Fungal expression systems with reduced volatile compounds

Abstract

The present invention relates to Aspergillus mutants with reduced or eliminated expression and / or activity of an endogenous psi-producing oxygenase C (PpoC) comprising in its genome one or more polynucleotide encoding a polypeptide of interest heterologous to the mutant cell, methods of producing a polypeptide of interest using said host cells, as well as the use of said cells in production methods to provide fermentation products comprising reduced volatile agents.

Description

[0001] FUNGAL EXPRESSION SYSTEMS WITH REDUCED VOLATILE COMPOUNDS

[0002] Reference to a Sequence Listing

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

[0004] Background of the Invention

[0005] Field of the Invention

[0006] The present invention relates to Aspergillus mutants with reduced or eliminated expression and / or activity of an endogenous psi-producing oxygenase C (PpoC) comprising in its genome one or more polynucleotide encoding a polypeptide of interest heterologous to the mutant, methods of producing a polypeptide of interest using said host cells, as well as the use of said cells in production methods to provide fermentation products comprising reduced volatile agents.

[0007] Description of the Related Art

[0008] Natural features of fungal cell biology have led to wide adoption of fungal species for use as host cell factories in industrial biotechnology production settings. For example, Aspergillus is ubiquitously used in the production of organic acids and high-value secondary metabolites. It’s high capacity for protein sectetion also makes it a profitable host for production or recombinant enzymatic and non-enzymatic proteins. Centuries of using Aspergillus in traditional food fermentations and addition of GRAS status (generally recognized as safe) for some key production host species have also made Aspergillus and attractive production organism for applications within the food and beverage industry. Considering these factors, Aspergillus has rapidly become an important production host to meet the increasing demand for highly concentrated, precision-fermented proteins for food applications.

[0009] The fermentation of fungal cells produces volatile compounds that contribute to undesirable odor and flavor profiles, especially in (but not limited to) food and beverage applications. The sensory profile of the production host becomes especially relevant when high concentrations of a precision protein is needed, for example when applied as a major ingredient in a food or beverage product. Carryover of flavor and odor through protein recovery processes can be substantial depending on the recovery scheme and molecular properties of the flavor compounds. Processes used for removal of odor and flavor can add substantial cost to production, often becoming prohibitively expensive for bulk protein ingredients where high concentrations and low-price points are requirements to be competitive in the market. Despite attempts of removing the volatile compounds, said compounds and resulting odor often remains associated with the final protein product through typical downstream processing strategies. Thus, there is a need to provide means and methods for lowering the amount of undesired volatile compounds in above-described systems and processes.

[0010] Summary of the Invention

[0011] The present invention provides fungal expression systems with reduced volatile compounds. The most prominent volatile odor compound identified in Aspergillus hosts is 1- octen-3-ol (mushroom alcohol). The system of the invention depicts a novel production host strain with improved sensory properties through the disruption of the central metabolic pathway forming 1-octen-3-ol and other volatiles. Surprisingly, said pathway has successfully been disrupted by reducing expression of a single gene, namely a gene encoding the psi-producing oxygenase C (PpoC), without impacting the host cell’s performance, e.g. cell growth. The major benefit over current methods for odor improvement is in cost reduction during production and down-stream purification of recombinant protein.

[0012] In a 1staspect, the invention relates to a mutant Aspergillus cell comprising in its genome one or more polynucleotide encoding a polypeptide of interest (POI), wherein the polynucleotide encoding the polypeptide of interest is heterologous to the mutant cell, and wherein expression and / or activity of one or more endogenous psi-producing oxygenase C (PpoC) is reduced or eliminated compared to a non-mutated otherwise isogenic cell or parent cell when cultivated under identical conditions.

[0013] In a 2ndaspect the invention relates to a method for producing one or more polypeptide of interest (POI) the method comprising providing a mutant cell according to the 1staspect, and cultivating said cell under conditions conducive for expression of the one or more POI.

[0014] In a 3rdaspect the invention relates to a whole broth formulation or cell culture composition comprising the mutant of the 1staspect.

[0015] In a 4thaspect the invention relates to a composition comprising the mutant of the 1staspect, and / or the whole broth formulation or cell culture composition according to the 3rdaspect.

[0016] In a 5thaspect the invention relates to use of the mutant of the 1staspect and / or the composition according to the 4thaspect in a method for producing a POI, e.g., a food or feed product.

[0017] In a 6thaspect the invention relates to a method of producing the mutant of the 1staspect.

[0018] In a 7thaspect the invention relates to the mutant obtained or obtainable by the method of the 6thaspect.

[0019] Brief Description of the Drawings

[0020] Figure 1 shows a schematic overview of the PpoC knock-out strategy in Aspergillus niger. Figure 2 shows a comparison of cell growth for PpoC wildtype cells and PpoC deletion mutants.

[0021] Figure 3 shows a comparison of a GCMS chromatogram for PpoC wildtype cells (A) and PpoC deletion mutants (B), and the identification of 1-octen-3-ol (C).

[0022] Figure 4 shows a schematic overview of the PpoC knock-out strategy in Aspergillus oryzae.

[0023] Definitions

[0024] In accordance with this detailed description, the following definitions apply. Note that the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0025] Unless defined otherwise or clearly indicated by context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0026] AlphaFold structure calculation: AlphaFold version 2 (AlphaFold2, AF2) is a computational method for calculating the three-dimensional structure of a polypeptide from its amino acid sequence (Jumper et al., 2021 , Nature 596: 583-589). Three-dimensional structures of millions of polypeptides deposited in the UniProt database have been calculated and deposited in the AlphaFold Protein Structure Database, using the AlphaFold Monomer v2.0 algorithm (Varadi et al., 2021 , Nucleic Acids Res. 50(D1):D439-D444). In the AlphaFold Protein Structure Database, the three-dimensional structure of a polypeptide can be obtained by searching for the UniProt accession number of the polypeptide.

[0027] In addition to the many three-dimensional structures that are already publicly available, code is available for reproducing and calculating structures of new polypeptides at source code repositories such as Github.com under deepmind / alphafold / , using notebooks / AlphaFold.ipynb, which uses AlphaFold v2.3.1 or newer. Additionally, it can be found in Github.com under sokrypton / ColabFold using v1.5.2 or newer, using AlphaFold2.ipynb. For technical details, please see Jumper et al. (vide supra).

[0028] AlphaFold 2 produces a per-residue estimate of its confidence on a scale from 0 to 100. This confidence measure is called pLDDT and corresponds to the model’s predicted score on the IDDT-Ca metric. It is stored in the B-factor fields of the mmCIF and PDB files available for download (although unlike a B-factor, higher pLDDT is better). Regions with pLDDT score of more than 90 are expected to be modelled to high accuracy. These should be suitable for any application that benefits from high accuracy (e.g., characterization of binding sites). Regions with a pLDDT score between 70 and 90 are expected to be modelled well, corresponding to a generally good backbone prediction. cDNA: The term "cDNA" means a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial, primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before appearing as mature spliced mRNA.

[0029] Coding sequence: The term “coding sequence” means a polynucleotide, which directly specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon, such as ATG, GTG, or TTG, and ends with a stop codon, such as TAA, TAG, or TGA. The coding sequence may be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0030] Control sequences: The term “control sequences” means nucleic acid sequences involved in regulation of expression of a polynucleotide in a specific organism or in vitro. Each control sequence may be native ( / .e., from the same gene) or heterologous ( / .e., from a different gene) to the polynucleotide encoding the polypeptide, and native or heterologous to each other. Such control sequences include, but are not limited to leader, polyadenylation, prepropeptide, propeptide, signal peptide, promoter, terminator, enhancer, and transcription or translation initiator and terminator sequences. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding a polypeptide.

[0031] Expression: The term “expression” means any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0032] Expression vector: An "expression vector" refers to a linear or circular DNA construct comprising a DNA sequence encoding a polypeptide, which coding sequence is operably linked to a suitable control sequence capable of effecting expression of the DNA in a suitable host. Such control sequences may include a promoter to effect transcription, an optional operator sequence to control transcription, a sequence encoding suitable ribosome binding sites on the mRNA, enhancers and sequences which control termination of transcription and translation.

[0033] Food or feed product: The term "food or feed product" refers to any consumable substance intended to provide nutritional support and sustainment for humans (food) or animals (feed). This product is characterized by the inclusion of one or more polypeptides, e.g. one or more polypeptide of interest (POI). The food or feed product may take various forms, including but not limited to solid, semi-solid, liquid, or powdered forms. It can be composed of raw, processed, or formulated ingredients designed to meet specific dietary requirements or to deliver health benefits. These products may be enriched or fortified with polypeptides to enhance their nutritional profile, provide functional properties such as improved texture or stability, or deliver specific bioactive effects, such as enzyme activity, immunomodulation, or metabolic regulation.

[0034] Additionally, the term "food or feed product" includes any intermediate thereof. An "intermediate thereof" refers to a composition that comprises one or more polypeptides and is intended to undergo further processing, formulation, or refinement before becoming a final food or feed product. Such intermediates are not yet consumable in their current form but will be used to produce the final consumable product.

[0035] The food or feed product, and any intermediate thereof, may be targeted for consumption by specific groups, such as infants, children, adults, elderly individuals, pets, livestock, or other animals, and can be tailored to address particular dietary needs or health conditions.

[0036] Fusion polypeptide: The term “fusion polypeptide” is a polypeptide in which one polypeptide is fused at the N-terminus and / or the C-terminus of a polypeptide of the present invention. A fusion polypeptide is produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the present invention, or by fusing two or more polynucleotides of the present invention together. Techniques for producing fusion polypeptides are known in the art, and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fusion polypeptide is under control of the same promoter(s) and terminator. Fusion polypeptides may also be constructed using intein technology in which fusion polypeptides are created post-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawson et al., 1994, Science 266: 776-779). A fusion polypeptide can further comprise a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, the sites disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000, J. Biotechnol. 7Q: 245-251 ; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; and Contreras et al., 1991 , Biotechnology 9: 378-381 ; Eaton et al., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995, Biotechnology 13: 982-987; Carter eta / ., 1989, Proteins: Structure, Function, and Genetics 6: 240-248; and Stevens, 2003, Drug Discovery World 4: 35-48.

[0037] Heterologous: The term "heterologous" means, with respect to a host cell, that a polypeptide or nucleic acid does not naturally occur in the host cell. The term "heterologous" means, with respect to a polypeptide or nucleic acid, that a control sequence, e.g., promoter, of a polypeptide or nucleic acid is not naturally associated with the polypeptide or nucleic acid, i.e., the control sequence is from a gene other than the gene encoding the mature polypeptide.

[0038] Host Strain or Host Cell: A "host strain" or "host cell" is an organism into which an expression vector, phage, virus, or other DNA construct, including a polynucleotide encoding a polypeptide of interest (e.g., an amylase) has been introduced. Exemplary host strains are microorganism cells (e.g., bacteria, filamentous fungi, and yeast) capable of expressing the polypeptide of interest and / or fermenting saccharides. The term "host cell" includes protoplasts created from cells.

[0039] Introduced: The term "introduced" in the context of inserting a nucleic acid sequence into a cell, means "transfection", "transformation" or "transduction," as known in the art.

[0040] Isolated: The term “isolated” means a polypeptide, nucleic acid, cell, or other specified material or component that has been separated from at least one other material or component, including but not limited to, other proteins, nucleic acids, cells, etc. An isolated polypeptide, nucleic acid, cell or other material is thus in a form that does not occur in nature. An isolated polypeptide includes, but is not limited to, a culture broth containing the secreted polypeptide expressed in a host cell.

[0041] Mature polypeptide: The term “mature polypeptide” means a polypeptide in its mature form following N-terminal and / or C-terminal processing (e.g., removal of signal peptide). In one aspect, the mature polypeptide is SEQ ID NO: 2.

[0042] Mature polypeptide coding sequence: The term “mature polypeptide coding sequence” means a polynucleotide that encodes a mature polypeptide having PpoC activity. In one aspect, the mature polypeptide coding sequence is shown in SEQ ID NO: 1.

[0043] Native: The term "native" means a nucleic acid or polypeptide naturally occurring in a host cell.

[0044] Nucleic acid: The term "nucleic acid" encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a polypeptide. Nucleic acids may be single stranded or double stranded, and may be chemical modifications. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and the present compositions and methods encompass nucleotide sequences that encode a particular amino acid sequence. Unless otherwise indicated, nucleic acid sequences are presented in 5'-to-3' orientation.

[0045] Nucleic acid construct: The term "nucleic acid construct" means a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, and which comprises one or more control sequences operably linked to the nucleic acid sequence.

[0046] Operably linked: The term "operably linked" means that specified components are in a relationship (including but not limited to juxtaposition) permitting them to function in an intended manner. For example, a regulatory sequence is operably linked to a coding sequence such that expression of the coding sequence is under control of the regulatory sequence.

[0047] PpoC: The term “PpoC” refers to a psi factor producing oxygenase C, or a psi-producing oxyngase C, which oxygenases are fusion proteins consisting of a peroxidase-like functionality in the N-terminus and a P450-fold in the C-terminal part of the polypeptide chain. Typically, PpoC polypeptides comprise a catalytic active site of the cytochrome P450 domain. Such catalytic active site is exemplified in SEQ ID NO: 8 showing the active site of the A. niger PpoC.

[0048] Typically, native PpoC polypeptides also comprise a proline knot motif, for example a proline knot motif as shown in SEQ ID NO:6 of the A. niger PpoC.

[0049] PpoC plays a critical role in the biosynthesis of volatile compounds. For example, the PpoC enzyme is involved in the metabolic pathway responsible for the formation of 1-octen-3-ol, a volatile compound with mushroom-like odor. The PpoC is part of a broader family of oxygenases that catalyze the oxidation of fatty acids, leading to the production of various volatile organic compounds.

[0050] Purified: The term “purified” means a nucleic acid, polypeptide or cell that is substantially free from other components as determined by analytical techniques well known in the art (e.g., a purified polypeptide or nucleic acid may form a discrete band in an electrophoretic gel, chromatographic eluate, and / or a media subjected to density gradient centrifugation). A purified nucleic acid or polypeptide is at least about 50% pure, usually at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, about 99.6%, about 99.7%, about 99.8% or more pure (e.g., percent by weight or on a molar basis). In a related sense, a composition is enriched for a molecule when there is a substantial increase in the concentration of the molecule after application of a purification or enrichment technique. The term "enriched" refers to a compound, polypeptide, cell, nucleic acid, amino acid, or other specified material or component that is present in a composition at a relative or absolute concentration that is higher than a starting composition.

[0051] In one aspect, the term "purified" as used herein refers to the polypeptide or cell being essentially free from components (especially insoluble components) from the production organism. In other aspects, the term "purified" refers to the polypeptide being essentially free of insoluble components (especially insoluble components) from the native organism from which it is obtained. In one aspect, the polypeptide is separated from some of the soluble components of the organism and culture medium from which it is recovered. The polypeptide may be purified ( / .e., separated) by one or more of the unit operations filtration, precipitation, or chromatography.

[0052] Accordingly, the polypeptide may be purified such that only minor amounts of other proteins, in particular, other polypeptides, are present. The term "purified" as used herein may refer to removal of other components, particularly other proteins and most particularly other enzymes present in the cell of origin of the polypeptide. The polypeptide may be "substantially pure", i.e., free from other components from the organism in which it is produced, e.g., a host organism for recombinantly produced polypeptide. In one aspect, the polypeptide is at least 40% pure by weight of the total polypeptide material present in the preparation. In one aspect, the polypeptide is at least 50%, 60%, 70%, 80% or 90% pure by weight of the total polypeptide material present in the preparation. As used herein, a "substantially pure polypeptide" may denote a polypeptide preparation that contains at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, even more preferably at most 2%, most preferably at most 1%, and even most preferably at most 0.5% by weight of other polypeptide material with which the polypeptide is natively or recombinantly associated.

[0053] It is, therefore, preferred that the substantially pure polypeptide is at least 92% pure, preferably at least 94% pure, more preferably at least 95% pure, more preferably at least 96% pure, more preferably at least 97% pure, more preferably at least 98% pure, even more preferably at least 99% pure, most preferably at least 99.5% pure by weight of the total polypeptide material present in the preparation. The polypeptide of the present invention is preferably in a substantially pure form ( / .e., the preparation is essentially free of other polypeptide material with which it is natively or recombinantly associated). This can be accomplished, for example by preparing the polypeptide by well-known recombinant methods or by classical purification methods.

[0054] Recombinant: The term "recombinant" is used in its conventional meaning to refer to the manipulation, e.g., cutting and rejoining, of nucleic acid sequences to form constellations different from those found in nature. The term recombinant refers to a cell, nucleic acid, polypeptide or vector that has been modified from its native state. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell, or express native genes at different levels or under different conditions than found in nature. The term “recombinant” is synonymous with “genetically modified” and “transgenic”.

[0055] Recover: The terms "recover" or “recovery” means the removal of a polypeptide from at least one fermentation broth component selected from the list of a cell, a nucleic acid, or other specified material, e.g., recovery of the polypeptide from the whole fermentation broth, or from the cell-free fermentation broth, by polypeptide crystal harvest, by filtration, e.g. depth filtration (by use of filter aids or packed filter medias, cloth filtration in chamber filters, rotary-drum filtration, drum filtration, rotary vacuum-drum filters, candle filters, horizontal leaf filters or similar, using sheed or pad filtration in framed or modular setups) or membrane filtration (using sheet filtration, module filtration, candle filtration, microfiltration, ultrafiltration in either cross flow, dynamic cross flow or dead end operation), or by centrifugation (using decanter centrifuges, disc stack centrifuges, hyrdo cyclones or similar), or by precipitating the polypeptide and using relevant solid-liquid separation methods to harvest the polypeptide from the broth media by use of classification separation by particle sizes. Recovery encompasses isolation and / or purification of the polypeptide.

[0056] Sequence difference: The term "sequence difference" means the percent of amino acid differences between a polypeptide and the polypeptide of any given SEQ ID NO: 2, and is calculated as follows:

[0057] (Different Residues x 100) / (Length of SEQ ID NO: of given polypeptide) wherein the different residues comprise any substitution, deletion, or insertion (e.g., an extension at the N-terminus and / or C-terminus) in the sequence.

[0058] Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity”.

[0059] 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:

[0060] (Identical Residues x 100) / (Length of Alignment - Total Number of Gaps in Alignment)

[0061] For purposes of the present invention, the sequence identity between two polynucleotide sequences is determined as the output of “longest identity” using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), 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 EDNAFULL (EMBOSS version of NCBI NUC4.4) 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:

[0062] (Identical Deoxyribonucleotides x 100) / (Length of Alignment - Total Number of Gaps in Alignment)

[0063] Signal Peptide: A "signal peptide" is a sequence of amino acids attached to the N- terminal portion of a protein, which facilitates the secretion of the protein outside the cell. The mature form of an extracellular protein lacks the signal peptide, which is cleaved off during the secretion process.

[0064] Structural Similarity: For purposes of the present invention, the relatedness between the three-dimensional structure of two polypeptides is described by the parameter “structural similarity”.

[0065] A three-dimensional structure of any polypeptide may be obtained experimentally via, e.g., X-ray crystallography or calculated using in silico methods such as AlphaFold2 (vide supra). The structural similarity between three-dimensional structures may then be determined by the TM- score, which is calculated using the following general formula (Zhang & Skolnick, 2004, Proteins 57:702-710):

[0066] TM-score where LN is the length of the native structure, LT is the length of the aligned residues to the template structure, dj is the distance between pair / of aligned residues and do is a scale to normalize the match difference. ‘Max’ denotes the maximum value after optimal spatial superposition.

[0067] For the purposes of the present invention, LN is the length of the reference polypeptide: TM score

[0068] A structural alignment of the three-dimensional structures of two polypeptides is necessary before the TM-score can be calculated. This is achieved via algorithms that optimize the structural overlap, and several methods are available, such as CEalign (Shindyalov and Bourne, 1998, Protein Eng., 11:739-747), DALI (Holm and Sander, 1995, Trends Biochem. Sci., 20:478-480), or TM-align (Zhang and Skolnick, 2005, Nucleic Acids Res. 33(7):2302-2309).

[0069] For the purposes of the present invention, TM-align is applied. For convenience, TM- score is integrated in the TM-align software, which is available from the author’s website (zhanggroup.org / TM-score / ). The version of TM-align is preferably updated 2019-08-22 or later, and the TM-score between a reference and a query protein is determined by running this command:

[0070] TMalign <query.pdb> <reference.pdb> -L <length of reference> where <query.pdb> is the name of the PDB file containing coordinates of the query polypeptide, <reference.pdb> is the name of the PDB file containing coordinates of the reference polypeptide. The TM-score is calculated and reported in the output, along with several other parameters from the alignment.

[0071] The maximal TM-score is 1 , e.g., 1.0, corresponding to identical three-dimensional structures.

[0072] Volatile agent: The term "volatile agent" refers to any compound that can readily vaporize at room temperature and atmospheric pressure, thereby contributing to the aroma or odor profile of a substance. Volatile agents are typically organic molecules with low molecular weight and high vapor pressure, which facilitate their transition from a liquid or solid state into a gaseous state.

[0073] In the context of producing recombinant proteins, particularly for use in food or feed products and intermediates thereof, volatile agents are considered undesirable due to their potential to impart off-flavors, odors, or other sensory characteristics that may negatively affect the quality and acceptability of the final product. Specifically, the invention addresses the reduction of such volatile agents through gene modification techniques.

[0074] One prominent example of a volatile agent relevant to this invention is 1-octen-3-ol, an eight-carbon alcohol commonly associated with a mushroom-like odor. The presence of 1-octen- 3-ol and other similar volatile agents in recombinant protein production can lead to unfavorable sensory properties in food or feed products or their intermediates.

[0075] Thus, the term "volatile agent" encompasses, but is not limited to, 1-octen-3-ol and any other volatile compounds that may arise during the production, processing, or storage of recombinant proteins, including 1-octen-3-one, (E)-Oct-2-enal, 1-octen-3-ol (Oct-1-en-3-ol), 1 ,3 Ocatdiene, 3-Octanol, 3-Octanone, 3-cyclohexene-1-carboxaldehyde, 4-methyl-Triepoxydecane, 1 ,1 O-Dimethyl, and trans-9-decahnol. The reduction or elimination of these volatile agents through genetic modifications aims to enhance the overall quality, stability, and consumer acceptance of the resultant food or feed products and their intermediates.

[0076] Volatile agents can be measured using various analytical techniques that allow for the detection and quantification of these compounds. Common methods include gas chromatography-mass spectrometry (GC-MS), which combines the separation capabilities of gas chromatography with the detection power of mass spectrometry, as well as solid-phase microextraction (SPME) coupled with GC-MS, and headspace analysis. These techniques provide precise identification and quantification of volatile compounds in complex mixtures.

[0077] Wild-type: The term "wild-type" in reference to an amino acid sequence or nucleic acid sequence means that the amino acid sequence or nucleic acid sequence is a native or naturally- occurring sequence. As used herein, the term "naturally-occurring" refers to anything (e.g., proteins, amino acids, or nucleic acid sequences) that is found in nature. Conversely, the term "non-naturally occurring" refers to anything that is not found in nature (e.g., recombinant nucleic acids and protein sequences produced in the laboratory or modification of the wild-type sequence).

[0078] Detailed Description of the Invention

[0079] Host Cells

[0080] In a 1staspect, the present invention relates to mutant Aspergillus cells comprising in its genome one or more polynucleotide encoding a polypeptide of interest (POI), wherein the polynucleotide encoding the polypeptide of interest is heterologous to the mutant cell, and wherein expression and / or activity of one or more endogenous psi-producing oxygenase C (PpoC) is reduced or eliminated compared to a non-mutated otherwise isogenic cell or parent cell when cultivated under identical conditions.

[0081] In one embodiment, the one or more first polynucleotide is encoding the PpoC, preferably the one or more first polynucleotide is endogenous to the mutant cell, more preferably the one or more first polynucleotide is endogenous to the mutant cell and the parent cell.

[0082] In one embodiment, the one or more PpoC is involved in the formation of one or more volatile agent, and wherein the mutant forms less of the one or more volatile agent compared to the non-mutated otherwise isogenic cell or parent cell when cultivated under identical conditions.

[0083] In one embodiment, the one or more first polynucleotide encoding the PpoC comprises at least one alteration selected from the list of: a) a partial or full deletion of the one or more first polynucleotide, b) a first heterologous promoter operably linked to the one or more first polynucleotide, c) a mutated Shine-Dalgarno sequence operably linked to the one or more first polynucleotide and derived from a parent Shine-Dalgarno sequence, d) one or more nucleic acid insertion, deletion, or substitution, and / or e) a premature stop codon.

[0084] In one embodiment, expression of the polynucleotide encoding the PpoC is decreased by a CRISPR inhibition construct, and / or expression of the polynucleotide encoding the PpoC is decreased by RNA interference.

[0085] In one embodiment, expression and / or activity of the one or more PpoC is decreased by at least 10%, e.g., by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%, compared to expression of the one or more PpoC in the parent cell when cultivated under identical conditions.

[0086] In one embodiment, the mutant is an Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae cell.

[0087] In one embodiment, the mutant cell is an Aspergillus niger cell.

[0088] In one embodiment, the mutant cell is an Aspergillus oryzae cell.

[0089] In one embodiment, the PpoC polypeptide comprises or consists of an amino acid sequence having a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence of SEQ ID NO: 10 or 12.

[0090] In one embodiment, the one or more first polynucleotide comprises or consists of a nucleic acid sequence having a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the nucleic acid sequence of SEQ I D NO: 9 or 11.

[0091] In one embodiment, the PpoC polypeptide comprises or consists of an amino acid sequence having a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence of SEQ ID NO: 2.

[0092] In one embodiment, the PpoC polypeptide comprises a motif comprising or consisting of an amino acid sequence having a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence of any one of SEQ ID NOs: 6, 7, or 8.

[0093] In one embodiment, the one or more first polynucleotide encoding the one or more PpoC polypeptide comprises or consists of a nucleic acid sequence having a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the nucleic acid sequence of SEQ ID NO: 1.

[0094] In one embodiment, the PpoC polypeptide comprises or consists of an amino acid sequence having a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence of a Trichoderma reesei PpoC polypeptide.

[0095] In one embodiment, the one or more first polynucleotide comprises or consists of a nucleic acid sequence having a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the nucleic acid sequence of a Trichoderma reesei ppoC coding sequence.

[0096] In one embodiment, transcription and / or translation of the one or more PpoC is decreased at least 1 %, e.g., at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11 %, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, relative to the transcription and / or translation of the one or more PpoC of the parent cell.

[0097] In one embodiment, expression of the one or more PpoC is decreased at least 1 %, e.g., at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11 %, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least

[0098] 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least

[0099] 37%, at least 38%, at least 39%, at least 40%, at least 41 %, at least 42%, at least 43%, at least

[0100] 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least

[0101] 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least

[0102] 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least

[0103] 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least

[0104] 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least

[0105] 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least

[0106] 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least

[0107] 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, relative to the expression of the one or more PpoC of the parent cell.

[0108] In one embodiment, activity of the one or more PpoC is decreased at least 1%, e.g., at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11 %, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21 %, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31 %, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41 %, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51 %, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, relative to the activity of the one or more PpoC of the parent cell.

[0109] In one embodiment, the one or more second heterologous promoter is linked to the one or more polynucleotide encoding the polypeptide of interest.

[0110] In one embodiment, the one or more second heterologous promoter is heterologous to the one or more polynucleotide encoding the polypeptide of interest.

[0111] In one embodiment, the mutant cell comprises at least two copies, e.g., at least three, at least four, or at least five, or at least six, or more copies of the polynucleotide encoding the polypeptide of interest in its genome.

[0112] In one embodiment, the first heterologous promoter results in decreased expression of the one or more PpoC, compared to PpoC expression controlled by the native promoter of the first polynucleotide in the parent cell when cultivated under identical conditions. In one embodiment, the first heterologous promoter results in decreased transcription of the first polynucleotide, relative to the transcription of the first polynucleotide when being operably linked to its native or endogenous promoter.

[0113] In one embodiment, the mutated Shine-Dalgarno sequence operably linked to the first polynucleotide results in decreased transcription of the first polynucleotide, relative to the transcription of the first polynucleotide when being operably linked to its native or endogenous Shine-Dalgarno sequence.

[0114] In one embodiment, the polypeptide of interest comprises an enzyme; preferably the enzyme is selected from the group consisting of hydrolase, isomerase, ligase, lyase, oxidoreductase, or transferase; more preferably an aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, endolase, esterase, alphagalactosidase, beta-galactosidase, alpha-glucosidase, beta-glucosidase, invertase, laccase, lipase, mannosidase, mutanase, nuclease, oxidase, pectinolytic enzyme, peroxidase, phosphodiesterase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, xylanase, and beta-xylosidase.

[0115] In one embodiment, the polypeptide of interest is selected from the list of ovalbumin, alpha-lactalbumin, alkaline phosphatase, saponin hydrolase, alpha-galactosidase C, alpha galactosidase 3, glycosyl hydrolase 71 (GH71), glycosyl hydrolase 20 (GH20), glycosyl hydrolase 13, beta-xylosidase, and phytase.

[0116] In one embodiment, the polypeptide of interest comprises a therapeutic polypeptide selected from the group consisting of an antibody, an antibody fragment, an antibody-based drug, a Fc fusion protein, an anticoagulant, a blood factor, a bone morphogenetic protein, an engineered protein scaffold, an enzyme, a growth factor, a blood clotting factor, a hormone, an interferon (such as an interferon alpha-2b), an interleukin, a lactoferrin, an alpha-lactalbumin, a beta-lactalbumin, an ovomucoid, an ovostatin, a cytokine, an obestatin, a human galactosidase (such as an human alpha-galactosidase A), a vaccine, a protein vaccine, and a thrombolytic.

[0117] In one embodiment, formation of the one or more volatile agent is decreased by at least 10%, e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, compared to the formation of the one or more volatile agent by the non-mutated otherwise isogenic cell or parent cell when cultivated under identical conditions, preferably decreased by at least 50%, more preferably decreased by at least 80%.

[0118] In one embodiment, the formation of the one or more volatile agent is decreased after at least 24 hours of cultivation, e.g., at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, or at least 144 hours of cultivation.

[0119] In one embodiment, the cultivation is a fed-batch, batch, or continuous cultivation process, preferably a fed-batch cultivation process. In one embodiment, the one or more volatile agent is selected from the list of 1-octen-3- one, (E)-Oct-2-enal, 1-octen-3-ol (Oct-1 -en-3-ol), 1 ,3 Ocatdiene, 3-Octanol, 3-Octanone, 3- cyclohexene-1-carboxaldehyde, 4-methyl-Triepoxydecane, 1 ,10-Dimethyl, or trans-9-decalinol.

[0120] In a preferred embodiment, the one or more volatile agent comprises or consists of 1- octen-3-ol.

[0121] In one embodiment, the formation of 1-octen-3-ol is decreased by at least 10%, e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, compared to the formation of 1-octen-3-ol by the non-mutated otherwise isogenic cell or parent cell when cultivated under identical conditions, preferably decreased by at least 50%, even more preferably decreased by at least 80%.

[0122] In one embodiment, the one or more volatile agent is selected from the list of Amyl vinyl carbinol, 1 -Vinylhexanol, Matsutake alcohol, Vinyl amyl carbinol, Vinyl hexanol, Matsuica alcohol, Mushroom alcohol, 3-Hydroxy-1 -octene, and Octenol.

[0123] In one embodiment, one or more volatile agent has a chemical structure corresponding to the following chemical formula:

[0124] In one embodiment, one or more volatile agent has the chemical formula CsHieO.

[0125] In one embodiment, the one or more volatile agent comprises or consists of one or more enantiomer selected from the list of ( / ?)-(-)- 1-octen-3-ol and (S)-(+)-1-octen-3-ol.

[0126] In one embodiment, a precursor of the one or more volatile agent comprises or consists of linoleic acid or linoleic acid 10(S)-hydroperoxide.

[0127] In one embodiment, the PpoC has a TM-score of at least 0.80, e.g., at least 0.85, at least 0.90, at least 0.905, at least 0.910, at least 0.915, at least 0.920, at least 0.925, at least 0.930, at least 0.935, at least 0.940, at least 0.945, at least 0.950, at least 0.955, at least 0.960, at least 0.965, at least 0.970, at least 0.975, at least 0.980, at least 0.985, at least 0.990, at least 0.995, or even 1.0, to the three-dimensional structure of the polypeptide of any of SEQ ID NO: 2, or SEQ ID NO: 10 or 12, wherein the three-dimensional structure is calculated by Alphafold.

[0128] In one embodiment, sequence identity is determined as described under “Sequence Identity” in the Definition section.

[0129] In one embodiment, the mutant is isolated.

[0130] In one embodiment, the mutant is purified. A construct or vector comprising a polynucleotide encoding a POI is introduced into a host cell so that the construct or vector is maintained as a chromosomal integrant or as a selfreplicating extra-chromosomal vector as described earlier. The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source. The POI can be native or heterologous to the mutant cell. Also, at least one of the one or more control sequences can be heterologous to the polynucleotide encoding the POI. The recombinant host cell may comprise a single copy, or at least two copies, e.g., three, four, five, or more copies of the polynucleotide encoding the POI.

[0131] The host cell may be any fungal cell useful in the recombinant production of a POI.

[0132] “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).

[0133] Fungal cells may be transformed by a process involving protoplast-mediated transformation, Agrobacterium-mediated transformation, electroporation, biolistic method and shock-wave-mediated transformation as reviewed by Li et al., 2017, Microbial Cell Factories 16: 168 and procedures described in EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81 : 1470-1474, Christensen etal., 1988, Bio / TechnologyQ: 1419-1422, and Lubertozzi and Keasling, 2009, Biotechn. Advances 27: 53-75. However, any method known in the art for introducing DNA into a fungal host cell can be used, and the DNA can be introduced as linearized or as circular polynucleotide.

[0134] The fungal mutant 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.

[0135] Methods of Production

[0136] In a 2ndaspect the invention relates to a method for producing one or more polypeptide of interest (POI) the method comprising providing a mutant cell according to the 1staspect, and cultivating said cell under conditions conducive for expression of the one or more POI.

[0137] In the 2ndaspect, the invention relates to a method for producing one or more polypeptides of interest, the method comprising a) providing a mutant cell according to the 1staspect, b) cultivating said mutant cell under conditions conducive for expression of the one or more polypeptides of interest; and, c) optionally recovering the one or more polypeptide of interest.

[0138] In one embodiment, formation of the one or more volatile agent is decreased by at least 10%, e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, compared to the formation of the one or more volatile agent by the non-mutated otherwise isogenic cell or parent cell when cultivated under identical conditions, preferably decreased by at least 50%, more preverably decreased by at least 80%.

[0139] In one embodiment, formation of 1-octen-3-ol is decreased by at least 10%, e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, compared to the formation of 1-octen-3-ol by the non-mutated otherwise isogenic cell or parent cell when cultivated under identical conditions, preferably decreased by at least 50%, more preferably decreased by at least 80%.

[0140] In one embodiment, the formation of the one or more volatile agent, e.g., 1-octen-3-ol, is decreased after at least 24 hours of cultivation, e.g., at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, or at least 144 hours of cultivation.

[0141] In one embodiment, the cultivation is a fed-batch, batch, or continuous cultivation process, preferably a fed-batch cultivation process.

[0142] In one embodiment the cultivation is carried out in a medium comprising at least 0.01% acetate (v / v).

[0143] In one embodiment the cultivation is carried out in a medium comprising acetate as the sole carbon source.

[0144] In one embodiment the cultivation is carried out in a medium comprising acetate as the predominant carbon source.

[0145] In one embodiment the cultivation is carried out in a medium free, or substantially free of glucose.

[0146] In one embodiment the cultivation is carried out in a medium comprising at least 0.1% glucose (v / v).

[0147] In one embodiment the cultivation is carried out in a medium comprising glucose as the sole carbon source.

[0148] In one embodiment the cultivation is carried out in a medium comprising glucose as the predominant carbon source.

[0149] In one embodiment the cultivation is carried out in a medium free, or substantially free of acetate.

[0150] In one embodiment the cultivation is carried out in a medium comprising at least 0.1% sucrose (v / v).

[0151] In one embodiment the cultivation is carried out in a medium comprising sucrose as the sole carbon source. In one embodiment the cultivation is carried out in a medium comprising sucrose as the predominant carbon source.

[0152] In one aspect, the cell is an Aspergillus cell. In another aspect, the cell is an Aspergillus niger cell. In another aspect, the cell is an Aspergillus oryzae cell.

[0153] In another aspect the cell is a Trichoderma cell.

[0154] In another aspect, the cell is a Trichoderma reesei cell.

[0155] The host cell is cultivated in a nutrient medium suitable for production of the POI using methods known in the art. For example, the cell may be cultivated by shake flask cultivation, or small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid-state, and / or microcarrier-based fermentations) in laboratory or industrial fermentors in a suitable medium and under conditions allowing the POI to be expressed and / or isolated. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the POI is secreted into the nutrient medium, the POI can be recovered directly from the medium. If the POI is not secreted, it can be recovered from cell lysates.

[0156] The POI may be detected using methods known in the art that are specific for the polypeptide, including, but not limited to, the use of specific antibodies, formation of an enzyme product, disappearance of an enzyme substrate, or an assay determining the relative or specific activity of the polypeptide.

[0157] The POI may be recovered from the medium using methods known in the art, including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation. In one aspect, a whole fermentation broth comprising the POI is recovered. In another aspect, a cell-free fermentation broth comprising the POI is recovered.

[0158] The POI may be purified by a variety of procedures known in the art to obtain substantially pure polypeptides and / or polypeptide fragments (see, e.g., Wingfield, 2015, Current Protocols in Protein Science-, 80(1): 6.1.1-6.1.35; Labrou, 2014, Protein Downstream Processing, 1129: 3-10).

[0159] In an alternative aspect, the POI is not recovered.

[0160] Fermentation Broth Formulations, Compositions, or Cell Compositions

[0161] In a 3rdaspect the invention relates to a whole broth formulation or cell culture composition comprising the mutant of the 1staspect.

[0162] In a 4thaspect the invention relates to a composition comprising the mutant of the 1staspect, and / or the whole broth formulation or cell culture composition according to the 3rdaspect.

[0163] In a 5thaspect the invention relates to use of the mutant of the 1staspect and / or the composition according to the 4thaspect in a method for producing a POI, e.g., a food or feed product.

[0164] The fermentation broth formulation or the cell composition further comprises additional ingredients used in the fermentation process, such as, for example, cells (including, the host cells containing the gene encoding the POI which are used to produce the POI), cell debris, biomass, fermentation media and / or fermentation products. In some embodiments, the composition is a cell-killed whole broth containing organic acid(s), killed cells and / or cell debris, and culture medium.

[0165] The term "fermentation broth" as used herein refers to a preparation produced by cellular fermentation that undergoes no or minimal recovery and / or purification. For example, fermentation broths are produced when microbial cultures are grown to saturation, incubated under carbon-limiting conditions to allow protein synthesis (e.g., expression of enzymes by host cells) and secretion into cell culture medium. The fermentation broth can contain unfractionated or fractionated contents of the fermentation materials derived at the end of the fermentation. Typically, the fermentation broth is unfractionated and comprises the spent culture medium and cell debris present after the microbial cells (e.g., filamentous fungal cells) are removed, e.g., by centrifugation. In some embodiments, the fermentation broth contains spent cell culture medium, extracellular enzymes, and viable and / or nonviable microbial cells.

[0166] In some embodiments, the fermentation broth formulation or the cell composition comprises a first organic acid component comprising at least one 1-5 carbon organic acid and / or a salt thereof and a second organic acid component comprising at least one 6 or more carbon organic acid and / or a salt thereof. In some embodiments, the first organic acid component is acetic acid, formic acid, propionic acid, a salt thereof, or a mixture of two or more of the foregoing and the second organic acid component is benzoic acid, cyclohexanecarboxylic acid, 4-methylvaleric acid, phenylacetic acid, a salt thereof, or a mixture of two or more of the foregoing.

[0167] In one aspect, the composition contains an organic acid(s), and optionally further contains killed cells and / or cell debris. In some embodiments, the killed cells and / or cell debris are removed from a cell-killed whole broth to provide a composition that is free of these components.

[0168] The fermentation broth formulation or cell composition may further comprise a preservative and / or anti-microbial (e.g., bacteriostatic) agent, including, but not limited to, sorbitol, sodium chloride, potassium sorbate, and others known in the art.

[0169] The cell-killed whole broth or cell composition may contain the unfractionated contents of the fermentation materials derived at the end of the fermentation. Typically, the cell-killed whole broth or cell composition contains the spent culture medium and cell debris present after the microbial cells (e.g., filamentous fungal cells) are grown to saturation, incubated under carbon- limiting conditions to allow protein synthesis. In some embodiments, the cell-killed whole broth or cell composition contains the spent cell culture medium, extracellular enzymes, and killed filamentous fungal cells. In some embodiments, the microbial cells present in the cell-killed whole broth or composition can be permeabilized and / or lysed using methods known in the art.

[0170] A whole broth or cell composition as described herein is typically a liquid, but may contain insoluble components, such as killed cells, cell debris, culture media components, and / or insoluble enzyme(s). In some embodiments, insoluble components may be removed to provide a clarified liquid composition.

[0171] The whole broth formulations and cell compositions of the present invention may be produced by a method described in WO 90 / 15861 or WO 2010 / 096673.

[0172] Removal or Reduction of PpoC Activity

[0173] In a 6thaspect the invention relates to a method of producing the mutant of the 1staspect, i.e. a method of producing a mutant of a parent cell, comprising mutating one or more first polynucleotide encoding a psi-producing oxygenase C (PpoC), which results in the mutant producing less of the PpoC than the parent cell.

[0174] In a 7thaspect the invention relates to the mutant obtained or obtainable by the method of the 6thaspect.

[0175] The methods of producing a mutant of a parent cell, comprise disrupting, modifying, or deleting a first polynucleotide, or a portion thereof, encoding a psi-producing oxygenase C (PpoC), which results in the mutant cell producing less of the PpoC than the parent cell when cultivated under the same conditions.

[0176] The mutant cell may be constructed by reducing or eliminating expression of the first polynucleotide using methods well known in the art, for example, one or more nucleotide insertions, one or more gene disruptions, one or more nucleotide replacements, or one or more nucleotide deletions.

[0177] The first polynucleotide to be modified or inactivated may be, for example, the coding region or a part thereof essential for activity, or a regulatory or control element required for expression of the coding region, e.g., a functional part of a promoter sequence, and / or a regulatory or control element required for the transcription or translation of the first polynucleotide. Other control sequences for possible modification include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, signal peptide sequence, transcription terminator, and transcriptional activator.

[0178] In one embodiment the first polynucleotide comprises a modification or deletion of the sequence encoding the proline knot motif.

[0179] In one embodiment the first polynucleotide comprises a modification or deletion of the sequence encoding the proximal His domain.

[0180] In one embodiment the first polynucleotide comprises a modification or deletion of the sequence encoding the catalytic active site of the cytochrome P450 domain.

[0181] In one embodiment, the PpoC comprises a catalytic active site of the cytochrome P450 domain comprising or consisting of an amino acid sequence having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least

[0182] 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99%, or 100% sequence identity to SEQ ID NO: 8.

[0183] Modification or inactivation of the first polynucleotide may be performed by subjecting the parent cell to mutagenesis and selecting for mutant cells in which expression of the first polynucleotide has been reduced or eliminated. The mutagenesis, which may be specific or random, may be performed, for example, by use of a suitable physical or chemical mutagenizing agent, by use of a suitable oligonucleotide, or by subjecting the DNA sequence to PCR generated mutagenesis. Furthermore, the mutagenesis may be performed by use of any combination of these mutagenizing agents.

[0184] Examples of a physical or chemical mutagenizing agent include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxyl amine, nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formic acid, and nucleotide analogues (see J. L. Bose, Springer Protocols 2016, Methods in Molecular Biology, The Genetic Manipulation of Staphylococci).

[0185] Additionally or alternatively, nucleotides may be inserted or removed so as to result in the introduction of a stop codon, the removal of the start codon, or a change in the open reading frame. Such modification or inactivation may be accomplished by site-directed mutagenesis or PCR generated mutagenesis in accordance with methods known in the art, or by targeted gene editing using one or more nucleases, e.g., zinc-finger nucleases or CRISPR-associated nucleases. Additionally or alternatively, the modification or inactivation may be achieved by gene silencing, genetic repression, genetic activation, and / or post-translational mutagenesis, e.g., by methods employing non-coding RNA, RNAi, siRNA, miRNA, ribozymes, catalytically inactive nucleases, CRISPRi, nucleotide methylation, and / or histone acetylation. The modification may be transient and / or reversible, irreversible and / or stable, or the modification may be dependent on chemical inducers or dependent on cultivation conditions, such as the cultivation temperature.

[0186] The modification may be performed in vivo, i.e., directly on the cell expressing the firist polynucleotide to be modified, or the modification be performed in vitro.

[0187] Examples for reducing or inactivating PpoC activity and / or expression include: a) a partial or full deletion of the one or more first polynucleotide encoding the PpoC, b) a first heterologous promoter operably linked to the one or more first polynucleotide encoding the PpoC wherein the first heterologous promoter confers a weaker expression of PpoC relative to the native PpoC promoter when cultivated under identical conditions, c) a mutated Shine-Dalgarno sequence operably linked to the one or more first polynucleotide encoding the PpoC and derived from a parent Shine-Dalgarno sequence which mutated Shine-Dalgarno sequence confers a weaker expression of PpoC relative to the native Shine- Dalgarno sequence when cultivated under identical conditions, and d) one or more nucleic acid insertion, deletion, or substitution in the polynucleotide encoding the PpoC to cause a frame-shift mutation and / or a premature stop codon. An example of a convenient way to eliminate or reduce expression of the first polynucleotide is based on techniques of gene replacement, gene deletion, or gene disruption (as shown in Example 1). For example, in the gene disruption method, a nucleic acid sequence corresponding to the endogenous first polynucleotide is mutagenized in vitro to produce a defective nucleic acid sequence that is then transformed into the parent cell to produce a defective gene. By homologous recombination, the defective nucleic acid sequence replaces the endogenous first polynucleotide. It may be desirable that the defective polynucleotide also encodes a marker that may be used for selection of transformants in which the first polynucleotide has been modified or destroyed. In an aspect, the first polynucleotide is disrupted with a selectable marker such as those described herein.

[0188] The present invention further relates to a mutant cell of a parent cell that comprises a disruption or deletion of a firsrt polynucleotide encoding the PpoC or a control sequence thereof or a silenced gene encoding the PpoC, which results in the mutant cell producing less of the PpoC or no PpoC compared to the parent cell.

[0189] The PpoC-deficient mutant cells are useful as host cells for expression of native and heterologous polypeptides of interest (PO). Therefore, the present invention further relates to methods of producing a native or heterologous POI, comprising (a) cultivating the mutant cell under conditions conducive for production of the POI; and optionally (b) recovering the POI. The term "heterologous polypeptides" or “heterologous POI” means polypeptides that are not native to the host cell, e.g., a variant of a native protein. The host cell may comprise more than one copy of a polynucleotide encoding the native or heterologous POI.

[0190] The methods of the present invention for producing an essentially 1-octen-3-ol-free product or a product with reduced 1-octen-3-ol content are of interest in the production of polypeptides, e.g., proteins such as enzymes, and / or polypeptides used in feed or food products. The PpoC-deficient cells may also be used to express heterologous proteins of pharmaceutical interest such as hormones, growth factors, receptors, and the like.

[0191] In some embodiments, the present invention relates to a protein product with reduced 1- octen-3-ol content, such as a protein product being essentially free from 1-octen-3-ol that is produced by a method of the present invention.

[0192] The present invention also relates to reduction and / or elimination of polypeptides having psi-producing oxygenase C (PpoC) activity. In an aspect, the PpoC polypeptides having PpoC activity, are selected from the group consisting of:

[0193] (a) a polypeptide having at least 80% sequence identity to SEQ ID NO: 2;

[0194] (b) a polypeptide having at least 80% sequence identity to SEQ ID NO: 10 or 12; or

[0195] (c) a polypeptide encoded by a polynucleotide having at least 80% sequence identity to the mature polypeptide coding sequence of any one of SEQ ID NOs: 1 , or 9, or the cDNA sequence thereof. In an aspect, the polypeptide has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99%, or 100% to SEQ ID NO: 2 or a mature polypeptide of SEQ ID NO: 2.

[0196] In an aspect, the polypeptide has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99%, or 100% to SEQ ID NO: 10 or 12 or a mature polypeptide of SEQ ID NO: 10 or 12.

[0197] In an aspect, the polypeptide has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99%, or 100% to a Trichoderma reesei ppoC polypeptide.

[0198] The polypeptide preferably comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 2 or a mature polypeptide thereof.

[0199] The polypeptide preferably comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 10 or 12 or a mature polypeptide thereof.

[0200] The polypeptide preferably comprises, consists essentially of, or consists of the amino acid sequence of a Trichoderma reesei ppoC polypeptide.

[0201] In some embodiments, the polypeptide is encoded by a polynucleotide having a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99%, or 100% to the mature polypeptide coding sequence of SEQ ID NO: 1 , 9, or 11 , or the cDNA sequence thereof.

[0202] Essential amino acids or activity relevant motifs of a PpoC are suitable targets to modify / remove in order to obain a mutant with reduced or eliminated PpoC activity or expression.

[0203] 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). 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.

[0204] In one embodiment, the PpoC comprises a catalytic active site comprising or consisting of an amino acid sequence having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99%, or 100% sequence identity to SEQ ID NO: 8.

[0205] In one embodiment, the PpoC comprises a proline knot motif comprising or consisting of an amino acid sequence having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99%, or 100% sequence identity to SEQ ID NO: 6.

[0206] In one embodiment, the PpoC comprises a proximal His domain comprising or consisting of an amino acid sequence having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99%, or 100% sequence identity to SEQ ID NO: 7.

[0207] In one embodiment, the catalytic active site is inactivated or removed from one or more PpoC.

[0208] Nucleic Acid Constructs

[0209] The present invention also relates to mutants according to the 1staspect, which mutants comprise one or more nucleic acid constructs comprising a polynucleotide encoding one or more POI, wherein the polynucleotide is operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.

[0210] The polynucleotide may be manipulated in a variety of ways to provide for expression of the POI. Manipulation of the polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. Techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art. Promoters

[0211] The control sequence may be a promoter, a polynucleotide that is recognized by a host cell for expression of a polynucleotide encoding a POI. The promoter contains transcriptional control sequences that mediate the expression of the polypeptide. The promoter may be any polynucleotide that shows transcriptional activity in the host cell including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

[0212] Examples of suitable promoters for directing transcription of the polynucleotide of a POI in a filamentous fungal host cell are promoters obtained from Aspergillus, Fusarium, Rhizomucor and Trichoderma cells, such as the promoters described in Mukherjee etal., 2013, “Trichoderma: Biology and Applications”, and by Schmoll and Dattenbdck, 2016, “Gene Expression Systems in Fungi: Advancements and Applications”, Fungal Biology.

[0213] Terminators

[0214] The control sequence may also be a transcription terminator, which is recognized by a host cell to terminate transcription. The terminator is operably linked to the 3’-terminus of the polynucleotide encoding the POI. Any terminator that is functional in the host cell may be used in the present invention.

[0215] Preferred terminators for filamentous fungal host cells may be obtained from Aspergillus or Trichoderma species, such as obtained from the genes for Aspergillus niger glucoamylase, Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, and Trichoderma reesei endoglucanase I, such as the terminators described in Mukherjee et al., 2013, “Trichoderma: Biology and Applications”, and by Schmoll and Dattenbdck, 2016, “Gene Expression Systems in Fungi: Advancements and Applications”, Fungal Biology. mRNA Stabilizers

[0216] The control sequence may also be an mRNA stabilizer region downstream of a promoter and upstream of the coding sequence of a gene which increases expression of the gene.

[0217] Examples of suitable mRNA stabilizer regions are obtained from a Bacillus thuringiensis crylllA gene (WO 94 / 25612) and a Bacillus subtilis SP82 gene (Hue etal., 1995, J. Bacteriol. 177: 3465-3471).

[0218] Examples of mRNA stabilizer regions for fungal cells are described in Geisberg et al., 2014, Cell 156(4): 812-824, and in Morozov et al., 2006, Eukaryotic Ce / / 5(11): 1838-1846.

[0219] Leader Sequences

[0220] The control sequence may also be a leader, a non-translated region of an mRNA that is important for translation by the host cell. The leader is operably linked to the 5’-terminus of the polynucleotide encoding the POI. Any leader that is functional in the host cell may be used. Preferred leaders for filamentous fungal host cells may be obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

[0221] Polyadenylation Sequences

[0222] The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3’-terminus of the polynucleotide which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence that is functional in the host cell may be used.

[0223] Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

[0224] Signal Peptides

[0225] The control sequence may also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of a polypeptide and directs the polypeptide into the cell’s secretory pathway. The 5’-end of the coding sequence of the polynucleotide may inherently contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence that encodes the polypeptide. Alternatively, the 5’-end of the coding sequence may contain a signal peptide coding sequence that is heterologous to the coding sequence. A heterologous signal peptide coding sequence may be required where the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, a heterologous signal peptide coding sequence may simply replace the natural signal peptide coding sequence to enhance secretion of the polypeptide. Any signal peptide coding sequence that directs the expressed polypeptide into the secretory pathway of a host cell may be used.

[0226] Effective signal peptide coding sequences for filamentous fungal host cells are the signal peptide coding sequences obtained from the genes for Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicola insolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucor miehei aspartic proteinase, such as the signal peptide described by Xu et al., 2018, Biotechnology Letters 40: 949-955

[0227] Propeptides

[0228] The control sequence may also be a propeptide coding sequence that encodes a propeptide positioned at the N-terminus of a polypeptide. The resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases). A propolypeptide is generally inactive and can be converted to an active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding sequence may be obtained from the genes for Myceliophthora thermophila laccase (WO 95 / 33836), Rhizomucor miehei aspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

[0229] Where both signal peptide and propeptide sequences are present, the propeptide sequence is positioned next to the N-terminus of a polypeptide and the signal peptide sequence is positioned next to the N-terminus of the propeptide sequence. Additionally or alternatively, when both signal peptide and propeptide sequences are present, the polypeptide may comprise only a part of the signal peptide sequence and / or only a part of the propeptide sequence. Alternatively, the final or isolated polypeptide may comprise a mixture of mature polypeptides and polypeptides which comprise, either partly or in full length, a propeptide sequence and / or a signal peptide sequence.

[0230] Regulatory Sequences

[0231] It may also be desirable to add regulatory sequences that regulate expression of the polypeptide relative to the growth of the host cell. Examples of regulatory sequences are those that cause expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. In filamentous fungi, the Aspergillus n / gerglucoamylase promoter, Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzae glucoamylase promoter, Trichoderma reesei cellobiohydrolase I promoter, and Trichoderma reesei cellobiohydrolase II promoter may be used. Other examples of regulatory sequences are those that allow for gene amplification. In fungal systems, these regulatory sequences include the dihydrofolate reductase gene that is amplified in the presence of methotrexate, and the metallothionein genes that are amplified with heavy metals.

[0232] Expression Vectors

[0233] The present invention also relates to mutants of the 1staspect comprising one or more recombinant expression vectors, the vectors comprising a polynucleotide encoding one or more POI, a promoter, and transcriptional and translational stop signals. The various nucleotide and control sequences may be joined together to produce a recombinant expression vector that may include one or more convenient restriction sites to allow for insertion or substitution of the polynucleotide encoding the POI at such sites. Alternatively, the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.

[0234] The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the polynucleotide encoding the POI. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.

[0235] The vector may be an autonomously replicating vector, i.e., a vector that 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. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon, may be used.

[0236] The vector preferably contains one or more selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells. 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.

[0237] The vector preferably contains at least one element that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.

[0238] For integration into the host cell genome, the vector may rely on the polynucleotide’s sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous recombination, such as homology-directed repair (HDR), or non- homologous recombination, such as non-homologous end-joining (NHEJ).

[0239] For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell. The term “origin of replication” or “plasmid replicator” means a polynucleotide that enables a plasmid or vector to replicate in vivo.

[0240] More than one copy of a polynucleotide encoding the POI may be inserted into a host cell to increase production of a POI. For example, 2 or 3 or 4 or 5 or more copies are inserted into a host cell. An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.

[0241] The present invention is further described by the following examples that should not be construed as limiting the scope of the invention. Examples

[0242] Example 1 : Generation of ppoC knockout strains

[0243] Starting with the Aspergillus niger strain O253R1 , the native ppoC gene body (SEQ ID NO:1) encoding the PpoC polypeptide of SEQ ID NO:2 was removed by donor-directed repair following a DNA double-strand break targeted to ppoC exon 3. Specifically, a 600-bp doublestranded DNA fragment composed of 300 bp 5' homology (5' HOM; SEQ ID NO:3) and 300 bp 3' homology (3' HOM; SEQ ID NO:4) to the chromosomal gene body flanks was co-transformed with a plasmid (pGMER280) expressing MAD7, a MAD7 guide RNA (SEQ ID NO:5) targeting ppoC, and hph encoding Hygromycin B resistance. PEG-mediated transformants were selected on hygromycin agar plates, genotyped by PCR and Illumina amplicon sequencing, spore-isolated (2x), and again genotyped by PCR and whole-genome Illumina sequencing. This way, transformants with confirmed deletion of the ppoC gene were obtained. One of the obtained A. niger ppoC deletion transformants was named “O253R1 ppoC-A”. The same approach was used in to obtain A. oryzae ppoC deletion mutants except that selection was based on ammonium glufosinate (basta)-containing plates from a MAD7 plasmid co-expressing bar. A schematic overview of the deletion approach is shown in Fig. 1. An analogous deletion approach for Aspergillus oryzae is provided in Fig. 4.

[0244] Example 2: PpoC deletion does not impact mutant growth

[0245] A. niger strain O253R1 with intact ppoC gene, and A. niger transformant O253R1 ppoC- A were both plated in spot assays at defined spore count dilutions. Plates were incubated at 30 degC for 3 days in a Ziploc bag, and then imaged. Images are shown in Fig. 2 (left image: pale background; right image: dark background).

[0246] As can be seen in Fig. 2, for each spore count dilution the ppoC deletion mutant (squared circles) sporulated and growed similarly to the parental reference strain (round circles) indicating that the ppoC gene is not essential for A. niger. Also, Fig. 2 shows that ppoC deletion has no negative impact on cell growth or sporulation.

[0247] Example 3: PpoC deletion removes volatile agents

[0248] Fig. 3 shows GCMS chromatograms (A-B) and mass spectrum (C) of samples obtained from fermentations of the control strain “O253R1” with intact ppoC gene (Fig. 3(A)) and from fermentatios of the ppoC knock-out strain “O253R1 ppoC-A” (Fig. 3(B)), revealing a significant reduction of several volatile agents in the ppoC deletion mutant. For example, the peak with retention time at ca. 14.7 mins present in the control strain appears to be significantly reduced or absent in the ppoC deletion mutant.

[0249] As shown in Fig. 3(C) the peak with retention time of 14.7 mins was identified as 1-octen- 3-ol by mass spectrum matching to NIST library and Rl (retention time index) confirmation. The differential abundance of 1-octen-3-ol was determined by comparing the total ion chromatograms of the two strains, revealing that the ppoC deletion strain exhibited reduced 1-octen-3-ol levels.

[0250] The inventors surprisingly identified that a single ppoC deletion resulted in significant reduction of 1-octen-3-ol levels and other volatile agents without impacting cell growth.

[0251] Example 4: PpoC deletion impact on heterologous protein yield

[0252] Cultivations of AppoC knockout strains were compared to the parental strain that retained ppoC in 2L scale fed-batch fermentations. A defined volume was sampled on day 7 from the fed- batch fermentations. The dry cell weight (grams) of the biomass was measured to determine if biomass accumulation was affected by the deletion of ppoC.

[0253] The AppoC knock-out strain had lower dry cell weight after fermentation compared to the control strain, suggesting the deletion leads to decreased growth under certain cultivation conditions. To test whether heterologous protein production was affected in AppoC strains, alpha lactalbumin was quantified by UV-Size exclusion chromatography (LIV-SEC) from the same day- 7 fermentation broths. No reduction in heterologous protein production was observed in AppoC strains. The results are shown in table 1 below.

[0254] Table 1. The invention described and claimed herein is not to be limited in scope by the specific aspects herein disclosed, since these aspects are intended as illustrations of several aspects of the invention. Any equivalent aspects are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will control.

[0255] The invention is further defined by the following numbered paragraphs:

[0256] 1. A fungal mutant cell comprising in its genome one or more polynucleotide encoding a polypeptide of interest (POI), wherein expression and / or activity of one or more endogenous psi-producing oxygenase C (PpoC) is reduced or eliminated compared to a non-mutated otherwise isogenic cell or parent cell when cultivated under identical conditions.

[0257] 2. The mutant according to paragraph 1 , wherein one or more first polynucleotide is encoding the PpoC, preferably the one or more first polynucleotide is endogenous to the mutant cell, more preferably the one or more first polynucleotide is endogenous to the mutant cell and the parent cell.

[0258] 3. The mutant according to any one of paragraphs 1-2, wherein the one or more PpoC is involved in the formation of one or more volatile agent, and wherein the mutant forms less of the one or more volatile agent compared to the non-mutated otherwise isogenic cell or parent cell when cultivated under identical conditions.

[0259] 4. The mutant according to any one of the preceding paragraphs, wherein the one or more first polynucleotide encoding the PpoC comprises at least one alteration selected from the list of: a) a partial or full deletion of the one or more first polynucleotide, b) a first heterologous promoter operably linked to the one or more first polynucleotide, c) a mutated Shine-Dalgarno sequence operably linked to the one or more first polynucleotide and derived from a parent Shine-Dalgarno sequence, d) one or more nucleic acid insertion, deletion, or substitution, and / or e) a premature stop codon.

[0260] 5. The mutant according to any one of the preceding paragraphs, wherein expression of the polynucleotide encoding the PpoC is decreased by a CRISPR inhibition construct, and / or expression of the polynucleotide encoding the PpoC is decreased by RNA interference. The mutant according to any one of the preceding paragraphs, wherein expression and / or activity of the one or more PpoC is decreased by at least 10%, e.g., by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%, compared to expression of the one or more PpoC in the parent cell when cultivated under identical conditions. The mutant according to any one of the preceding paragraphs, which is a yeast recombinant host cell, e.g., a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cell. The mutant according to any one of the preceding paragraphs, which is a filamentous fungal recombinant host cell, e.g., an 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, or Trichoderma cell, in particular, an 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, or Trichoderma vi ride cell.

[0261] 9. The mutant according to any one of the preceding paragraphs, wherein the mutant cell is an Aspergillus cell.

[0262] 10. The mutant according to any one of the preceding paragraphs, wherein the mutant cell is an Aspergillus niger cell.

[0263] 11 . The mutant according to any one of the preceding paragraphs, wherein the mutant cell is an Aspergillus oryzae cell.

[0264] 12. The mutant according to any one of the preceding paragraphs, wherein the mutant cell is a Trichoderma cell.

[0265] 13. The mutant according to any one of the preceding paragraphs, wherein the mutant cell is a Trichoderma reesei cell.

[0266] 14. The cell according to any one of the preceding paragraphs, wherein the PpoC polypeptide comprises or consists of an amino acid sequence having a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence of SEQ ID NO: 10 or 12.

[0267] 15. The cell according to any one of the preceding paragraphs, wherein the one or more first polynucleotide comprises or consists of a nucleic acid sequence having a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the nucleic acid sequence of SEQ ID NO: 9 or 11.

[0268] 16. The mutant according to any one of the preceding paragraphs, wherein the PpoC polypeptide comprises or consists of an amino acid sequence having a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence of SEQ ID NO: 2. 17. The mutant according to any one of the preceding paragraphs, wherein the PpoC polypeptide comprises a motif comprising or consisting of an amino acid sequence having a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence of any one of SEQ ID NOs: 6, 7, or 8.

[0269] 18. The mutant according to any one of the preceding paragraphs, wherein the one or more first polynucleotide encoding the one or more PpoC polypeptide comprises or consists of a nucleic acid sequence having a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the nucleic acid sequence of SEQ ID NO: 1.

[0270] 19. The cell according to any one of the preceding paragraphs, wherein the PpoC polypeptide comprises or consists of an amino acid sequence having a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence of a Trichoderma reesei PpoC polypeptide.

[0271] 20. The cell according to any one of the preceding paragraphs, wherein the one or more first polynucleotide comprises or consists of a nucleic acid sequence having a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the nucleic acid sequence of a Trichoderma reesei ppoC coding sequence.

[0272] 21. The mutant according to any one of the preceding paragraphs, wherein transcription and / or translation of the one or more PpoC is decreased at least 1%, e.g., at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11 %, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31 %, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51 %, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, relative to the transcription and / or translation of the one or more PpoC of the parent cell.

[0273] 22. The mutant according to any one of the preceding paragraphs, wherein expression of the one or more PpoC is decreased at least 1 %, e.g., at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11 %, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31 %, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51 %, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, relative to the expression of the one or more PpoC of the parent cell.

[0274] 23. The mutant according to any one of the preceding paragraphs, wherein activity of the one or more PpoC is decreased at least 1%, e.g., at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11 %, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31 %, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51 %, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, relative to the activity of the one or more PpoC of the parent cell.

[0275] 24. The mutant according to any one of the preceding paragraphs, wherein the polynucleotide encoding the polypeptide of interest is heterologous to the mutant cell.

[0276] 25. The mutant according to any one of the preceding paragraphs, wherein one or more second heterologous promoter is linked to the one or more polynucleotide encoding the polypeptide of interest.

[0277] 26. The mutant according to any one of the preceding paragraphs, wherein the one or more second heterologous promoter is heterologous to the one or more polynucleotide encoding the polypeptide of interest.

[0278] 27. The mutant according to any one of the preceding paragraphs, wherein the polypeptide of interest is heterologous to the mutant.

[0279] 28. The mutant according to any one of the preceding paragraphs, wherein the mutant cell comprises at least two copies, e.g., at least three, at least four, or at least five, or at least six, or more copies of the polynucleotide encoding the polypeptide of interest in its genome.

[0280] 29. The mutant according to any one of the preceding paragraphs, wherein the first heterologous promoter results in decreased expression of the one or more PpoC, compared to PpoC expression controlled by the native promoter of the first polynucleotide in the parent cell when cultivated under identical conditions.

[0281] 30. The mutant according to any one of the preceding paragraphs, wherein the first heterologous promoter results in decreased transcription of the first polynucleotide, relative to the transcription of the first polynucleotide when being operably linked to its native or endogenous promoter.

[0282] 31. The mutant according to any one of the preceding paragraphs, wherein the mutated Shine-Dalgarno sequence operably linked to the first polynucleotide results in decreased transcription of the first polynucleotide, relative to the transcription of the first polynucleotide when being operably linked to its native or endogenous Shine-Dalgarno sequence. . The mutant according to any one of the preceding paragraphs, wherein the polypeptide of interest comprises an enzyme; preferably the enzyme is selected from the group consisting of hydrolase, isomerase, ligase, lyase, oxidoreductase, or transferase; more preferably an aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, endolase, esterase, alpha-galactosidase, beta-galactosidase, alphaglucosidase, beta-glucosidase, invertase, laccase, lipase, mannosidase, mutanase, nuclease, oxidase, pectinolytic enzyme, peroxidase, phosphodiesterase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, xylanase, and beta-xylosidase. . The mutant according to any one of the preceding paragraphs, wherein the polypeptide of interest is selected from the list of ovalbumin, alpha-lactalbumin, alkaline phosphatase, saponin hydrolase, alpha-galactosidase C, alpha galactosidase 3, glycosyl hydrolase 71 (GH71), glycosyl hydrolase 20 (GH20), glycosyl hydrolase 13, beta-xylosidase, and phytase. . The mutant according to any one of the preceding paragraphs, wherein the polypeptide of interest comprises a therapeutic polypeptide selected from the group consisting of an antibody, an antibody fragment, an antibody-based drug, a Fc fusion protein, an anticoagulant, a blood factor, a bone morphogenetic protein, an engineered protein scaffold, an enzyme, a growth factor, a blood clotting factor, a hormone, an interferon (such as an interferon alpha- 2b), an interleukin, a lactoferrin, an alpha-lactalbumin, a beta-lactalbumin, an ovomucoid, an ovostatin, a cytokine, an obestatin, a human galactosidase (such as an human alphagalactosidase A), a vaccine, a protein vaccine, and a thrombolytic. . The mutant according to any one of the preceding paragraphs, wherein formation of the one or more volatile agent is decreased by at least 10%, e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, compared to the formation of the one or more volatile agent by the non-mutated otherwise isogenic cell or parent cell when cultivated under identical conditions, preferably decreased by at least 50%, more preferably decreased by at least 80%. . The mutant according to any one of the preceding paragraphs, wherein the formation of the one or more volatile agent is decreased after at least 24 hours of cultivation, e.g., at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, or at least 144 hours of cultivation. 37. The mutant according to any one of the preceding paragraphs, wherein the cultivation is a fed-batch, batch, or continuous cultivation process, preferably a fed-batch cultivation process.

[0283] 38. The mutant according to any one of the preceding paragraphs, wherein the one or more volatile agent is selected from the list of 1-octen-3-one, (E)-Oct-2-enal, 1-octen-3-ol (Oct-1 - en-3-ol), 1 ,3 Ocatdiene, 3-Octanol, 3-Octanone, 3-cyclohexene-1-carboxaldehyde, 4-methyl- Triepoxydecane, 1 ,10-Dimethyl, or trans-9-decalinol.

[0284] 39. The mutant according to any one of the preceding paragraphs, wherein the one or more volatile agent comprises or consists of 1-octen-3-ol.

[0285] 40. The mutant according to any one of the preceding paragraphs, wherein formation of 1- octen-3-ol is decreased by at least 10%, e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, compared to the formation of 1-octen-3-ol by the non-mutated otherwise isogenic cell or parent cell when cultivated under identical conditions, preferably decreased by at least 50%, even more preferably decreased by at least 80%.

[0286] 41 . The mutant according to any one of the preceding paragraphs, wherein the one or more volatile agent is selected from the list of Amyl vinyl carbinol, 1 -Vinylhexanol, Matsutake alcohol, Vinyl amyl carbinol, Vinyl hexanol, Matsuica alcohol, Mushroom alcohol, 3-Hydroxy-1 -octene, and Octenol.

[0287] 42. The mutant according to any one of the preceding paragraphs, wherein one or more volatile agent has a chemical structure corresponding to the following chemical formula:

[0288] 43. The mutant according to any one of the preceding paragraphs, wherein one or more volatile agent has the chemical formula CsHieO.

[0289] 44. The mutant according to any one of the preceding paragraphs, wherein the one or more volatile agent comprises or consists of one or more enantiomer selected from the list of ( / ?)- (-)-1-octen-3-ol and (S)-(+)-1-octen-3-ol. 45. The mutant according to any one of the preceding paragraphs, wherein a precursor of the one or more volatile agent comprises or consists of linoleic acid or linoleic acid 10(S)- hydroperoxide.

[0290] 46. The mutant according to any one of the preceding paragraphs, wherein the PpoC has a TM-score of at least 0.80, e.g., at least 0.85, at least 0.90, at least 0.905, at least 0.910, at least 0.915, at least 0.920, at least 0.925, at least 0.930, at least 0.935, at least 0.940, at least 0.945, at least 0.950, at least 0.955, at least 0.960, at least 0.965, at least 0.970, at least 0.975, at least 0.980, at least 0.985, at least 0.990, at least 0.995, or even 1.0, to the three- dimensional structure of the polypeptide of any of SEQ ID NO: 2, or SEQ ID NO: 10, or SEQ ID NO: 12, wherein the three-dimensional structure is calculated by Alphafold.

[0291] 47. The mutant according to any one of the preceding paragraphs, wherein sequence identity is determined as described under “Sequence Identity” in the Definition section.

[0292] 48. The mutant according to any one of the preceding paragraphs, which is isolated.

[0293] 49. The mutant according to any one of the preceding paragraphs, which is purified.

[0294] 50. A method for producing one or more polypeptides of interest, the method comprising a) providing a mutant cell according to any one of the previous paragraphs, b) cultivating said mutant cell under conditions conducive for expression of the one or more polypeptides of interest; and, c) optionally recovering the one or more polypeptide of interest.

[0295] 51. The method according to paragraph 50, wherein formation of the one or more volatile agent is decreased by at least 10%, e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, compared to the formation of the one or more volatile agent by the non-mutated otherwise isogenic cell or parent cell when cultivated under identical conditions, preferably decreased by at least 50%, more preverably decreased by at least 80%.

[0296] 52. The method according to paragraph 51 , wherein formation of 1-octen-3-ol is decreased by at least 10%, e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, compared to the formation of 1-octen-3-ol by the non-mutated otherwise isogenic cell or parent cell when cultivated under identical conditions, preferably decreased by at least 50%, more preferably decreased by at least 80%. 53. The method according to any one of the preceding paragraphs, wherein the formation of the one or more volatile agent, e.g., 1-octen-3-ol, is decreased after at least 24 hours of cultivation, e.g., at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, or at least 144 hours of cultivation.

[0297] 54. The method according to any one of the preceding paragraphs, wherein the cultivation is a fed-batch, batch or continuous cultivation process, preferably a fed-batch cultivation process.

[0298] 55. The method according to any one of the preceding paragraphs, wherein the cultivation is carried out in a medium comprising at least 0.01% acetate (v / v).

[0299] 56. The method according to any one of the preceding paragraphs, wherein the cultivation is carried out in a medium comprising acetate as the sole carbon source.

[0300] 57. The method according to any one of the preceding paragraphs, wherein the cultivation is carried out in a medium comprising acetate as the predominant carbon source.

[0301] 58. The method according to any one of the preceding paragraphs, wherein the cultivation is carried out in a medium free, or substantially free of glucose.

[0302] 59. The method according to any one of the preceding paragraphs, wherein the cultivation is carried out in a medium comprising at least 0.1 % glucose (v / v).

[0303] 60. The method according to any one of the preceding paragraphs, wherein the cultivation is carried out in a medium comprising glucose as the sole carbon source.

[0304] 61 . The method according to any one of the preceding paragraphs, wherein the cultivation is carried out in a medium comprising glucose as the predominant carbon source.

[0305] 62. The method according to any one of the preceding paragraphs, wherein the cultivation is carried out in a medium free, or substantially free of acetate.

[0306] 63. The method according to any one of the preceding paragraphs, wherein the cultivation is carried out in a medium comprising at least 0.1 % sucrose (v / v). The method according to any one of the preceding paragraphs, wherein the cultivation is carried out in a medium comprising sucrose as the sole carbon source. The method according to any one of the preceding paragraphs, wherein the cultivation is carried out in a medium comprising sucrose as the predominant carbon source. A whole broth formulation or cell culture composition comprising the mutant cell of any one of paragraphs 1-49. A composition comprising the mutant cell of any one of paragraphs 1 -49, and / or the whole broth formulation or cell culture composition according to paragraph 66. The composition of paragraph 67, wherein the composition is a food or feed product, or intermediate thereof. Use of the mutant and / or composition according to any one of the preceding paragraps in a method for production of a polypeptide of interest, e.g., a food or feed product. A method of producing a mutant of a parent cell, comprising mutating one or more first polynucleotide encoding a psi-producing oxygenase C (PpoC), which results in the mutant producing less of the PpoC than the parent cell. The method of paragraph 70, wherein the mutant is a mutant according to any one or paragraphs 1-49. A mutant cell produced by the method of paragraph 71.

Claims

Claims1. A mutant Aspergillus cell comprising in its genome one or more polynucleotide encoding a polypeptide of interest (POI), wherein the polynucleotide encoding the polypeptide of interest is heterologous to the mutant cell, and wherein expression and / or activity of one or more endogenous psi-producing oxygenase C (PpoC) is reduced or eliminated compared to a non-mutated otherwise isogenic cell or parent cell when cultivated under identical conditions.

2. The mutant according to claim 1, wherein the PpoC polypeptide comprises or consists of an amino acid sequence having a sequence identity of at least 80% to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 10, or SEQ ID NO: 12.

3. The mutant according to any one of the preceding claims, wherein the one or more PpoC is involved in the formation of one or more volatile agent, and wherein the mutant forms less of the one or more volatile agent compared to the non-mutated otherwise isogenic cell or parent cell when cultivated under identical conditions.

4. The mutant according to claim 5, wherein formation of the one or more volatile agent is decreased by at least 10%, e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, compared to the formation of the one or more volatile agent by the non-mutated otherwise isogenic cell or parent cell when cultivated under identical conditions, preferably decreased by at least 50%, more preferably decreased by at least 80%.

5. The mutant according to any one of claims 3-4, wherein the one or more volatile agent is selected from the list of 1-octen-3-one, (E)-Oct-2-enal, 1-octen-3-ol (Oct-1 -en-3-ol), 1 ,3 Ocatdiene, 3-Octanol, 3-Octanone, 3-cyclohexene-1-carboxaldehyde, 4-methyl- Triepoxydecane, 1,10-Dimethyl, or trans-9-decalinol.

6. The mutant according to any one of claims 3-5, wherein formation of 1-octen-3-ol is decreased by at least 10%, e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, compared to the formation of 1-octen-3-ol by the non-mutated otherwise isogenic cell or parent cell when cultivated under identical conditions, preferably decreased by at least 50%, even more preferably decreased by at least 80%.

437. The mutant according to any one of the preceding claims, wherein one or more polynucleotide encoding the PpoC comprises at least one alteration selected from the list of: a) a partial or full deletion of the one or more polynucleotide, b) a first heterologous promoter operably linked to the one or more polynucleotide, c) a mutated Shine-Dalgarno sequence operably linked to the one or more polynucleotide and derived from a parent Shine-Dalgarno sequence, d) one or more nucleic acid insertion, deletion, or substitution, and / or e) a premature stop codon.

8. A method for producing one or more polypeptides of interest, the method comprising a) providing a mutant cell according to any one of the preceding claims, b) cultivating said mutant cell under conditions conducive for expression of the one or more polypeptides of interest; and, c) optionally recovering the one or more polypeptide of interest.

9. A whole broth formulation or cell culture composition comprising the mutant cell of any one of claims 1-7.

10. A composition comprising the mutant cell of any one of claims 1-7, and / or the whole broth formulation or cell culture composition according to claim 9.

11. Use of the mutant and / or composition according to any one of the claims 1-7 and / or 9- 10 in a method for production of a polypeptide of interest, e.g., to produce a food or feed product.44

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