Modified gene improving protein secretion

Abstract

he present disclosure describes a genetically modified microbial host cell expressing and secreting a compound of interest, wherein the cell comprises a native gene involved in the regulation of secretion from the cell of the compound of interest with a signal peptide and / or without a signal peptide wherein the native gene has been modified to alter its functionality in the cell whereby the secretion and yield of the compound of interest from the cell is increased compared to a cell where the gene has not been modified.

Description

Case Ref. P268WO IPTector®Modified gene improving protein secretionTechnical Field

[0001] The present disclosure describes a genetically modified host cell comprising a modified native gene which greatly improves secretion and yields of proteins produced by the host cell.Background

[0002] UGT51 / atg26 has been described as a gene involved in autophagy / recycling of cellular components by many publications as well as involved in pathogenic processes in some plant-pathogen interactions (Asakura et al., 2009). There are significant differences in the role of the UGT51 / Atg26 protein in recycling of cellular components such as cytoplasm to vacuole transport, macroautophagy or pexophagy (Cao and Klionsky, 2007). In the yeast S. cerevisiae, UGT51 is responsible for the synthesis of ergosteryl beta-glycoside (SG). SG is a membrane component and SG levels increase in stress conditions in yeast. Interestingly, modification of the gene in Aspergillus oryzae (full gene deletion or deletions of the different protein domains of Atg26), affected autophagy and resulted in severe growth defects. Atg26 is involved in autophagy of different organelles in A. oryzae (Kikuma et al., 2016). No study has been published on the effect of sequence modification including single mutations in the Atg26 protein sequence, nor on the effect on the titer of secreted proteins.Summary

[0003] The present disclosure describes the identification of mutations in a gene corresponding to atg26 - AoAtg26 (herein referred to as UGT51) in the A. oryzae (Kikuma et al., 2017)) that result in an increase of protein production (titer) for proteins secreted using either the conventional (using a signal peptide) or unconventional (without) secretion pathway. This is the first report of a single gene in which sequence modifications increase protein titer on both secretion pathways.

[0004] The need for increased protein titers in precision fermentation is a critical aspect to make this technology cost-competitive with traditional farming, agriculture for bulk proteins like food ingredients, etc.

[0005] The present disclosure combines the identified modifications of UTG51 in comprehensively optimized strains that have been developed for decades and that already have a very high titer.

[0006] Accordingly, in a first aspect this disclosure describes a genetically modified microbial host cell expressing and secreting a compound of interest, wherein the cell comprises a native gene involved in the regulation of secretion from the cell of the compound of interest with a signal peptide and / orCase Ref. P268WO IPTector® without a signal peptide wherein the native gene has been modified to alter its functionality in the cell whereby the secretion of the compound of interest by the cell is increased compared to a cell where the gene has not been modified.

[0007] In another aspect this disclosure describes a cell culture, comprising the host cells described herein and a growth medium.

[0008] In another aspect this disclosure describes a method for producing a compound of interest comprising: a) culturing the cell culture described herein at conditions allowing the cell to produce the compound of interest; and b) optionally recovering and / or isolating the compound of interest.

[0009] In another aspect this disclosure describes a fermentation composition comprising the cell culture described herein and / or the compound of interest comprised in the cell culture.Description of drawings and figures

[0010] The figures included herein are illustrative and simplified for clarity, and they merely show details which are essential to the understanding of the invention, while other details may have been left out.

[0011] Figure 1 shows fluorescence used to identify mutant strains producing more protein (citrine).

[0012] Figure 2 shows the UGT51 G946S mutation identified by mutagenesis that increases protein titers of BLG. Protein titer of strain A0OIO8 (single copy fluorescent (citrine) strain), expression cassette with no signal peptide) and A0OI6O (isolated as a strong fluorescent UV-induced mutant of A0OIO8).

[0013] Figure 3 shows that the UGT51 G946S mutation identified by mutagenesis increases protein titers of BLG. Protein titer of strain A0OI6I (single copy BLG strain) and Ao0352 (same BLG cassette in the A0OI6O derived strain containing the UGT51 / Atg26 G946S mutation). The strains were run in duplicates.

[0014] Figure 4 shows the AoUGT51 locus and modifications.

[0015] Figure 5 shows forward engineering UGT51 mutations and full length UGT51 deletion. Lane 1: A0OIO8 AUGT51, Lane 2: Ao0353 (G946S mutation in A0OIO8), Lane 3: Ao0354 (C-term deletion in A0OIO8), Lane 4: Ao0355 (G1147fs mutation in A0OIO8), Lane 5: A0OIO8 (Single copy citrine strain), Lane 6: A0OI6O (UV mutant from A0OIO8).

[0016] Figure 6 shows that the UGT51 mutation in strain Ao0024 results in increased BLG titers. Relative copy number of the BLG gene in A. oryzae strains (A) and BLG titer in DWP cultivation as seen in SDS-PAGE (B). Lane M: Molecular Weight Marker; Lane 2: Ao0250; Lane 3: Ao0251; Lane 4: Ao0252;Case Ref. P268WO IPTector®Lane 5: Ao0024 (containing the G1147fs mutation in UGT51 / Atg26).Figure 7A shows that G946 is a highly conserved residue in UGT51 homologues. The disclosed sequences are UGT51's from Saccharomyces cerevisiae Q06321 (SEQ ID NO 13), Yarrowia lipolytica Q6C8M8 (SEQ ID NO 14), Komagataella phaffii Q9Y751 (SEQ ID NO 15), Aspergillus niger A2QNQ5 (SEQ ID NO 10), Aspergillus oryzae Q2U0C3QS (SEQ ID NO 16), Emericella nidulans Q5B4C9 (SEQ ID NO 17), Penicillium rubens A7KAN4 (SEQ ID NO 18) and Hypocrea jecorina G0RK64 (SEQ ID NO 19).Figure 7B shows that UGT51 is also conserved in other phylae like plants. The disclosed sequences are UGT's from Arabidopsis thaliana Q9M8Z7 (SEQ ID NO 20), Arabidopsis thaliana Q9XIG1 (SEQ ID NO 21), Nicotiana tabacum A0A1S4AH95 (SEQ ID NO 22), Nicotiana tabacum A0A1S4DRF0 (SEQ ID NO 23), Solanum tuberosum M1BAL7 (SEQ ID NO 24), Solanum tuberosum M1BAL8 (SEQ ID NO 25) and Chlamydomonas reinhardtii A8JIV0 (SEQ ID NO 26).

[0017] Figure 8 shows the level of identity already shown within fungi but especially in less related phyla / taxa

[0018] Figure 9 shows the UGT51 G951S mutation which results in a significant increase in production of bovine alpha-lactalbumin (a-LA) in A. niger. The position of non-glycosylated and glycosylated a-LA is shown by arrows.

[0019] Figure 10 shows the UGT51 G1152 stop mutation, truncation the expressed protein from amino acid 1152 which results in a significant increase in production of bovine alpha-lactalbumin (a- LA) in A. niger. The position of non-glycosylated and glycosylated a-LA is shown by arrows.

[0020] Figure 11 shows production of plectasin in the UGT51-1 strain background Ao0380. Culture supernatant from 4 day cultivation in YPM in deep well plate was run on an SDS PAGE. See the text in example 7 for strain information.Incorporation by reference

[0021] All publications, patents, and patent applications referred to herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event of a conflict between a term herein and a term in an incorporated reference, the term herein prevails and controls.Detailed Description

[0022] The technical features and advantages of the invention described herein are readily apparent to a person skilled in the art by the below detailed description of embodiments and examples with reference to the figures and drawings included herein.Case Ref. P268WO IPTector®Definitions

[0023] The term "heterologous" or "recombinant" or "genetically modified" and their grammatical equivalents as used herein interchangeably about nucleotides, polypeptides and cells refers to entities "derived from a different species or cell". For example, a heterologous or recombinant polynucleotide gene is a gene in a host cell not naturally containing that gene, i.e. the gene is from a different species or cell type than the host cell. A heterologous or recombinant polypeptide is a polypeptide produced in a host cell not naturally containing the polypeptide, i.e. the polypeptide is from a different species or cell type than the host cell. Where the terms as used herein about host cells, they refer to host cells comprising and expressing heterologous or recombinant polynucleotides.

[0024] The term "% identity" is used herein about the relatedness between two amino acid sequences or between two nucleotide sequences usings standard alignment software known in the art, and applying settings as instructed for the software, including gaps, to achieve the maximum percent identity / similarity / homology and, if necessary, considering any conservative substitutions according to the NCIUB rules (hftp: / / www.chem. qmul.ac.uk / iubmb / misc / naseq.html; NC-IUB, Eur. J. Biochem. (1985)), as part of the sequence identity. 5' or 3' extensions nor insertions (for nucleic acids) or N' or C' extensions nor insertions (for polypeptides) usually result in a reduction of identity, similarity or homology using such standard software. For example the "% sequence identity", as used herein, can be calculated from e.g. two amino acid sequences as follows: The sequences are aligned using Version 9 of the Genetic Computing Groups GAP (global alignment program), using the default BLOSUIVI62 matrix (see below) with a gap open penalty of -12 (for the first null of a gap) and a gap extension penalty of -4 (for each additional null in the gap). After alignment, percentage identity is calculated by expressing the number of matches as a percentage of the number of amino acids in the reference amino acid sequence using the following BLOSUIVI62 matrix:Case Ref. P268WO IPTector®SerThrTv-rVaiHis lie Leu Lys Met Phe Pro Ser Thr Trp Tyr Vat

[0025] "Reference sequence" refers to a defined sequence to which another sequence is compared.

[0026] The term "host cell" refers to any cell type that is susceptible to transformation, transfection, transduction, or the like with a nucleic acid construct or expression vector comprising a polynucleotide to be expressed in the host cell. Host cell encompasses any progeny of a parent cell including those that are not identical to the parent cell due to mutations that occur during replication.

[0027] "Polypeptide" and "protein" are used interchangeably herein to denote a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post- translational modification (e.g., glycosylation, phosphorylation, lipidation, myristylation, ubiquitination, etc.). Included within this definition are D- and L-amino acids, and mixtures of D- and L-amino acids.

[0028] The term "comprise" and "include" as used throughout the specification and the accompanying items as well as variations such as "comprises", "comprising", "includes" and "including" are to be interpreted inclusively. These words are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context allows.

[0029] The articles "a" and "an" are used herein refer to one or to more than one (i.e. to one or at least one) of the grammatical object of the article. By way of example, "an element" may mean one element or more than one element.

[0030] Terms like "preferably", "commonly", "particularly", and "typically" are not utilized herein to limit the scope of the itemized invention nor to imply that certain features are critical, essential, or even important to the structure or function of the itemed invention. Rather, these terms are merely intended to highlight alternative attractive or additional features that may or may not be utilized in a particular embodiment of the present invention.

[0031] The term "cell culture" as used herein refers to a culture medium comprising a plurality of theCase Ref. P268WO IPTector® host cells described herein. A cell culture may comprise a single strain of host cells or may comprise two or more distinct host cell strains. The culture medium may be any medium that may comprise a recombinant host, e.g., a liquid medium (i.e., a culture broth) or a semi-solid medium, and may comprise additional components, e.g., a carbon source; a nitrogen source; a phosphate source; vitamins; trace elements; salts; amino acids; nucleobases; and the like.

[0032] Term "endogenous" or "native" as used herein refers to a gene or a polypeptide in a host cell which originates from the same host cell.

[0033] The terms "substantially" or "approximately" or "about", as used herein refers to a reasonable deviation around a value or parameter such that the value or parameter is not significantly changed. These terms of deviation from a value should be construed as including a deviation of the value where the deviation would not negate the meaning of the value deviated from. For example, in relation to a reference numerical value the terms of degree can include a range of values plus or minus 10% from that value. For example, deviation from a value can include a specified value plus or minus a certain percentage from that value, such as plus or minus 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from the specified value.

[0034] Where a numerical limit or range is stated herein, the endpoints are included. Also, all values and sub ranges within a numerical limit or range are specifically included as if explicitly written out.

[0035] The term "and / or" as used herein is intended to represent an inclusive "or". The wording X and / or Y is meant to mean both X or Y and X and Y. Further the wording X, Y and / or Z is intended to mean X, Y and Z alone or any combination of X, Y, and Z.

[0036] The term "isolated" as used herein about a compound, refers to any compound, which by means of human intervention, has been put in a form or environment that differs from the form or environment in which it is found in nature. Isolated compounds include but are not limited to compounds of the disclosure for which the ratio of the compounds relative to other constituents with which they are associated in nature is increased or decreased. In an important embodiment the amount of compound is increased relative to other constituents with which the compound is associated in nature. In an embodiment the compound of the disclosure may be isolated into a pure or substantially pure form. In this context a substantially pure compound means that the compound is separated from other extraneous or unwanted material present from the onset of producing the compound or generated in the manufacturing process. Such a substantially pure compound preparation contains less than 10%, such as less than 8%, such as less than 6%, such as less than 5%, such as less than 4%, such as less than 3%, such as less than 2%, such as less than 1 %, such as less than 0.5% by weight of other extraneous or unwanted material usually associated with the compound when expressed natively or recombinantly. In an embodiment the isolated compound is at least 90%Case Ref. P268WO IPTector® pure, such as at least 91% pure, such as at least 92% pure, such as at least 93% pure, such as at least 94% pure, such as at least 95% pure, such as at least 96% pure, such as at least 97% pure, such as at least 98% pure, such as at least 99% pure, such as at least 99.5% pure, such as 100 % pure by weight.

[0037] The term "deletion" as used herein in the context of polynucleotides and genes refers to the manipulation of a gene so that it is no longer expressed in a host cell.

[0038] The term "disruption" as used herein refers to manipulation of a gene or any of the machinery participating in the expression the gene, so that it is no longer expressed in a host cell.

[0039] The term "attenuation" as used herein refers to manipulation of a gene or any of the machinery participating in the expression the gene, so that it the expression of the gene is reduced as compared to expression without the manipulation.

[0040] All methods described herein can be performed in any suitable order of steps unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0041] All percentages, ratios and proportions herein are by weight, unless otherwise specified. A weight percent (weight %, also as wt. %) of a component, unless specifically stated to the contrary, is based on the total weight of the composition in which the component is included (e.g., on the total amount of the reaction mixture).

[0042] Unless specifically defined herein, all technical and scientific terms used have the same meaning as commonly understood by a skilled person in the fields of biochemistry, genetics, and microbiology.

[0043] All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, with suitable methods and materials being described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, shall prevail. Further, the materials, methods, and examples are illustrative only and are not intended to be limiting, unless otherwise specified.

[0044] Practice of the invention described herein employs, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, and recombinant DNA methodologies, which are available to the person skilled in the art. Such techniques are explained fully in the literature. See, for example, Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and son Inc, Library of Congress, USA); Molecular Cloning: ACase Ref. P268WO IPTector®Laboratory Manual, Third Edition, (Sambrook et al, 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Harries & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the series, Methods In ENZYMOLOGY (J. Abelson and M. Simon, eds. -in-chief, Academic Press, Inc., New York), specifically, Vols.154 and 155 (Wu et al. eds.) and Vol. 185, "Gene Expression Technology" (D. Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes l-IV (D. M. Weir and C. C. Blackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

[0045] The term "paralogs" as used herein about genes refers to genes that has arisen through duplication within the same genome and has evolved to perform a similar function compared to its original gene counterpart. These genes, known as paralogous genes, are typically part of a gene family within the same organism and often retain some sequence similarity due to their shared ancestry.

[0046] The term "orthologs" as used herein about genes refers to genes in different species that originated from a common ancestral gene through a speciation event. These genes generally retain similar sequences and functions across different organisms, as they continue to perform equivalent roles in their respective species.

[0047] Any reference in a claim or embodiment to a range of other embodiments or claims is to be interpreted as an independent reference to each single member or group of members in the said range of embodiments or claims.Genetically modified microbial host cells

[0048] As described supra, in a first aspect a genetically modified microbial host cell is provided expressing and secreting a compound of interest, wherein the cell comprises a native gene involved in the regulation of secretion from the cell of the compound of interest with a signal peptide and / or without a signal peptide wherein the native gene has been modified to alter its functionality in the cell whereby the secretion of the compound of interest by the cell is increased compared to a cell where the gene has not been modified.

[0049] In some embodiments the modification of the native gene in the host cell comprises deletion, mutation, and / or attenuation of the expression of the gene.

[0050] In some embodiments the reduction in expression of the native gene comprises sequenceCase Ref. P268WO IPTector® modifications (base pair change, small deletions or insertions or even disruption of the full-length gene.

[0051] In some embodiments the modification of the native gene increases secretion of the compound of interest both with and without a signal peptide.

[0052] In some embodiments the native gene is involved in secretion or the autophagy of the host cell, optionally the recycling of cellular components and / or organelles.

[0053] In some embodiments the native gene corresponds to UGT51 (atg26 - AoAtg26) in a fungal cell, optionally a filamentous fungus or a yeast cell or any of its paralogs or orthologs.

[0054] In some embodiments the native gene corresponds to UGT51(atg26 - AoAtg26) in Aspergillus oryzae (SEQ ID NO 1) or in Aspergillus niger (SEQ ID NO: 9) or any of their paralogs or orthologs.

[0055] Relevant paralogs or orthologs of a UGT51 gene can suitably be a paralog within the species, genus, family, order, class, phylum, kingdom or domain of the microbial host. Relevant paralogs or orthologs of a UGT51 in Aspergillus oryzae or Aspergillus niger, can be those of other aspergilly, of other filamentous fungi, of other fungi such as yeast, or of bacteria and so on.

[0056] In some embodiments the native gene has at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 1 or SEQ ID NO: 9.

[0057] In some embodiments the native UGT51 gene comprises a mutation causing an amino acid mutation in the expressed protein at a position corresponding to G946 in SEQ ID NO: 2 or G951 in SEQ ID NQ:10.

[0058] In some embodiments the modification of the native UGT51 gene comprises a mutation causing an amino acid mutation in the expressed protein corresponding to G946S in SEQ ID NO: 2 or G951S in SEQ ID NQ:10. SEQ ID NO. 3 or 29 and 4 respectively shows the modified gene and expressed modified protein with the G946S substitution in A. oryzae UGT51, while SEQ ID NO. 27 and 28 respectively shows the modified gene and expressed modified protein with the G951S subsstitution in A. niger UGT51.

[0059] In some embodiments the modification of the native gene comprises a mutation corresponding to G946 or any of its paralogs or orthologs having at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 1.

[0060] In another embodiment the modification of the native UGT51 gene comprises a mutation corresponding to nucleotide position 2886 in the UGT51 gene of SEQ ID NO: 1 or nucleotide positionCase Ref. P268WO IPTector®2901 in the UGT51 gene of SEQ ID NO:9 or any of their paralogs or orthologs having at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 1 or SEQ ID NO: 9 respectively.

[0061] In some embodiments the modification of the native gene comprises a mutation corresponding to G946S of SEQ ID NO: 3 or 29 or any of its paralogs or orthologs having at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 3 or 29. In some embodiments the modification of the native UGT51 gene is a G to A substitution in the nucleotide corresponding to nucleotide position G2886A in the UGT51 gene of SEQ ID NO: 1 or nucleotide position G2901A in the UGT51 gene of SEQ ID NO:9 or any of their paralogs or orthologs having at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 1, or 9 respectively.

[0062] A substition of nucleotide position G2886A in the UGT51 gene in A. Oryzae (SEQ ID NO: 1 (wt)) gives protein mutation G946S found in SEQ ID NO: 4. The corresponding mutant gene sequence is found in SEQ ID NO: 3 or 29.

[0063] A substition of nucleotide position G2901A in the UGT51 gene in A. niger (SEQ ID NO:9 (wt)) gives protein mutation G951S found in SEQ ID NO: 28. The corresponding mutant gene sequence is found in SEQ ID NO: 27.

[0064] In some embodiments the modification of the native UGT51 gene comprises a frameshift mutation causing a truncation of the expressed protein.

[0065] In some embodiments the modified expressed protein comprises a truncation from a position corresponding to amino acid 1157 of SEQ ID NO: 2. The modified UGT51 gene encodig such protein may suitably comprise a nucleotide sequence as set forth in SEQ ID NO: 7 and the expressed protein may comprise an amino acid sequence as set forth in SEQ ID NO: 8. The modification of the native UGT51 gene suitably comprises a mutation (base pair deletion) corresponding to nucleotide position G3491 in the UGT51 gene of SEQ ID NO: 1 or any of its paralogs or orthologs having at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 1, said mutation causing a truncation in the expressed protein from a position corresponding to 1157 of SEQ ID NO: 2. This nucleotide deletion causes a frameshift and thereby a trucation of the expressed protein from amino acid 1157.

[0066] In another embodiment the modified expressed protein comprises a truncation from aCase Ref. P268WO IPTector® position corresponding to amino acid 1152 of SEQ ID NO: 10. The modified UGT51 gene encodig such protein may suitably comprise a nucleotide sequence as set forth in SEQ ID NO: 11 and the expressed protein may comprise an amino acid sequence as set forth in SEQ ID NO: 12. The modification of the native UGT51 gene suitably comprises a substitution corresponding to nucleotide position G3504T in the UGT51 gene of SEQ ID NO: 9 or any of its paralogs or orthologs having at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 9, said mutation causing a truncation in the expressed protein from a position corresponding to 1152 of SEQ ID NO: 10. This nucleotide deletion also causes a frameshift and thereby a trucation of the expressed protein from amino acid 1152.

[0067] In some embodiments the modification of the native gene comprises a mutation corresponding to deletion of nucleotide positions 946 to 1904 (SEQ ID NO: 5) in the UGT51 gene of SEQ ID NO: 1 or any of its paralogs or orthologs having at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 1.

[0068] In some embodiments the modification of the native gene comprises a G to A substitution of the nucleotide corresponding to position 2886 of SEQ ID NO: 1 or any of its paralogs or orthologs having at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 1.

[0069] In some embodiments the modification of the native gene comprises a deletion of nucleotide G corresponding to position 3491 (SEQ ID NO: 7) of SEQ ID NO: 1 or any of its paralogs or orthologs having at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 1, resulting in expression of a truncated UGT51 protein.

[0070] In some embodiments the modification of the native gene comprises a deletion of the entire gene corresponding to SEQ ID NO: 1 or SEQ ID NO: 9 or any of their paralogs or orthologs having at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 1 or SEQ ID NO: 9, repectively.

[0071] In some embodiments the modification of the native gene increases the secretion of the compound of interest by at least 10%, such as at least 25%, such as at least 50%, such as at least 50%, such as at least 50%, such as at least 50%, such as at least 50%, such as at least 50%, such as at least 50%, such as at least 50%.Case Ref. P268WO IPTector®

[0072] In another aspect, the compound of interest is selected from proteins optionally proteins selected from dairy proteins, egg proteins, enzymes, antibodies, hormones, biocides, antimicrobial peptides, structural proteins, and functional proteins.

[0073] In some embodiments the protein is recombinant protein.

[0074] In some embodiments the protein is a dairy protein.

[0075] In some embodiments the dairy protein is a lactoglobulin or a casein.

[0076] In some embodiments the lactoglobulin is p-lactoglobulin, a-lactalbumin, and / or Immunoglobulin.

[0077] In some embodiments the casein is asl-Casein (Alpha-Si Casein), as2-Casein (Alpha-S2 Casein), P-Casein (Beta-Casein), K-Casein (Kappa-Casein), and / or y-Casein (Gamma-Casein).

[0078] In some embodiments the protein is selected from albumin, alpha-lactalbumin, betalactoglobulin, caseins, collagen, hemoglobin, myoglobin, insulin, lysozyme, ovalbumin, lactoferrin, osteopontin, Nisin, Pediocin, Reuterin, Colicin, Enterocin, Natamycin, Defensin, Bacillomycin, Erythropoietin (EPO), Human Growth Hormone (hGH), Interferons (IFNs), Monoclonal Antibodies (mAbs), Granulocyte Colony-Stimulating Factor (G-CSF), Factor VIII, Tumor Necrosis Factor (TNF),Thyroid Hormone, Alteplase (tPA), Etanercept, Foil itropin. Antithrombin.

[0079] In some embodiments the microbial host cell is a eukaryotic, a prokaryotic or an algal cell.

[0080] In some embodiments the eukaryotic cell is a fungal cell, optionally a filamentous fungus or a yeast cell.

[0081] In some embodiments the filamentous fungus is selected from the genus of Aspergillus,Trichoderma, or Rhizopus.

[0082] In some embodiments the filamentous fungus is selected from the species Aspergillus sp, Aspergillus oryzae, Aspergillus niger, Trichoderma sp, or Rhizopus sp.

[0083] In some embodiments the yeast is selected from the genus of Komagataella, Pichia, Saccharomyces, Yarrowia, or Hansenula.

[0084] In some embodiments the yeast is selected from the species of Komagataella phaffii, S. cerevisiae, Y. lipolytica, or Hansenula sp.

[0085] In some embodiments the composition comprises a host cell and a growth medium.

[0086] In some embodiments is comprised a method for producing a compound of interest:a. culturing the cell culture at conditions allowing the cell to produce the compound of interest; and b. optionally recovering and / or isolating the compound of interest.

[0087] In some embodiments the method further comprises one or more elements selected from: a) culturing the cell culture in a nutrient medium; b) culturing the cell culture under aerobic or anaerobic conditionsCase Ref. P268WO IPTector® c) culturing the cell culture under agitation; d) culturing the cell culture at a temperature of between 25 to 50 °C; e) culturing the cell culture at a pH of between 3-9; and f) culturing the cell culture for between 10 hours to 30 days.

[0088] In some embodiments the method further comprises recovery and / or isolation step comprises separating a liquid phase of the cell or cell culture from a solid phase of the cell or cell culture to obtain a supernatant comprising the compound of interest and / or subjecting the supernatant to one or more steps selected from: a) separating the supernatant from the solid phase of the cell culture, such as by filtration or gravity separation; b) contacting the supernatant with one or more adsorbent resins to obtain at least a portion of the produced protein; c) contacting the supernatant with one or more ion exchange or reversed-phase chromatography columns to obtain at least a portion of the protein; d) extracting the modified protein;; and / or e) precipitating the modified protein; by crystallization or evaporating the solvent of the liquid phase; and optionally isolating the modified protein; by filtration or gravity separation; thereby recovering and / or isolating the protein.

[0089] In some embodiments the fermentation composition comprising the cell culture and / or the compound of interest comprised therein.

[0090] In some embodiments at least 50%, such as at least 75%, such as at least 95%, such as at least 99% of solid cell material has been separated from the fermentation composition.

[0091] In some embodiments the fermentation composition comprises one or more compounds selected from trace metals, vitamins, salts, yeast nitrogen base, carbon source, YNB, and / or amino acids of the fermentation; wherein the concentration of the compound of interest is at least 1 mg / kg composition.

[0092] In some embodiments the fermentation composition further comprising one or more carriers, agents, additives and / or excipients.

[0093] Having generally described the inventions contained herein, a further understanding can be obtained by reference to specific examples, which are provided below for purposes of illustration only, not intended to be limiting unless otherwise specified.ExamplesCase Ref. P268WO IPTector®Materials and methods

[0094] Chemicals used in the examples herein, e.g. for buffers and substrates, are commercial products of at least reagent grade.

[0095] General used methods of PCR, cloning, etc. were well-known in the art and can for example be found in in " Molecular cloning: A laboratory manual ", Sambrook et al. (1989), Cold Spring Harbor lab., Cold Spring Harbor, N.Y.; Ausubel, F.M. et al. (eds.); " Current protocols in Molecular Biology ", John Wiley and Sons, (1995); Harwood , C. R. , and Cutting, S. M. (eds.). Copy number determination was performed using a QIAcuity digital PCR system, according to the manufacturer's recommendations (Qiagen).Example 1 - A mutation in the A. oryzae UGT51 / Atg26 encoding gene results in an unexpectedly high titer increase.

[0096] Classical mutagenesis is a well-established approach to increase protein yields. This requires a method providing an easy, HTS-friendly read-out, such as a fluorescent signal. We used UV- mutagenesis of spores of A. oryzae strain A0OIO8 that contains a single copy, signal peptide-less expression cassette for citrine, a GFP-like fluorescent protein. Spores were plated after mutagenesis and treatments resulting in 95-99 % killing were examined to identify colonies showing strong fluorescence (Fig.l). One of these clones, strain A0OI6O, was shown to produce more than a 2-fold increase in secrete citrine, as judged from SDS-PAGE of culture supernatants derived from Deep Well plate cultivation (Fig. 2). Illumina DNA sequencing and genome analysis of strain A0OI6O, A0OIO8 and the common predecessor of both strains was performed. In this way, analysis of SNPs identified a point mutation in the coding region of the Aspergillus oryzae UGT51 / atg26 gene in strain A0OI6O. This mutation results in a single amino acid substitution (Gly946Ser or G946S) in the catalytic (CAT) domain of UGT51 / Atg26. This domain is involved in autophagy in A. oryzae (Kikuma et al., 2017). The G946S mutation (SEQ ID NO: 3 or 29) in UGT51 results in an increase in protein titers for proteins following the unconventional (e.g., citrine) and the conventional (e.g., bovine lactoglobulin, BLG) pathway.Example 2. A single base pair (bp) deletion in the A. oryzae UGT51 / Atg26 encoding gene identified in a high producer strain containing multiple copies of a gene encoding bovine lactoglobulin (BLG).

[0097] Genome analysis of a BLG highly producer A. oryzae strain identified a single bp deletion (G1147fs) (SEQ ID NO: 7) in the same gene (UGT51 / atg26) that results in a change (due to spontaneous mutation) from Glyll47 to a frameshift, leading to a short C-terminal extension og the UGT51 / Atg26 protein. A comparison between highly producer BLG strains containing a similar orCase Ref. P268WO IPTector® higher number of copies of the BLG gene integrated at the same chromosomal location showed that the presence of the G1147fs mutation resulted in a significant increase in protein titers (Fig. 3).Thus, the G1147fs is the result of a spontaneous mutation that was identified in the same gene / function (UGT51 / Atg26) as the G846S mutation identified using classical mutagenesis. Taken together, these results indicate that 1) UGT51 is involved in a key function in secretion for both conventional and unconventional pathways and 2) moderate alterations of the amino acid sequence of UGT51 / Atg26 can lead to unexpectedly higher protein titers.Example 3. Reverse engineering the G946S mutation demonstrates the titer increase.

[0098] The UGT51 / Atg26 G946S mutation present in strain A0OI6O resulted in a significant increase in protein titer for a protein that is secreted through the unconventional secretion pathway (e.g., citrine with no signal peptide). To investigate whether this mutation also resulted in titer changes for a protein that is secreted through the conventional secretion pathway, the citrine cassette present in strain A0OI6O was removed using chlorate resistance resulting in strain Ao0303. Shortly the citrine gene is integrated between the niaD and niiA genes. The strain can grow in nitrate as a sole nitrogen source. Plating in medium containing chlorate selects strains that have undergone a complete excision of the citrine gene. Strain Ao0303 is therefore chlorate resistant and cannot grow in nitrate. Then, an expression cassette for bovine lactoglobulin (BLG) with a signal peptide was inserted at this location in Ao0303 resulting in strain Ao0352. Remarkably, comparing BLG titers in a near isogenic strain containing the BLG gene and the same SP integrated at the same location (A0OI6I) with Ao0352 demonstrated that the same mutation in UGT51 / Atg26 also resulted in an increase of BLG titer (Fig. 4).To our knowledge this is the first demonstration of a single mutation that contributes to significant titer increase for proteins that follow the conventional and unconventional pathway. UGT51 / Atg26 has been involved in recycling of cellular components and organelles (peroxisomes, mitochondria, etc.; Kikuma et al., 2017). Truncation or deletion of UGT51 / atg26 in A. oryzae affects autophagy, growth and sporulation (Kikuma et al., 2017). It Is tempting to speculate that the identified mutation affects the decision between degradation of vesicles containing the protein at the vacuole or secretion. It is possible to speculate that since both secretion pathways require protein transport vesicles and the G946S mutation increases titers of both citrine and BLG, this change may alter (and increase) the share of protein vesicles that proceed through secretion and escape degradation.An overview of the modifications of the UGT51 gene studied is shown in Fig. 5 and Table 1.Table 1: Overview of the modifications of the UGT51 geneCase Ref. P268WO IPTector®Example 4. Deletion of the full coding sequence of UGT1 does not affect growth and still results in high protein yields.

[0099] It has previously been shown that a deletion of the UGT51 gene / atg26 resulted in severe growth defects including sporulation in A. oryzae (Kikuma et al., 2017). Surprisingly, no growth defect was observed for strain Ao0108mutl that contains a full-length deletion (AUGT51) of the UGT51 gene (Fig. 6). Additionally, a higher titer of citrine production was observed in strain Ao0108mutl carrying a deletion of the full-length coding sequence of the UGT51 gene. Taken together, these results demonstrate that strains carrying a deletion of the UGT51 gene lead to a higher production of recombinant proteins in fungal cell factories like A. oryzae.The G1147fs mutation in UGT51 does not seem to affect protein titers for proteins following the unconventional pathway (e.g., citrine). In fact, the deletion of the gene increases protein titers at least for proteins secreted through the unconventional pathway.Example 5. Forward engineering the UGT51 G946S mutation results in high protein titer

[0100] To unambiguously define the role of the UGT51 G946S mutation on protein titer, we introduced the SNP responsible for the mutation in strain A0108. The resulting strain (Ao0353) produced remarkably higher levels of citrine compared to A0OIO8 but also higher than A0OI6O. These results suggest that other SNPs present in the genome of A0OI6O may have a negative effect on titers and that the G946S mutation alone leads to a severe increase in protein titers. Similarly, introducing a deletion in the CAT, C-terminal domain of UGT51, a moderate increase in citrine was observed (strain Ao0354, lane 3, Fig. 5). Additionally, no effect was evident when introducing the G1147fs mutation into A0OIO8 (strain Ao0355, lane 4, Fig. 5), indicating that this modification leadsCase Ref. P268WO IPTector® to increased protein titers mainly for proteins secreted through the conventional pathway.G946 is a highly conserved residue in UGT51 homologues (figure 7A). UGT51 is also conserved in other phylae like plants (figure 7B). Fig. 8 shows % identity to illustrate the identity already shown within fungi but especially in less related phyla / taxa..Example 6. Modification of the UGT51 gene in Aspergillus niger results in improved bovine alphalactalbumin protein titer

[0101] As demonstrated in the previous examples the different exemplified mutations in the UGT51 gene resulted in significant increase in protein titer for bovine beta-lactoglobulin in Aspergillus oryzae. The UGT51 amino acid sequence is highly conserved in yeast, fungi and plants, tin this example, the effect of mutation in UGT51 gene (SEQ NO: 9) in Aspergillus niger was investigated. The A. niger UGT51 coding sequence was modified to introduce an amino acid substitution, G951S, in A. niger strain, An21LS044, expressing the bovine alpha-lactalbumin (a-LA, Uniprot entry P00711). G951 is a highly conserved residue present in UGT51 corresponding to G946 in the A. oryzae UGT51 protein sequence (see figure 7A) and the G951S modified protein sequence from A. niger corresponds to the G946S UGT51-1 G946S modified protein from A. oryzae (SEQ ID NO: 4 . The G951S modification was done by co-transforming 96 bp double-stranded DNA fragment and CRISPR plasmid containing protospacer, PS2, that directed DNA cleavage at the coding region of the wild-type UGT51 sequence. Strains containing the correct G951S modification were identified. Micro-titer plate (MTP) cultivation was performed on strains with the G951S modification of the UGT51 and wild-type UGT51 strains for 7 days at 30 °C and samples from culture supernatants were run on SDS-PAGE (see figure 9). The relative improvement in bovine alpha-lactalbumin titer in the strains with the G951S substitution of the UGT51 was approximately two times greater compared to parent strain, An21LS044, containing the wild-type UGT51.

[0102] Similarly, the Aspergillus niger UGT51 coding sequence (SEQ ID NO: 9) was modified to introduce amino acid substitution, G1151stop in A. niger strain, An21LS044, expressing the bovine alpha-lactalbumin. This modification corresponded to the modification G1147fs of SEQ ID NO: 8 (UGT51-3) which showed increased production of protein in A. oryzae. The modification was done by co-transforming 95 bp double-stranded DNA fragment and CRISPR plasmid containing protospacer, PS3, that directs DNA cleavage at the coding region of the wild-type UGT51 sequence. Strains containing the correct G1151stop modification were identified. Micro-titer plate (MTP) cultivation was performed on strains with the G1151 stop modification of the UGT51 and wild-type UGT51 strains for 7 days at 30°C and samples from culture supernatants were run on SDS-PAGE (Figure 10). The relative improvement in bovine alpha-lactalbumin titer in the strains with the G1151stop substitution of theCase Ref. P268WO IPTector®UGT51 was approximately two times greater compared to parent strain, An21LS044, containing the wild-type A. niger UGT51. Thus, as shown in Fig 10, the UGT51 sequence in Aspergill i and other fungi (here represented by A. oryzae and A. niger) is highly conserved, changes in the UGT51 protein sequence is contemplated to have similar effect in multiple fungal species and others, where such modifications in UGT51 is believed to increase protein titers.Example 7. Production of small peptides is significantly increased in a strain containing a mutation in the UGT51gene

[0103] In this example the effect the UGT51-1 mutation (SEQ ID NO: 3 / 29 and 4) on the production and secretion of a small antimicrobial peptide (plectasin from the fungus Pseudoplectania nigrella, Uniprot entry Q53I06). Strains of Aspergillus oryzae expressing 1 (Ao0580) or 2 copies (Ao0581) in a wildtype UGT51 background produce plectasin in correlation to the copy number, as the two-copy strain produces approximately two-fold higher amount of plectasin (Figure 11). Both Ao0580 and Ao0581 had a wildtype UGT51 gene. A strain Ao557 was constructed from strain Ao0580 was the background strain containing modification corresponding to the G946S mutation in the UGT51 gene (SEQ ID NO: 3 / 29 UGT51-1). Strain Ao0557 was constructed by introduction of a single copy of the plectasin gene into Ao0380. Strain Ao0557 produced more than a two-fold higher amount of plectasin compared to Ao0581 (two copy plectasin strain) or more than 4x compared to the single copy strain Ao0580 at lab scale (deep well plates, Figure 11). The plectasin expression cassette(s) was integrated by homologous recombination at the same chromosomal location to ensure comparability of the protein titer.ReferencesAsakura et al. (2009) Atg26-mediated pexophagy is required for host invasion by the plant pathogenic fungus Colletotrichum orbiculare. Plant Cell 21:1291-1304Cao and Klioonsky (2007) Atg26 is Not Involved in Autophagy-Related Pathways in Saccharomyces cerevisiae. Autophagy 3:17-20Kikuma et al. (2017) AoAtg26, a putative sterol glucosyltransferase, is required for autophagic degradation of peroxisomes, mitochondria, and nuclei in the filamentous fungus Aspergillus oryzae. Biosci Biotech Biochem 81:384-395Rudinskiy and Molinari (2023) ER-to-lysosome-associated degradation in a nutshell: mammalian,Case Ref. P268WO IPTector® yeast, and plant ER-phagy as induced by misfolded proteins. FEBS Lett 597:1928-1945Watanabe et al. (2015) Ergosteryl-b-glucosidase (Eghl) involved in sterylglucoside catabolism and vacuole formation in Saccharomyces cerevisiae. Glycobiol 25:1079-1089Sequence listings

[0104] The present application contains a listing of sequences described in the below table 2 submitted electronically in ST26 format which is hereby incorporated by reference in its entirety. Table 2. Sequences:Case Ref. P268WO IPTector®

Claims

Case Ref. P268WO IPTector®Claims1. A genetically modified microbial host cell expressing and secreting a compound of interest, wherein the cell comprises a native gene involved in the regulation of secretion from the cell of the compound of interest with a signal peptide and / or without a signal peptide wherein the native gene has been modified to alter its functionality in the cell whereby the secretion of the compound of interest by the cell is increased compared to a cell where the gene has not been modified.

2. The host cell of claim 1 wherein the modification of the native gene comprises deletion, mutation, and / or attenuation of the expression gene.

3. The host cell of any precedingclaim wherein the reduction in expression of the native gene comprises sequence modifications (base pair change, small deletions or insertions or even disruption of the full-length gene.

4. The host cell of any preceding claim wherein the modification of the native gene increases secretion of the compound of interest both with and without a signal peptide.

5. The host cell of any preceding claim wherein the native gene is involved in secretion or the autophagy of the host cell, optionally the recycling of cellular components and / or organelles.

6. The host cell of any preceding claim wherein the native gene corresponds to UGT51 (atg26 - AoAtg26) in a fungal cell, optionally a filamentous fungus or a yeast cell or any of its paralogs or orthologs.

7. The host cell of claim 6 wherein the native gene corresponds to UGT51 (atg26 - AoAtg26) in Aspergillus oryzae (SEQ ID NO 1) or in Aspergillus niger (SEQ ID NO: 9) or any of their paralogs or orthologs.

8. The host cell of claim 7 wherein the native gene has at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 1 or SEQ ID NO: 9.Case Ref. P268WO IPTector®9. The host cell of any one of claims 7 to 8 wherein the modification of the native UGT51 gene comprises a mutation causing an amino acid mutation in the expressed protein at a position corresponding to G946 in SEQ ID NO: 2 or G951 in SEQ ID NO:10.

10. The host cell of any one of claims 9 wherein the modification of the native UGT51 gene comprises a mutation causing an amino acid mutation in the expressed protein corresponding to G946S in SEQ ID NO: 2 or G951S in SEQ ID NQ:10.

11. The host cell of any one of claims 7 to 10 wherein the modification of the native UGT51 gene comprises a mutation corresponding to nucleotide position G2886 in the UGT51 gene of SEQ ID NO: 1 or nucleotide position G2901 in the UGT51 gene of SEQ ID NO:9 or any of their paralogs or orthologs having at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 1 or SEQ ID NO: 9 respectively.

12. The host cell of any one of claims 11 wherein the modification of the native UGT51 gene is a G to A substitution in the nucleotide corresponding to nucleotide position G2886A in the UGT51 gene of SEQ ID NO: 1 or nucleotide position G2901A in the UGT51 gene of SEQ ID NO:9 or any of their paralogs or orthologs having at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 1, or 9 respectively.

13. The host cell of any preceding claim wherein the modification of the native UGT51 gene comprises a frameshift mutation causing a truncation of the expressed protein.

14. The host cell of claim 13 wherein the expressed protein comprises a truncation from a position corresponding to amino acid 1157 of SEQ ID NO: 2.

15. The host cell of claim 14 wherein the modified UGT51 gene comprises a nucleotide sequence as set forth in SEQ ID NO: 7 and the expressed protein comprises an amino acid sequence as set forth in SEQ ID NO: 8.

16. The host cell of any one of claims 13 to 15 wherein the modification of the native UGT51 gene comprises a mutation (base pair deletion) corresponding to nucleotide position G3491 in the UGT51Case Ref. P268WO IPTector® gene of SEQ ID NO: 1 or any of its paralogs or orthologs having at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 1, said mutation causing a truncation in the expressed protein from a position corresponding to G1157 of SEQ ID NO: 2.

17. The host cell of claim 13 wherein expressed protein comprises a truncation from a position corresponding to amino acid 1152 of SEQ ID NO: 10.

18. The host cell of claim 17 wherein the modified UGT51 gene comprises a nucleotide sequence as set forth in SEQ ID NO: 11 and the expressed protein comprises an amino acid sequence as set forth in SEQ ID NO: 12.

19. The host cell of any one of claims 17 to 18 wherein the modification of the native gene comprises a substitution corresponding to nucleotide position G3504T in the UGT51 gene of SEQ ID NO: 9 or any of its paralogs or orthologs having at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 9, said mutation causing a truncation in the expressed protein from a position corresponding to 1152 of SEQ ID NO: 10.

20. The host cell of any one of claims 7 to 8 wherein the modification of the native gene comprises a mutation corresponding to deletion of nucleotide positions 946 to 1904 (SEQ ID NO: 5) in the UGT51 gene of SEQ ID NO: 1 or any of its paralogs or orthologs having at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 1.

21. The host cell of any one of claims 7 to 8 wherein the modification of the native gene comprises a deletion of the entire gene corresponding to SEQ ID NO: 1 or SEQ ID NO: 9 or any of its paralogs or orthologs having at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 1 or 9, respectively.Case Ref. P268WO IPTector®22. The host cell of any one of claim 7 to 21 wherein the modification of the native gene increases the secretion of the compound of interest by at least 10%, such as at least 25%, such as at least 50%, such as at least 50%, such as at least 50%, such as at least 50%, such as at least 50%, such as at least 50%, such as at least 50%, such as at least 50%.

23. The host cell of any preceding claim wherein the compound of interest is selected from proteins optionally proteins selected from dairy proteins, egg proteins, enzymes, antibodies, hormones, biocides, antimicrobial peptides, structural proteins, and functional proteins.

24. The host cell of claim 23 wherein the protein is recombinant protein.

25. The host cell of any one of claims 23 to 24 wherein the protein is a dairy protein.

26. The host cell of claim 25 wherein the dairy protein is a lactoglobulin or a casein.

27. The host cell of claim 26 wherein the lactoglobulin is p-lactoglobulin, a-lactalbumin, and / orImmunoglobulin.

28. The host cell of claim 26 wherein the casein is asl-Casein (Alpha-Si Casein), as2-Casein (Alpha- 52 Casein), P-Casein (Beta-Casein), K-Casein (Kappa-Casein), and / or y-Casein (Gamma-Casein).

29. The host cell of any one of claims 23 to 28 wherein the protein is selected from albumin, alphalactalbumin, beta-lactoglobulin, caseins, collagen, hemoglobin, myoglobin, insulin, lysozyme, ovalbumin, lactoferrin, osteopontin, Nisin, Pediocin, Reuterin, Colicin, Enterocin, Natamycin, Defensin, Bacillomycin, Erythropoietin (EPO), Human Growth Hormone (hGH), Interferons (IFNs), Monoclonal Antibodies (mAbs), Granulocyte Colony-Stimulating Factor (G-CSF), Factor VIII, Tumor Necrosis Factor (TNF), Thyroid Hormone, Alteplase (tPA), Etanercept, Fol litropin, Antithrombin.

30. The host cell of any preceding claim, wherein the microbial host cell is a eukaryotic, a prokaryotic or an algal cell.

31. The host cell of claim 30, wherein the eukaryotic cell is a fungal cell, optionally a filamentous fungus or a yeast cell.Case Ref. P268WO IPTector®32. The host cell of claim 31, wherein the filamentous fungus is selected from the genus of Aspergillus, Trichoderma, or Rhizopus.

33. The host cell of claim 32, wherein the filamentous fungus is selected from the species Aspergillus sp, Aspergillus oryzae, Aspergillus niger, Trichoderma sp, or Rhizopus sp.

34. The host cell of claim 31, wherein the yeast is selected from the genus of Komagataella, Pichia, Saccharomyces, Yarrowia, or Hansenula.

35. The host cell of claim 34, wherein the yeast is selected from the species of Komagataella phaffii, S. cerevisiae, Y. lipolytica, or Hansenula sp.

36. A cell culture, comprising the host cell of any one of claims 1 to 28 and a growth medium.

37. A method for producing a compound of interest: a) culturing the cell culture of claim 36at conditions allowing the cell to produce the compound of interest; and b) optionally recovering and / or isolating the compound of interest.

38. The method of claim 37 further comprising one or more elements selected from: a) culturing the cell culture in a nutrient medium; b) culturing the cell culture under aerobic or anaerobic conditions c) culturing the cell culture under agitation; d) culturing the cell culture at a temperature of between 25 to 50 °C; e) culturing the cell culture at a pH of between 3-9; and f) culturing the cell culture for between 10 hours to 30 days.

39. The method of any one of claims 37 to 38 wherein the recovery and / or isolation step comprises separating a liquid phase of the cell or cell culture from a solid phase of the cell or cell culture to obtain a supernatant comprising the compound of interest and / or subjecting the supernatant to one or more steps selected from: a) separating the supernatant from the solid phase of the cell culture, such as by filtration or gravity separation;Case Ref. P268WO IPTector® b) contacting the supernatant with one or more adsorbent resins to obtain at least a portion of the produced BLG; c) contacting the supernatant with one or more ion exchange or reversed-phase chromatography columns to obtain at least a portion of the BLG; d) extracting the modified BLG; and / or e) precipitating the modified BLG by crystallization or evaporating the solvent of the liquid phase; and optionally isolating the modified BLG by filtration or gravity separation; thereby recovering and / or isolating the BLG.

40. A fermentation composition comprising the cell culture of claim 36 and / or the compound of interest comprised therein.

41. The fermentation composition of claim 40, wherein at least 50%, such as at least 75%, such as at least 95%, such as at least 99% of solid cell material has been separated from the composition.

42. The fermentation composition of claim 40 to 41, further comprising one or more compounds selected from trace metals, vitamins, salts, yeast nitrogen base, carbon source, YNB, and / or amino acids of the fermentation; wherein the concentration of the compound of interest is at least 1 mg / kg composition.

43. The fermentation composition of claim 40 to 42 further comprising one or more carriers, agents, additives and / or excipients.* * *

Images