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WO2025133003A2 - Non-animal bovine beta lactoglobulin - Google Patents

Non-animal bovine beta lactoglobulin Download PDF

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Publication number
WO2025133003A2
WO2025133003A2 PCT/EP2024/087733 EP2024087733W WO2025133003A2 WO 2025133003 A2 WO2025133003 A2 WO 2025133003A2 EP 2024087733 W EP2024087733 W EP 2024087733W WO 2025133003 A2 WO2025133003 A2 WO 2025133003A2
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WO
WIPO (PCT)
Prior art keywords
blg
cell
host
modified
seq
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Pending
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PCT/EP2024/087733
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French (fr)
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WO2025133003A3 (en
Inventor
José Arnau MARTINEZ
Jan Lehmbeck
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21stBio AS
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21stBio AS
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Publication of WO2025133003A2 publication Critical patent/WO2025133003A2/en
Publication of WO2025133003A3 publication Critical patent/WO2025133003A3/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4717—Plasma globulins, lactoglobulin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/38—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from Aspergillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C12N15/625—DNA sequences coding for fusion proteins containing a sequence coding for a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/58—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi
    • C12N9/62—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi from Aspergillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/66—Aspergillus
    • C12R2001/69—Aspergillus oryzae

Definitions

  • the! BLG! comprises! at! least! one! substitution! of! a! non- essential!and/or!non-branched!amino!acid!with!an!essential!and/or!branched!amino!acid!compared! to!a!parent!unmodified!BLG.! [0006] In!another!aspect!this!disclosure!describes!a!modified!nucleotide!sequence!which!is!at!least!
  • polynucleotide ! ⁇ substitution ⁇ ! or! ⁇ substituted ⁇ ! refers! to! modification! of! the! polynucleotide! by! replacing!one!nucleotide!with!another,!for!instance!the!replacement!of!a!cytosine!with!a!thymine!in!a! polynucleotide!sequence!is!a!nucleotide!substitution.! [0038]
  • The! term!expression”! includes!any!step! involved! in! the!production!of!a!polypeptide! (e.g.,! encoded! enzyme)! including,! but! not! limited! to,! transcription,! post-transcriptional!
  • the! modified! BLG! may! contain! a! Histidine! and! a! Valine! at! positions! corresponding!to!position!59!and!118!respectively!in!anyone!of!the!BLG ⁇ s!comprised!in!SEQ!ID!NO:!1,! 2!or!3.!Such!a!modified!BLG!can!be!a!BLG!comprising!an!amino!acid!sequence!which!is!at!least!50%! ! ! !
  • the! heterologous! gene! encoding! the! BLG! in! the! host! cell! further! comprises!a!moiety!coding!for!a!signal!peptide!fused!to!the!BLG.!This!signal!peptide!and!the!moiety!
  • promoters! include! engineered! synthetic! promoters.! The! promoter! is! preferably! an! inducible!promoter,!inducible!e.g.!by!nutrients!such!as!starch,!sugars,!maltose,!glucose!and!the!like.!In! some!embodiments!the!promoter! is!an!amylase!promoter.!Examples!of!such!useful!promoters!are!
  • the! host! cell! can! advantageously!be!modified!by!one!or!more!further!genetic!modifications!improving!the!expression! and/or!secretion!of!the!BLG!selected!from! a ) attenuation,! disruption! and/or! deletion! the! expression! of! one! or! more! genes! which! are! endogenous/native!to!the!host!cell;!and! b) mutation!of!one!or!more!genes!native!to!the!host!cell.! [0081] In! some!
  • the! attenuated,! disrupted! and/or! deleted! native! genes! encodes! a! protease!capable!of!digesting!BLG!or!its!signal!peptide.! [0082] It! has! been! found! that! the! attenuation,! disruption! and/or! deletion! of! native! genes! advantageously!include!genes!that!encode!proteases!capable!of!digesting!BLG!or!its!signal!peptide,! ! ! Case Ref.
  • The!host!cell! is!preferably!a!eukaryotic,!a!prokaryotic!or!an!algal!cell.!
  • the! method! includes! a! recovery! and/or! isolation! step! comprising!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!BLG!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!
  • the!modified!BLG! comprises! at! least!one! amino! acid! substitution! compared! to! anyone! of! bovine! BLG! A,! bovine! BLG! B! and! bovine! BLG! C,! optionally! at! least! two! substitutions.!In!some!embodiments!the!modified!BLG!is!a!modified!bovine!BLG!C!comprising!at!least!
  • the! modified! BLG! is! a! modified! BLG! A! comprising! at! least! one! ! ! Case Ref. P238WO IPTector® substitution!of!Glutamine!with!Histidine!at!a!position!corresponding!to!position!59!of!the!BLG!A!in!SEQ!

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Mycology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Toxicology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The present disclosure describes a genetically modified microbial host cell expressing one or more heterologous genes encoding a modified beta-lactoglobulin (BLG), thereby producing the said modified BLG, wherein the modified BLG comprises at least one substitution of a non-essential and/or non-branched amino acid with an essential and/or branched amino acid compared to a parent unmodified BLG.

Description

Case Ref. P238WO IPTector® Non-animal!bovine!beta!lactoglobulin!! Technical!Field! [0001] The!present!disclosure!describes!genetically!modified!host!cell!producing!beta-lactoglobulin! (BLG).!Also!described!herein!are!gene!construct!and!methods!useful!for!producing!such!BLG!as!well!as! modified! bovine! BLG! having! improved! nutritional! value.! Also! described! herein! are! composition! comprising!the!BLG!and!use!of!the!BLG!in!food!and/or!feed.! ! Background!! [0002] Recombinantly! made! beta! lactoglobulins! (BLG^s)! and! their! use! as! substitute! milk! protein! components!are!known!in!the!art!e.g.!from!WO2020/219596!or!WO2021/168343.! [0003] However,! there! is! a! continuous! need! for! finding! and! developing! better! ways! for! the! recombinant!production!of!such!BLG^s!and!for!identifying!BLG^s!with!improved!properties!compared! to!the!naturally!available!BLG^s!such!as!BLG^s!from!cow^s!milk.!! ! Summary! [0004] Over!this!background!art!one!objective!of!this!disclosure!is!to!present!an!improved!microbial! expression!host!offering!better!and!more!efficient!production!of!BLGs.!Another!objective!is!to!present! a!modified!bovine!BLG!which!has!a!higher!nutritional!value!than!the!natural!bovine!BLG.!! [0005] Accordingly,!in!an!aspect!this!disclosure!describes!a!genetically!modified!microbial!host!cell! expressing!one!or!more!heterologous!genes!encoding!a!modified!beta-lactoglobulin!(BLG),!thereby! producing! the! said!modified! BLG,!wherein! the! BLG! comprises! at! least! one! substitution! of! a! non- essential!and/or!non-branched!amino!acid!with!an!essential!and/or!branched!amino!acid!compared! to!a!parent!unmodified!BLG.! [0006] In!another!aspect!this!disclosure!describes!a!modified!nucleotide!sequence!which!is!at!least! 50%!identical!to!the!BLG!encoding!gene!comprised!in!anyone!of!SEQ!ID!NO:!5,!6,!7,!8,!9,!10,!11!12,!57,! 59,!61,!63,!65,!67,!69,!70,!72,!74,!76,!78,!80,!82,!84,!86,!88,!90,!92,!94,!96,!98,!100,!102,!104,!106,!110,! 112,!114,!116,!118,!120,!122,!124,!126,!128,!130,!132,!134,!136,!138,!140,!142,!144,!146,!148,!149,!150,! 152,!154,!156,!158,!160,!162,!164,!167,!168,!170!and/or!171!and!encodes!a!modified!BLG!having!at! least!one!additional!essential!amino!acid!compared!to!a!parent!BLG.! [0007] In!another!aspect!this!disclosure!describes!a!polynucleotide!construct!comprising!a!nucleotide! sequence!which!is!at!least!50%!identical!to!the!BLG!encoding!gene!comprised!in!anyone!of!SEQ!ID!NO:! 5,!6,!7,!8,!9,!10,!11!12,!57,!59,!61,!63,!65,!67,!69,!70,!72,!74,!76,!78,!80,!82,!84,!86,!88,!90,!92,!94,!96,! ! ! Case Ref. P238WO IPTector® 98,!100,!102,!104,!106,!110,!112,!114,!116,!118,!120,!122,!124,!126,!128,!130,!132,!134,!136,!138,!140,! 142,!144,!146,!148,!149,!150,!152,!154,!156,!158,!160,!162,!164,!167,!168,!170!and/or!171,!operably! linked!to!an!inducible!promoter!which!is!optionally!at!least!50!%!identical!to!the!promoter!comprised! in!SEQ!ID!NO:!15.! [0008] In!another!aspect!this!disclosure!describes!a!cell!culture,!comprising!the!host!cells!described! herein!and!a!growth!medium.! [0009] In!another!aspect!this!disclosure!describes!a!method!for!producing!a!BLG!comprising:! a) culturing! the! cell! culture! described! herein! at! conditions! allowing! the! cell! to! produce! the! modified!BLG;!and!! b) optionally!recovering!and/or!isolating!the!BLG.! [0010] In!another!aspect! this!disclosure!describes!a! fermentation!composition!comprising!the!cell! culture!described!herein!and/or!the!BLG!comprised!in!the!cell!culture.! [0011] In!another!aspect!this!disclosure!describes!a!modified!bovine!BLG!polypeptide!comprising!at! least!one!substitution!of!a!non-essential!and/or!non-branched!amino!acid!with!an!essential!and/or! branched!amino!acid!compared!to!a!parent!unmodified!BLG.! [0012] In!final!aspects!this!disclosure!describes!a!food!or!feed!composition!comprising!the!modified! bovine!BLG!polypeptide!described!herein!and!a!method!of!enriching!a!food!or!feed!composition!with! essential!amino!acids!comprising!mixing!one!or!more!food!or!feed!ingredients!with!the!modified!BLG.! ! Description!of!drawings!and!figures! [0013] 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.!! [0014] Figure! 1! shows! the! sequence! comparison! between! the! amino! acid! sequence! of! BLG! manufactured!in!A.!oryzae!(AoBLG)!with!the!three!major!BLG!variants!present!in!consumer!milk!(BLG! A,!B!and!C).!The!amino!acid!modifications!introduced!in!a!BLG!B!background!are!highlighted!as!white! text!with!black!background!(H!and!V).!The!only!additional!amino!acid!difference!between!BLG!A,!B!and! C!is!shown!as!a!grey!background!(D!in!BLG!A)!and!is!not!included!in!the!manufactured!AoBLG.!The! sequences!shown!in!figure!1!is!included!in!SEQ!ID!NOs:!1,!2,!3!and!4!! [0015] Figure!2!shows!the!SDS!PAGE!of!culture!supernatants!of!A.!oryzae!strains!cultivated!in!YPM! for!4!days!at!30!C.!Lane!M:!Molecular!Weight!Marker;!lane!1:!strain!TFB-Ao0138;!lane!2:!TFB-Ao0144;! lane!3:!strain!TFB-Ao0145.!All!strains!contain!the!same!copy!number!of!the!AoBLG!gene!inserted!at! the!same!chromosomal!location!(niaD).! [0016] Figure!3!shows!the!SDS!PAGE!of!culture!supernatants!of!BLG!strains!constructed!in!control! ! ! Case Ref. P238WO IPTector® strain! TFB-Ao0094! (derived! from! TFB-Ao0010! to! only! integrate! one! copy)! using! different! codon! optimized!sequences.!Strain!names! indicate!the!codon!optimized!sequence!used! in!each!case!e.g.,! strains!186TA!and!186TB!are!strains!that!contain!the!AoBLG-D!sequence!in!either!2!(186TA)!or!4!copies! (186TB,!see!Table!1).! [0017] Figure!4!shows!the!SDS!PAGE!of!culture!supernatants!of!BLG!strains!constructed!in!control! strain!TFB-Ao0094!using!different!signal!peptides,!cultivated!in!duplicate.!Lane!M:!Molecular!Weight! Marker;! Lanes! 1! and! 2:! Plectasin! signal! peptide;! Lanes! 3! and! 4:! Cutinase! signal! peptide! (see!WO! 2005/121333);!Lanes!5!and!6:!TL!lipase!signal!peptide!(Thermomyces!lanuginosus!lipase,!identical!to! sequence! 4! from! patent! US8586330);! Lanes! 7! and! 8:! SP0034! signal! peptide! (from! an! A.! niger! hypothetical!glycoside!hydrolase);!Lanes!9!and!10:!SP0002!signal!peptide!(from!the!A.!niger!mannan! endo-1,4-beta-mannosidase).! [0018] Figure!5!shows!the!strategy!applied!for!the!construction!of!strains!for!testing!SP!efficiency,! illustrated! for! the!plectasin!signal!peptide,! (Ple!SP).!Shortly,!Fragment!1! is!specific! for!each!SP!and! contains!the!upstream!flank!of!the!wA!gene!(Fragment!FI-wA).!Fragment!2!contains!the!AoBLG!coding! sequence.! Fragment! 3! contains! the! Tamg! terminator! and! the! downstream! flank! of! the! wA! gene! (Fragment! FIII-wA).! The!overlapping! regions! between! Fragment!1! and!2! (Overlap! 1)! and!between! Fragment!2!and!3!(Overlap!2)!are!depicted.!! [0019] Figure!6!shows!the!SDS!PAGE!of!culture!supernatants!of!BLG!strains!constructed!in!strain!TFB- Ao0094!using!different!signal!peptides,!cultivated! in!duplicate.!Lane!M:!Molecular!Weight!Marker;! Lanes!1!and!2:!strain!L-BLG-21!(Ple!SP);!Lanes!3!and!4:!strain!L-BLG-23!(Cut!SP);!Lanes!5!and!6:!strain! L-BLG-25!(Lip!SP);!Lanes!7!and!8:!strain!L-BLG-27!(SP034);!Lanes!9!and!10:!strain!L-BLG-29!(SP002).! [0020] Figure!7!shows!the!prtT!locus!in!A.!oryzae!in!strain!TFB-Ao0010!and!the!deletion!present!in! strain!TFB-Ao0127!and!TFB-Ao0131.!The!coding!region!of!wild!type!prtT!gene!is!depicted!(prtT!grey! arrow),! together!with! the! sequences!used! for!MAD7!cleavage! (PS_AoprtT1!and!PS_AoprtT2!white! arrows).!The!deleted!region!is!depicted!as!a!black!box!(^Deletion^).!The!position!of!primers!used!as! donor!DNA!(P0459A!and!P0459B)!and!primers!for!identification!of!the!deletion!(P0457!and!P0458)!are! depicted!above.!! [0021] Figure!8!shows!a!map!of!plasmid!pTFB-0075!used!containing!the!AoBLG!expression!cassette,! including!the!Ple!SP!(included!in!the!depicted!AoBLG)!for!the!construction!of!multicopy!strains.!! [0022] Figure!9!shows!the!distribution!of!essential!and!non-essential!amino!acids!in!BLG!B!and!new! BLG!variants!AoBLG,!SuperBLG!and!LeuMax.! [0023] Figure!10!shows!the!sequence!alignment!of!new!BLG!variants!AoBLG,!SuperBLG!and!LeuMax! using!Muscle!(Snapgene)!using!cow!milk!BLG!B!variant!as!reference.!The!sequences!shown!in!figure!10! are!included!in!SEQ!ID!NOs:!2,!4,!59!and!58.! ! ! Case Ref. P238WO IPTector® [0024] Figure!11! shows! the!branched!amino!acid!content! in!BLG!B!and!new!BLG!variants!AoBLG,! SuperBLG!and!LeuMax.! [0025] Figure!12!shows!prediction!of!protein!folding!for!bovine!BLG!B,!AoBLG,!SuperBLG!and!LeuMore! 2!using!AlphaFold.!The!graphs!depict!the!probability!of!having!a!structure.!Values!above!90%!strongly! predict!a!structured!form!of!the!protein.!Lower!values!predict!unstructured!regions!of!the!protein!(e.g.! SuperBLG).! [0026] Figure! 13! shows! Leucine! content! of! new! BLG!molecules! containing! increasing! number! of! Leucine!residues!LeuMore1!(3!additional!Leu)!(SEQ!ID!NO:!60)!to!LeuMore5!(5!additional!Leu)!(SEQ!ID! NO:68)!compared!to!both!bovine!BLG!B!and!AoBLG.! [0027] Figure!14! shows!SDS!PAGE!of! selected!strains!expressing! the!different!AoBLG!variants.! (A)! Strains!(8)!containing!several!copies!of!the!SuperBLG!gene!(left),!a!triplicate!cultivation!of!a!single!copy! AoBLG!strain!(middle)!and!eight!LeuMax!multicopy!strains!(right);!(B)!From!left!to!right:!Single!copy! AoBLG!strain,!single!copy!LeuMore1!(SEQ!ID!NO:!60)!to!LeuMore!5!(SEQ!ID!NO:!68)!strains!(2-3!strains! per!variant).! [0028] Figure!15!shows!protease!content!in!the!supernatant!from!a!strain!with!prtT!deletion!and!a! ^wild!type^!strain!(^wt^)!without!deletion.!The!left!column!shows!the!number!of!proteases!in!a!pilot! fermentation!with!wt!strain!and!the!two!columns!to!the!right!are!duplicates!of!the!strain!with!prtT! deletion.! [0029] Figure!16!shows!expression!of!AoBLG!in!different!A.!niger!strains!(Lanes!1-4)!and!P.!pastoris! strains! (Lanes!5-8).! Lanes!9! and! 10:! Control! A.! niger! and!P.! pastoris! strains,! respectively.! Lane!M:! Molecular!weight!marker! ! ! Incorporation!by!reference! [0030] 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! [0031] 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. P238WO IPTector® Definitions! [0032] The! term! ^BLG^! or! ^parent! natural! BLG^! as! used! herein! refers! to! native! or! natural! beta! lactoglobulin,!in!general!a!protein!well!known!in!the!art!e.g.!from!cow^s!milk.!! [0033] The!term!^modified!BLG^!or!^modified!bovine!BLG^!as!used!herein!refers!to!a!non-natural!BLG! which!comprises!one!or!more!modifications!of!a!naturally!occurring!BLG.!A!modified!bovine!BLG!may! for!example!be!a!bovine!BLG!in!which!one!or!more!amino!acids!have!been!deleted!or!substituted!with! other! amino! acids! to! produce! an! amino! acid! sequence! or! protein! configuration! distinct! from! the! bovine!BLG!and!not!found!in!nature.! [0034] 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.!! [0035] 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! BLOSUM62!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!BLOSUM62!matrix:! ! ! Case Ref. P238WO IPTector®
Figure imgf000007_0001
! [0036] "Reference!sequence"!refers!to!a!defined!sequence!to!which!another!sequence!is!compared.!! [0037] ^Substitution^!or! ^substituted^! refers! to!modification! of! the! polypeptide! by! replacing! one! amino!acid!residue!with!another,!for!instance!the!replacement!of!a!Serine!residue!with!a!Glycine!or! Alanine!residue!in!a!polypeptide!sequence!is!an!amino!acid!substitution.!When!used!with!reference!to! a! polynucleotide,! ^substitution^! or! ^substituted^! refers! to! modification! of! the! polynucleotide! by! replacing!one!nucleotide!with!another,!for!instance!the!replacement!of!a!cytosine!with!a!thymine!in!a! polynucleotide!sequence!is!a!nucleotide!substitution.! [0038] The! term!"expression"! includes!any!step! involved! in! the!production!of!a!polypeptide! (e.g.,! encoded! enzyme)! including,! but! not! limited! to,! transcription,! post-transcriptional! modification,! translation,!post-translational!modification,!and!secretion.! [0039] The! term!"expression!vector"! refers! to!a!DNA!molecule,!either!single-!or!double! stranded,! either! linear!or!circular,!which!comprises!a!polynucleotide!encoding!a!polypeptide!and! is!operably! linked! to! control! sequences! that! provide! for! its! expression.! Expression! vectors! include! expression! cassettes! for! the! integration! of! genes! into! a! host! cell! as! well! as! plasmids! and/or! chromosomes! comprising!such!genes.!! [0040] 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.! [0041] The! term! ^nucleotide! sequence^! or! "polynucleotide"! are! used! interchangeably! herein! to! denote!a!polymer!of!at!least!two!nucleic!acid!monomer!units!or!bases!(e.g.,!adenine,!cytosine,!guanine,! ! ! Case Ref. P238WO IPTector® thymine)!covalently!linked!by!a!phosphodiester!bond,!regardless!of!length!or!base!modification.! [0042] The! term! "polynucleotide! construct"! refers! to! a! polynucleotide,! 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!a!polynucleotide!encoding!a!polypeptide!and!one!or!more!control!sequences.!! [0043] The!term!"operably!linked"!refers!to!a!configuration!in!which!a!control!sequence,!such!as!a! promoter,!is!placed!at!an!appropriate!position!relative!to!a!coding!polynucleotide!such!that!the!control! sequence! directs! expression! of! the! coding! polynucleotide.! More! generally,! a! control! sequence! "operably!linked"!to!a!coding!sequence!is!ligated!in!such!a!way!that!expression!of!the!coding!sequence! is!achieved!under!conditions!compatible!with!the!control!sequence.!A!promoter!sequence!is!"operably! linked"!to!a!gene!when!it!is!in!sufficient!proximity!to!the!transcription!start!site!of!a!gene!to!regulate! transcription!of!the!gene.! [0044] As!used!herein,!"promoter"!refers!to!a!sequence!of!DNA,!usually!upstream!(5')!of!the!coding! region!of!a!structural!gene,!which!controls!the!expression!of!the!coding!region!by!providing!recognition! and! binding! sites! for! RNA! polymerase! and! other! factors! which!may! be! required! for! initiation! of! transcription.!The!selection!of!the!promoter!will!depend!upon!the!nucleic!acid!sequence!of!interest.!A! suitable!^promoter"!is!generally!one!which!is!capable!of!supporting!the!initiation!of!transcription!in!a! bacterium!of!the!invention,!causing!the!production!of!an!mRNA!molecule.! [0045] ^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.! [0046] 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.! [0047] 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.! [0048] 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.! ! ! Case Ref. P238WO IPTector® [0049] The!term!^cell!culture^!as!used!herein!refers!to!a!culture!medium!comprising!a!plurality!of!the! 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.! [0050] 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.! [0051] 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.!! [0052] 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.! [0053] 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.! [0054] 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! ! ! Case Ref. P238WO IPTector® when!expressed!natively!or!recombinantly.!In!an!embodiment!the!isolated!compound!is!at!least!90%! 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.! [0055] 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.!! [0056] 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.! [0057] 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.! [0058] 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.! [0059] 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).! [0060] 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.! [0061] 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.! [0062] 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! ! ! Case Ref. P238WO IPTector® (Frederick!M.!AUSUBEL,! 2000,!Wiley! and! son! Inc,! Library! of! Congress,!USA);!Molecular! Cloning:!A! 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!I-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).! ! Genetically!modified!microbial!host!cell! 1. As!described!supra,!in!a!first!aspect!a!genetically!modified!microbial!host!cell!is!provided!expressing! one!or!more!heterologous!genes!encoding!a!modified!beta-lactoglobulin!(BLG),!thereby!producing!the! said!modified!BLG,!wherein!the!modified!BLG!comprises!at!least!one!substitution!of!a!non-essential! and/or!non-branched!amino!acid!with!an!essential!and/or!branched!amino!acid!compared!to!a!parent! unmodified!BLG.!! [0063] In!an!embodiment!the!the!modified!BLG!comprises!an!amino!acid!sequence!which!is!at!least! 50%,!such!as!at!least!55%,!such!as!at!least!60%,!such!as!at!least!65%,!such!as!at!least!70%,!such!as!at! least!75%,!such!as!at!least!80%,!such!as!at!least!85%,!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%!identical! to!the!BLG!comprised!in!anyone!of!SEQ!ID!NO:!1,!2,!3,!4,!56,!58,!60,!62,!64,!66,!68,!71,!73,!75,!77,!79,! 81,!83,!85,!87,!89,!91,!93,!95,!97,!99,!101,!103,!105,!107,!109,!111,!113,!115,!117,!119,!121,!123,!125,! 127,!129,!131,!133,!135,!137,!139,!141,!143,!145,!147,!149,!151,!153,!155,!157,!159,!161,!163,!165,!167! and/or!169.! [0064] The!one!or!more!heterologous!genes!encoding!the!BLG!can!in!some!embodiments!comprise!a! nucleotide!sequence!which!is!at!least!50%!identical!to!anyone!of!the!BLG!encoding!genes!comprised! in!SEQ!ID!NO:!2,!4!or!6,!such!as!at!least!55%,!such!as!at!least!60%,!such!as!at!least!65%,!such!as!at!least! 70%,!such!as!at!least!75%,!such!as!at!least!80%,!such!as!at!least!85%,!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%!identical!to!anyone!of!the!BLG!encoding!genes!comprised!in!SEQ!ID!NO:!5,!6,!7,!8,!9,!10,!11! ! ! Case Ref. P238WO IPTector® 12,!57,!59,!61,!63,!65,!67,!69,!70,!72,!74,!76,!78,!80,!82,!84,!86,!88,!90,!92,!94,!96,!98,!100,!102,!104,! 106,!110,!112,!114,!116,!118,!120,!122,!124,!126,!128,!130,!132,!134,!136,!138,!140,!142,!144,!146,!148,! 149,!150,!152,!154,!156,!158,!160,!162,!164,!167,!168,!170!and/or!171.!In!some!embodiments!the!one! or!more! heterologous! genes! encoding! the! BLG! comprise! a! nucleotide! sequence!which! is! the!BLG! encoding!gene!set!forth!in!anyone!of!SEQ!ID!NO:!5,!6,!7,!8,!9,!10,!11,!12,!57,!59,!61,!63,!65,!67,!69,!70,! 72,!74,!76,!78,!80,!82,!84,!86,!88,!90,!92,!94,!96,!98,!100,!102,!104,!106,!110,!112,!114,!116,!118,!120,! 122,!124,!126,!128,!130,!132,!134,!136,!138,!140,!142,!144,!146,!148,!149,!150,!152,!154,!156,!158,!160,! 162,!164,!166,!168,!170!and/or!171.! [0065] In!some!embodiments!the!modified!BLG!expressed!by!the!host!cell!is!modified!BLG!to!have!a! higher!fraction!of!essential!and/or!branched!amino!acids!compared!to!the,!optionally!natural,!parent! BLG.!Such!modified!BLGs!are!more!attractive!than!natural!BLGs!because!they!have!a!higher!nutritional! value!which!can!be!critical!when!using!the!BLG!e.g.!as!ingredient!in!food!or!feed!compositions,!for! example!infant!formulas!for!feeding!malnourished!infants.!In!some!embodiments!the!modified!BLG! has!an! increased! fraction!of!His,! Leu!and/or!Val!compared! to! the!natural!parent!BLG.!A!preferred! modified!BLG!is!a!modified!bovine!BLG.!In!useful!embodiments!the!modified!BLG!comprises!one!or! more!moieties!taken!from!bovine!BLG!A!(SEQ!ID!NO:!1),!bovine!BLG!B!(SEQ!ID!NO:!2)!and/or!bovine! BLG!C!(SEQ!ID!NO:!3).!Further,!the!modified!BLG!can!include!one!or!more!amino!acid!substitutions! compared!to!the!parent!natural!BLG,!such!as!substitutions!of!amino!acid!in!anyone!of!bovine!BLG!A,! bovine! BLG! B! and! bovine! BLG! C.! In! some! embodiments! the! modified! BLG! includes! at! least! two! substitutions!compared!to!a!natural!parent!BLG,!such!as!exactly! two!substitutions.!Particularly! the! modified!BLG!may!comprise!at!least!one!substitution!of!a!non-essential!and/or!non-branched!amino! acid!with!an!essential!and/or!branched!amino!acid,!optionally!compared!to!a!parent!unmodified!BLG.! More!particularly!the!modified!BLG!may!comprise!at!least!at!least!2,!such!as!at!least!5,!such!as!at!least! 10,!such!as!at!least!15,!such!as!at!least!20,!such!as!at!least!25!substitutions!of!a!non-essential!and/or! non-branched!amino!acid!with!an!essential!and/or!branched!amino!acid,!optionally!compared!to!a! parent!unmodified!BLG.!! [0066] Additionally!or!alternatively,!the!modified!BLG!may!comprise!at!least!at!least!1,!such!as!at!least! 2,!such!as!at!least!3,!such!as!at!least!4,!such!as!5,!such!as!at!least!6,!such!as!at!least!7!substitutions!of! a!non-leucine!amino!acid!with!a!leucine!amino!acid.!! [0067] In!a!further!embodiment!the!modified!BLG!comprises!up!to!7!substitutions!of!a!non-leucine! amino!acid!with!a!leucine!amino!acid!corresponding!to!anyone!of!position!1,!5,!21,!96,!108,!120,!139,! 150!and/or!153!of!the!modified!BLG!set!forth!in!anyone!SEQ!ID!NO:!73,!75,!77,!79,!81,!83,!85,!87,!89,! 91,!93,!95,!97,!99,!101,!103,!105,!107,!109,!111,!113,!115,!117,!119,!121,!123,!125,!127,!129,!131,!133,! 135,!137,!139,!141,!143,!145,!147,!149,!151,!153,!155,!157,!159,!161,!163,!165,!167!and/or!169.! ! ! Case Ref. P238WO IPTector® [0068] In!a!further!embodiment!the!modified!BLG!comprises!one!substitution!of!a!non-leucine!amino! acid!with!a!leucine!amino!acid!in!a!position!corresponding!to!anyone!of!position!1,!21,!96,!108,!120,! 139,!150!and/or!153!of!the!modified!BLG!set!forth!in!anyone!of!SEQ!ID!NO:!75,!77,!79,!81,!83,!85!and/or! 87.! [0069] In!a!further!embodiment!the!modified!BLG!comprises!2!substitutions!of!a!non-leucine!amino! acid!with!a!leucine!amino!acid!corresponding!to!anyone!of!position!1,!5,!21,!96,!108,!120,!139,!150! and/or!153!of!the!modified!BLG!set!forth!in!anyone!of!SEQ!ID!NO:!73,!89,!91,!93,!95,!97,!99,!101,!103,! 105,!107!and/or!109.! [0070] In!a!further!embodiment!the!modified!BLG!comprises!3!substitutions!of!a!non-leucine!amino! acid!with!a!leucine!amino!acid!corresponding!to!anyone!of!position!21,!96,!108,!120,!139,!150!and/or! 153!of!the!modified!BLG!set!forth!in!anyone!of!SEQ!ID!NO:!111,!113,!115,!117,!119,!121,!123,!125,!127,! 129,!131!and/or!133.! [0071] In!a!further!embodiment!the!modified!BLG!comprises!4!substitutions!of!a!non-leucine!amino! acid!with!a!leucine!amino!acid!corresponding!to!anyone!of!position!21,!96,!108,!120,!139,!150!and/or! 153!of!the!modified!BLG!set!forth!in!anyone!of!SEQ!ID!NO:!135,!137,!139,!141,!143,!145!and/or!147.! [0072] In!a!further!embodiment!the!modified!BLG!comprises!5!substitutions!of!a!non-leucine!amino! acid!with!a!leucine!amino!acid!corresponding!to!anyone!of!position!1,!21,!96,!108,!120,!139,!150!and/or! 153!of!the!modified!BLG!(SEQ!ID!NO:!149,!151,!153,!155,!157,!159!and/or!161).! [0073] In!a!further!embodiment!the!modified!BLG!comprises!6!substitutions!of!a!non-leucine!amino! acid!with!a!leucine!amino!acid!corresponding!to!anyone!of!position!21,!96,!108,!120,!139,!150!and/or! 153!of!the!modified!BLG!set!forth!in!anyone!of!SEQ!ID!NO:!163,!165!and/or!167.! [0074] In!a!further!embodiment!the!modified!BLG!comprises!7!substitutions!of!a!non-leucine!amino! acid!with!a!leucine!amino!acid!corresponding!to!position!21,!96,!108,!120,!139,!150!and!153!of!the! modified!BLG!set!forth!in!SEQ!ID!NO:!169.!! [0075] In!a! further!embodiment! the!modified!BLG!comprises!at! least!one!amino!acid!substitution! compared! to! anyone! of! bovine! BLG! A,! bovine! BLG! B! and! bovine! BLG! C,! optionally! at! least! two! substitutions.! [0076] In! a! preferred! embodiment! the! BLG! is! a!modified! bovine! BLG! C! comprising! at! least! one! substitution!of!Alanine!with!Valine!in!a!position!corresponding!to!position!118!of!the!BLG!C!in!SEQ!ID! NO:!3.!In!another!preferred!embodiment!the!modified!BLG!is!a!modified!BLG!A!comprising!at!least!one! substitution!of!Glutamine!with!Histidine!at!a!position!corresponding!to!position!59!of!the!BLG!A!in!SEQ! ID! NO:! 1.! In! particular! the! modified! BLG! may! contain! a! Histidine! and! a! Valine! at! positions! corresponding!to!position!59!and!118!respectively!in!anyone!of!the!BLG^s!comprised!in!SEQ!ID!NO:!1,! 2!or!3.!Such!a!modified!BLG!can!be!a!BLG!comprising!an!amino!acid!sequence!which!is!at!least!50%! ! ! Case Ref. P238WO IPTector® identical!to!the!BLG!sequence!comprised!in!SEQ!ID!NO:!4,!56,!58,!60,!62,!64,!66,!68,!73,!75,!77,!79,!81,! 83,!85,!87,!89,!91,!93,!95,!97,!99,!101,!103,!105,!107,!109,!111,!113,!115,!117,!119,!121,!123,!125,!127,! 129,!131,!133,!135,!137,!139,!141,!143,!145,!147,!149,!151,!153,!155,!157,!159,!161,!163,!165,!167! and/or!169,!such!as!at!least!55%,!such!as!at!least!60%,!such!as!at!least!65%,!such!as!at!least!70%,!such! as!at!least!75%,!such!as!at!least!80%,!such!as!at!least!85%,!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%! identical!to!the!BLG!sequence!comprised!in!SEQ!ID!NO:!4,!56,!58,!60,!62,!64,!66,!68,!73,!75,!77,!79,!81,! 83,!85,!87,!89,!91,!93,!95,!97,!99,!101,!103,!105,!107,!109,!111,!113,!115,!117,!119,!121,!123,!125,!127,! 129,!131,!133,!135,!137,!139,!141,!143,!145,!147,!149,!151,!153,!155,!157,!159,!161,!163,!165,!167! and/or! 169.! In! further! embodiments! the! one! or!more! heterologous! genes! encoding! the! BLG! can! comprise!a!nucleotide!sequence!which!is!at!least!50%!identical!to!the!BLG!encoding!gene!comprised! in!anyone!of!SEQ!ID!NO:!5,!6,!7,!8,!9,!10,!11!12,!57,!59,!61,!63,!65,!67,!69,!70,!72,!74,!76,!78,!80,!82,!84,! 86,!88,!90,!92,!94,!96,!98,!100,!102,!104,!106,!110,!112,!114,!116,!118,!120,!122,!124,!126,!128,!130,! 132,!134,!136,!138,!140,!142,!144,!146,!148,!149,!150,!152,!154,!156,!158,!160,!162,!164,!167,!168,!170! and/or!171,!such!as!at!least!55%,!such!as!at!least!60%,!such!as!at!least!65%,!such!as!at!least!70%,!such! as!at!least!75%,!such!as!at!least!80%,!such!as!at!least!85%,!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%! identical!to!the!BLG!encoding!gene!comprised!in!anyone!of!SEQ!ID!NO:!5,!6,!7,!8,!9,!10,!11!12,!57,!59,! 61,!63,!65,!67,!69,!70,!72,!74,!76,!78,!80,!82,!84,!86,!88,!90,!92,!94,!96,!98,!100,!102,!104,!106,!110,!112,! 114,!116,!118,!120,!122,!124,!126,!128,!130,!132,!134,!136,!138,!140,!142,!144,!146,!148,!149,!150,!152,! 154,!156,!158,!160,!162,!164,!167,!168,!170!and/or!171.! [0077] In! further! embodiments! the! heterologous! gene! encoding! the! BLG! in! the! host! cell! further! comprises!a!moiety!coding!for!a!signal!peptide!fused!to!the!BLG.!This!signal!peptide!and!the!moiety! encoding!it!may!also!be!heterologous!to!the!host!cell!and/or!to!the!BLG.!In!some!embodiments!the! signal!peptide!comprises!an!amino!acid!sequence!which!is!at!least!50%!identical!to!the!signal!peptide! comprised!in!SEQ!ID!NO:!13,!such!as!at!least!55%,!such!as!at!least!60%,!such!as!at!least!65%,!such!as! at!least!70%,!such!as!at!least!75%,!such!as!at!least!80%,!such!as!at!least!85%,!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%!identical!to!the!signal!peptide!comprised!in!SEQ!ID!NO:!13.!In!other!embodiments!the! nucleotide!sequence!of!the!moiety!encoding!the!signal!peptide!has!a!nucleotide!sequence!which!is!at! least!50%!identical!to!the!signal!peptide!encoding!sequence!comprised!in!SEQ!ID!NO:!14,!such!as!at! least!55%,!such!as!at!least!60%,!such!as!at!least!65%,!such!as!at!least!70%,!such!as!at!least!75%,!such! as!at!least!80%,!such!as!at!least!85%,!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%!identical!to!the!signal! ! ! Case Ref. P238WO IPTector® peptide!encoding!sequence!comprised!in!SEQ!ID!NO:!14.!It!has!been!found!that!advantageously,!the! signal!peptide!is!in!preferred!embodiments!derived!from!a!natural!defensin!including!but!not!limited! to!fungal!defensins.!One!particularly!useful!example!of!such!fungal!defensins!is!plectasin,!in!particular! plectasin!derived!from!a!strain!of!the!genus!of!Pseudoplectania,!such!as!Pseudoplectania!nigrella.! [0078] In! further!embodiments! the!heterologous!gene!encoding! the!BLG! in! the!host!cell! is!under! control!of!a!promoter,!preferably!a!promoter!that!is!heterologous!to!host!cell!and/or!to!the!BLG.!! [0079] Such! promoters! include! engineered! synthetic! promoters.! The! promoter! is! preferably! an! inducible!promoter,!inducible!e.g.!by!nutrients!such!as!starch,!sugars,!maltose,!glucose!and!the!like.!In! some!embodiments!the!promoter! is!an!amylase!promoter.!Examples!of!such!useful!promoters!are! promoters!which!have!a!nucleotide!sequence!which!is!at!least!50%!identical!to!the!promoter!sequence! comprised!in!SEQ!ID!NO:!15,!such!as!at!least!55%,!such!as!at!least!60%,!such!as!at!least!65%,!such!as! at!least!70%,!such!as!at!least!75%,!such!as!at!least!80%,!such!as!at!least!85%,!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%!identical!to!the!promoter!sequence!comprised!in!SEQ!ID!NO:!15.! [0080] Production!of!bulk!proteins!like!modified!bovine!BLG!requires!a!very!efficient!expression!and! secretion!in!the!microbial!system!of!choice!(such!as!fungal,!yeast!or!bacterial!host!cells)!and!-!at!the! same!time!-!the!lowest!possible!level!of!endogenous!proteases!that!enable!growth!but!also!increases! protein!degradation.!Host!cell!improvements!were!made!on!e.g.,!identifying!efficient!signal!peptides! to!ease!secretion,!and!where!possible!deleting!or!downregulating!protease!genes!to!increase!yields! and!protein!stability.!Deletion!of!protease!genes!may!however!severely!affect!the!fitness!of!the!strain.! The!choices!for!yield! improvement! in!the!art!are!numerous!but!not!widely!applicable!as!high!yield! production!of!heterologous!proteins!is!dependent!on!many!traits!starting!from!the!primary!amino!acid! sequence! and! including! secretion,! folding,! post-translational!modifications! to!mention! just! a! few.! Furthermore,! a! system! for! multicopy! strain! construction! was! used! in! this! work! that! enabled! construction! of! highly! producing! strains.! Accordingly,! in! further! embodiments! the! host! cell! can! advantageously!be!modified!by!one!or!more!further!genetic!modifications!improving!the!expression! and/or!secretion!of!the!BLG!selected!from! a) attenuation,! disruption! and/or! deletion! the! expression! of! one! or! more! genes! which! are! endogenous/native!to!the!host!cell;!and!! b) mutation!of!one!or!more!genes!native!to!the!host!cell.! [0081] In! some! embodiment! the! attenuated,! disrupted! and/or! deleted! native! genes! encodes! a! protease!capable!of!digesting!BLG!or!its!signal!peptide.! [0082] It! has! been! found! that! the! attenuation,! disruption! and/or! deletion! of! native! genes! advantageously!include!genes!that!encode!proteases!capable!of!digesting!BLG!or!its!signal!peptide,! ! ! Case Ref. P238WO IPTector® and!thereby!weaking!the!production,!secretion!and/or!folding!of!the!BLG!protein.!It!is!known!in!the! art!that!endogenous!proteases!may!be!a!challenge!for!production!of!recombinant!proteins!at!high! titers!(Arnau!et!al.,!2020).!Therefore,!a!main!approach,! to! increase!protein!titers! for! the!host!cells! described!herein,!was!the! inactivation/deletion!of!protease!genes.!But!endogenous!proteases!may! have!an!essential!function!in!the!cell,!making!it!challenging!to!obtain!loss!of!function!strains.!The!initial! strain! (TFB-Ao0010)! used! for! BLG! production! in! particular! was! engineered! to! provide! a! very! low! protease!level!using!several!approaches.!The!genes!encoding!the!two!major!secreted!proteases,!alpA! (alkaline!protease)!and!nprA!(neutral!metalloprotease!I)!were!inactivated.!In!addition,!pepE!(aspartic! endopeptidase)! was! inactivated! and! pepC! (encoding! a! serine! protease! present! in! vacuoles! and! involved!in!degradation!of!e.g.,!unfolded!proteins)!was!downregulated.!Thus,!in!the!best!performing! strains! selective! protease! activities! were! not! present! or! down! regulated.! The! best! performing! A.! oryzae!strain!was!constructed!by!deleting!alpA,!nprA,!pepE!and!down!regulating!pepC.!Additionally!or! alternatively,! the! prtT! gene! in! host! cells! encodes! a! transcription! factor! (PrtT)! that! functions! as! a! general! protease! regulator.! Interestingly,! it!was! found! that! the!expression!of! the!major!A.! oryzae! proteases! encoded! by! alpA! and! nprA! was! indeed! regulated! by! PrtT.! Moreover,! although! having! inactivated!major! proteases! in! the! host! cell,! it!was!unexpectedly! found! that! deletion! of! prtT! in! a! production!host! cell! results! in!a! remarkable! increase! in!modified!BLG,! such!as!AoBLG,! titers!when! comparing!to!control!strains!containing!the!same!copy!number!of!the!expression!cassette!(see!Figure! 2).!Accordingly,!proteases!that!are!useful!to!attenuate,!disrupt!and/or!delete!include!alpA!(alkaline! protease),! nprA! (neutral! metalloprotease! I),! pepE! (aspartic! endopeptidase),! and! pepC! (serine! protease).!Suitable!alpAs!are!the!alpAs!having!an!amino!acid!sequence!as!set!forth!in!SEQ!ID!NO:!16! or!18,!and!accordingly!in!a!further!embodiment!an!alpA!(alkaline!protease)!comprised!in!SEQ!ID!NO:! 16!or!18!is!attenuated,!disrupted!and/or!deleted.! [0083] Suitable!nprAs!are!the!nprAs!having!an!amino!acid!sequence!as!set!forth!in!SEQ!ID!NO:!20!or! 22!and!accordingly!a!nprA!(neutral!metalloprotease!I)!comprised!in!SEQ!ID!NO:!20!or!22!is!attenuated,! disrupted!and/or!deleted.! [0084] Suitable!pepEs!are!pepEs!having!an!amino!acid!sequence!as!set!forth!in!SEQ!ID!NO:!36!or!38,! and!accordingly!a!pepE!having!an!amino!acid!sequence!comprised!in!SEQ!ID!NO:!36!or!38!is!attenuated,! disrupted!and/or!deleted.! [0085] Suitable!pepCs!are!pepCs!having!an!amino!acid!sequence!as!set!forth!in!SEQ!ID!NO:!32!or!34! accordingly!a!pepC!having!an!amino!acid!sequence!comprised!in!SEQ!ID!NO:!32!or!34!is!attenuated,! disrupted!and/or!deleted.! [0086] Further!protease!that!are!suitably!deleted!or!down!regulated!is!described!in!WO2015/025055.! In!some!embodiments!the!native!protease!is!attenuated,!disrupted!and/or!deleted!by!attenuation,! ! ! Case Ref. P238WO IPTector® disruption! and/or! deletion! of! a! native! endogenous! protease! transcription! factor! controlling! the! expression!the!native!proteases!in!the!host!cell.!Such!protease!transcription!factors!include!prtT!or! any! analogue! or! ortholog! thereof,! that! may! be! present! in! the! host! cell.! The! gene! encoding! the! transcription! factor! suitably!has!a! nucleotide! sequence!which! is! at! least! 50%! identical! to! the!prtT! sequence!comprised!in!SEQ!ID!NO:!24!or!26,!such!as!at!least!55%,!such!as!at!least!60%,!such!as!at!least! 65%,!such!as!at!least!70%,!such!as!at!least!75%,!such!as!at!least!80%,!such!as!at!least!85%,!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%!identical!to!the!prtT!sequence!comprised!in!SEQ!ID!NO:!24!or!26.! [0087] The! folding! and! assembly! of! secreted! proteins! occurs! at! the! Endoplasmic! Reticulum! (ER).! Physiological!stimuli!generate!stress!in!the!ER!through!the!accumulation!of!misfolded!proteins.!Under! such! circumstances,! a! signaling! network! termed! the! Unfolded! Protein! Response! (UPR)! becomes! activated! to! rebalance! ER! homeostasis.! At! the! initial! phase! of! the! UPR,! the! adaptive! response! is! triggered!to!relieve!this!stress.!Upon!activation,!the!synthesis!of! ER!resident!chaperones!occurs!to! increase!the!ER!folding!capacity,!whereas!the!folding!load!in!the!ER!is!decreased!through!attenuation! of! the! global! mRNA! translation! and! increased! clearance! of! misfolded! proteins! by! ER-associated! degradation! (ERAD;! Siwecka! et! al.,! 2021).! These!mechanisms! are! widely! conserved! in! eukaryotic! organisms!such!as!S.!cerevisiae!and!A.!oryzae.!Accordingly,!where!the!host!cell!is!modified!by!further! genetic!modifications!by!mutation!of!one!or!more!genes!native! to! the!host! cell! for! improving! the! expression!and/or!secretion!of!the!BLG,!in!some!embodiments!the!mutated!native!genes!preferably! encode!an!enzyme! involved! in! activation!of! a! transcription! factor! regulating! the!Unfolded!Protein! Response! (UPR)! in! the!host! cell.! In! some!embodiments! the!mutation!of! the!enzyme! increases! the! activation!of!the!UPR!transcription!factor.! [0088] One!such!factor! is!HAC1!(or!HAC1p!or! its!ortholog!HacA)!which! is! involved!in!the!unfolded! protein! response! (UPR)! pathway! in! eukaryotic! cells,! particularly! in! yeast! such! as! Saccharomyces! cerevisiae! or! other! fungi.! When! unfolded! proteins! accumulate! in! the! ER,! the! HAC1/HacA! mRNA! undergoes! a! unique! splicing! event! that! produces! an! active! transcription! factor.! This! splicing! is! catalyzed! by! an! endonuclease,! Ire1! (or! Ire1p),! which! senses! the! ER! stress! and! activates! the! UPR! signaling!cascade.!The!spliced!form!of!HAC1!is!then!translated!into!an!active!transcription!factor!that! translocates! to! the! nucleus! and! activates! the! expression! of! genes! involved! in! ER! protein! folding.! Accordingly,!in!a!preferred!embodiment!the!transcription!factor!activated!by!the!mutated!enzymes!is! HAC1.!Ire1!(inositol!responsive!enzyme!1),!and!its!ortholog!in!A.!oryzae!IreA,!is!one!such!enzyme!which! is! an! ER-resident! transmembrane! kinase! and! endoribonuclease! that! activates! HAC1.! HAC1! is! a! conserved!transcription!factor!that!serves!as!the!master!regulator!involved!in!UPR!and!IreA!mediates! unconventional!intron!splicing!from!HAC1!mRNA,!as!said,!leading!to!the!production!of!functional!HAC1! ! ! Case Ref. P238WO IPTector® protein!that!induces!genes!encoding!ER!chaperones!and!ER!associated!degradation!functions!(Zhou!et! al.,!2016).! [0089] UPR!is!typically!associated!with!high!level!expression!of!secreted!proteins!that!may!result!in! high!levels!of!unfolded!protein!in!the!ER.!Accordingly,!in!further!embodiments!the!mutated!enzyme!is! an!ER-resident!transmembrane!kinase!and/or!endoribonuclease,!in!particular!an!inositol!responsive! enzyme.!The!inositol!responsive!enzyme!is!suitably!Ire1!or!an!Ire1!Type!enzyme!or!any!of!its!analogues! or!orthologues.!Preferred!mutated!enzymes!are!those!having!an!amino!acid!sequence!which!is!at!least! 50%!identical!to!the!inositol!responsive!enzyme!comprised!in!SEQ!ID!NO:!28!or!30,!such!as!at!least! 55%,!such!as!at!least!60%,!such!as!at!least!65%,!such!as!at!least!70%,!such!as!at!least!75%,!such!as!at! least!80%,!such!as!at!least!85%,!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%! identical! to! the! inositol! responsive!enzyme!comprised!in!SEQ!ID!NO:!28!or!30.!In!a!specific!embodiment!the!mutated!enzyme! comprises!a!mutation!at!the!positions!corresponding!to!position!A80!and/or!A83!of!SEQ!ID!NO:!28,! more!particularly!the!mutations!are!A80T!and/or!A83T,!such!as!the!enzyme!comprised!in!SEQ!ID!NO:! 28,!also!denoted!as!IreA!(the!Ire1!of!A.!oryzae).!Preferably!the!mutated!inositol!responsive!enzyme! comprised!an!amino!acid!sequence!as!set!forth!in!SEQ!ID!NO:!30.!In!special!embodiments!the!mutated! inositol!responsive!enzyme!is!encoded!by!a!gene!having!a!nucleotide!sequence!which!is!at!least!50%! identical!to!the!inositol!responsive!enzyme!encoding!sequence!comprised!in!SEQ!ID!NO:!29!or!31,!such! as!at!least!55%,!such!as!at!least!60%,!such!as!at!least!65%,!such!as!at!least!70%,!such!as!at!least!75%,! such!as!at!least!80%,!such!as!at!least!85%,!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%!identical!to!the! inositol!responsive!enzyme!encoding!sequence!comprised!in!SEQ!ID!NO:!29!or!31.!However,!IreA!does! not!always!contribute!to!increased!yields.!On!the!contrary,!IreA!is!also!involved!in!the!degradation!of! mRNAs!of! secreted! proteins.! Regulated! Ire1-dependent! decay! (RIDD)! is! a! feedback!mechanism! in! which!Ire1!cleaves!endoplasmic!reticulum!(ER)-localized!mRNAs!encoding!secretory!and!membrane! proteins!in!eukaryotic!cells!under!ER!stress!(Tanaka!et!al.,!2023).!Accordingly,!it!was!a!surprise!that! using! a!mutated! IreA! and! in! particular! in! combination!with! prtT! deletion! generated! an! increased! production!of!BLG!in!the!host!cell.!As!shown!in!figure!2!the!use!of!a!mutated!IreA!and!the!deletion!of! prtT!both!contributed!to!higher!yield!of!BLG.!In!further!embodiments!the!host!cell!comprises!at!least! 2!copies!of!the!heterologous!genes!encoding!the!BLG,!to!allow!for!overexpression!of!this!product.!! [0090] Also,! heterologous! genes! encoding! the! BLG! are! codon! optimized! for! expression! in! a! filamentous!fungus,!optionally!A.!oryzae,!such!as!the!codon!optimized!genes!set!forth!in!anyone!of! SEQ!ID!NO:!5,!6,!7,!8,!9,!10,!11,!12,!57,!59,!61,!63,!65,!67,!69,!70,!72,!74,!76,!78,!80,!82,!84,!86,!88,!90,! 92,!94,!96,!98,!100,!102,!104,!106,!110,!112,!114,!116,!118,!120,!122,!124,!126,!128,!130,!132,!134,!136,! ! ! Case Ref. P238WO IPTector® 138,! 140,! 142,! 144,! 146,! 148,! 149,! 150,! 152,! 154,! 156,! 158,! 160,! 162,! 164,! 167,! 168,! 170! and/or! 171which!have!shown!particularly!useful!and!effective!for!the!recombinant!expression!of!modified! bovine!BLG.!Further,!the!host!cell!may!comprise! further!modifications! increasing!expression,!post- translational!modification,!secretion!and/or!folding!of!the!BLG.! [0091] Several! codon-optimized! gene! sequences! for! AoBLG! (see! table! 1)! were! investigated! all! resulting!in!similar!high!BLG!titers.!The!codon!optimized!DNA!sequences!encoding!the!AoBLG!were! used!to!assess!protein!titers!are!shown!below.!All!constructions!were!introduced!in!multiple!copies!in! strain!TFB-Ao0010.!To!enable!assessment!of!the!expression!level!of!each!codon!optimized!sequence,! strains!containing!identical!copy!number!were!obtained!and!used!in!cultivation!in!DWP!(see!figure!3).! ! Table!1! Codon! optimized! DNA!sequence sequence! (plasmid! name) AoBLG-A TTGATAGTAACTCAGACCATGAAAGGTCTTGATATTCAGAAGGTTGCCGGCACCTGGTACTCATTG (pTFB-75)! GCTATGGCGGCGTCGGACATTTCTCTCTTGGATGCACAGTCCGCGCCCTTGCGTGTTTACGTGGAG (SEQ!ID!NO:!5) GAACTTAAACCTACGCCAGAAGGCGATTTGGAAATATTGCTTCATAAATGGGAAAACGGCGAATG TGCCCAAAAGAAGATTATCGCTGAGAAAACAAAGATCCCCGCGGTATTCAAAATTGATGCCCTCAA TGAAAACAAGGTATTGGTCCTTGATACGGATTACAAGAAATACCTGCTGTTCTGTATGGAGAATAG CGCAGAGCCAGAACAATCTCTTGTTTGTCAATGCCTGGTAAGGACGCCGGAAGTGGATGACGAAG CCCTTGAGAAATTTGACAAGGCTCTGAAGGCGCTTCCTATGCACATCAGACTGTCTTTCAACCCTAC GCAATTGGAGGAGCAGTGTCACATT! AoBLG-B TTGATAGTGACGCAAACTATGAAAGGGTTGGACATTCAGAAGGTGGCGGGGACTTGGTATAGTCT (pTFB-77)! CGCCATGGCAGCCTCGGACATTAGCTTGCTGGACGCGCAATCTGCCCCATTGCGTGTCTATGTGGA (SEQ!ID!NO:!6)! GGAATTGAAGCCAACTCCCGAGGGGGATCTGGAGATTCTTCTCCACAAATGGGAAAATGGAGAAT GTGCACAAAAGAAGATAATCGCAGAAAAAACCAAAATTCCTGCCGTATTTAAAATTGACGCGCTG AACGAGAACAAAGTATTGGTACTTGACACTGACTATAAGAAGTATCTGCTCTTCTGCATGGAGAAT AGCGCGGAGCCAGAGCAAAGTCTTGTCTGTCAGTGCCTGGTCCGCACACCAGAGGTAGATGATGA AGCACTTGAGAAGTTCGATAAGGCCTTGAAAGCCCTTCCCATGCACATACGACTCAGTTTTAACCC CACACAGCTTGAGGAGCAATGCCATATT! AoBLG-C CTTATAGTCACACAAACGATGAAGGGGCTGGACATCCAGAAGGTCGCAGGTACCTGGTATTCATT (pTFB-79)! GGCTATGGCGGCATCCGATATATCCCTTCTGGACGCCCAATCGGCCCCACTGAGAGTGTACGTTGA (SEQ!ID!NO:!7)! GGAGCTCAAGCCAACCCCGGAAGGAGACCTTGAGATTCTGTTGCATAAGTGGGAGAATGGAGAG TGCGCTCAAAAGAAAATTATAGCGGAGAAGACTAAGATACCGGCGGTTTTCAAGATCGATGCCCT TAATGAAAATAAGGTTCTTGTCCTTGATACGGACTATAAAAAATACCTGTTGTTCTGCATGGAGAA CTCCGCTGAACCAGAGCAATCGCTTGTTTGCCAGTGCTTGGTGAGGACACCAGAAGTAGACGATG AGGCCCTTGAAAAATTTGACAAAGCGCTCAAAGCGCTGCCTATGCACATAAGGTTGAGCTTCAACC CGACACAACTGGAAGAACAATGCCATATA! AoBLG-D CTCATTGTGACTCAGACTATGAAGGGCTTGGATATCCAGAAAGTGGCAGGCACTTGGTACTCGCTC (pTFB-186)! GCCATGGCAGCATCGGACATTTCGTTGCTCGATGCACAGTCCGCACCTTTGCGCGTCTATGTGGAG (SEQ!ID!NO:!8)! GAGTTGAAGCCCACACCCGAAGGCGATTTGGAAATCCTCTTGCATAAGTGGGAGAACGGAGAGT GTGCACAGAAAAAGATCATCGCGGAAAAGACCAAGATTCCGGCAGTCTTCAAGATTGACGCCCTC AACGAGAACAAAGTGTTGGTCCTCGACACCGACTACAAAAAGTACCTCCTCTTCTGTATGGAAAAC TCCGCAGAGCCCGAGCAGTCGCTCGTGTGTCAGTGTTTGGTGCGAACACCCGAGGTCGACGACGA GGCCTTGGAGAAATTCGACAAGGCGCTCAAGGCCCTCCCCATGCACATTAGGCTCTCGTTCAACCC GACTCAGTTGGAAGAGCAGTGTCATATC! ! ! Case Ref. P238WO IPTector® Codon! optimized! DNA!sequence sequence! (plasmid! name) AoBLG-E! TTGATCGTCACTCAGACTATGAAGGGCCTCGACATCCAGAAAGTGGCAGGTACCTGGTATTCCTTG (pTFB-188)! GCAATGGCAGCGTCGGACATTTCCCTCTTGGACGCCCAGTCCGCACCTCTCCGCGTCTATGTGGAA (SEQ!ID!NO:!9)! GAGTTGAAGCCCACACCTGAAGGAGATTTGGAGATCCTCTTGCACAAATGGGAAAACGGCGAGT GTGCCCAGAAGAAAATCATCGCGGAAAAGACCAAGATTCCTGCGGTCTTCAAGATTGATGCGCTC AACGAAAACAAGGTGCTCGTGTTGGACACCGACTACAAAAAGTACCTCCTCTTCTGTATGGAAAAC TCGGCAGAACCCGAACAGTCCCTCGTGTGTCAGTGTCTCGTGAGGACACCCGAAGTGGATGACGA GGCCCTCGAAAAGTTCGATAAGGCACTCAAGGCCTTGCCGATGCACATCCGCCTCTCGTTCAACCC TACTCAGTTGGAGGAACAGTGTCATATC! AoBLG-F! TTGATCGTTACCCAGACCATGAAGGGCTTGGACATCCAGAAGGTTGCCGGTACCTGGTATTCTCTG (pTFB-217! GCCATGGCCGCTTCTGACATCTCTTTGTTGGACGCCCAGTCTGCTCCACTTCGCGTTTACGTTGAGG (SEQ!ID!NO:!10)! AGCTTAAGCCAACCCCAGAGGGTGATCTGGAGATCTTGTTGCACAAGTGGGAGAATGGCGAGTG TGCCCAGAAGAAGATCATCGCTGAGAAGACCAAGATCCCTGCTGTTTTCAAGATCGACGCTCTTAA CGAGAATAAGGTGCTGGTGCTGGACACCGATTACAAGAAGTATTTGTTGTTCTGTATGGAGAACT CTGCTGAGCCAGAGCAGTCTTTGGTTTGCCAGTGCCTTGTTCGCACCCCTGAGGTGGACGACGAG GCTCTTGAGAAGTTCGATAAGGCTTTGAAGGCTCTGCCAATGCACATCCGCTTGAGCTTCAATCCT ACCCAGTTGGAGGAGCAGTGTCACATCTGA! AoBLG-G CTTATCGTTACCCAGACCATGAAGGGCTTGGATATCCAGAAGGTGGCTGGTACCTGGTACTCTCTT (pTFB-219)! GCCATGGCTGCCTCTGACATCTCTCTTCTTGACGCCCAGTCCGCCCCTCTTCGTGTGTATGTTGAAG (SEQ!ID!NO:!11)! AATTGAAGCCGACCCCTGAAGGTGATCTTGAGATCCTGTTGCATAAGTGGGAAAATGGTGAATGT GCCCAGAAGAAGATCATCGCTGAAAAGACCAAGATCCCTGCCGTTTTCAAGATCGACGCTTTGAAT GAAAATAAGGTTCTTGTTTTGGATACCGATTATAAGAAGTACCTGCTGTTCTGCATGGAGAATAGC GCCGAACCCGAACAGAGCCTGGTGTGCCAGTGCCTTGTTCGTACCCCGGAAGTTGACGATGAGGC TTTGGAGAAGTTCGATAAGGCCCTGAAGGCCTTGCCTATGCACATCCGTTTGTCCTTCAATCCTACC CAGCTTGAGGAACAGTGCCACATCTGA! AoBLG-H TTGATCGTGACCCAGACTATGAAGGGATTGGATATCCAGAAGGTGGCTGGAACCTGGTACTCCTT (pTFB-221)! GGCTATGGCCGCTAGCGACATCTCTCTGTTGGACGCCCAGTCCGCCCCTTTGCGCGTGTACGTTGA (SEQ!ID!NO:!12)! GGAACTGAAGCCTACCCCTGAGGGTGATTTGGAAATCTTGCTTCACAAGTGGGAAAACGGTGAAT GCGCTCAAAAGAAGATCATCGCTGAAAAGACCAAGATCCCGGCCGTTTTCAAGATCGACGCCTTG AACGAAAACAAGGTTTTGGTTCTGGATACTGATTACAAGAAGTACTTGCTGTTCTGTATGGAAAAT TCTGCCGAACCGGAACAGTCTCTGGTTTGTCAGTGCCTGGTTCGGACTCCCGAAGTTGATGACGAG GCCCTTGAGAAGTTCGACAAGGCCCTTAAGGCCCTGCCCATGCACATCCGTCTGTCTTTCAACCCTA CTCAACTGGAGGAGCAGTGCCATATCTGA! ! [0092] The!host!cell!is!preferably!a!eukaryotic,!a!prokaryotic!or!an!algal!cell.!Among!eukaryotic!host! cells,!fungal!cells,!optionally!a!filamentous!fungus!or!yeast!are!particularly!useful!and!productive!when! it!comes!to!production!of!BLG.!Particularly,!the!filamentous!fungus,!such!as!those!from!the!genus!of! Aspergillus,!Trichoderma,!or!Rhizopus!are!preferred.!Such! filamentous! fungi! include! the!species!of! Aspergillus!sp,!Aspergillus!oryzae,!Aspergillus!niger,!Trichoderma!sp,!or!Rhizopus!sp.!Also!yeast,!e.g.!of! the!genus!of!Pichia,!Saccharomyces,!Yarrowia,!or!Hansenula,!can!advantageously!be!used!as!host!cell,! including!particularly!yeast!from!the!species!of!Pichia!pastoris,!Pichia!sp.,!Saccharomyces!cerevisiae,! Yarrowia!sp.,!Yarrowia!lipolytica,!Hansenula!polymorpha,!or!Hansenula!sp.! [0093] More!specifically!described!herein!is!a!genetically!modified!A.!oryzae!host!cells!expressing!a! modified!BLG!comprising!an!amino!acid!sequence!as!comprised!in!or!set!forth!in!SEQ!ID!NO:!4,!56,!58,! 60,!62,!64,!66,!68,!73,!75,!77,!79,!81,!83,!85,!87,!89,!91,!93,!95,!97,!99,!101,!103,!105,!107,!109,!111,! 113,!115,!117,!119,!121,!123,!125,!127,!129,!131,!133,!135,!137,!139,!141,!143,!145,!147,!149,!151,!153,! ! ! Case Ref. P238WO IPTector® 155,!157,!159,!161,!163,!165,!167!and/or!169,!having!a!higher!fraction!of!essential!and/or!branched! amino!acids! compared! to!a!parent,! optionally!natural,! BLG,! said!host! cell! expressing!one!or!more! codon!optimized!heterologous!genes!as!comprised!in!or!set!forth!in!anyone!of!SEQ!ID!NO:!5,!6,!7,!8,!9,! 10,!11!12,!57,!59,!61,!63,!65,!67,!69,!70,!72,!74,!76,!78,!80,!82,!84,!86,!88,!90,!92,!94,!96,!98,!100,!102,! 104,!106,!110,!112,!114,!116,!118,!120,!122,!124,!126,!128,!130,!132,!134,!136,!138,!140,!142,!144,!146,! 148,!149,!150,!152,!154,!156,!158,!160,!162,!164,!167,!168,!170!and/or!171,!encoding!the!modified! BLG,!wherein!the!host!cell!is!further!modified!by!attenuating,!disrupting!or!deleting!one!or!more!native! genes! encoding! a! protease! selected! from! the! group! of! alpA! (alkaline! protease),! nprA! (neutral! metalloprotease!I),!pepE!(aspartic!endopeptidase),!and!pepC!(serine!protease),!and!wherein!the!host! cell! is! further! modified! by! mutating! a! native! inositol! responsive! enzyme! having! two! mutations! corresponding!to!positions!A80T!and!A83T!as!set!forth!in!SEQ!ID!NO:!28!thereby!increasing!activation! of!transcription!factor!HAC1!in!the!host!cell.! ! Nucleotides! [0094] As!described!supra,!in!a!further!aspect!a!modified!nucleotide!sequence!is!provided!which!is!at! least!50%!identical!to!the!BLG!encoding!gene!comprised!in!anyone!of!SEQ!ID!NO:!5,!6,!7,!8,!9,!10,!11! 12,!57,!59,!61,!63,!65,!67,!69,!70,!72,!74,!76,!78,!80,!82,!84,!86,!88,!90,!92,!94,!96,!98,!100,!102,!104,! 106,!110,!112,!114,!116,!118,!120,!122,!124,!126,!128,!130,!132,!134,!136,!138,!140,!142,!144,!146,!148,! 149,!150,!152,!154,!156,!158,!160,!162,!164,!167,!168,!170!and/or!171,!such!as!at!least!55%,!such!as!at! least!60%,!such!as!at!least!65%,!such!as!at!least!70%,!such!as!at!least!75%,!such!as!at!least!80%,!such! as!at!least!85%,!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%!identical!to!the!BLG!encoding!gene!comprised! in!anyone!of!SEQ!ID!NO:!5,!6,!7,!8,!9,!10,!11!12,!57,!59,!61,!63,!65,!67,!69,!70,!72,!74,!76,!78,!80,!82,!84,! 86,!88,!90,!92,!94,!96,!98,!100,!102,!104,!106,!110,!112,!114,!116,!118,!120,!122,!124,!126,!128,!130,! 132,!134,!136,!138,!140,!142,!144,!146,!148,!149,!150,!152,!154,!156,!158,!160,!162,!164,!167,!168,!170! and/or!171and!encodes!a!modified!BLG!having!at!least!one!additional!essential!amino!acid!compared! to! a! parent! BLG.! Further! a! polynucleotide! construct! is! provided! which! comprises! the! nucleotide! sequence!which!is!at!least!50%!identical!to!the!BLG!encoding!gene!comprised!in!anyone!of!SEQ!ID!NO:! 5,!6,!7,!8,!9,!10,!11!12,!57,!59,!61,!63,!65,!67,!69,!70,!72,!74,!76,!78,!80,!82,!84,!86,!88,!90,!92,!94,!96,! 98,!100,!102,!104,!106,!110,!112,!114,!116,!118,!120,!122,!124,!126,!128,!130,!132,!134,!136,!138,!140,! 142,!144,!146,!148,!149,!150,!152,!154,!156,!158,!160,!162,!164,!167,!168,!170!and/or!171,!such!as!at! least!55%,!such!as!at!least!60%,!such!as!at!least!65%,!such!as!at!least!70%,!such!as!at!least!75%,!such! as!at!least!80%,!such!as!at!least!85%,!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%!identical!to!the!BLG! ! ! Case Ref. P238WO IPTector® encoding!gene!comprised!in!anyone!of!SEQ!ID!NO:!5,!6,!7,!8,!9,!10,!11!12,!57,!59,!61,!63,!65,!67,!69,!70,! 72,!74,!76,!78,!80,!82,!84,!86,!88,!90,!92,!94,!96,!98,!100,!102,!104,!106,!110,!112,!114,!116,!118,!120,! 122,!124,!126,!128,!130,!132,!134,!136,!138,!140,!142,!144,!146,!148,!149,!150,!152,!154,!156,!158,!160,! 162,!164,!167,!168,!170!and/or!171.!The!BLG!encoding!gene!preferably!also!encodes!a!signal!peptide! fused!to!the!BLG.!The!signal!peptide!is!preferably!heterologous!to!host!cell!and/or!to!the!BLG.!Several! signal!peptides!of!fungal!origin!were!compared!to!identify!the!optimal!sequence!for!secretion!of!BLG.! No!secretion!was!detected!using!the!native!bovine!signal!peptide!(not!shown).!As!shown!(see!figure! 4),! the! use! of! an! engineered! fungal! signal! peptide! derived! from! plectasin! resulted! in! the! highest! amount!of!BLG!present!in!the!culture!supernatant!in!a!strain!containing!a!single!copy!of!the!AoBLG! gene!(AoBLG-A),!while!when!using!a!cutinase!SP,!titers!comparable!to!the!use!of!plectasin!SP!were! observed!in!single!copy!strains.!Lower!titers!were!obtained!for!the!remaining!SP!tested.!Accordingly,! in!some!embodiments!the!encoded!signal!peptide!comprises!an!amino!acid!sequence!which!is!at!least! 50%!identical!to!the!signal!peptide!comprised!in!SEQ!ID!NO:!13,!such!as!at!least!55%,!such!as!at!least! 60%,!such!as!at!least!65%,!such!as!at!least!70%,!such!as!at!least!75%,!such!as!at!least!80%,!such!as!at! least!85%,!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%!identical!to!the!signal!peptide!comprised!in!SEQ!ID! NO:!13.!The!polynucleotide!moiety!encoding!the!signal!peptide!may!be!at!least!50%!identical!to!the! signal!peptide!encoding!sequence!comprised!in!SEQ!ID!NO:!14,!such!as!at!least!55%,!such!as!at!least! 60%,!such!as!at!least!65%,!such!as!at!least!70%,!such!as!at!least!75%,!such!as!at!least!80%,!such!as!at! least!85%,!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%!identical!to!the!signal!peptide!encoding!sequence! comprised! in! SEQ! ID! NO:! 14.! In! other! embodiments! the! signal! peptide! is! derived! from! a! natural! defensin.!Natural!defensins!are!known!in!the!art!and!include!for!example!fungal!defensin.!One!such! fungal!defensin!signal!peptide!which!has!shown!particularly!useful!for!this!use!is!the!plectasin!signal! peptide,!in!particular!the!signal!peptide!from!the!plectasin!from!the!genus!of!Pseudoplectania,!such! as!Pseudoplectania!nigrella.!! [0095] The!gene!encoding!the!BLG!and!optionally!the!signal!peptide!is!preferably!operably!linked!to! a!promoter.!The!said!promoter!can!be!native!or!heterologous!to!the!host!cell!and/or!to!the!BLG.!In! preferred!embodiments!the!promoter!is!heterologous!to!both!the!host!cell!and!to!the!BLG!for!optimal! performance.! In! some! embodiments! the! promoter! is! an! engineered! synthetic! promoter,! in! other! embodiments!the!promoter!is!an!inducible!promoter!for!example!by!nutrients!such!as!starch,!sugars,! maltose,!glucose!and!the!like.!In!still!other!embodiments,!the!promoter!is!an!amylase!promoter.!The! nucleotide!sequence!of!the!promoter!is!preferably!at!least!50%!identical!to!the!promoter!sequence! comprised!in!SEQ!ID!NO:!15,!such!as!at!least!55%,!such!as!at!least!60%,!such!as!at!least!65%,!such!as! ! ! Case Ref. P238WO IPTector® at!least!70%,!such!as!at!least!75%,!such!as!at!least!80%,!such!as!at!least!85%,!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%!identical!to!the!promoter!comprised!in!SEQ!ID!NO:!15.!The!said!polynucleotide!construct! is!preferably!comprised!in!an!expression!vector,!and/or!is!in!itself!and!expression!vector.!Furthermore,! the!host!cell!described!herein!also!preferably!comprises!the!aforesaid!polynucleotide!construct.!! ! Cell!culture!and!method!of!culturing! [0096] As!described!supra,!in!a!further!aspect!a!cell!culture!is!provided!which!comprises!the!host!cell! described! herein! and! a! growth!medium,! as! well! as!methods! are! provided! for! producing! BLG! by! culturing! the! cell! culture! at! conditions! allowing! the! host! cell! to! produce! the! BLG;! and! optionally! recovering!and/or!isolating!the!BLG.!Suitable!growth!mediums!for!host!cell!described!herein!are!well! known!in!the!art.!The!cell!culture!can!be!cultivated!in!a!nutrient!medium!using!methods!known!in!the! art!at!conditions!suitable!for!production!of!the!BLG!and/or!for!propagating!cell!count.!For!example,! the!culture!may!be!cultivated!by! shake! flask!cultivation,!or! small-scale!or! large-scale! fermentation! (including! continuous,! batch,! fed-batch,! or! solid-state! fermentations)! in! laboratory! or! industrial! fermenters! in! a! suitable! medium! and! under! conditions! allowing! the! host! cells! to! grow! and/or! propagate,!optionally!to!be!recovered!and/or!isolated.! [0097] The!cultivation!can!take!place!in!a!suitable!nutrient!medium!comprising!carbon!and!nitrogen! sources!and!inorganic!salts,!using!procedures!known!in!the!art.!The!medium!will!usually!contain!all! nutrients!necessary!for!the!growth!and!survival!of!the!respective!host!cell,!such!as!carbon!and!nitrogen! sources!and!other!inorganic!salts.!Suitable!media,!e.g.!minimal!or!complex!media,!are!available!from! commercial!suppliers,!or!may!be!prepared!according!to!published!receipts,!e.g.!the!American!Type! Culture! Collection! (ATCC)! Catalogue! of! strains.! Non-limiting! standard!medium!well! known! to! the! skilled! person! include! Luria! Bertani! (LB)! broth,! Sabouraud! Dextrose! (SD)! broth,! MS! broth,! Yeast! Peptone! Dextrose,! BMMY,! GMMY,! or! Yeast!Malt! Extract! (YM)! broth,! which! are! all! commercially! available.!The!selection!of!an!appropriate!medium!may!be!based!on!the!choice!of!host!cell!and/or! based!on!the!regulatory!requirements!for!the!host!cell.!The!medium!may,!if!desired,!contain!additional! components! favoring! the! host! cells! over! other! potentially! contaminating! microorganisms.! Accordingly,!in!an!embodiment!a!suitable!nutrient!medium!comprises!a!carbon!source!(e.g.!C6!sugars! (such! as! glucose),! maltose,! molasses,! starch,! cellulose,! xylan,! pectin,! lignocellolytic! biomass! hydrolysate,! C5! sugars! (such! as! arabinose! or! xylose),! acetate,! glycerol,! plant! oils,! sucrose,! yeast! extract,!peptone,!casamino!acids!or!mixtures!thereof),!a!nitrogen!source!(e.g.!ammonia,!ammonium! sulphate,!ammonium!nitrate,!ammonium!chloride,!etc.),!an!organic!nitrogen!source!(e.g.!amines!yeast! extract,!malt!extract,!peptone,!soybean-hydrolysate,!or!digested!fermentative!microorganisms!etc.)! ! ! Case Ref. P238WO IPTector® and!inorganic!nutrient!sources!(e.g.!phosphate,!magnesium,!potassium,!zinc,!iron,!etc.).!In!particular! embodiments!the!medium!also!contains!an!inducer!inducing!expression!of!the!BLG!in!the!host!cell.! [0098] Culturing!of!the!host!cell!may!be!performed!over!a!period!of!about!0.5!to!about!30!days.!The! cultivation!process!may!be!a!batch!process,!continuous!or!fed-batch!process,!suitably!performed!at!a! temperature!in!the!range!of!0-100!°C!or!10-80!°C,!for!example,!from!about!20°C!to!about!50!°C!and/or! at!a!pH,!for!example,!from!about!2!to!about!10.!Preferred!fermentation!conditions!for!fungal!host!cells! are!a!temperature!in!the!range!of!from!about!25!°C!to!about!55!°C!and!at!a!pH!of!from!about!3!to! about!9.!The!appropriate!conditions!are!usually!selected!based!on!the!choice!of!host!cell.!Accordingly,! in!an!embodiment!the!method!may!further!comprise!one!or!more!elements/steps!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.! [0099] In!some!embodiments!the!method!further!comprises!feeding!the!cell!culture!with!one!or!more! precursors!or!substrates!or! inducers! leading!to!the!expression!of!the!BLG.!The!method!can!further! comprise! recovering! the!BLG!and!mixing! it!with!one!or!more!carriers,!agents,!additives,!adjuvants! and/or!excipients.!The!cell!culture!and/or!the!BLG!comprised!in!the!cell!culture!and/or!the!medium! om! the! cell! culture!may! also! be! recovered! and/or! isolated! using!methods! known! in! the! art.! For! example,!the!BLG!may!be!recovered!or!purified!from!the!nutrient!medium!by!conventional!procedures! including,! but! not! limited! to,! centrifugation,! filtration,! extraction,! precipitation,! spray-drying,! or! lyophilization.! In! a! particular! embodiment! the! method! includes! a! recovery! and/or! isolation! step! comprising!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!BLG!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!BLG;!! c)! contacting!the!supernatant!with!one!or!more!ion!exchange!or!reversed-phase!chromatography! columns!in!order!to!obtain!at!least!a!portion!of!the!BLG;!! d)! extracting!the!BLG;!and/or! e)! precipitating! the! BLG! by! crystallization! or! evaporating! the! solvent! of! the! liquid! phase;! and! ! ! Case Ref. P238WO IPTector® optionally!isolating!the!BLG!by!filtration!or!gravity!separation;! thereby!recovering!and/or!isolating!the!BLG.! ! Fermentation!compositions! [0100] In!a!further!aspect!a!fermentation!composition!is!provided!which!comprises!the!BLG!obtained! from! the! culturing! of! the! cell! culture.! In! some! embodiments! a! majority! of! the! solid! cellular! material/debris!has!been!separated,!such!as!at!least!50%,!such!as!at!least!75%,!such!as!at!least!95%,! such! as! at! least! 99%! of! solid! cellular! material! has! been! separated! from! the! composition.! The! fermentation!composition!may!further!comprise!one!or!more!further!compounds!or!metabolites!from! the!cell!culture.!Such!compounds!and/or!metabolites!of!the!cell!culture!include!precursors!for!the!BLG! as!well!as!compounds!selected!from!trace!metals,!vitamins,!salts,!yeast!nitrogen!base,!carbon!source,! YNB,!and/or!amino!acids!of!the!fermentation.!In!particular!the!composition!comprises!a!concentration! of!the!BLG!of!at!least!1!mg/kg!composition,!such!as!at!least!5!mg/kg,!such!as!at!least!10!mg/kg,!such! as!at!least!20!mg/kg,!such!as!at!least!50!mg/kg,!such!as!at!least!100!mg/kg,!such!as!at!least!500!mg/kg,! such!as!at!least!1.000!mg/kg,!such!as!at!least!5.000!mg/kg,!such!as!at!least!10.000!mg/kg,!such!as!at! least!50.000!mg/kg.!The!composition!may!also!further!comprise!one!or!more!carriers,!agents,!additives! and/or!excipients.! ! Modified!BLG!polypeptides!and!compositions! [0101] As!described! supra,! in! a! further!aspect! a!modified!BLG!polypeptide,!optionally! a!modified! bovine!BLG!polypeptides,!such!as!AoBLG,!is!provided!comprising!at!least!one!substitution!of!a!non- essential!and/or!non-branched!amino!acid!with!an!essential!and/or!branched!amino!acid!compared! to!a!parent!unmodified!BLG.!In!some!embodiments!the!modified!BLG!polypeptide!comprises!at!least! at!least!2,!such!as!at!least!5,!such!as!at!least!10,!such!as!at!least!15,!such!as!at!least!20,!such!as!at!least! 25!substitutions!of!a!non-essential!and/or!non-branched!amino!acid!with!an!essential!and/or!branched! amino!acid.!In!some!embodiments!the!modified!BLG!polypeptide!comprises!at!least!1,!such!as!at!least! 2,!such!as!at!least!3,!such!as!at!least!4,!such!as!5,!such!as!at!least!6,!such!as!at!least!7!substitutions!of! a! non-leucine! amino! acid! with! a! leucine! amino! acid.! In! some! embodiments! the! modified! BLG! comprises!an!amino!acid!sequence!which!is!at!least!50%,!such!as!at!least!55%,!such!as!at!least!60%,! such!as!at!least!65%,!such!as!at!least!70%,!such!as!at!least!75%,!such!as!at!least!80%,!such!as!at!least! 85%,!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%!identical!to!the!BLG!comprised!in!anyone!of!SEQ!ID!NO:! 1,!2,!3,!4,!56,!58,!60,!62,!64,!66,!68,!71,!73,!75,!77,!79,!81,!83,!85,!87,!89,!91,!93,!95,!97,!99,!101,!103,! 105,!107,!109,!111,!113,!115,!117,!119,!121,!123,!125,!127,!129,!131,!133,!135,!137,!139,!141,!143,!145,! ! ! Case Ref. P238WO IPTector® 147,!149,!151,!153,!155,!157,!159,!161,!163,!165,!167!and/or!169.!In!some!embodiments!the!modified! BLG!comprises!an!amino!acid!sequence!as!set!forth!in!SEQ!ID!NO:!4,!56,!58,!60,!62,!64,!66,!68,!73,!75,! 77,!79,!81,!83,!85,!87,!89,!91,!93,!95,!97,!99,!101,!103,!105,!107,!109,!111,!113,!115,!117,!119,!121,!123,! 125,!127,!129,!131,!133,!135,!137,!139,!141,!143,!145,!147,!149,!151,!153,!155,!157,!159,!161,!163,!165,! 167!and/or!169.!In!some!embodiments!the!modified!BLG!comprises!up!to!7!substitutions!of!a!non- leucine!amino!acid!with!a!leucine!amino!acid!corresponding!to!anyone!of!position!1,!5,!21,!96,!108,! 120,!139,!150!and/or!153!of!the!modified!BLG!set!forth!in!anyone!SEQ!ID!NO:!73,!75,!77,!79,!81,!83,! 85,!87,!89,!91,!93,!95,!97,!99,!101,!103,!105,!107,!109,!111,!113,!115,!117,!119,!121,!123,!125,!127,!129,! 131,!133,!135,!137,!139,!141,!143,!145,!147,!149,!151,!153,!155,!157,!159,!161,!163,!165,!167!and/or! 169.!In!some!embodiments!the!modified!BLG!comprises!one!substitution!of!a!non-leucine!amino!acid! with!a!leucine!amino!acid!in!a!position!corresponding!to!anyone!of!position!1,!21,!96,!108,!120,!139,! 150!and/or!153!of!the!modified!BLG!set!forth!in!anyone!of!SEQ!ID!NO:!75,!77,!79,!81,!83,!85!and/or!87.! In!some!embodiments!the!modified!BLG!comprises!2!substitutions!of!a!non-leucine!amino!acid!with!a! leucine!amino!acid!corresponding!to!anyone!of!position!1,!5,!21,!96,!108,!120,!139,!150!and/or!153!of! the!modified!BLG!set!forth!in!anyone!of!SEQ!ID!NO:!73,!89,!91,!93,!95,!97,!99,!101,!103,!105,!107!and/or! 109.!In!some!embodiments!the!modified!BLG!comprises!3!substitutions!of!a!non-leucine!amino!acid! with!a!leucine!amino!acid!corresponding!to!anyone!of!position!21,!96,!108,!120,!139,!150!and/or!153! of!the!modified!BLG!set!forth!in!anyone!of!SEQ!ID!NO:!111,!113,!115,!117,!119,!121,!123,!125,!127,! 129,!131!and/or!133.! In!some!embodiments!the!modified!BLG!comprises!4!substitutions!of!a!non- leucine!amino!acid!with!a!leucine!amino!acid!corresponding!to!anyone!of!position!21,!96,!108,!120,! 139,!150!and/or!153!of!the!modified!BLG!set!forth!in!anyone!of!SEQ!ID!NO:!135,!137,!139,!141,!143,! 145!and/or!147.!In!some!embodiments!the!modified!BLG!comprises!5!substitutions!of!a!non-leucine! amino!acid!with!a!leucine!amino!acid!corresponding!to!anyone!of!position!1,!21,!96,!108,!120,!139,! 150!and/or!153!of!the!modified!BLG!as!set!forth!in!SEQ!ID!NO:!149,!151,!153,!155,!157,!159!and/or! 161.!In!some!embodiments!the!modified!BLG!comprises!6!substitutions!of!a!non-leucine!amino!acid! with!a!leucine!amino!acid!corresponding!to!anyone!of!position!21,!96,!108,!120,!139,!150!and/or!153! of!the!modified!BLG!as!set!forth!in!anyone!of!SEQ!ID!NO:!163,!165!and/or!167.!In!some!embodiments! the!modified!BLG!comprises!7!substitutions!of!a!non-leucine!amino!acid!with!a! leucine!amino!acid! corresponding!to!position!21,!96,!108,!120,!139,!150!and!153!of!the!modified!BLG!set!forth!in!SEQ!ID! NO:!169.! In! some!embodiments! the!modified!BLG! comprises! at! least!one! amino! acid! substitution! compared! to! anyone! of! bovine! BLG! A,! bovine! BLG! B! and! bovine! BLG! C,! optionally! at! least! two! substitutions.!In!some!embodiments!the!modified!BLG!is!a!modified!bovine!BLG!C!comprising!at!least! one!substitution!of!Alanine!with!Valine!in!a!position!corresponding!to!position!118!of!the!BLG!C!in!SEQ! ID! NO:! 3.! In! other! embodiments! the! modified! BLG! is! a! modified! BLG! A! comprising! at! least! one! ! ! Case Ref. P238WO IPTector® substitution!of!Glutamine!with!Histidine!at!a!position!corresponding!to!position!59!of!the!BLG!A!in!SEQ! ID!NO:!1.!In!some!embodiments!the!modified!BLG!may!contain!a!Histidine!and!a!Valine!at!positions! corresponding!to!position!59!and!118!respectively!in!anyone!of!the!BLG^s!comprised!in!SEQ!ID!NO:!1,! 2!or!3.!! [0102] The!BLG!is!or!can!be!a!combination!of!the!three!major!BLG!variants!present!in!cow!milk!(A,!B! and! C)! in! a! single!molecule,! incorporating! the! best! possible! composition! of! essential! amino! acids! without!introducing!any!non-natural!modification!i.e.,!two!amino!acid!substitutions!are!introduced!in! the!bovine!BLG!B!backbone,!one!from!BLG!A!and!one!from!BLG!C.!Contrary!to!all!other!developed! recombinant!BLGs!that!are!based!solely!on!BLG!B,!the!BLG!described!herein!is!therefore!nutritionally! superior! to! known! recombinant! BLG! products,! while! still! being! representative! of/equivalent! to! drinking!cow^s!milk!than!a!single!variant.!The!amino!acid!sequence!of!the!AoBLG!has!a!His!at!position! 59!and!Val!at!position!118!replacing!Gln59!and!Ala118,!respectively!(see!figure!1).!These!modifications! were!selected!to!achieve!a!combination!of!not!altering!natural!BLG!sequences!but!combining!them!in! a!way!that!enhances!nutritional!value!^!using!essential!amino!acids!(His,!Leu!and/or!Val)!instead!of! non-essential!amino!acid!(Gln!and!Ala).!In!this!way,!the!AoBLG!product!has!a!higher!level!of!essential! amino!acids!than!BLG!A,!B!or!C!in!a!single!molecule,!and!has!a!more!equivalent!sequence!compared! to!the!major!BLG!variants!present!in!milk.!The!modified!BLG!polypeptide!is!also!suitably!incorporated! and/or! mixed! into! a! food! or! feed! composition,! optionally! thereby! enriching! the! food! or! feed! composition!with!essential!amino!acids!from!the!modified!BLG.!! [0103] 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.! ! Sequence!listings! [0104] The! present! application! contains! a! listing! of! sequences! included! in! the! below! table! A! submitted!electronically!in!ST26!format!which!is!hereby!incorporated!by!reference!in!its!entirety.!! Table!A! SEQ!ID!NO:!1!->!protein!sequence!of!BLG!A! SEQ!ID!NO:!2!->!protein!sequence!of!BLG!B! SEQ!ID!NO:!3!->!protein!sequence!of!BLG!C! SEQ!ID!NO:!4!->!protein!sequence!of!AoBLG! SEQ!ID!NO:!5!->!DNA!sequence!encoding!AoBLG-A! SEQ!ID!NO:!6!->!DNA!sequence!encoding!AoBLG-B! SEQ!ID!NO:!7!->!DNA!sequence!encoding!AoBLG-C! SEQ!ID!NO:!8!->!DNA!sequence!encoding!AoBLG-D! SEQ!ID!NO:!9!->!DNA!sequence!encoding!AoBLG-E! ! ! Case Ref. P238WO IPTector® SEQ!ID!NO:!10!->!DNA!sequence!encoding!AoBLG-F! SEQ!ID!NO:!11!->!DNA!sequence!encoding!AoBLG-G! SEQ!ID!NO:!12!->!DNA!sequence!encoding!AoBLG-H! SEQ!ID!NO:!13!->!protein!sequence!of!Plectasin!Signal! SEQ!ID!NO:!14!->!DNA!sequence!of!Plectasin!Signal! SEQ!ID!NO:!15!->!DNA!sequence!of!PNA2!promoter! SEQ!ID!NO:!16!->!protein!sequence!of!AlpA!protease!WILD!TYPE! SEQ!ID!NO:!17!->!DNA!sequence!of!AlpA!protease!WILD!TYPE! SEQ!ID!NO:!18!->!protein!sequence!of!AlpA!protease!AFTER!DELETION! SEQ!ID!NO:!19!->!DNA!sequence!of!AlpA!protease!AFTER!DELETION! SEQ!ID!NO:!20!->!protein!sequence!of!nprA!protease!WILD!TYPE! SEQ!ID!NO:!21!->!DNA!sequence!of!nprA!protease!WILD!TYPE! SEQ!ID!NO:!22!->!protein!sequence!of!nprA!protease!AFTER!DELETION! SEQ!ID!NO:!23!->!DNA!sequence!of!nprA!protease!AFTER!DELETION! SEQ!ID!NO:!24!->!protein!sequence!of!prtT!transcription!factor!WILD!TYPE! SEQ!ID!NO:!25!->!DNA!sequence!of!prtT!transcription!factor!WILD!TYPE! SEQ!ID!NO:!26!->!protein!sequence!of!prtT!transcription!factor!AFTER!DELETION! SEQ!ID!NO:!27!->!DNA!sequence!of!prtT!transcription!factor!AFTER!DELETION! SEQ!ID!NO:!28!->!protein!sequence!of!IreA!WILD!TYPE! SEQ!ID!NO:!29!->!DNA!sequence!of!IreA!WILD!TYPE! SEQ!ID!NO:!30!->!protein!sequence!of!IreA!MUTANT!(A80T/A83T)! SEQ!ID!NO:!31!->!DNA!sequence!of!IreA!MUTANT!(A80T/A83T)! SEQ!ID!NO:!32!->!protein!sequence!of!pepC!WILD!TYPE!! SEQ!ID!NO:!33!->!DNA!sequence!of!pepC!WILD!TYPE!(own!promoter!DNA!in!green)! SEQ!ID!NO:!34!->!protein!sequence!of!pepC!AFTER!PROMOTER!EXCHANGE! SEQ!ID!NO:!35!->!DNA!sequence!of!pepC!AFTER!PROMOTER!EXCHANGE! SEQ!ID!NO:!36!->!protein!sequence!of!pepE!protease!WILD!TYPE! SEQ!ID!NO:!37!->!DNA!sequence!of!pepE!protease!WILD!TYPE! SEQ!ID!NO:!38!->!protein!sequence!of!pepE!AFTER!DELETION! SEQ!ID!NO:!39!->!DNA!sequence!of!pepE!AFTER!DELETION! SEQ!ID!NO:!40!->!DNA!sequence!of!Brick-007! SEQ!ID!NO:!41!->!DNA!sequence!of!Fragment!FI-wA! SEQ!ID!NO:!42!->!DNA!sequence!of!Fragment!FII-wA! SEQ!ID!NO:!43!->!DNA!sequence!of!Fragment!FIII-wA! SEQ!ID!NO:!44!->!DNA!sequence!of!Ple!SP! SEQ!ID!NO:!45!->!DNA!sequence!of!Cut!SP! SEQ!ID!NO:!46!->!DNA!sequence!of!Lip!SP! SEQ!ID!NO:!47!->!DNA!sequence!of!SP002! SEQ!ID!NO:!48!->!DNA!sequence!of!SP034! SEQ!ID!NO:!49!->!DNA!sequence!of!P0200! SEQ!ID!NO:!50!->!DNA!sequence!of!P0201! SEQ!ID!NO:!51!->!DNA!sequence!of!P0202! SEQ!ID!NO:!52!->!DNA!sequence!of!P0203! SEQ!ID!NO:!53!->!DNA!sequence!of!P0459! SEQ!ID!NO:!54!->!DNA!sequence!of!P0460! SEQ!ID!NO:!55!->!DNA!sequence!of!P0461! ! ! Case Ref. P238WO IPTector® SEQ!ID!NO:!56!->!Protein!sequence!of!SuperBLG! SEQ!ID!NO:!57!->!DNA!sequence!of!SuperBLG! SEQ!ID!NO:!58!->!Protein!sequence!of!LeuMax! SEQ!ID!NO:!59!->!DNA!sequence!of!LeuMax!! SEQ!ID!NO:!60!->!LeuMore!1!Protein!(Mature!sequence,!no!signal!peptide!^!identical!as!for!AoBLG)! SEQ!ID!NO:!61!->!LeuMore!1!DNA!(no!signal!peptide!or!start!ATG!^!identical!as!for!AoBLG)! SEQ!ID!NO:!62!->!LeuMore!2!Protein!(Mature!sequence,!no!signal!peptide!^!identical!as!for!AoBLG)! SEQ!ID!NO:!63!->!LeuMore!2!DNA!(no!signal!peptide!or!start!ATG!^!identical!as!for!AoBLG)! SEQ!ID!NO:!64!->!LeuMore!3!Protein!(Mature!sequence,!no!signal!peptide!^!identical!as!for!AoBLG)! SEQ!ID!NO:!65!->!LeuMore!3!DNA!(no!signal!peptide!or!start!ATG!^!identical!as!for!AoBLG)! SEQ!ID!NO:!66!->!LeuMore!4!Protein!(Mature!sequence,!no!signal!peptide!^!identical!as!for!AoBLG)! SEQ!ID!NO:!67!->!LeuMore!4!DNA!(no!signal!peptide!or!start!ATG!^!identical!as!for!AoBLG)! SEQ!ID!NO:!68!->!LeuMore!5!Protein!(Mature!sequence,!no!signal!peptide!^!identical!as!for!AoBLG)! SEQ!ID!NO:!69!->!LeuMore!5!DNA!(no!signal!peptide!or!start!ATG!^!identical!as!for!AoBLG)! SEQ!ID!NO:!70!->!DNA!sequence!of!BLG-Leu1! SEQ!ID!NO:!71!->!protein!sequence!of!BLG-Leu1!->!Mutation!L1Q! SEQ!ID!NO:!72->!DNA!sequence!of!BLG-Leu2! SEQ!ID!NO:!73->!protein!sequence!of!BLG-Leu2!->!Mutation!L1Q, Q5L! SEQ!ID!NO:!74!->!DNA!sequence!of!BLG-Leu3! SEQ!ID!NO:!75!->!protein!sequence!of!BLG-Leu3!->!Mutation!S150L ! SEQ!ID!NO:!76!->!DNA!sequence!of!BLG-Leu4! SEQ!ID!NO:!77!->!protein!sequence!of!BLG-Leu4!->!Mutation!S21L ! SEQ!ID!NO:!78->!DNA!sequence!of!BLG-Leu5! SEQ!ID!NO:!79!->!protein!sequence!of!BLG-Leu5!->!Mutation!A139L ! SEQ!ID!NO:!80!->!DNA!sequence!of!BLG-Leu6! SEQ!ID!NO:!81!->!protein!sequence!of!BLG-Leu6!->!Mutation!P153L ! SEQ!ID!NO:!82->!DNA!sequence!of!BLG-Leu7! SEQ!ID!NO:!83!->!protein!sequence!of!BLG-Leu7!->!Mutation!D96L ! SEQ!ID!NO:!84!->!DNA!sequence!of!BLG-Leu8! SEQ!ID!NO:!85!->!protein!sequence!of!BLG-Leu8!->!Mutation!Q120L ! SEQ!ID!NO:!86->!DNA!sequence!of!BLG-Leu9! SEQ!ID!NO:!87!->!protein!sequence!of!BLG-Leu9!->!Mutation!E108L ! SEQ!ID!NO:!88->!DNA!sequence!of!BLG-Leu10! SEQ!ID!NO:!89!->!protein!sequence!of!BLG-Leu10!->!Mutation!S150L, S21L! ! ! Case Ref. P238WO IPTector® SEQ!ID!NO:!90->!DNA!sequence!of!BLG-Leu11! SEQ!ID!NO:!91!->!protein!sequence!of!BLG-Leu11!->!Mutation!S150L, A139L! SEQ!ID!NO:!92->!DNA!sequence!of!BLG-Leu12! SEQ!ID!NO:!93!->!protein!sequence!of!BLG-Leu12!->!Mutation!S150L, P153L! SEQ!ID!NO:!94->!DNA!sequence!of!BLG-Leu13! SEQ!ID!NO:!95!->!protein!sequence!of!BLG-Leu13!->!Mutation!S150L, D96L! SEQ!ID!NO:!96->!DNA!sequence!of!BLG-Leu14! SEQ!ID!NO:!97!->!protein!sequence!of!BLG-Leu14!->!Mutation!S150L, Q120L! SEQ!ID!NO:!98->!DNA!sequence!of!BLG-Leu15! SEQ!ID!NO:!99!->!protein!sequence!of!BLG-Leu15!->!Mutation!S150L, E108L! SEQ!ID!NO:!100!->!DNA!sequence!of!BLG-Leu16! SEQ!ID!NO:!101->!protein!sequence!of!BLG-Leu16!->!Mutation!S21L, Q120L! SEQ!ID!NO:!102!->!DNA!sequence!of!BLG-Leu17! SEQ!ID!NO:!103!->!protein!sequence!of!BLG-Leu17!->!Mutation!D96L, A139L! SEQ!ID!NO:!104->!DNA!sequence!of!BLG-Leu18! SEQ!ID!NO:!105->!protein!sequence!of!BLG-Leu18!->!Mutation!D96L, Q120L! SEQ!ID!NO:!106!->!DNA!sequence!of!BLG-Leu19! SEQ!ID!NO:!107!->!protein!sequence!of!BLG-Leu19!->!Mutation!D96L, E108L! SEQ!ID!NO:!108!->!DNA!sequence!of!BLG-Leu20! SEQ!ID!NO:!109!->!protein!sequence!of!BLG-Leu20!->!Mutation!Q120L, E108L! SEQ!ID!NO:!110->!DNA!sequence!of!BLG-Leu21! SEQ!ID!NO:!111!->!protein!sequence!of!BLG-Leu21!->!Mutation!S150L, S21L, A139L! SEQ!ID!NO:!112->!DNA!sequence!of!BLG-Leu22! SEQ!ID!NO:!113!->!protein!sequence!of!BLG-Leu22!->!Mutation!S150L, S21L, P153L! SEQ!ID!NO:!114->!DNA!sequence!of!BLG-Leu23! SEQ!ID!NO:!115->!protein!sequence!of!BLG-Leu23!->!Mutation!S150L, A139, P153L! SEQ!ID!NO:!116->!DNA!sequence!of!BLG-Leu24! SEQ!ID!NO:!117->!protein!sequence!of!BLG-Leu24!->!Mutation!S21L, A139L, P153L! SEQ!ID!NO:!118->!DNA!sequence!of!BLG-Leu25! SEQ!ID!NO:!119!->!protein!sequence!of!BLG-Leu25!->!Mutation!S150L, S21L, Q120L! SEQ!ID!NO:!120->!DNA!sequence!of!BLG-Leu26! SEQ!ID!NO:!121!->!protein!sequence!of!BLG-Leu26!->!Mutation!S150L, S21L, D96L! SEQ!ID!NO:!122!->!DNA!sequence!of!BLG-Leu27! ! ! Case Ref. P238WO IPTector® SEQ!ID!NO:!123!->!protein!sequence!of!BLG-Leu27!->!Mutation!S150L, S21L, E108L! SEQ!ID!NO:!124->!DNA!sequence!of!BLG-Leu28! SEQ!ID!NO:!125!->!protein!sequence!of!BLG-Leu28!->!Mutation!S150L, D96L, Q120L! SEQ!ID!NO:!126->!DNA!sequence!of!BLG-Leu29! SEQ!ID!NO:!127!->!protein!sequence!of!BLG-Leu29!->!Mutation!S150L, E108L, D96L! SEQ!ID!NO:!128->!DNA!sequence!of!BLG-Leu30! SEQ!ID!NO:!129!->!protein!sequence!of!BLG-Leu30!->!Mutation!S150L, E108L, Q120L! SEQ!ID!NO:!130->!DNA!sequence!of!BLG-Leu31! SEQ!ID!NO:!131!->!protein!sequence!of!BLG-Leu31!->!Mutation!S21L, D96L, Q120L! SEQ!ID!NO:!132->!DNA!sequence!of!BLG-Leu32! SEQ!ID!NO:!133!->!protein!sequence!of!BLG-Leu32!->!Mutation!S21L, E108L, Q120L! SEQ!ID!NO:!134->!DNA!sequence!of!BLG-Leu33! SEQ!ID!NO:!135!->!protein!sequence!of!BLG-Leu33!->!Mutation!S150L, S21L, A139L, P153L! SEQ!ID!NO:!136->!DNA!sequence!of!BLG-Leu34! SEQ!ID!NO:!137!->!protein!sequence!of!BLG-Leu34!->!Mutation!S150L, S21L, D96L, Q120L! SEQ!ID!NO:!138!->!DNA!sequence!of!BLG-Leu35! SEQ!ID!NO:!139!->!protein!sequence!of!BLG-Leu35!->!Mutation!S150L, S21L, D96L, E108L! SEQ!ID!NO:!140->!DNA!sequence!of!BLG-Leu36! SEQ!ID!NO:!141!->!protein!sequence!of!BLG-Leu36!->!Mutation!S150L, D96L, Q120L, A139L! SEQ!ID!NO:!142->!DNA!sequence!of!BLG-Leu37! SEQ!ID!NO:!143!->!protein!sequence!of!BLG-Leu37!->!Mutation!S150L, S21L, E108L, Q120L! SEQ!ID!NO:!144!->!DNA!sequence!of!BLG-Leu38! SEQ!ID!NO:!145!->!protein!sequence!of!BLG-Leu38!->!Mutation!S150L, D96L, Q120L, P153L! SEQ!ID!NO:!146!->!DNA!sequence!of!BLG-Leu39! SEQ!ID!NO:!147!->!protein!sequence!of!BLG-Leu39!->!Mutation!S21L, D96L, E108L, Q120L! SEQ!ID!NO:!148->!DNA!sequence!of!BLG-Leu40! SEQ!ID!NO:!149!->!protein!sequence!of!BLG-Leu40!->!Mutation!S150L, S21L, A139L, P153L, D96L! SEQ!ID!NO:!150->!DNA!sequence!of!BLG-Leu41! SEQ!ID!NO:!151!->!protein!sequence!of!BLG-Leu41!->!Mutation!S150L, S21L, D96L, E108L, Q120L! SEQ!ID!NO:!152->!DNA!sequence!of!BLG-Leu42! SEQ!ID!NO:!153!->!protein!sequence!of!BLG-Leu42!->!Mutation!S21L, A139L, P153L, D96L, E108L! SEQ!ID!NO:!154->!DNA!sequence!of!BLG-Leu43! SEQ!ID!NO:!155!->!protein!sequence!of!BLG-Leu43!->!Mutation!S21L, A139L, D96L, E108L, Q120L! ! ! Case Ref. P238WO IPTector® SEQ!ID!NO:!156->!DNA!sequence!of!BLG-Leu44! SEQ!ID!NO:!157!->!protein!sequence!of!BLG-Leu44!->!Mutation!S150L, S21L, A139L, D96L, Q120L! SEQ!ID!NO:!158->!DNA!sequence!of!BLG-Leu45! SEQ!ID!NO:!159->!protein!sequence!of!BLG-Leu45!->!Mutation!S150L, S21L, A139L, D96L, E108L! SEQ!ID!NO:!160->!DNA!sequence!of!BLG-Leu46! SEQ!ID!NO:!161!->!protein!sequence!of!BLG-Leu46!->!Mutation!S150L, A139L, P153L, D96L, Q120L! SEQ!ID!NO:!162!->!DNA!sequence!of!BLG-Leu47! SEQ!ID!NO:!163!->!protein!sequence!of!BLG-Leu47!->!Mutation!S150L, S21L, A139L, D96L, Q120L, E108L! SEQ!ID!NO:!164!->!DNA!sequence!of!BLG-Leu48! SEQ!ID!NO:!165!->!protein!sequence!of!BLG-Leu48!->!Mutation!S150L, S21L, P153L, D96L, Q120L, E108L! SEQ!ID!NO:!166->!DNA!sequence!of!BLG-Leu49! SEQ!ID!NO:!167!->!protein!sequence!of!BLG-Leu49!->!Mutation!S150L, S21L, A139L, P153L, D96L, Q120L! SEQ!ID!NO:!168->!DNA!sequence!of!BLG-Leu50! SEQ!ID!NO:!169!->!protein!sequence!of!BLG-Leu50!->!Mutation!S150L, S21L, A139L, P153L, D96L, Q120L, E108L! SEQ!ID!NO:170!->!DNA!sequence!of!Pichia!pastoris!optimised!BLG! AoBLG!(no!signal!peptide!or!start!ATG)! SEQ!ID!NO:171!->!DNA!sequence!of!Aspergillus!niger!optimised!BLG! AoBLG!(no!signal!peptide!or!start!ATG)! ! *!!*!!*! ! ! ! ! Case Ref. P238WO IPTector® Examples! Materials!and!methods! [0105] Chemicals! used! in! the! examples! herein,! e.g.! for! buffers! and! substrates,! are! commercial! products!of!at!least!reagent!grade.! [0106] 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).! ! Strain!Cultivation! [0107] Deep!Well!Plates!(DWP)!containing!3!mL!YPM!medium!(2!g/L!yeast!extract,!2!g/L!peptone,!2! %!(w/v)!maltose)!were!inoculated!with!spores!from!a!transformant!strain!and!incubated!at!30°!C,!200! rpm!for!4!days.!To!select!candidates,!biological!replicates!and!duplicate!cultivation!was!performed.! This!cultivation!protocol!enables!the!prediction!of!yields!obtained!in!bioreactor.! ! Sucrose!Medium! [0108] 1M!Sucrose,!0.18!mM!Na2B407,!2.3!mM!CuSO4,!4.7!mM!FeSO4,!4.7!mM!MnSO4,!3.6!mM!NaMo04,! 45!mM!ZnSO4,!7!mM!KCl,!4.3!mM!MgSO4,!11.2!mM!KH2PO4.!Transformation!plates!contained!sucrose! medium,!supplemented!with!10!mM!NaNO3!and!2!%!agar.! ! Strain!Construction! [0109] Strain! construction!was! carried! out! by! transformation! of! Aspergillus! oryzae! essentially! as! described! in! Christensen! et! al.! (1988);!High-level! expression! of! recombinant! genes! in! Aspergillus! oryzae.!Biotechnology,!vol.!6,!pp.!1419-1422.!A.!oryzae!was!grown!in!a!rich!nutrient!broth.!The!biomass! was! separated! from! the! broth! by! filtration.! The! enzyme! preparation!Glucanex®! (Novozymes)!was! added!to!the!biomass!in!osmotically!stabilizing!buffer!such!as!1.2!M!MgSO4!buffered!to!pH!5.0!with! sodium!phosphate.!The!suspension!was!incubated!for!60!minutes!at!37°!C.!with!gentle!agitation!to! produce! protoplasts.! Protoplasts! were! filtered! through! miracloth! to! remove!mycelial! debris.! The! protoplasts!were!harvested!and!washed!twice!with!STC!(1.2!M!sorbitol,!10!mM!CaCl2,!10!mM!Tris-HCl! pH!7.5).!Protoplasts!were!finally!resuspended!in!200-1000!mL!STC.!! [0110] For!transformation,!2-10!mg!of!plasmid!DNA!was!added!to!100!mL!protoplast!suspension!and! then!200!mL!PEG!solution!(60!%!PEG!4000,!10!mM!CaCl2,!10!mM!Tris-HCl!pH!7.5)!was!added!and!the! ! ! Case Ref. P238WO IPTector® mixture!is!incubated!for!20!minutes!at!room!temperature.!Protoplasts!were!harvested!and!washed! twice! with! 1.2! M! sorbitol.! Protoplasts! were! finally! resuspended! in! 200! mL! 1.2! M! sorbitol.! Transformants!were!selected!for!its!ability!to!grow!on!minimal!sucrose!plates!using!nitrate!(NaNO3)!as! the! sole! nitrogen! source.! The! transforming! DNA! either! in! circular! or! linear! form! is! targeted! to! a! truncated!niaD!gene!that!is!restored!upon!correct!integration,!as!described!in!WO!2013/178674.!After! 5-7!days!of!growth!at!37°!C,!transformants!appeared!as!vigorously!growing!and!sporulating!colonies.! Transformants! were! purified! through! conidiation.! For! some! of! the! strain! modifications! (e.g.,! to! introduce!the!IreA!mutation),!the!CRISPR!MAD7!nuclease!was!used!as!described!elsewhere!using!one! or! two! guide! RNAs! and! a! donor! DNA! fragment! encoding! the! intended! sequence!modification,! if! required!(Vanegas!et!al.,!2022).! ! Strains! [0111] The!original!recipient!strain!used!in!this!work,!TFB-Ao0010,!was!derived!from!A.!oryzae!BECh2,! described! in!WO2000/39322.! The!modifications! introduced! in! the! strain! included! 1)! reduction! of! protease! levels! (e.g.,! alkaline!protease!AlpA,!Neutral! protease!NprA!among!others);! 2)!deletion!of! genes!encoding!major!secreted!endogenous!proteins!(including!neutral!amylase)!and!3)!inactivation! of! a! gene! (ligD)! involved! in! the! non-homologous! end-joining! (NHEJ)! pathway! ^! to! increase! the! frequency!of!homologous!recombination!and!facilitate!gene!editing!(Nakamura!et!al.,!^017).!! [0112] Using!TFB-Ao0010!mul#copy!strains!were!obtained!that!contained!di$erent!copy!numbers!of! the!expression!casse&e!integrated!at!the!niaD!locus!using!the!methods!described!in!WO2013/178674.! The!recipient!strain!used!for!comparison!of!e.g.,!signal!pep#de!e'ciency,!TFB-Ao0094,!was!a!further! modi*ca#on!that!resulted! in!strains!having!the!expression!casse&e! integrated! in!single!copy!at!the! same!niaD!locus.!The!list!of!strains!used!is!shown!in!Table!2.! Strain!Name! Recipient! Copy! no.! Comment/Property! Strain! (AoBLG)! TFB-Ao0010! -! -! Mul#copy!recipient!strain!! TFB-Ao0127! TFB-Ao0010! -! prtT-!! TFB-Ao0131! TFB-Ao0127! -! prtT-,!IreA! TFB-Ao0138! TFB-Ao0010! 5! Mul#copy!AoBLG!in!wt!prtT,!IreA!background! TFB-Ao0144! TFB-Ao0127! 5! Mul#copy!AoBLG!in!prtT-!background! TFB-Ao0145! TFB-Ao0131! 5! Mul#copy! AoBLG! in! prtT-,! IreA! muta#on! background! TFB-Ao0094! TFB-Ao0010! -! Single!copy!recipient!strain! L-BLG-21!! TFB-Ao0094! 1! Single!copy!AoBLG,!Plectasin!SP!! L-BLG-23!! TFB-Ao0094! 1! Single!copy!AoBLG,!Cu#nase!SP!! L-BLG-25!! TFB-Ao0094! 1! Single!copy!AoBLG,!Lipolase!SP!! L-BLG-27!! TFB-Ao0094! 1! Single!copy!AoBLG,!SP034!SP!! L-BLG-29!! TFB-Ao0094! 1! Single!copy!AoBLG,!SP002!SP!! ! ! Case Ref. P238WO IPTector® ! Table!2.!Used!strains!of!A.!oryzae.! ! [0113] For!multicopy!strain!construction,!plasmid!pTFB-0075!was!constructed!using!NEBuilder®!HiFi! DNA! assembly! following! the! manufacturer^s! protocol.! The! backbone! plasmid! was! a! shuttle! E.! coli/Aspergillus! vector! containing! a! complementing! niaD! region! (3^! niaD),! the! A.! oryzae! pyrG! as! selection!marker,!an!engineered!promoter!(Pna2)!of!the!Aspergillus!niger!neutral!amylase!gene!2!and! the! Tamg! terminator! of! the! glucoamylase! gene! as! described! in! WO2013/178674).! The! plasmid! contained! two! adjacent! SwaI! restriction! sites! located! between! the! Pna2! promoter! and! the! Tamg! terminator.! The! insert! (Brick-0007)! encoding! the! expression! cassette! for! AoBLG! and! 20/21! bp! overlapping!DNA!flanks!(to!the!backbone!plasmid)!was!obtained!as!a!gBlock!from!IDT!and!cloned!into! the!SwaI-digested!backbone!plasmid.! [0114] For!single!copy!strain!construc#on,!co-transforma#on!with!three!overlapping!PCR!fragments! was!performed.!Two!of!the!fragments!included!the!upstream!and!downstream!+anking!region!of!the! target!locus!and!the!third!one!included!AoBLG!(Figure!5,!see!Example!2).!As!above,!sequence!overlap! between!the!fragments!enables!in!vivo!liga#on/inser#on!at!the!target!locus!(Figure!5).! ! Plasmids! [0115] Plasmid!pTFB-0075!is!described!in!Example!3!below!(Figure!8)! ! Protein!yield!analysis! [0116] SDS-PAGE!precast!gels!(NuPAGE!Bis-Tris!4-12%)!were!used!run!and!stained!with!Coomassie! blue!as!recommended!by!the!manufacturer!(Invitrogen).! ! Example!1!^!Designing!an!improved!modified!bovine!beta-lactoglobulin!(BLG)!protein!sequence!to! be!produced!in!Aspergillus!oryzae.! [0117] A!modified!bovine!BLG!protein!sequence!was!designed!(AoBLG)!(SEQ!ID!NO:!4)!which!was!a! combination!of!the!three!major!BLG!variants!present!in!cow!milk!(A,!B!and!C)!in!a!single!molecule.!The! AoBLG! amino! acid! sequence! incorporates! the! best! possible! composition! of! essential! amino! acid! without!introducing!any!non-natural!modification!i.e.,!two!amino!acid!substitutions!were!made!in!the! bovine!BLG!B! ^backbone^,! one! from!BLG!A! and!one! from!BLG!C.! Contrary! to! all! other! developed! recombinant!BLGs!that!are!based!solely!on!BLG!B,!AoBLG!is!therefore!nutritionally!superior!to!current! recombinant!BLG!products.! Further,! ingestion/consumption!of!AoBLG! is!more!representative!of!or! ! ! Case Ref. P238WO IPTector® equivalent!to!intake!of!cow^s!milk!than!a!single!variant,!including!the!BLG!B!variant!typically!chosen! for!recombinant!production.!! [0118] The!amino!acid!sequence!of! the!designed!modified!bovine!BLG!manufactured! in!A.!oryzae! (AoBLG)!had!a!His!at!position!59!and!Val!at!position!118!replacing!Gln59!and!Ala118,!respectively!(see! Figure!1).!As!mentioned!above,!the!rationale!for!making!these!changes!is!a!combination!of!not!altering! natural!BLG!sequences!but!combining!them!in!a!way!that!enhances!nutritional!value!^!using!essential! amino!acids!(His,!LEu!and/or!Val)!instead!of!non-essential!amino!acids!(Gln!and!Ala).!In!this!way,!the! AoBLG!product!had!a!higher!level!of!essential!amino!acids!than!BLG!A,!B!or!C!in!a!single!molecule,!and! had!a!more!equivalent!sequence!compared!to!the!major!BLG!variants!present!in!milk.!This!modified! BLG!could!be!produced! in!commercially! feasible! levels! in!microbial!cell! factories!and!shown! in! the! following!examples.! ! Example!2!-!Signal!peptide!sequences!for!increased!AoBLG!yield.! [0119] The!purpose!of!this!experiment!was!to!screen!for!signal!peptides!to!investigate!and!identify! SPs!providing!the!best!yield!of!AoBLG!when!expressed!in!host!cells.!The!investigated!SPs!are!described! below!(Table!2)!and!include!heterologous!SP^s!Including!Ple!SP,!Cut!SP!and!Lip!SP!of!different!length! and!amino!acid!composition!(Table!3).! Signal!pep"de! Origin! Plectasin!(Ple!SP)! Engineered!SP!derived!from!the!Pseudoplectania!nigrella!plectasin!gene! Cu#nase!(Cut!SP)! Humicola!insolens!cu#nase!gene!(WO!2005/121333)! Lipase!(Lip!SP)! Thermomyces!lanuginosus!lipase!gene!(US8586330)! SP002! A.!niger!gene!puta#ve!glycoside!hydrolase! SP034! A.!niger!gene!encoding!mannan!endo-1,4-beta-mannosidase! Table!3.!Origin!of!the!used!SP!sequences!for!AoBLG!yield.! ! [0120] To!enable!comparison!of!BLG!yields!using!different!SP,!three!overlapping!DNA!fragments!were! co-transformed!into!strain!TFB-Ao0094,!for!each!SP.!Fragment!1!encodes!the!upstream!region!of!the! A.!oryzae!colony!color!gene!wA,!the!Pna2!promoter!derived!from!the!A.!niger!neutral!amylase!gene! and!the!specific!SP.!Fragment!2!encodes!the!mature!AoBLG.!Fragment!3!encodes!the!Tamg!terminator! derived!from!the!A.!niger!glucoamylase!gene!and!the!downstream!region!of!wA!(Figure!5).! [0121] In!all!cases,!correct!integration!of!the!AoBLG!cassette!could!be!monitored!by!the!change!from! green!to!white!colonies!that! resulted! from!disruption!of! the!wA!gene.!Sequence!confirmation!was! obtained! for! all! strains.! Cultivation! single! copy! AoBLG! strains!was! performed! in!Deep!Well! Plates! (DWP)!for!4!days!at!30°!C!and!samples!from!the!culture!supernatant!were!used!to!run!an!SDS-PAGE.! ! ! Case Ref. P238WO IPTector® As!shown!(Figure!6),!the!use!of!the!Ple!SP!derived!from!plectasin!resulted!in!the!highest!amount!of! BLG!present!in!the!culture!supernatant!in!a!strain!(L-BLG-21)!containing!a!single!copy!of!the!AoBLG! gene!inserted!at!the!wA!gene!(Figure!6).!Using!a!cutinase!SP!(Cut!SP,!described!in!WO!2005/121333,! Table!2),!AoBLG!levels!comparable!to!the!use!of!Ple!SP!were!observed!in!single!copy!strain!L-BLG-23.! Lower!titers!were!obtained!for!the!remaining!three!SP!tested!(strains!L-BLG-25,!L-BLG-27!and!L-BLG- 29),!including!a!frequently!used!lipase!SP!(Lip!SP,!Table!3,!Figure!6).!Thus,!the!use!of!Ple!SP!or!Cut!SP! resulted!in!the!highest!AoBLG!yields.! ! Example!3! -!A! combination!of!modifications!of! the!host! strain! that! result! in!higher!BLG!yield! in! multicopy!strains.! [0122] The!effects!on!BLG!yield!of!deletion!of!the!protease!regulator!prtT!(Punt!et!al.,!2008)!and!the! modification!of!the!Inositol!responsive,!endoplasmic!reticulum!located!ribonuclease/kinase!(IreA)!was! investigated.! ! PrtT!deletion! [0123] Strain!TFB-Ao0010!was!co-transformed!using!two!MAD7!plasmids!as!described!(Vanegas!et! al.,! 2023)! in!which! two! protospacer! sequences! were! cloned! (P0460! or! P0461)! and! a! donor! DNA! fragment!spanned!the!flanks!of!the!deleted!prtT!gene!(P0459).!The!expected!deletion!of!the!prtT!gene! including!most!of!the!coding!region!(Figure!7)!was!confirmed!in!strain!TFB-Ao0127.!! [0124] Initially!it!was!believed!that!the!main!part!of!proteases!would!be!removed!by!deletion!of!pitT! and!that!deletion!of!prtT!would!harm!the!prtT!deleted!A.!oryzae!strain.!However,!surprisingly,!although! >25! %! of! the! proteases! found! in! the! fermentation! broth! of! a! prtT! deletion! mutant! (Figure! 15)! disappears,!the!mutant!strain!still!performed!well.! ! IreA!mutation! [0125] The!modification!of! IreA!(also!referred!to!as! Ire1)!is!described!in!WO2020/112911.!Shortly,! the!A.!oryzae!IreA!coding!sequence!was!modified!to!result!in!two!amino!acid!substitutions!(A80T!and! A83T).!The!modification!was!obtained!using!a!588-bp!repair!DNA!fragment!generated!using!primers! P0200!and!P0201!and!genomic!DNA!from!strain!TFB-Ao0010.!The!repair!fragment!was!used!to!co- transform!strain!TFB-Ao0127!using!two!MAD7!plasmids!directing!DNA!cleavage!at!the!coding!region! of!the!wt! IreA!sequence!(using!protospacers!P0202!and!P0203).!Strain!TFB-Ao0131,!containing!the! correct!sequence!modification!of!IreA!was!selected.!! [0126] Strain!TFB-Ao0010,!TFB-Ao0127!(prtT-)!and!TFB-Ao0131!(prtT-,!IreA)!were!transformed!with! pTFB-0075!containing!the!AoBLG!expression!cassette!that!contained!the!Ple!SP!(Figure!8).!Integration! ! ! Case Ref. P238WO IPTector® of! pTFB-0075! occurred! by! homologous! recombination! between! the! deleted! niaD! regions! of! the! chromosomal! locus! and! the! 3^! niaD! included! in! the! plasmid! (Figure! 8).! Transformants! containing! different! copies! numbers! were! obtained.! Selected! transformants! were! used! for! copy! number! estimation.!Strains!obtained!in!the!three!recipient!strains!expressing!AoBLG!and!having!the!same!copy! number! (5)!were!used! to!compare!yields.!DWP!cultivation! for!4!days!at! 30°!C!was!performed!and! samples!from!culture!supernatants!were!run!on!SDS-PAGE!(Figure!2).!! [0127] As!shown,!the!prtT!deletion!had!an!unexpected!positive!effect!on!AoBLG!levels!resulting!in! more!than!a!2-fold!higher!yield!(strain!TFB-Ao0144,!Figure!2).!Thus,!a!significant!reduction!of!protease! levels! could! be! obtained! even! in! strains! like! TFB-Ao0010/TFB-Ao0094,!where! genes! encoding! the! major!secreted!proteases!like!AlpA!and!NprA!have!been!inactivated,!resulting!in!higher!AoBLG!yields.! These!results!provide!evidence!of!a!prtT-controlled!set!of!genes!that!code!for!proteases!that!affect! the! yield! of! recombinant! protein! production! in! already! optimized! fungal! strains!where! the!major! proteases!were!inactivated.!! [0128] Furthermore,!the!yields!observed!in!the!prtT-,!IreA!strain!TFB-Ao0145!were!more!than!4-fold! higher!compared!to!the!non-modified!strain!TFB-Ao0138!(Figure!2).!These!results!demonstrated!that! the!IreA!mutation!increases!the!ER!capacity!without!indication!of!any!measurable!negative!effect!like! AoBLG!mRNA!degradation!as!described!elsewhere! (regulated! Ire1-dependent!mRNA!decay! (RIDD),! Tanaka!et!al.,!2023).!! [0129] Overall,! these!results!provided!the!basis! for! improved!yield!of! recombinant!BLG!that!were! required!for!large!scale!manufacturing!of!this!nutritionally!enhanced!protein!to!establish!a!sustainable! process!for!protein!production.!! ! Example!4!^!Expression!of!nutritionally!improved!BLG!sequences.! [0130] As!BLG!is!an!important!protein!for!human!nutrition!and!protein!sources!are!scarce,!two!further! variant!BLG!proteins!(SEQ!ID!NO:!56!and!58)!encoded!by!synthetic!genes!(SEQ!ID!NO:!57!and!59)!were! expressed!using!the!method!described!in!Materials!and!Methods.!These!BLG!proteins!were!enriched! not!only!in!essential!amino!acids!(EAA)!but!also!in!branched!amino!acids!(BCAA)!compared!not!only!to! bovine!BLG!B!(SEQ!ID!NO:!2)!but!also!to!the!AoBLG!(SEQ!ID!NO:4)!described!herein.!! [0131] Using!AoBLG!as!starting!point,!for!each!position,!not!having!an!essential!amino!acid,!but!where! another!BLG!were!found!to!have!an!essential!amino!acid,!the!non-essential!amino!acid!was!substituted! with! the! essential! amino! acid.! This! generated! the! SuperBLG! variant,! which! maintained! the! characteristics!of!a!BLG,!but!which!possessed!a!much!higher!content!of!EAA!(107!aa!or!66%)!compared! to!AoBLG!(78!or!48%)!(see!figure!9).!! [0132] Further,!also!using!AoBLG!as!starting!point!and!using!the!same!principle!as!for!SuperBLG,!any! ! ! Case Ref. P238WO IPTector® position!in!AoBLG!not!having!a!leucine,!but!where!another!BLG!were!found!to!have!a!leucine,!the!non- leucine!amino!acid!in!AoBLG!was!substituted!with!leucine.!This!generated!the!LeuMax!variant,!which! maintained!the!characteristics!of!a!BLG,!but!which!contained!higher!levels!of!BCAA!and!Leucine!(Fig.! 9).!BLG!B!has!25%!BCAA,!while!AoBLG!has!26%,!SuperBLG!30%!and!LeuMax!33%.!Leucine!content!was! maintained!from!BLG!B!to!AoBLG!and!further!to!SuperBLG!(22!residues!or!14%)!while!LeuMax!contains! 27!Leucine!residues!(17%).!The!amount!of!branched!amino!acids!in!the!BLGs!are!shown!in!figure!11! and!alignment!between!AoBLG,!SuperBLG!and!LeuMax!using!BLG!B!as!reference!is!shown!in!figure!10.! ! Example!5.!Generation!of!further!nutritionally!improved!variant!BLG!molecules.! [0133] Identifying!comprehensively!modified!BLG!sequences!which!can!be!microbially!produced!is! challenging!(see!e.g.!fig.!14).!One!challenge!to!overcome!was!that!the!synthesis,!folding!and!secretion! of!the!BLG!variant,!such!as!SuperBLG!and!LeuMax!could!be!compromised!if!the!structure!of!the!protein! deviates!extensively!from!BLG.!Accordingly,!a!expression!of!variant!BLG^s!having!increased!content!of! both! SuperBLG! and! LeuMax! may! be! significantly! affected! by! the! comprehensive! number! of! aa! changes.! Using! AlphaFoldstructural! similarities! was! predicted! between! BLG! B! and! AoBLG,! while! significant!structural!differences!was!predicted!between!BLG!B!and!SuperBLG!(Fig.!12).!This!software! package!was! used! to! analyse! AoBLG! sequence! variants! containing! increasing! amounts! of! Leucine! (LeuMore!1!to!LeuMore!5,!containing!from!3!to!6!extra!Leucine!residues!(see!fig.!13).!In!fact,!LeuMore! 5! contains! one!more! Leucine! compared! to! LeuMax!but! has! a!much! lower! number! of! amino! acid! changes!(6!compared!to!40).! [0134] As! mentioned! above,! multicopy! strains! containing! either! the! SuperBLG! or! the! LeuMax! expression! cassette! produced! very! low! amounts! of! the! protein! while! significant! expression! of! AoBLGwas!detected!in!single!copy!strains!(fig.!14!A).!Therefore,!LeuMore!1!to!5!were!expressed! in! single!copy!strains,!using!the!same!strain!background!used!for!expression!of!AoBLG!in!single!copy.!As! shown,!we!detected!expression!of!all!the!LeuMore!variants!in!single!copy,!supporting!the!fact!that! structure!prediction!using!AlphaFold! could!be!used! for!predicting!protein! sequences! for! improved! expression!(fig.!14!B).!The!expression!levels!varied!among!the!LeuMore!variants.! ! Example!6.!BLG!variants!with!increased!essential!amino!acids!content.! [0135] Additional!BLG!variants!were!designed!by!additional!substitution!of!non-essential!amino!acid! (non-EAA)!residues!with!Leu.!The!design! included!calculation!of!molecular!dynamics!and!structural! stability!using!<<G!values,!building!structural!models!using!AlphaFold!2!and!calculating!<<G!for!all! non-EAA!to!Leu!substitutions.!These!substitutions!were!combined!with!amino!acid!changes!derived! from!structure-based!conservation.! In!a!number!of!cases,! the!two!approaches! identified!the!same! ! ! Case Ref. P238WO IPTector® amino!acid!change!to!Leu!and!resulted!in!a!list!of!seven!consolidated!changes!from!either!one!or!both! approaches!(S21L,!D96L,!E108L,!Q120L,!A139L,!S150L!and!P153L).!Combinations!of!one!or!more!of! these!substitutions!were!used!to!design!a!new!set!of!50!BLG!variants!with!increased!Leu!content!(SEQ! ID!NO:!73,!75,!77,!79,!81,!83,!85,!87,!89,!91,!93,!95,!97,!99,!101,!103,!105,!107,!109,!111,!113,!115,!117,! 119,!121,!123,!125,!127,!129,!131,!133,!135,!137,!139,!141,!143,!145,!147,!149,!151,!153,!155,!157,!159,! 161,!163,!165,!167!and/or!169).! ! Example!7.!BLG!variants!with!increased!essential!amino!acids!content.! [0136] As!shown!in!previous!examples,!discrete!changes!in!the!amino!acid!sequence!of!BLG!can!result! in!very!different!BLG!protein!titers!(Example!5).!Therefore,!the!expression!potential!for!AoBLG!was! investigated!in!well-known!alternative!microorganisms!that!are!industrially!relevant.! Two!hosts!organisms!were!tested.!One,!Aspergillus!niger,!was!a!strain!nowadays!developed!to!a!very! advanced! level!which! used! for!manufacture! of! numerous! food! proteins! as! described! in! e.g.,!WO! 2012/160093.!Another,!Pichia!pastoris,!was!a!universally!used,!but!much!less!optimized!strain!derived! from!the!Alt-Host!MIT-Industry!Consortium.! [0137] For!A.!niger,!the!host!strain!contained!both!the!prtT!deletion!and!the!same!ireA!mutation!as! described!for!A.!oryzae.!The!same!promoter,!transcriptional!terminator!and!signal!peptide!was!used! as! for! A.! oryzae! (Examples! 1-5)! and! the! same! sequence! encoding! the! AoBLG! protein! sequence.! Multicopy!strains!were!obtained!and!tested!in!24!well!MTP!using!glucose!as!C!source!(induction!of! expression).!Thus,!protein!titers!in!A.!niger!are!comparable!to!the!results!obtained!in!A.!oryzae.! [0138] For!P.!pastoris,!strain!S774!was!used!as!host.!S774!is!an!optimized!Pichia!strain.!It!has!deletions! in! 6! non-essential! secretory! protein! encoding! genes! to! enhance! the! production! capacity! for! recombinant!proteins.!The!AoBLG!expression!cassette!used!for!strain!construction!consisted!of!the! standard!elements!used!in!Pichia!e.g.,!the!AOX1!promoter,!using!the!well-characterized!S.!cerevisiae! alpha!mating!factor!signal!peptide.!Transformation!was!performed!using!zeocin!to!facilitate!multicopy! strain! selection.! 24!well!MTP! cultivation!was! performed,! using! 4%! glycerol! for! growth! and! 1.5!%! methanol!for! induction!as!standard.!SDS-PAGE!of!samples!from!culture!supernatants!from!A.!niger! and!P.!pastoris!was!used!for!qualitative!analysis!of!titer.! [0139] For!both!A.!niger!or!P.!pastoris,!no!optimization!work!for!production!of!AoBLG!(e.g.,!codon! optimization,!growth!conditions,!induction!regime,!etc.)!was!performed,!for!the!preliminary!screening.! In!a!separate!experiment!the!BLG!(AoBLG!)!encoding!genes!were!codon!optimized!for!Pichia!pastoris! SEQ!ID!NO:!170)!and!for!Aspergillus!niger!SEQ!ID!NO:!171.! [0140] As!shown!in!figure!16,!a!high!expression!level!of!AoBLG!in!individual!strains!of!both!A.!niger! and!P.!pastoris!was!obtained.!These!results!strongly!indicate!that!1)!the!use!of!Aspergillus!sp.!strains! ! ! Case Ref. P238WO IPTector® containing!the!prtT!and!ireA!modifications!described!here!result!in!optimal!BLG!protein!titers,!and!2)! high!expression!levels!are!also!obtained!in!the!less!developed!P.!pastoris!host!strain,!supporting!the! fact!that!the!BLG!protein!sequence!is!universally!well!expressed!in!microbial!cell!factories!described! herein.!! ! References! Arnau!et!al.!(2020);!Strategies!and!challenges!for!the!development!of!industrial!enzymes!using!fungal! cell!factories.!in:!H.!Nevalainen!(ed.);!Grand!challenges!in!fungal!biotechnology,!grand!challenges!in! biology!and!biotechnology.! ! Liu!et!al.! (2022);!High-yield!production!of!acidic!pectin! lyase!pnlzj5b!for! juice!processing;!Lett!Appl! Microbiol!75:1055-1062.! ! Mygind!et!al.!(2005);!Plectasin!is!a!peptide!antibiotic!with!therapeutic!potential!from!a!saprophytic! fungus;!Nature!437:975-980.! ! Nakamura!et!al.!(2017);!Highly!efficient!gene!targeting!in!aspergillus!oryzae!industrial!strains!under! ligd!mutation!introduced!by!genome!editing:!strain-specific!differences!in!the!effects!of!deleting!ecdr,! the!negative!regulator!of!sclerotia!formation;!J.!Gen.!Appl.!Microbiol.!63:172-178.! ! Punt!et!al.! (2008);!Characterization!of!the!aspergillus!niger!prtt,!a!unique!regulator!of!extracellular! protease!encoding!genes;!Fungal!Genet!Biol!45:1591-1599.! ! Siwecka!et!al.!(2021)!the!structure,!activation!and!signaling!of!ire1!and!its!role!in!determining!cell!fate.! biomedicines!9:156! ! Tanaka! et! al.! (2014)! effects! of! codon! optimization! on! the! mrna! levels! of! heterologous! genes! in! filamentous!fungi.!appl!microbiol!biotechnol!98:3859-3867! ! Vanegas!et!al.!(2023)!a!mad7!system!for!genetic!engineering!of!filamentous!fungi.!j.!fungi!9:16! ! Yoon!et!al.!(2011);!Disruption!of!ten!protease!genes!in!the!filamentous!fungus!aspergillus!oryzae!highly! improves!production!of!heterologous!proteins;!Appl.!Microbiol.!Biotech.;!89:747-759.! ! ! ! Case Ref. P238WO IPTector® Zhou! et! al.! (2016);! Functional! and! transcriptomic! analysis! of! the! key! unfolded! protein! response! transcription!factor!hacA!in!aspergillus!oryzae;!Gene;!593:143-153.! ! *!!*!!*! ! ! ! !

Claims

Case Ref. P238WO IPTector® ! Claims! 1.!A!genetically!modified!microbial!host!cell!expressing!one!or!more!heterologous!genes!encoding!a! modified!beta-lactoglobulin!(BLG),!thereby!producing!the!said!modified!BLG,!wherein!the!modified! BLG!comprises!at!least!one!substitution!of!a!non-essential!and/or!non-branched!amino!acid!with!an! essential!and/or!branched!amino!acid!compared!to!a!parent!unmodified!BLG.! ! 2.!The!genetically!modified!microbial!host!cell!of!claim!1!wherein!the!BLG!comprises!an!amino!acid! sequence!which!is!at!least!50%!identical!to!the!BLG!comprised!in!anyone!of!SEQ!ID!NO:!4,!163,!165,! 167,!1,!2,!3,!56,!58,!60,!62,!64,!66,!68,!71,!73,!75,!77,!79,!81,!83,!85,!87,!89,!91,!93,!95,!97,!99,!101,!103,! 105,!107,!109,!111,!113,!115,!117,!119,!121,!123,!125,!127,!129,!131,!133,!135,!137,!139,!141,!143,!145,! 147,!149,!151,!153,!155,!157,!159,!161!and/or!169.! ! 3.!The!host!cell!of!any!preceding!claim,!wherein!the!BLG!is!a!modified!BLG,!modified!to!have!a!higher! fraction!of!essential!and/or!branched!amino!acids!compared!to!a!parent!natural!BLG.! ! 4.!The!host!cell!of!any!preceding!claim,!wherein!the!modified!BLG!have!an!increased!fraction!of!Leu,! His!and/or!Val!compared!to!a!natural!parent!BLG.! ! 5.!The!host!cell!of!anyone!of!claim!3!to!4,!wherein!the!modified!BLG!is!a!modified!bovine!BLG.! ! 6.!The!host!cell!of!anyone!of!claim!3!to!5,!wherein!the!modified!BLG!comprises!one!or!more!moieties! from!bovine!BLG!A!as!set!forth!in!SEQ!ID!NO:!1,!bovine!BLG!B!as!set!forth!in!SEQ!ID!NO:!2!and/or!bovine! BLG!C!as!set!forth!in!SEQ!ID!NO:!3.! ! 7.!The!host!cell!of!claim!6,!wherein!the!modified!BLG!comprises!at!least!one!amino!acid!substitution! compared! to! anyone! of! bovine! BLG! A,! bovine! BLG! B! and! bovine! BLG! C,! optionally! at! least! two! substitutions.! ! 8.!The!host!cell!of!anyone!of!claim!3!to!7!wherein!the!modified!BLG!comprises!at!least!one!substitution! of!a!non-essential!and/or!non-branched!amino!acid!with!an!essential!and/or!branched!amino!acid.! ! ! ! Case Ref. P238WO IPTector® 9.!The!host!cell!of!claim!8!wherein!the!modified!BLG!comprises!at!least!at!least!2,!such!as!at!least!5,! such!as!at!least!10,!such!as!at!least!15,!such!as!at!least!20,!such!as!at!least!25!substitutions!of!a!non- essential!and/or!non-branched!amino!acid!with!an!essential!and/or!branched!amino!acid.! ! 10.!The!host!cell!anyone!of!claim!8!to!9!wherein!the!modified!BLG!comprises!at!least!at!least!1,!such! as!at! least!2,!such!as!at! least!3,!such!as!at! least!4,!such!as!5,!such!as!at! least!6,!such!as!at! least!7! substitutions!of!a!non-leucine!amino!acid!with!a!leucine!amino!acid.! ! 11.!The!host!cell!of!claim!10!wherein!the!modified!BLG!comprises!up!to!7!substitutions!of!a!non-leucine! amino!acid!with!a!leucine!amino!acid!corresponding!to!anyone!of!positions!1,!5,!21,!96,!108,!120,!139,! 150!and/or!153!of!the!modified!BLG!as!set!forth!in!anyone!of!SEQ!ID!NO:!73,!75,!77,!79,!81,!83,!85,!87,! 89,!91,!93,!95,!97,!99,!101,!103,!105,!107,!109,!111,!113,!115,!117,!119,!121,!123,!125,!127,!129,!131,! 133,!135,!137,!139,!141,!143,!145,!147,!149,!151,!153,!155,!157,!159,!161,!163,!165,!167!and/or!169.! ! 12.!The!host!cell!of!claim!10!wherein!the!modified!BLG!comprises!1!substitution!of!a!non-leucine!amino! acid!with!a!leucine!amino!acid!in!a!position!corresponding!to!anyone!of!positions!1,!21,!96,!108,!120,! 139,!150!and/or!153!of!the!modified!BLG!as!set!forth!in!anyone!of!SEQ!ID!NO:!75,!77,!79,!81,!83,!85! and/or!87.! ! 13.!The!host!cell!of!claim!10!wherein!the!modified!BLG!comprises!2!substitutions!of!a!non-leucine! amino!acid!with!a!leucine!amino!acid!corresponding!to!anyone!of!positions!1,!5,!21,!96,!108,!120,!139,! 150!and/or!153!of!the!modified!BLG!as!set!forth!in!anyone!of!SEQ!ID!NO:!73,!89,!91,!93,!95,!97,!99,! 101,!103,!105,!107!and/or!109.! ! 14.!The!host!cell!of!claim!10!wherein!the!modified!BLG!comprises!3!substitutions!of!a!non-leucine! amino!acid!with!a!leucine!amino!acid!corresponding!to!anyone!of!positions!21,!96,!108,!120,!139,!150! and/or!153!of!the!modified!BLG!as!set!forth!in!anyone!of!SEQ!ID!NO:!111,!113,!115,!117,!119,!121,!123,! 125,!127,!129,!131!and/or!133.! ! 15.!The!host!cell!of!claim!10!wherein!the!modified!BLG!comprises!4!substitutions!of!a!non-leucine! amino!acid!with!a!leucine!amino!acid!corresponding!to!anyone!of!positions!21,!96,!108,!120,!139,!150! and/or!153!of!the!modified!BLG!as!set!forth!in!anyone!of!SEQ!ID!NO:!135,!137,!139,!141,!143,!145! and/or!147.! ! ! ! Case Ref. P238WO IPTector® 16.!The!host!cell!of!claim!10!wherein!the!modified!BLG!comprises!5!substitutions!of!a!non-leucine! amino!acid!with!a!leucine!amino!acid!corresponding!to!anyone!of!positions!1,!21,!96,!108,!120,!139,! 150!and/or!153!of!the!modified!BLG!as!set!forth!in!anyone!of!SEQ!ID!NO:!149,!151,!153,!155,!157,!159! and/or!161.! ! 17.!The!host!cell!of!claim!10!wherein!the!modified!BLG!comprises!6!substitutions!of!a!non-leucine! amino!acid!with!a!leucine!amino!acid!corresponding!to!anyone!of!positions!21,!96,!108,!120,!139,!150! and/or!153!of!the!modified!BLG!as!set!forth!in!anyone!of!SEQ!ID!NO:!163,!165!and/or!167.! ! 18.!The!host!cell!of!claim!10!wherein!the!modified!BLG!comprises!7!substitutions!of!a!non-leucine! amino!acid!with!a!leucine!amino!acid!corresponding!to!position!21,!96,!108,!120,!139,!150!and/or!153! of!the!modified!BLG!as!set!forth!in!anyone!of!SEQ!ID!NO:!169.! ! 19.!The!host!cell!of!anyone!of!claim!7!to!10,!wherein!the!BLG!is!a!modified!bovine!BLG!C!comprising!at! least!one!substitution!of!Alanine!with!Valine!in!a!position!corresponding!to!position!118!of!the!bovine! BLG!C!in!SEQ!ID!NO:!3.! ! 20.!The!host!cell!anyone!of!claim!7!to!10!wherein!the!BLG!is!a!modified!bovine!BLG!A!comprising!at! least!one!substitution!of!Glutamine!with!Histidine!at!a!position!corresponding!to!position!59!of!the! bovine!BLG!A!in!SEQ!ID!NO:!1.! ! 21.!The!host!cell!of!claim!anyone!of!claim!7!to!10,!wherein!the!BLG!comprises!a!Histidine!and!a!Valine! at!positions!corresponding!to!position!59!and!118!respectively!in!anyone!of!the!BLG^s!comprised!in! SEQ!ID!NO:!1,!2!or!3.!! ! 22.!The!host!cell!of!any!preceding!claim,!wherein!the!BLG!comprises!an!amino!acid!sequence!as!set! forth!in!anyone!of!SEQ!ID!NO:!4,!163,!165,!167,!56,!58,!60,!62,!64,!66,!68,!71,!73,!75,!77,!79,!81,!83,!85,! 87,!89,!91,!93,!95,!97,!99,!101,!103,!105,!107,!109,!111,!113,!115,!117,!119,!121,!123,!125,!127,!129,! 131,!133,!135,!137,!139,!141,!143,!145,!147,!149,!151,!153,!155,!157,!159,!161!and/or!169.! ! 23.!The!host!cell!of!any!preceding!claim,!wherein!the!one!or!more!heterologous!genes!encoding!the! BLG! comprise! a! nucleotide! sequence! which! is! at! least! 50%! identical! to! the! BLG! encoding! gene! comprised!in!anyone!of!SEQ!ID!NO:!5,!6,!7,!8,!9,!10,!11,!12,!57,!59,!61,!63,!65,!67,!69,!70,!72,!74,!76,!78,! 80,!82,!84,!86,!88,!90,!92,!94,!96,!98,!100,!102,!104,!106,!110,!112,!114,!116,!118,!120,!122,!124,!126,! ! ! Case Ref. P238WO IPTector® 128,!130,!132,!134,!136,!138,!140,!142,!144,!146,!148,!149,!150,!152,!154,!156,!158,!160,!162,!164,!166,! 168,!170!and/or!171.! ! 24.!The!host!cell!of!claim!23,!wherein!the!one!or!more!heterologous!genes!encoding!the!BLG!comprise! a!nucleotide!sequence!which!is!the!BLG!encoding!gene!set!forth!in!anyone!of!SEQ!ID!NO:!5,!6,!7,!8,!9,! 10,!11,!12,!57!or,!59,!61,!63,!65,!67,!69,!70,!72,!74,!76,!78,!80,!82,!84,!86,!88,!90,!92,!94,!96,!98,!100,! 102,!104,!106,!110,!112,!114,!116,!118,!120,!122,!124,!126,!128,!130,!132,!134,!136,!138,!140,!142,!144,! 146,!148,!149,!150,!152,!154,!156,!158,!160,!162,!164,!166,!168,!170!and/or!171.! ! 25.!The!host!cell!of!any!preceding!claim,!wherein!the!one!or!more!heterologous!genes!encoding!the! BLG!further!encodes!a!signal!peptide!fused!to!the!BLG.! ! 26.!The!host!cell!of!claim!25,!wherein!the!signal!peptide!is!heterologous!to!host!cell!and/or!to!the!BLG.!! ! 27.!The!host!cell!of!claim!24!to!26,!wherein!the!signal!peptide!comprises!an!amino!acid!sequence!which! is!at!least!50%!identical!to!the!signal!peptide!comprised!in!SEQ!ID!NO:!13.! ! 28.!The!host!cell!of!claim!25!to!27,!comprising!a!nucleotide!sequence!encoding!the!signal!peptide,! wherein!said!nucleotide!sequence!is!at!least!50%!identical!to!the!signal!peptide!encoding!sequence! comprised!in!SEQ!ID!NO:!14.! ! 29.!The!host!cell!of!anyone!of!claim!25!to!28,!wherein!the!signal!peptide! is!derived!from!a!natural! defensin.! ! 30.!The!host!cell!of!claim!29,!wherein!the!natural!defensin!is!a!fungal!defensin.! ! 31.!The!host!cell!of!claim!30!wherein!the!fungal!defensin!is!plectasin.! ! 32.! The! host! cell! of! claim! 31,! wherein! the! plectasin! is! derived! from! a! strain! of! the! genus! of! Pseudoplectania.! ! 33.!The!host!cell!of!claim!32,!wherein!the!strain!of!Pseudoplectania!is!Pseudoplectania!nigrella.! ! ! ! Case Ref. P238WO IPTector® 34.!The!host!cell!of!any!preceding!claim,!wherein!the!one!or!more!heterologous!genes!encoding!the! BLG!is!under!control!of!a!promoter.! ! 35.!The!host!cell!of!claim!34,!wherein!the!promoter!is!heterologous!to!host!cell!and/or!to!the!BLG.! ! 36.! The! host! cell! of! anyone! of! claim! 34! to! 35! wherein! the! promoter! is! an! engineered! synthetic! promoter.! ! 37.!The!host!cell!of!claim!34!to!36,!wherein!the!promoter!is!an!inducible!promoter.! ! 38.!The!host!cell!of!claim!342!to!37!wherein!the!nucleotide!sequence!of!the!promoter!is!at!least!50%! identical!to!the!promoter!sequence!comprised!in!SEQ!ID!NO:!15.!! ! 39.! The! host! cell! of! any! preceding! claim,! comprising! one! or! more! further! genetic! modifications! improving!the!expression!and/or!secretion!of!the!BLG!selected!from! a) attenuation,! disruption! and/or! deletion! the! expression! of! one! or! more! genes! which! are! endogenous/native!to!the!host!cell;!and!! b) mutation!of!one!or!more!genes!native!to!the!host!cell.! ! 40.!The!host!cell!of!claim!39,!wherein!the!attenuated,!disrupted!and/or!deleted!native!genes!encodes! a!protease!capable!of!digesting!BLG!or!its!signal!peptide.! ! 41.!The!host!cell!of!claim!40,!wherein!the!protease!is!an!alpA!(alkaline!protease)!comprised!in!SEQ!ID! NO:!16!or!18.! ! 42.!The!host!cell!of!claim!40,!wherein!the!protease!is!a!nprA!(neutral!metalloprotease!I)!comprised!in! SEQ!ID!NO:!20!or!22.! ! 43.!The!host!cell!of!anyone!of!claim!40!to!42,!wherein!the!native/endogenous!protease!is!attenuated,! disrupted!and/or!deleted!by!attenuation,!disruption!and/or!deletion!of!a!native!endogenous!protease! transcription!factor!controlling!the!expression!the!one!or!more!proteases!endogenous/native!to!the! host!cell.! ! ! ! Case Ref. P238WO IPTector® 44.! The! host! cell! of! claim!43,!wherein! the! protease! transcription! factor! is! prtT! or! an! analogue! or! ortholog!thereof.! ! 45.!The!host!cell!of!claim!44,!wherein!the!prtT!is!encoded!by!a!gene!having!a!nucleotide!sequence! which!is!at!least!50%!identical!to!the!prtT!sequence!comprised!in!SEQ!ID!NO:!24!or!26.! ! 46.!The!host!cell!of!anyone!of!claim!39!to!45,!wherein!the!mutated!native!genes!encode!an!enzyme! involved!in!activation!of!a!transcription!factor!regulating!the!Unfolded!Protein!Response!(UPR)!in!the! host!cell.! ! 47.!The!host!cell!of!claim!46,!wherein!the!transcription!factor!is!HAC1.! ! 48.!The!host! cell!of! anyone!of! claim!46! to!47,!wherein! the!mutation!of! the!enzyme! increases! the! activation!of!the!UPR!transcription!factor.! ! 49.!The!host!cell!of!anyone!of!claim!46!to!48,!wherein!the!enzyme!is!an!ER-resident!transmembrane! kinase!and/or!endoribonuclease.! ! 50.!The!host!cell!of!claim!49,!wherein!the!enzyme!is!an!inositol!requiring!enzyme.! ! 51.!The!host!cell!of!claim!50,!wherein!the!enzyme!is!Ire1!or!any!of!its!analogues!or!orthologues.! ! 52.!The!host!cell!of!claim!51,!wherein!the!enzyme!is!IreA.! ! 53.!The!host!cell!of!anyone!of!claim!46!to!52,!wherein!the!enzyme!has!an!amino!acid!sequence!which! is!at!least!50%!identical!to!the!inositol!requiring!enzyme!comprised!in!SEQ!ID!NO:!28!or!30.! ! 54.! The! host! cell! of! anyone! of! claim! 46! to! 53,! wherein! the! enzyme! comprises! a!mutation! at! the! positions!corresponding!to!position!A80!and/or!A83!of!SEQ!ID!NO:!28!or!30.! ! 55.!The!host!cell!of!claim!54,!wherein!the!mutations!are!A80T!and/or!A83T.! ! ! ! Case Ref. P238WO IPTector® 56.!The!host!cell!of!anyone!of!claim!50!to!53!wherein!the!inositol!requiring!enzyme!is!encoded!by!a! gene!having!a!nucleotide!sequence!which! is!at! least!50%!identical!to!the! inositol!requiring!enzyme! encoding!sequence!comprised!in!anyone!of!SEQ!ID!NO:!29!or!31.! ! 57.!The!host!cell!of!any!preceding!claim,!comprising!at!least!2!copies!of!the!one!or!more!heterologous! genes!encoding!the!BLG.! ! 58.!The!host!cell!of!any!preceding!claim,!wherein!the!one!or!more!heterologous!genes!encoding!the! BLG!are!codon!optimized!for!expression!in!a!filamentous!fungus,!optionally!A.!Oryzae.! ! 59.!The!host!cell!of!any!preceding!claim!further!comprising!one!or!more!modifications!increasing!the! expression,!the!post-translational!modification,!the!secretion!and/or!the!folding!of!the!BLG.! ! 60.!The!host!cell!of!any!preceding!claims,!wherein!the!microbial!host!cell!is!a!eukaryotic,!a!prokaryotic! or!an!algal!cell.! ! 61.!The!host!cell!of!claim!60,!wherein!the!eukaryotic!cell!is!a!fungal!cell,!optionally!a!filamentous!fungus! or!a!yeast!cell.! ! 62.!The!host!cell!of!claim!61,!wherein!the!filamentous!fungus!is!selected!from!the!genus!of!Aspergillus,! Trichoderma,!or!Rhizopus.! ! 63.!The!host!cell!of!claim!62,!wherein!the!filamentous!fungus!is!selected!from!the!species!Aspergillus! sp,!Aspergillus!oryzae,!Trichoderma!sp,!or!Rhizopus!sp.! ! 64.!The!host!cell!of!claim!61,!wherein!the!yeast!is!selected!from!the!genus!of!Pichia,!Saccharomyces,! Yarrowia,!or!Hansenula.! ! 65.!The!host!cell!of!claim!64,!wherein!the!yeast!is!selected!from!the!species!of!P.!pastoris,!S.!cerevisiae,! Y.!lipolytica,!or!Hansenula!sp.! ! 66.!A!genetically!modified!A.!oryzae!host!cell!expressing!a!modified!BLG!comprising!an!amino!acid! sequence!as!set!forth!in!anyone!of!SEQ!ID!NO:!4,!163,!165,!167!56,!58,!60,!62,!64,!66,!68,!71,!73,!75,! 77,!79,!81,!83,!85,!87,!89,!91,!93,!95,!97,!99,!101,!103,!105,!107,!109,!111,!113,!115,!117,!119,!121,!123,! ! ! Case Ref. P238WO IPTector® 125,!127,!129,!131,!133,!135,!137,!139,!141,!143,!145,!147,!149,!151,!153,!155,!157,!159,!161!and/or! 169!having!a!higher!fraction!of!essential!amino!acids!compared!to!a!parent!natural!BLG,!said!host!cell! expressing!one!or!more!codon!optimized!heterologous!genes!as!set!forth!in!anyone!of!SEQ!ID!NO:!5,! 6,!7,!8,!9,!10,!11,!12,!57,!59,!61,!63,!65,!67,!69,!70,!72,!74,!76,!78,!80,!82,!84,!86,!88,!90,!92,!94,!96,!98,! 100,!102,!104,!106,!110,!112,!114,!116,!118,!120,!122,!124,!126,!128,!130,!132,!134,!136,!138,!140,!142,! 144,!146,!148,!149,!150,!152,!154,!156,!158,!160,!162,!164,!166,!168,!170!and/or!171!encoding!the! modified!BLG,!wherein!the!host!cell!is!further!modified!by!deleting!one!or!more!native!genes!encoding! a!protease!selected!from!the!group!of!alpA!(alkaline!protease),!nprA!(neutral!metalloprotease!I),!pepE! (aspartic!endopeptidase),!and!pepC!(serine!protease),!and!wherein!the!host!cell!is!further!modified!by! mutating!a!native!inositol!requiring!enzyme!having!two!mutations!corresponding!to!positions!A80T! and!A83T!as!set!forth!in!SEQ!ID!NO:!28!thereby!increasing!activation!of!transcription!factor!HAC1!in! the!host!cell.! ! 67.! A! modified! nucleotide! sequence! which! is! at! least! 50%! identical! to! the! BLG! encoding! gene! comprised!in!anyone!of!SEQ!ID!NO:!5,!6,!7,!8,!9,!10,!11,!12,!57,!59,!61,!63,!65,!67,!69,!70,!72,!74,!76,!78,! 80,!82,!84,!86,!88,!90,!92,!94,!96,!98,!100,!102,!104,!106,!110,!112,!114,!116,!118,!120,!122,!124,!126,! 128,!130,!132,!134,!136,!138,!140,!142,!144,!146,!148,!149,!150,!152,!154,!156,!158,!160,!162,!164,!167! 168,!170!and/or!171!and!encodes!a!modified!BLG!having!at!least!one!additional!essential!amino!acid! compared!to!a!parent!BLG.! ! 68.!A!polynucleotide!construct!comprising!the!nucleotide!sequence!of!claim!65,!operably!linked!to!an! inducible!promoter!which!is!at!least!50!%!identical!to!the!promoter!comprised!in!SEQ!ID!NO:!15.! ! 69.!The!polynucleotide!construct!of!claim!68,!wherein!the!construct!is!an!expression!vector.! ! 70.!The!host!cell!of!anyone!of!claim!1!to!66!comprising!the!polynucleotide!construct!of!claims!68!to! 69.!! ! 71.!A!cell!culture,!comprising!the!host!cell!of!claim!anyone!of!1!to!65!or!claim!70!and!a!growth!medium.! ! 72.!A!method!for!producing!a!BLG!comprising:! a) culturing!the!cell!culture!of!claim!71!at!conditions!allowing!the!cell!to!produce!the!modified! BLG;!and!! b) optionally!recovering!and/or!isolating!the!BLG.! ! ! Case Ref. P238WO IPTector® ! 73.!The!method!of!claims!72,!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.! ! 74.!The!method!of!anyone!of!claim!72!to!73!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!modified!BLG!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!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.! ! 75.!A! fermentation!composition!comprising!the!cell!culture!of!claim!71!and/or! the! BLG!comprised! therein.! ! 76.!The!fermentation!composition!of!claims!75,!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.! ! 77.! The! fermentation! composition! of! anyone! of! claim! 75! to! 76,! 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!BLG! is!at! least!1!mg/kg! composition.! ! ! Case Ref. P238WO IPTector® ! 78.!The!fermentation!composition!of!anyone!of!claim!75!to!77!further!comprising!one!or!more!carriers,! agents,!additives!and/or!excipients.! ! 79.!A!modified!BLG!polypeptide!comprising!at!least!one!substitution!of!a!non-essential!and/or!non- branched!amino!acid!with!an!essential!and/or!branched!amino!acid!compared!to!a!parent!unmodified! BLG.! ! 80.!The!modified!BLG!polypeptide!of!claim!79!comprising!an!amino!acid!sequence!which!is!at! least! 50%!identical!to!the!BLG!comprised!in!anyone!of!SEQ!ID!NO:!4,!163,!165,!167,!1,!2,!3,!56,!58,!60,!62,! 64,!66,!68,!71,!73,!75,!77,!79,!81,!83,!85,!87,!89,!91,!93,!95,!97,!99,!101,!103,!105,!107,!109,!111,!113,! 115,!117,!119,!121,!123,!125,!127,!129,!131,!133,!135,!137,!139,!141,!143,!145,!147,!149,!151,!153,!155,! 157,!159,!161,!and/or!169.!! ! 81.!The!modified!BLG!polypeptide!of!claim!80!comprising!an!amino!acid!sequence!as!set!forth!in!SEQ! ID!NO:!4,!163,!165,!167,!56,!58,!73,!75,!77,!79,!81,!83,!85,!87,!89,!91,!93,!95,!97,!99,!101,!103,!105,!107,! 109,!111,!113,!115,!117,!119,!121,!123,!125,!127,!129,!131,!133,!135,!137,!139,!141,!143,!145,!147,!149,! 151,!153,!155,!157,!159,!161,!and/or!169.!! ! 82.!The!modified!BLG!polypeptide!of!claim!79!or!80!comprising!at!least!at!least!2,!such!as!at!least!5,! such!as!at!least!10,!such!as!at!least!15,!such!as!at!least!20,!such!as!at!least!25!substitutions!of!a!non- essential!and/or!non-branched!amino!acid!with!an!essential!and/or!branched!amino!acid.! ! 83.!The!modified!BLG!polypeptide!of!claim!82!comprising!at!least!1,!such!as!at!least!2,!such!as!at!least! 3,!such!as!at!least!4,!such!as!5,!such!as!at!least!6,!such!as!at!least!7!substitutions!of!a!non-leucine!amino! acid!with!a!leucine!amino!acid.! ! 84.!The!modified!BLG!polypeptide!of!anyone!of!claim!83!comprising!up!to!7!substitutions!of!a!non- leucine!amino!acid!with!a!leucine!amino!acid!corresponding!to!anyone!of!position!1,!5,!21,!96,!108,! 120,!139,!150!and/or!153!of!the!modified!BLG!set!forth!in!anyone!SEQ!ID!NO:!73,!75,!77,!79,!81,!83,! 85,!87,!89,!91,!93,!95,!97,!99,!101,!103,!105,!107,!109,!111,!113,!115,!117,!119,!121,!123,!125,!127,!129,! 131,!133,!135,!137,!139,!141,!143,!145,!147,!149,!151,!153,!155,!157,!159,!161,!163,!165,!167!and/or! .!! ! ! ! Case Ref. P238WO IPTector® 85.!The!modified!BLG!polypeptide!of!claim!84!comprising!one!substitution!of!a!non-leucine!amino!acid! with!a!leucine!amino!acid!in!a!position!corresponding!to!anyone!of!position!1,!21,!96,!108,!120,!139,! 150!and/or!153!of!the!modified!BLG!set!forth!in!anyone!of!SEQ!ID!NO:!75,!77,!79,!81,!83,!85!and/or!87.!! ! 86.!The!modified!BLG!polypeptide!of!claim!84!comprising!two!substitutions!of!a!non-leucine!amino! acid!with!a!leucine!amino!acid!corresponding!to!anyone!of!position!1,!5,!21,!96,!108,!120,!139,!150! and/or!153!of!the!modified!BLG!set!forth!in!anyone!of!SEQ!ID!NO:!73,!89,!91,!93,!95,!97,!99,!101,!103,! 105,!107!and/or!109.!! ! 87.!The!modified!BLG!polypeptide!of!claim!84!comprising!three!substitutions!of!a!non-leucine!amino! acid!with!a!leucine!amino!acid!corresponding!to!anyone!of!position!21,!96,!108,!120,!139,!150!and/or! 153!of!the!modified!BLG!set!forth!in!anyone!of!SEQ!ID!NO:!111,!113,!115,!117,!119,!121,!123,!125,!127,! 129,!131!and/or!133.!! ! 88.!The!modified!BLG!polypeptide!of!claim!84!comprising!four!substitutions!of!a!non-leucine!amino! acid!with!a!leucine!amino!acid!corresponding!to!anyone!of!position!21,!96,!108,!120,!139,!150!and/or! 153!of!the!modified!BLG!set!forth!in!anyone!of!SEQ!ID!NO:!135,!137,!139,!141,!143,!145!and/or!147.!! ! 89.!The!modified!BLG!polypeptide!of!claim!84!comprising!five!substitutions!of!a!non-leucine!amino! acid!with!a!leucine!amino!acid!corresponding!to!anyone!of!position!1,!21,!96,!108,!120,!139,!150!and/or! 153!of!the!modified!BLG!as!set!forth!in!anyone!of!SEQ!ID!NO:!149,!151,!153,!155,!157,!159!and/or!161.!! ! 90.!The!modified!BLG!polypeptide!of!claim!84!comprising!six!substitutions!of!a!non-leucine!amino!acid! with!a!leucine!amino!acid!corresponding!to!anyone!of!position!21,!96,!108,!120,!139,!150!and/or!153! of!the!modified!BLG!set!forth!in!anyone!of!SEQ!ID!NO:!163,!165!and/or!167.!! ! 91.!The!modified!BLG!polypeptide!of!claim!84!comprising!7!substitutions!of!a!non-leucine!amino!acid! with!a!leucine!amino!acid!corresponding!to!position!21,!96,!108,!120,!139,!150!and!153!of!the!modified! BLG!set!forth!in!SEQ!ID!NO:!169.!! ! 92.! The! modified! BLG! polypeptide! of! claim! 84! comprising! at! least! one! amino! acid! substitution! compared! to! anyone! of! bovine! BLG! A,! bovine! BLG! B! and! bovine! BLG! C,! optionally! at! least! two! substitutions.!! ! ! ! Case Ref. P238WO IPTector® 93.!The!modified!BLG!polypeptide!of!claim!92!comprising!at! least!one!substitution!of!Alanine!with! Valine!in!a!position!corresponding!to!position!118!of!the!BLG!C!of!SEQ!ID!NO:!3.!! ! 94.!The!modified!BLG!polypeptide!of!claim!92!comprising!at!least!one!substitution!of!Glutamine!with! Histidine!at!a!position!corresponding!to!position!59!of!the!BLG!A!of!SEQ!ID!NO:!1.!! ! 95.! The! modified! BLG! polypeptide! of! claim! 92! comprising! a! Histidine! and! a! Valine! at! positions! corresponding!to!position!59!and!118!respectively!in!anyone!of!the!BLG^s!comprised!in!SEQ!ID!NO:!1,! 2!or!3.!! 96.!A!food!or!feed!composition!comprising!the!modified!bovine!BLG!polypeptide!of!anyone!of!claim! 79!to!95.! ! 97.!A!method!of!enriching!a!food!or!feed!composition!with!essential!amino!acids!comprising!mixing! one!or!more!food!or!feed!ingredients!with!the!modified!bovine!BLG!polypeptide!of!anyone!of!claim! 79!to!95.! ! *!!*!!*! ! ! ! ! ! ! !
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