice (an intermediate of sugar beet processing) or a syrup comprising isomaltose and maltodextrines, such as a 95 DE syrup. One example with glucose includes a 30-95DE syrup comprising, the maltose isomaltose as inducing compound of the glucoamylase promoter.

The pH of the fermentation may be maintained between 3 to 8 pH, most likely between 4 to 7 pH. Depending on the type of the expressed protein, however, this range may be further finetuned. For example, the optimal pH for the production of homologous enzymes using A. niger is pH 3.5 - 5.5. In the case of heterologous expression of animal proteins a higher pH range is preferred. E.g. for ovalbumin, having the pl value around 4.8 - 5, a pH of 5 to 7 pH is preferred. Furthermore, by monitoring the pH during the fermentation the induction spectrum of proteases can be altered. Consequently, in one embodiment, the host cell is cultured at a pH that is equal to or higher than pH 5.0, 5.5, 6.0, 6.5 or 7. Linear polymeric antifoaminq agent

Antifoaming agents are generally well-known in the art. An antifoaming agent is a chemical additive that reduces and hinders the formation of foam in industrial process liquids, such as in fermentation broths. Strictly speaking, defoamers eliminate existing foam and antifoamers prevent the formation of (further) foam. In the art, the terms defoaming agent and antifoaming agent are sometimes used interchangeably.

Generally, an antifoaming agent is insoluble in the foaming medium and has surface active properties. An antifoaming agent for use in for instance the chemically defined aqueous culture media of the invention will therefore usually have a hydrophobic character, i.e. comprising or consisting of hydrophobic molecules. Furthermore, the agent preferably is compatible with a fermentation process in the sense that the agent is not toxic to the host cell or otherwise negatively impacts its growth and preferably does not interact with medium component in a way that negatively impacts the process or the fermentation products produced thereby. Antifoaming agents that are suitable for application in fermentation processes are generally well known in the art.

The antifoaming agent for use in a process of the invention is a linear polymeric antifoaming agent. A linear polymer is a polymer with minimal, preferably no branching or crosslinking. In effect, a linear polymer is a single chain without branches or interconnections. Examples of linear polymeric antifoaming agents include mineral oils, polyalkylene glycols, or silicones such as polydimethylsiloxane. In preferred embodiments the antifoaming agent is a polyalkylene glycol.

Polyalkylene glycols are broadly known. Examples include polypropylene glycol (PPG), polyethylene glycol (PEG), polytetramethylene glycol (PTMEG), poly(p-phenylene oxide) (PPG), and poloxamers. Excellent results were obtained when the polyalkylene glycol is polypropylene glycol.

The polypropylene glycol can have terminal hydroxyl moieties. The polypropylene glycol can have terminal alkyl ethers such as methyl, ethyl, or propyl ethers. The nature of the terminal moieties is not critical and a skilled person can easily assess the suitability of a particular polypropylene glycol.

An important feature of the linear polymeric antifoaming agent is its length, which is correlated with its weight. In preferred embodiments is provided the process according to the invention, wherein the linear polymeric antifoaming agent has a molecular weight of 0.2 to 20 kDa, preferably 0.4 to 10 kDa, more preferably 0.5 to 5 kDa, most preferably 0.6 to 3 kDa. In some embodiments the linear polymeric antifoaming agent has a molecular weight of 0.3 to 15 kDa, of 0.7 to 13 kDa, of 0.8 to 12 kDa, of 0.9 to 11 kDa, of 1 .1 to 9 kDa, of 1 .2 to 8 kDa, of 1 .3 to 7 kDa, of 1 .4 to 6 kDa, or of 1.5 to 4 kDa. In some embodiments the linear polymeric antifoaming agent has a molecular weight of 0.7 to 2.5 kDa, preferably 1 to 2 kDa. Preferred linear polymeric antifoaming agents are PPG with an Mw of 2 kDa or of 1 kDa, most preferably 1 kDa.

In the process according to the invention, the linear polymeric antifoaming agent is preferably present at a concentration of 0.01 to 10 g/L, preferably 0.05 to 5 g/L, more preferably 0.08 to 3 g/L, most preferably 0.1 to 2.5 g/L. Good results were obtained with at least 0.2 g/L. More preferably, at least 0.3 g/L is used, still more preferably at least 0.4 g/L. The agent can also be present at a concentration of 0.5 to 5 g/L, 0.6 to 4 g/L, 0.7 to 3 g/L, 0.8 to 2.7 g/L, 0.9 to 2.2 g/L, and 1 to 1 .5 g/L. In some embodiments at least 0.5 g/L is used.

The antifoaming agent preferably is a food-grade antifoaming agent. A preferred food-grade antifoaming agent is a clean-label antifoaming agent. In preferred embodiments, no additional antifoaming agent is used.

In preferred embodiments, no defoaming agent is used. Optionally a defoaming agent is used. A preferred vegetable oil for use as defoaming agent in a process of the invention is olive oil or high-oleic sunflower oil, of which olive oil is most preferred. Only a small amount of the defoaming agent needs to be present during the culturing of the host cell, for instance to maintain the dispersed morphology of a fungal strain, which supports good growth. Preferably, the defoaming agent is present and/or is maintained in the chemically defined medium at a concentration of at least 1 , 2, 5, 10, 25, 50, 75,100, 125 or 135 ppm (w/v). Use of a defoamer can be as described in WO 2021/148592.

Recovering the protein of interest

The protein of interest can be produced as a secreted protein, which, by virtue of the presence of the secretory signal sequence in the expression cassette, is secreted from the host cell into the fermentation medium. Such secreted proteins of interest can be recovered from the fermentation broth in different ways. Therefore, in one embodiment, the method optionally includes step iii) recovering the protein of interest. In one embodiment, the recovery is during fermentation. In one embodiment, the recovery is post fermentation. In one embodiment, the recovery is both during and post fermentation. The recovery of the protein of interest preferably at least includes separation of the biomass from the medium comprising the (dissolved) protein of interest. One of the possibilities to separate the microbial biomass is by centrifugation. Even more preferred the fermented broth may be set for release of the protein at the end of the fermentation, which may be following the separation of the released protein directly by centrifugation of the biomass. Therefore, in one embodiment, the recovery is by centrifugation. After the separation by centrifugation the supernatant usually contains most of the secreted protein. Consequently, in a further embodiment, the supernatant may require further processing. In one embodiment, the supernatant may be microfiltrated using a 0.2 pm filter. Such practice will be known to one skilled in the art to remove remaining fragments of (fungal) biomass. In addition the supernatant can be ultra-filtrated through a membrane with variable molecular weight cut-off values, typically 10 kDa or 20 kDa, to further separate small molecular weight components from the ultrafiltrate. In one embodiment, such further processing includes ultrafiltration, wherein the supernatant is also concentrated. However, depending on the nature of the protein, hydrophobic proteins usually have the tendency to stick to the biomass. Such an example may be for instance found in US 8703463 B2, which describes expression of a lipase in a yeast Kluyveromyces lactis and a filamentous fungus Aspergillus niger. In this example, the protein was released from the biomass by changing the pH of the solution. Suitable means for recovering ovalbumin are described in WO2021144342. Optionally, after microfiltration and ultrafiltration of the fermentation broth containing the expressed protein, the next step may involve purification. Alternatively, microfiltration and ultrafiltration may not be required. The choice of a purification method may influence the protein stability, purity and yield, but the costs, sustainability and waste streams associated with different methods differ. One of the most important criteria during purification is reduction of protein losses due to aggregation or denaturation. A person skilled in art will recognize that the following methods being typically applied for purification of proteins. In one embodiment, the purification is one or more of ammonium sulfate precipitation; an aqueous biphasic system (ABS); and chromatography methods. In a further embodiment, purification is chromatography selected form one or more of anion-exchange; cation-exchange; hydrophobic-interaction; and size-exclusion chromatography.

In one embodiment, the process for purifying an animal-derived food-protein of interest from a spent culture medium that was obtained in a process for producing the protein of interest as herein defined. In one embodiment, the process for purifying an animal-derived food-protein of interest comprises at least a first anion exchange step. The ion exchange resin can be prepared according to known methods. Typically, an equilibration buffer, which allows the resin to bind its counter ions, can be passed through the ion exchange resin prior to loading the sample or composition comprising the polypeptide and one or more contaminants onto the resin. Conveniently, the equilibration buffer can be the same as the loading buffer, but this is not required.

Expression cassette

In preferred embodiments the host cell comprises an expression cassette, wherein the expression cassette comprises a first nucleotide sequence coding for the protein of interest, wherein the first nucleotide sequence is operably linked to at least one regulatory sequence that is capable of effecting expression of the encoded protein of interest by the host cell.

The expression cassette comprises a first nucleotide sequence coding for the protein of interest such as for an animal-derived food-protein of interest. The coding nucleotide sequence is operably linked to at least one regulatory sequence that effects or controls expression of the encoded protein of interest by the host cell. The expression regulatory sequence preferably at least includes a transcription regulatory sequence or promoter operably linked to the coding sequence. The expression cassette further preferably includes regulatory sequences such as translation initiation sequences, secretion signal sequences, transcription termination sequences, polyadenylation signals. Additional factors necessary or helpful in effecting expression may also be present, such as expression enhancer elements. In one embodiment the regulatory sequences are the regulatory sequences of a highly expressed fungal protein.

The promoter that is operably linked to the coding sequence in the expression cassette according to the invention can be a constitutive promoter, an inducible promoter or a hybrid promoter. Examples of preferred inducible promoters that can be used are a starch-, copper-, oleic acid-inducible promoters.

A preferred promoter is a promoter of a highly expressed fungal protein. Preferred promoters from highly expressed fungal genes include the promoters obtained from the genes encoding fungal or filamentous fungal acid a-amylase, a-amylase, TAKA-amylase, glucoamylase, xylanase, cellobiohydrolase, pyruvate kinase, glyceraldehyde-phosphate dehydrogenase, alcohol dehydrogenase, aldehyde dehydrogenase, sucrase, acetamidase and superoxide dismutase genes. Specific examples thereof include the promoters from genes encoding the A. oryzae TAKA amylase, the A. niger neutral alpha-amylase, the A. niger acid stable alpha-amylase, the A. niger or A. awamori glucoamylases (glaA). Particularly preferred promoters for use in filamentous fungal cells are the A. niger and A. awamori glucoamylase (glaA) promoters, or a functional part thereof. In a preferred embodiment, not only the promoter but also the further regulatory sequences such as translation initiation sequences, secretion signal sequences, transcription termination sequences, polyadenylation signals are from a highly expressed fungal protein as indicated, of which the A. niger and A. awamori glucoamylase (glaA) regulatory sequences are most preferred.

The expression cassette is preferably part of an expression vector, which in addition to the expression cassette can comprise additional sequence elements such as selectable marker genes, sequences for targeting integration at specific loci in the host cell’s genome and/or for autonomous replication.

The expression vector may be any vector (e.g., a plasmid or virus), which can be conveniently subjected to recombinant DNA procedures and can bring about the expression of the polynucleotide of interest. The choice of the vector will typically depend on the compatibility of the vector with host cell into which the vector is to be introduced. The vectors may be linear or closed circular plasmids. The vector may be an autonomously replicating vector, i.e., a vector, which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. An autonomously maintained cloning vector may comprise the AMA1 -sequence (see e.g. Aleksenko and Clutterbuck (1997), Fungal Genet. Biol. 21 : 373-397). Alternatively, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Preferably, the integrative cloning vector comprises a DNA fragment, which is homologous to a DNA sequence in a predetermined target locus in the genome of the host cell for targeting the integration of the cloning vector to this predetermined locus. In order to promote targeted integration, the cloning vector is preferably linearized prior to transformation of the host cell. Linearization is preferably performed such that at least one but preferably either end of the cloning vector is flanked by sequences homologous to the target locus. The length of the homologous sequences flanking the target locus is preferably at least 30 bp, 50 bp, 0.1 kb, 0.2kb, 0.5 kb, 1 kb or 1.5 kb, preferably at least 2.0 kb, more preferably at least 2.5 kb or most preferably at least 3.0 kb.

Preferred homologous sequences for targeting the expression vector/cassette are sequences from highly expressed fungal genes such as sequences obtained from the genes encoding fungal or filamentous fungal acid a-amylase, a-amylase, TAKA-amylase, glucoamylase, xylanase, cellobiohydrolase, pyruvate kinase, glyceraldehyde-phosphate dehydrogenase, alcohol dehydrogenase, aldehyde dehydrogenase and sucrase. More preferably, the homologous sequences for targeting the expression vector/cassette are sequences that target to loci comprising highly expressed fungal genes, which loci are amplified in the fungal genome, such as the TAKA amylase genes in Aspergillus oryzae (e.g. in strain IF04177) or the amplified g/aA locus of Aspergillus niger (see US6432672B1 and US8734782B2), e.g. in strains CBS 513.88 and CICC2462. In a preferred embodiment, the expression cassette is integrated in a locus of a gene coding for a highly expressed fungal protein. Preferably, said locus is a locus that is amplified in the fungal genome, wherein more preferably, the locus is an A. niger glaA locus.

In one embodiment, the expression cassette is integrated by (homologous) recombination via a single cross-over. Preferably, said locus is a locus that is amplified in the fungal genome, wherein more preferably, the locus is an A. niger glaA locus.

In another embodiment, the expression cassette is integrated by (homologous) gene replacement in a locus of a gene coding for a highly expressed fungal protein (i.e. double crossover). Preferably, said locus is a locus that is amplified in the fungal genome and the expression cassette replaces several copies or each copy of the gene coding for the highly expressed fungal protein in the fungal host cell’s genome, wherein more preferably, the locus is an A. niger glaA locus.

In one embodiment, the fungal host cell comprises multiple copies of the expression cassette, preferably integrated into the genome of the fungal host cell. Preferably, the fungal host cell comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25 or 30 copies of the expression cassette, preferably integrated into the genome of the fungal host cell, more preferably at a predefined location, such as a locus comprising a highly expressed endogenous fungal gene.

The vectors preferably contain one or more selectable markers, which permit easy selection of transformed cells. Using the method of co-transformation, one vector may contain the selectable marker whereas another vector may contain the polynucleotide of interest or the nucleic acid construct of interest; the vectors are simultaneously used for transformation of the host cell. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. A selectable marker for use in a filamentous fungal host cell may be selected from the group including, but not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), bleA (phleomycin binding), hygB (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents from other species. Preferred for use in an Aspergillus host cell are the amdS (US5876988, US6548285B1) and pyrG genes of A. nidulans or A. oryzae and the bar gene of Streptomyces hygroscopicus. More preferably an amdS gene is used, even more preferably an amdS gene from A. nidulans or A. niger. A most preferred selection marker gene is the A. nidulans amdS coding sequence fused to the A. nidulans gpdA promoter (see EP 0635574 B1). AmdS genes from other filamentous fungi may also be used (US 6548285 B1).

Means and methods for constructing the expression vectors and cassettes of the present invention are well known to one skilled in the art (see, e.g., Sambrook and Russell, supra; and Ausubel et al., Current Protocols in Molecular Biology, Wiley InterScience, NY, 1995).

In one embodiment, the nucleotide sequence encoding the protein of interest in the expression cassette of the invention preferably is adapted to optimize its codon usage to that of the host cell in question. The adaptiveness of a nucleotide sequence encoding an enzyme to the general codon usage of a host cell may be expressed as codon adaptation index (CAI). The codon adaptation index is herein defined as a measurement of the relative adaptiveness of the codon usage of a gene towards the codon usage of highly expressed genes in a particular host cell or organism. The relative adaptiveness (w) of each codon is the ratio of the usage of each codon, to that of the most abundant codon for the same amino acid. The CAI index is defined as the geometric mean of these relative adaptiveness values. Non-synonymous codons and termination codons (dependent on genetic code) are excluded. CAI values range from 0 to 1 , with higher values indicating a higher proportion of the most abundant codons (see Sharp and Li, 1987, Nucleic Acids Research 15: 1281-1295; also see: Jansen et al, 2003, Nucleic Acids Res. 3J_(8):2242-51). An adapted nucleotide sequence preferably has a CAI of at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 or 0.9.

In a preferred embodiment the nucleotide sequence encoding the protein of interest in the expression cassette of the invention preferably is adapted to optimize its codon usage to that of a highly expressed protein in the host cell in question, preferably to the codon usage of a highly expressed protein that is endogenous to the host cell in question. More preferably, the nucleotide sequence encoding the protein of interest in the expression cassette of the invention is adapted to optimize its codon usage to that of a highly expressed fungal protein selected from acid a-amylase, a-amylase, TAKA-amylase, glucoamylase, xylanase, cellobiohydrolase, pyruvate kinase, glyceraldehyde-phosphate dehydrogenase, alcohol dehydrogenase, aldehyde dehydrogenase and sucrase. The nucleotide sequence encoding the protein of interest can be adapted to the codon usage of a highly expressed fungal protein e.g. by using an on-line available DNA optimizing tool such as OPTIMISER (available on the internet at genomes.urv.es/OPTIMISER/; Puigbo P. et al., 2007; Nucl Acids Res 35; W126-W131) or commercial service providers of codon optimized synthetic genes (e.g. GenScript, USA).

In a further preferred embodiment, the expression cassette comprises a nucleotide sequence encoding the protein of interest that operably linked to a promoter, wherein the coding sequence is codon optimized with reference to the coding sequence that is native to the promoter, whereby preferably, the promoter is a promoter that is native to a gene for a highly expressed fungal protein, whereby, more preferably the highly expressed fungal protein preferably is a highly expressed fungal protein as indicated above, of which glucoamylase is the most preferred.

In yet a further preferred embodiment, the expression cassette further comprises a DNA fragment, which is homologous to a DNA sequence in a predetermined target locus in the genome of the host cell for targeting the integration of the cloning vector to this predetermined locus, whereby preferably the target locus is the locus of the highly expressed fungal protein native to the promoter in the expression cassette and to which the codon usage of the nucleotide sequence encoding the protein of interest is optimized whereby, preferably the highly expressed fungal protein preferably is a highly expressed fungal protein as indicated above, of which A.niger glucoamylase is most preferred.

In one embodiment, the expression cassette comprises an N-terminal secretion signal sequence that is operably linked to the coding sequence of the protein of interest for directing secretion of the protein of interest from the fungal host cell. A "signal sequence" is an amino acid sequence which when operably linked to the amino-terminus of a protein of interest permits the secretion of such protein from the host fungus. Such signal sequences may be the signal sequence normally associated with the protein of interest (i.e., a native signal sequence) or may be derived from other sources (i.e., a signal sequence foreign or heterologous to the protein of interest). Signal sequences are operably linked to a heterologous polypeptide either by utilizing a native signal sequence or by joining a DNA sequence encoding a foreign signal sequence to a DNA sequence encoding the protein of interest in the proper reading frame to permit translation of the signal sequence and protein of interest. Preferred signal sequences for use in the present invention include signals derived from highly expressed secreted fungal proteins such as acid a-amylase, a- amylase, TAKA-amylase, glucoamylase, xylanase, cellobiohydrolase and sucrase, of which A.niger glucoamylase is most preferred.

In a further preferred embodiment, the expression cassette encodes the protein of interest as part of a fusion protein. Preferably, the expression cassette encodes a fusion protein wherein the protein of interest is fused at its N-terminus with at least a part of highly expressed secreted fungal protein. More preferably, the expression cassette encodes a fusion protein wherein the protein of interest is fused at its N-terminus with at least an N-terminal part of the highly expressed secreted fungal protein, which preferably includes at least the signal sequence of the highly expressed secreted fungal protein. The fusion protein can further also comprise the pro-sequence of the highly expressed secreted fungal protein and/or or further parts of the mature highly expressed secreted fungal protein. Alternatively, the signal sequence may contain the pre- prosequences. If the protein of interest is a secreted protein, the N-terminal part of the highly expressed secreted fungal protein can be fused to the N-terminus of the mature secreted protein of interest. Alternatively, the N-terminal may be fused to the start of the mature protein (e.g. afterthe processing to avoid possible misprocessing in the fungal cell). If the protein of interest is not a secreted protein, the N-terminal part of the highly expressed secreted fungal protein can replace the N-terminal methionine of the protein of interest.

It is further preferred that the expression cassette encodes a fusion protein wherein the protein of interest is fused to its N-terminal fusion partner through of cleavable linker polypeptide such as the prosequence of glucoamylase, the prosequence of bovine chymosin, the prosequence of subtilisin, prosequences of retroviral proteases including HIV protease and DNA sequences encoding amino acid sequences recognized and cleaved by trypsin, factor Xa, collagenase, clostripin, subtilisin, chymosin, yeast KEX2 protease and Aspergillus KEXB. Particularly preferred cleavable linkers are the KEX2 protease recognition site (Lys-Arg), which can be cleaved by a native Aspergillus KEX2-like (KEXB) protease.

In a preferred embodiment, the expression cassette encodes a fusion protein comprising in a N- to C-terminal direction: an A. niger glucoamylase pre-pro sequence; optionally, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% of the mature A. niger glucoamylase amino acid sequence; optionally, a cleavable linker polypeptide and/or KEX2 (Lys-Arg) cleavage site; fused to the protein of interest. Examples thereof are described in the Examples of WO2021144342: the first 502 amino acids of A. niger glucoamylase including the pre-pro sequence and a synthetic peptide of 8 amino acids which includes the KEX2 (Lys-Arg) cleavage site fused to a protein of interest, or further truncations of the A. niger glucoamylase with only 54 or 100 amino acids including the pre-pro sequences and before the KEX2 (Lys-Arg) cleavage site fused to the protein of interest.

In embodiment where the protein of interest is ovalbumin, the ovalbumin that is encoded in the expression cassette of the invention comprises an amino acid sequence with at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100% identity to the amino acid sequence of an ovalbumin from a bird selected from the group consisting of chicken, pelican, quail, pigeon, ostrich, plover, turkey, duck, goose, gull, guinea fowl, jungle fowl, peafowl, partridge, pheasant, emu, rhea and kiwi, of which chicken, pigeon, pelican and quail are preferred.

In one embodiment, the ovalbumin that is encoded in the expression cassette of the invention comprises an amino acid sequence with at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100% identity to at least one of SEQ ID NO.’s: 1 - 6.

Alternative ovalbumin sequences from edible birds include: Genbank accession number AAC16664.1 and POI27989.1 , forturkey and partridge, respectively. The examples of duck, goose, guinea fowl, pheasant, emu and kiwi ovalbumin sequences can be found under the NCBI reference number NP_001298098.1 , XP_013056574.1 , XP_021241976.1 , XP_031445133.1 ,

XP_025956522.1 and XP_025932497.1 , respectively. In one embodiment, the ovalbumin may comprise amino acid sequences from more than one species.

In one embodiment, the amino acid sequence of the ovalbumin is modified so as to reduce or eliminate allergenicity. The allergenicity of an ovalbumin, e.g. chicken ovalbumin, can be reduced by replacing one or more amino acids in allergenic epitopes in the allergenic ovalbumin with different amino acid that are present in corresponding positions in the sequences of one or more ovalbumins from other bird species that are not allergenic. Methods used to make an assessment of allergenicity include but are not limited to initial bioinformatics techniques to later challenge testing.

Compositions

The invention provides a composition comprising a linear polymer antifoaming agent and a microbial host cell. The composition can be a fermentation broth, or it can be a storage composition. The composition can be a dried fermentation broth, comprising an amount of antifoaming agent as described above after reconstitution in water.

Also provided is a composition comprising a linear polymer antifoaming agent and an egg protein, preferably an egg white protein, most preferably ovalbumin. The ovalbumin is preferably as prepared using a process of the invention. Features and definitions have been described elsewhere. It was surprisingly found that the linear polymer antifoaming agent, particularly polypropylene glycols, stabilized the egg protein, particularly ovalbumin. This was not achieved with other antifoaming agents such as vegetable oils or polysorbates. Preferred compositions are liquid compositions comprising 0.05 to 10 g/L egg protein, preferably 0.1 to 8 g/L, more preferably 0.5 to 5 g/L, most preferably 1 to 3 g/L such as about 1 g/L. Preferably the solvent is aqueous, more preferably it is water such as food-grade water. The amount of linear polymer antifoaming agent is preferably as described above for use during step ii) of the process.

General definitions

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention.

In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one". As used herein, the term "and/or" indicates that one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases. As used herein, with "At least" a particular value means that particular value or more. For example, "at least 2" is understood to be the same as "2 or more" i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, ... ,etc., including fractions where appropriate. The word “about” or “approximately” when used in association with a numerical value (e.g. about 10) preferably means that the value may be the given value (of 10) more or less 5%, preferably 1 %, more preferably 0.1 % of the value.

The terms “homology”, “sequence identity” and the like are used interchangeably herein. Sequence identity is herein defined as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences. "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide. "Identity" and "similarity" can be readily calculated by known methods.

“Sequence identity” and “sequence similarity” can be determined by alignment of two peptide or two nucleotide sequences using global or local alignment algorithms, depending on the length of the two sequences. Sequences of similar lengths are preferably aligned using a global alignment algorithms (e.g. Needleman Wunsch) which aligns the sequences optimally over the entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g. Smith Waterman). Sequences may then be referred to as "substantially identical” or “essentially similar” when they (when optimally aligned by for example the programs GAP or BESTFIT using default parameters) share at least a certain minimal percentage of sequence identity (as defined below). GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length (full length), maximizing the number of matches and minimizing the number of gaps. A global alignment is suitably used to determine sequence identity when the two sequences have similar lengths. Generally, the GAP default parameters are used, with a gap creation penalty = 50 (polynucleotides) I 8 (proteins) and gap extension penalty = 3 (nucleotides) Z 2 (proteins). For nucleotides the default scoring matrix used is nwsgapdna and for proteins the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). Sequence alignments and scores for percentage sequence identity may be determined using computer programs, such as the GCG Wisconsin Package, Version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752 USA, or using open source software, such as the program “needle” (using the global Needleman Wunsch algorithm) or “water” (using the local Smith Waterman algorithm) in EmbossWIN version 2.10.0, using the same parameters as for GAP above, or using the default settings (both for ‘needle’ and for ‘water’ and both for protein and for DNA alignments, the default Gap opening penalty is 10.0 and the default gap extension penalty is 0.5; default scoring matrices are Blosum62 for proteins and DNAFull for DNA). When sequences have a substantially different overall lengths, local alignments, such as those using the Smith Waterman algorithm, are preferred.

Alternatively percentage similarity or identity may be determined by searching against public databases, using algorithms such as FASTA, BLAST, etc. Thus, the nucleic acid and protein sequences of the present invention can further be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the BLASTn and BLASTx programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403 — 10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed with the BLASTx program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17): 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTx and BLASTn) can be used. See the homepage of the National Center for Biotechnology Information at www.ncbi.nlm.nih.gov/.

A "nucleic acid construct" or "nucleic acid vector" is herein understood to mean a man-made nucleic acid molecule resulting from the use of recombinant DNA technology. The term "nucleic acid construct" therefore does not include naturally occurring nucleic acid molecules although a nucleic acid construct may comprise (parts of) naturally occurring nucleic acid molecules. The terms "expression vector" or “expression construct" refer to nucleotide sequences that are capable of effecting expression of a gene in host cells or host organisms compatible with such sequences. These expression vectors typically include at least suitable transcription regulatory sequences and optionally, 3' transcription termination signals. Additional factors necessary or helpful in effecting expression may also be present, such as expression enhancer elements. The expression vector will be introduced into a suitable host cell and be able to effect expression of the coding sequence in an in vitro cell culture of the host cell. The expression vector will be suitable for replication in the host cell or organism of the invention.

As used herein, the term "promoter" or "transcription regulatory sequence" refers to a nucleic acid fragment that functions to control the transcription of one or more coding sequences, and is located upstream with respect to the direction of transcription of the transcription initiation site of the coding sequence, and is structurally identified by the presence of a binding site for DNA- dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter. A "constitutive" promoter is a promoter that is active in most tissues under most physiological and developmental conditions. An "inducible" promoter is a promoter that is physiologically or developmentally regulated, e.g. by the application of a chemical inducer. An inducible promoter may also be present but not induced.

The term "selectable marker" is a term familiar to one of ordinary skill in the art and is used herein to describe any genetic entity which, when expressed, can be used to select for a cell or cells containing the selectable marker. The term "reporter" may be used interchangeably with marker, although it is mainly used to refer to visible markers, such as green fluorescent protein (GFP). Selectable markers may be dominant or recessive or bidirectional.

As used herein, the term "operably linked" refers to a linkage of polynucleotide elements in a functional relationship. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For instance, a transcription regulatory sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein encoding regions, contiguous and in reading frame.

The terms "protein" or "polypeptide" are used interchangeably and refer to molecules consisting of a chain of amino acids, without reference to a specific mode of action, size, structure, or origin.

The term "gene" means a DNA fragment comprising a region (transcribed region), which is transcribed into an RNA molecule (e.g. an mRNA) in a cell, operably linked to suitable regulatory regions (e.g. a promoter). A gene will usually comprise several operably linked fragments, such as a promoter, a 5' leader sequence, a coding region, exons, introns and a 3'-nontranslated sequence (3'-end) e.g. comprising a polyadenylation- and/or transcription termination site.

"Expression of a gene" refers to the process wherein a DNA region which is operably linked to appropriate regulatory regions, particularly a promoter, is transcribed into an RNA, which is biologically active, i.e. which is capable of being translated into a biologically active protein or peptide.

The term "homologous" when used to indicate the relation between a given (recombinant) nucleic acid or polypeptide molecule and a given host organism or host cell, is understood to mean that in nature the nucleic acid or polypeptide molecule is produced by a host cell or organisms of the same species, preferably of the same variety or strain. If homologous to a host cell, a nucleic acid sequence encoding a polypeptide will typically (but not necessarily) be operably linked to another (heterologous) promoter sequence and, if applicable, another (heterologous) secretory signal sequence and/or terminator sequence than in its natural environment. It is understood that the regulatory sequences, signal sequences, terminator sequences, etc. may also be homologous to the host cell. In this context, the use of only "homologous" sequence elements allows the construction of "self-cloned" genetically modified organisms (GMO's) (self-cloning is defined herein as in European Directive 98/81/EC Annex II). When used to indicate the relatedness of two nucleic acid sequences the term "homologous" means that one single-stranded nucleic acid sequence may hybridize to a complementary single-stranded nucleic acid sequence. The degree of hybridization may depend on a number of factors including the amount of identity between the sequences and the hybridization conditions such as temperature and salt concentration as discussed earlier herein.

The terms "heterologous" and "exogenous" when used with respect to a nucleic acid (DNA or RNA) or protein refers to a nucleic acid or protein that does not occur naturally as part of the organism, cell, genome or DNA or RNA sequence in which it is present, or that is found in a cell or location or locations in the genome or DNA or RNA sequence that differ from that in which it is found in nature. Heterologous and exogenous nucleic acids or proteins are not endogenous to the cell into which it is introduced, but have been obtained from another cell or synthetically or recombinantly produced. Generally, though not necessarily, such nucleic acids encode proteins, i.e. exogenous proteins, that are not normally produced by the cell in which the DNA is transcribed or expressed. Similarly exogenous RNA encodes for proteins not normally expressed in the cell in which the exogenous RNA is present. Heterologous/exogenous nucleic acids and proteins may also be referred to as foreign nucleic acids or proteins. Any nucleic acid or protein that one of skill in the art would recognize as foreign to the cell in which it is expressed is herein encompassed by the term heterologous or exogenous nucleic acid or protein. The terms heterologous and exogenous also apply to non-natural combinations of nucleic acid or amino acid sequences, i.e. combinations where at least two of the combined sequences are foreign with respect to each other. The terms heterologous and exogenous specifically also apply to non-naturally occurring modified versions of otherwise endogenous nucleic acids or proteins.

The term “fermentation” or “fermentation process” is herein broadly defined in accordance with its common definition as used in industry as any (large-scale) microbial process occurring in the presence or absence of oxygen, comprising the cultivation of at least one microorganism whereby preferably the microorganism produces a useful product at the expense of consuming one or more organic substrates. The term “fermentation” is herein thus has a much broader definition than the more strict scientific definition wherein it is defined as being limited a microbial process wherein the microorganism extracts energy from carbohydrates in the absence of oxygen. Likewise, the term “fermentation product” is herein broadly defined as any useful product produced in a (large- scale) microbial process occurring in the presence or absence of oxygen. All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

Description of the figures

Fig. 1 - Fraction of remaining dissolved ovalbumin from an initial solution in water (1 g/L) over time during mixing and air sparging (0.63 wm) in a 1 L fermentation vessel. Different concentrations of linear polymeric antifoaming agent (here PPG) are used. For 0 g/L, no mixing and sparging was performed.

Fig. 2A - Fraction of remaining dissolved ovalbumin from an initial solution in water (1 g/L) over time during mixing (400 rpm) and air sparging (0.63 wm) in a 1 L fermentation vessel at different concentrations and types of linear polymeric antifoaming agent.

Fig. 2B - as for Fig. 2A, using different concentrations of PPG during mixing (600 rpm, 0.796 kW/m³) and air sparging (0.63 wm).

Fig. 2C - as for Fig. 2B, using different concentrations of PPG during mixing (800 rpm) and air sparging (0.63 wm).

Fig. 3 - SDS-Page gel of the supernatant of shake flask cultures using various antifoams. The shake flask cultures were performed in 100 mL non-baffled shake flasks with a recombinant Aspergillus niger strain, at a temperature of 32 °C and shaking rate of 220 rpm. The ovalbumin bands are highlighted by boxes. Only linear polymeric antifoaming agents led to visible bands.

Fig. 4A - Glucose and ovalbumin concentration profiles of the culture of a recombinant Aspergillus niger strain expressing ovalbumin. The fermentation was performed in 5 L stirred vessels (2.5 L working volume) with a maximum stirring rate of 350 rpm (0.31 kW/m³) and an air sparging rate of 1 wm. Fermentation A uses BT03, a hydroxylated vegetable oil, as antifoam (0.2 g/L).

Fig. 4B - As for Fig. 4A, except that fermentation B uses a linear polymeric agent (PPG 2000) as antifoam (0.2 g/L).

Examples

Example 1

1. 1 Introduction

Egg white is used in many food applications for its nutritional value and functional properties, such as binding, gelling, and foaming. Egg white is a complex mixture of water and various proteins, of which ovalbumin, ovotransferrin, ovomucoid, ovomucin, and lysozyme are the most abundant. Although egg white is a mixture of these proteins, pure ovalbumin has very similar functional properties as the mixture and could therefore be a proper substitute for egg white.

The production of egg white provides challenges in terms of sustainability, water use, land use, nitrogen emissions, and animal/human health (Salmonella, avian flu, antibiotics resistance). Precision fermentation can offer an alternative production route of ovalbumin, which can then be used as a substitute for animal egg white. Engineered yeasts (S. cerevisiae, P. pastoris) or filamentous fungi (Aspergillus, Trichoderma) can be used as cell factories for heterologous protein expression, where the latter have the potential to reach significantly higher protein concentrations during fermentation and secrete the produced proteins into the fermentation broth.

Synthesis of heterologous proteins occurs under aerobic conditions, meaning that oxygen has to be supplied to the fermentation mixture. In combination with the (functional) properties of proteins, this can lead to significant challenges in operating the fermentation process. One challenge in almost any aerobic fermentation process is to keep foam formation under control while supplying enough oxygen to the microorganism. As one of the key functional properties of ovalbumin is its capability to unfold at an air/water interface and thereby stabilize foams, the challenge of keeping foaming under control is enlarged for ovalbumin. Furthermore ovalbumin has the capability to form a network, which gives the molecule its excellent gelling/binding properties. Depending on physical process conditions, such as pH, temperature, and ionic strength, ovalbumin molecules can also interact with each other when in solution, forming aggregates and precipitates. In a fermentation process where the solid biomass has to be removed to obtain a pure product, such aggregation and precipitation processes result in loss of the complete product, as the ovalbumin that came out of solution will end up with the solid biomass waste.

Demonstrated here are means and methods to allow for heterologous production of ovalbumin through precision fermentation, overcoming the challenges caused by the conflict between functional properties of the protein product and the required conditions for an efficient fermentation process which would result in loss of product during fermentation. It was found that linear polymeric antifoaming agents such as polypropylene glycol (PPG) are able to fulfill both roles, acting both as an antifoam and a as an ovalbumin stabilizer. In fermentation processes using other antifoams such as the commercially available BT03 or polysorbate, produced ovalbumin was lost due to physical degradation and no ovalbumin could be recovered from the fermentation broth. Using PPG as antifoam resulting in ovalbumin remaining in solution and allowing for recovery of ovalbumin from the fermentation broth.

1.2 General Techniques

Unless indicated otherwise, methods used are standard techniques. Examples of suitable general methodology textbooks include Sambrook et al., Molecular Cloning, a Laboratory Manual (1989) and Ausubel et al., Current Protocols in Molecular Biology (1995), John Wiley & Sons, Inc. A description of ovalbumin production is provided in WO2021144342.

1.3 Sparging and mixing leads to ovalbumin loss

The inherent instability of ovalbumin under aerobic fermentation conditions (i.e. gas sparging and mixing) became apparent when an ovalbumin mixture (1 g/L in water) was stirred at an increasing stirring rate resemblant of a fermentation process and aerated at 1 wm. All conditions show a clear ovalbumin decrease (Fig. 1), with the exception of the condition with no antifoaming agent in which no air was sparged. This indicates that ovalbumin loss is induced by air bubbles, but that the severity of this effect can be mitigated by addition of a linear polymeric antifoaming agent. For instance, at higher concentrations of polypropylene glycol (PPG) the ovalbumin degradation is slowed down. 1.4 Different linear polymeric agents suitably protect at various doses

The interaction between protein and antifoaming agent can vary depending on the chain length of the linear polymeric molecule. An experiment as described in section 1 .3 was performed (stirring had a mechanical energy input of 0.244 kW/m³. It was found that a shorter molecule (1000 Da) was found to have better ovalbumin stabilizing capabilities (Fig. 2), as it was able to almost completely halt ovalbumin degradation as opposed to a larger molecule (2000 Da). Nonetheless both agents showed stabilization. When mechanical energy input was increased to 0.796 kW/m³, 1000 Da polymers still protected ovalbumin at various concentrations.

1.5 Linear polymeric agents are effective during fermentation

In shake flask cultures using a recombinant Aspergillus niger strain, ovalbumin could be expressed. However, despite the less severe mixing and aeration conditions in shake flasks as compared to stirred vessels, the produced ovalbumin could not be obtained without using a linear polymeric antifoaming agent. The conditions without antifoam (control), or using antifoaming agents that are not linear polymers (vegetable oil BT03 and polysorbate Tween80) showed no ovalbumin presence at 120 and 140 h, where the condition with linear polymeric agent (0.2 g/L) showed a clear ovalbumin band on the SDS-page gel for both timepoints (Error! Reference source not found.). Where for conventional protein products fermentations in shake flasks can be performed without antifoam due to the absence of air sparging, in stirred vessels antifoams are a necessity. As described above, the choice of antifoam has much impact on the successful formation of ovalbumin. BT03 is a polyol-based antifoam derived from vegetable oils. When it is used, no ovalbumin is produced while glucose is consumed (Fig. 4A). When the antifoam is switched to PPG 2000, a linear polymeric agent, ovalbumin remains stable in the fermentation broth and the ovalbumin concentration increases while glucose is being consumed (Fig. 4B). This absence and presence of ovalbumin in both fermentation processes is visible from SDS-page gels of the samples.

Claims

1 . A process for producing a protein of interest, the process comprising the steps of: i) providing a host cell capable of expressing the protein of interest; ii) culturing the host cell in a medium in a fermenter under conditions conducive to expression of the protein of interest, wherein the culturing is in the presence of a linear polymeric antifoaming agent; and, iii) optionally, recovering the protein of interest.

2. The process according to claim 1 , wherein the linear polymeric antifoaming agent is a polyalkylene glycol.

3. The process according to claim 2, wherein the polyalkylene glycol is polypropylene glycol.

4. The process according to any one of claims 1-3, wherein the protein of interest is a foodprotein, preferably an animal-derived food-protein.

5. The process according to claim 4, wherein the food-protein is a milk protein, a hemeprotein, or an egg protein, preferably an egg white protein, more preferably ovalbumin.

6. The process according to any one of claims 1-5, wherein the host cell comprises an expression cassette, wherein the expression cassette comprises a first nucleotide sequence coding for the protein of interest, wherein the first nucleotide sequence is operably linked to at least one regulatory sequence that is capable of effecting expression of the encoded protein of interest by the host cell.

7. The process according to any one of claims 1-6, wherein the host cell is a microbial host cell.

8. The process according to claim 7, wherein the microbial host cell is a yeast or a filamentous fungus, preferably Pichia pastoris or a filamentous fungus such as those belonging to the species Altemaria alternata, Apophysomyces variabilis, Aspergillus spp., Aspergillus fumigatus, Aspergillus flavus, Aspergillus oryzae, Aspergillus niger, Aspergillus awamori, Aspergillus nidulans, Aspergillus terreus, Cladosphialophora spp., Fonsecaea pedrosoi, Fusarium spp., Fusarium oxysporum, Fusarium solani, Lichtheimia spp., Lichtheimia corymbifera, Lichtheimia ramosa, Myceliophthora spp., Myceliophthora thermophila, Rhizopus spp., Rhizopus microsporus, Rhizomucor spp., Rhizomucor pusillus, Rhizomucor miehei, Trichoderma spp., Trichoderma reesei, Trichophyton spp., Trichophyton interdigitale, and Trichophyton rubrum, more preferably the microbial host cell belongs to the species Aspergillus, most preferably the microbial host cell belongs to the species Aspergillus niger.

9. The process according to any one of claims 1-8, wherein the fermenter is a stirred tank reactor.

10. The process according to claim 9, wherein input of mechanical power into the medium in the stirred tank reactor is no more than 2.5, 2.0, 1 .8, 1 .6, 1 .4, 1 .0, 0.5, 0.2 or 0.1 kW/m³.

11 . The process according to any one of claims 1-10, wherein the linear polymeric antifoaming agent has a molecular weight of 0.2 to 20 kDa, preferably 0.4 to 10 kDa, more preferably 0.5 to 5 kDa, most preferably 0.6 to 3 kDa.

12. The process according to any one of claims 1-11 , wherein the linear polymeric antifoaming agent is present at a concentration of 0.01 to 10 g/L, preferably 0.05 to 5 g/L, more preferably 0.08 to 3 g/L, most preferably 0.1 to 2.5 g/L.

13. Use of a linear polymer as an antifoaming agent in the microbial production of an animal- derived food-protein.

14. Composition comprising a linear polymer antifoaming agent and a microbial host cell.

15. Composition comprising a linear polymer antifoaming agent and an egg protein, preferably an egg white protein, most preferably ovalbumin.