EP4587565A1 - Peptides signal fongiques - Google Patents

Peptides signal fongiques

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Publication number
EP4587565A1
EP4587565A1 EP23769180.3A EP23769180A EP4587565A1 EP 4587565 A1 EP4587565 A1 EP 4587565A1 EP 23769180 A EP23769180 A EP 23769180A EP 4587565 A1 EP4587565 A1 EP 4587565A1
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EP
European Patent Office
Prior art keywords
seq
polypeptide
signal peptide
nucleic acid
polynucleotide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP23769180.3A
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German (de)
English (en)
Inventor
Hiromi AKEBOSHI
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Novozymes AS
Original Assignee
Novozymes AS
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Publication date
Application filed by Novozymes AS filed Critical Novozymes AS
Publication of EP4587565A1 publication Critical patent/EP4587565A1/fr
Pending legal-status Critical Current

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Classifications

    • 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)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • 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
    • C07K14/76—Albumins
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • 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
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y402/00—Carbon-oxygen lyases (4.2)
    • C12Y402/02—Carbon-oxygen lyases (4.2) acting on polysaccharides (4.2.2)
    • C12Y402/0201—Pectin lyase (4.2.2.10)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

  • the present invention relates to nucleic acid constructs comprising a first polynucleotide encoding a signal peptide, e.g., from a fungal glycosidase, and a second polynucleotide encoding an alphalactalbumin (ALAB) polypeptide; expression vectors and host cells comprising said nucleic acid constructs; methods for producing ALAB polypeptides; and fusion proteins comprising an ALAB polypeptide and a signal peptide.
  • ALAB alphalactalbumin
  • SPs Signal peptides
  • Signal peptides are short amino acid sequences present in the amino terminus of many newly synthesized polypeptides that target these into or across cellular membranes, thereby aiding maturation and secretion.
  • the amino acid sequence of the SP influences secretion efficiency and thereby the yield of the polypeptide manufacturing process.
  • Bioinformatic tools such as SignalP and SignalP5 can predict SPs from amino acid sequences, but most cannot distinguish between various types of SPs (Armenteros et al., Nat. Biotechnol. 37: 420-423, 2019).
  • a large degree of redundancy in the amino acid sequence of SPs makes it difficult to predict the efficiency of any given SP for production of recombinant proteins at industrial scale.
  • Alpha-lactalbumin is a principal protein of milk.
  • ALAB forms the regulatory subunit of the lactose synthase (LS) heterodimer and beta 1 ,4-galactosyltransferase (beta4Gal-T1) forms the catalytic component.
  • LS lactose synthase
  • beta4Gal-T1 beta 1 ,4-galactosyltransferase
  • alpha-lactalbumin strongly binds calcium and zinc ions and may possess bactericidal or antitumor activity.
  • a folding variant of alpha-lactalbumin, called HAMLET likely induces apoptosis in tumor and immature cells.
  • Recombinant ALAB has the potential to improve the nutritional value in food, beverages, and feed.
  • Recombinant ALAB expression has been reported previously in different organisms including transgenic goats (Yuan YG et al, J Anal Methods Chem. 2014; 2014:281031. doi: 10.1155/2014/281031).
  • Recombinantly produced phytase is widely used as a feed supplement to effectively improve phosphorous utilization and reduce fecal phosphorous excretion in animals as poultry and pig.
  • polypeptides ALAB and phytase there is a growing need for access thereto in the respective industries.
  • the present invention is based on the surprising and inventive finding that expression of difficult- to-express proteins (alpha-lactalbumin and phytase) with a fungal signal peptide provides increased yield when expressed in fungal host cells.
  • difficult- to-express proteins alpha-lactalbumin and phytase
  • MTP microtiter plates
  • SF shake flasks
  • laboratory fermentation tanks laboratory fermentation tanks.
  • the present invention relates to expression vectors comprising a nucleic acid construct of the first aspect.
  • SEQ ID NO:4 is the GH16 signal peptide (MRLPLVSSTVALLSSASLVAA).
  • SEQ ID NO:7 is the ALAB coding sequence, (with SP GH26)
  • SEQ ID NO:13 is the phytase coding sequence, (with SP GH26)
  • SEQ ID NO: 14 is the phytase amino acid sequence (with SP GH26)
  • SEQ ID NO:17 is the intron between SP GH16/ SP GH26 and the ALAB coding sequence
  • SEQ ID NO:22 is the GH16 polypeptide coding sequence from Aspergillus luchuensis
  • SEQ ID NO:23 is the GH16 polypeptide from A. luchuensis
  • SEQ ID NO:25 is the reference GH13 signal peptide
  • SEQ ID NO:26 is the reference cutinase (JSP004) signal peptide coding sequence
  • SEQ ID NO:27 is the reference cutinase (JSP004) signal peptide
  • SEQ ID NO:28 is the reference GH72 signal peptide coding sequence
  • SEQ ID NO:29 is the reference GH72 signal peptide
  • SEQ ID NO:30 is the SP GH16-ALAB expression cassette (promoter-SPGH16-intron-ALAB- terminator)
  • SEQ ID NO: 31 is the SP GH26-ALAB expression cassette (promoter-SPGH26-intron-ALAB- terminator)
  • SEQ ID NO:32 is the SP GH72-ALAB expression cassette (promoter-SPGH72-intron-ALAB- terminator)
  • SEQ ID NO:33 is the SP GH16-phytase expression cassette (promoter-SPGH16-phytase- terminator)
  • SEQ ID NO: 34 is HA442 primer
  • SEQ ID NO: 35 is HA451 primer
  • SEQ ID NO: 36 is HA283 primer
  • SEQ ID NO: 37 is HA444 primer
  • SEQ ID NO: 38 is HA450 primer
  • SEQ ID NO: 39 is HA445 primer
  • SEQ ID NO: 43 is HA489 primer
  • SEQ ID NO: 44 is the LYA1_4 (JSP017, Aspergillus luchuensis) signal peptide coding sequence
  • SEQ ID NO: 45 is the LYA1_4 (JSP017, Aspergillus luchuensis) signal peptide (MKYAAALTAVAALAARAAA)
  • SEQ ID NO: 48 is the GH28_9 endo-1 ,4-alpha-polygalacturonase (JSP008, Aspergillus luchuensis) signal peptide coding sequence
  • the mature polypeptide is amino acids 22 to 144 of SEQ ID NQ:10 and amino acids 1 to 21 of SEQ ID NQ:10 are a signal peptide.
  • nucleotides 1 to 54 of SEQ ID NO: 19 encode a signal peptide
  • nucleotides 55 to 100 of SEQ ID NO:19 is an intron.
  • the mature polypeptide coding sequence is nucleotides 1 to 1233 of SEQ ID NO:11.
  • the mature polypeptide coding sequence is nucleotides 55 to 1287 of SEQ ID NO: 13 and nucleotides 1 to 54 of SEQ ID NO: 13 encode a signal.
  • the mature polypeptide coding sequence is nucleotides 64 to 1296 of SEQ ID NO: 15 and nucleotides 1 to 63 of SEQ ID NO: 15 encode a signal peptide.
  • Native means a nucleic acid or polypeptide naturally occurring in a host cell.
  • Nucleic acid encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a polypeptide. Nucleic acids may be single stranded or double stranded, and may be chemical modifications. The terms “nucleic acid” and “polynucleotide” are used interchangeably. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and the present compositions and methods encompass nucleotide sequences that encode a particular amino acid sequence. Unless otherwise indicated, nucleic acid sequences are presented in 5'-to-3' orientation.
  • nucleic acid construct means a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, and which comprises one or more control sequences operably linked to the nucleic acid sequence.
  • Obtained polypeptide/peptide/polynucleotide The term “obtained” or “derived” when used in reference to a polynucleotide sequence, ALAB sequence, polypeptide sequence, mannanase sequence, glucanase sequence, phytase sequence, variant sequence or signal peptide sequence, means that the molecule originally has been isolated from the given source and that the molecule can either be utilized in its native sequence or that the molecule is modified by methods known to the skilled person.
  • operably linked means that specified components are in a relationship (including but not limited to juxtaposition) permitting them to function in an intended manner.
  • a regulatory sequence is operably linked to a coding sequence such that expression of the coding sequence is under control of the regulatory sequence.
  • parent means a polypeptide functioning as a signal peptide, or a polypeptide having phytase activity, or an ALAB polypeptide which forms a functional regulatory subunit of the lactose synthase (LS) heterodimer, to which an alteration is made to produce variants of the present invention.
  • the parent may be a naturally occurring (wild-type) polypeptide or a variant or fragment thereof.
  • Phytase In the present context, a preferred Phytase according to the invention is classified as belonging to the EC 3.1 .3.26 group. The EC numbers referto Enzyme Nomenclature 1992 from NC-IUBMB, Academic Press, San Diego, California, including supplements 1 -5 published in Eur. J.
  • Phytase activity is determined by the libertation of inorganic phosphate from Na-phytate solution, wherein one phytase activity unit is the amount of enzyme which liberates 1 pmol inorganic phosphate per min from a 0.0051 M Na-phytate solution in 0.25 M Na-acetate, pH 5.5 and at 37° C (Engelen, A. J., et al., 1994, "Simple and rapid determination of phytase activity", J. AOAC Int. 77:760-764).
  • Examples of activity unit names are: FYT, FTU and U.
  • the sequence identity between two polynucleotide sequences is determined as the output of “longest identity” using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 6.6.0 or later.
  • the parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix.
  • the nobrief option must be specified in the command line.
  • the output of Needle labeled “longest identity” is calculated as follows:
  • the third polynucleotide is a non-coding intron.
  • the third polynucleotide has a sequence identity of at least 80%, e.g. at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to SEQ ID NO:17 (gtaagtaacatccactctgttctagtgccatgctgagattgtacag).
  • the construct comprises a polynucleotide sequence with a sequence identity of at least 80%, e.g. at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to SEQ ID NO:18 or SEQ ID NO:19.
  • the promoter is a P3 promoter or a P3-based promoter, preferably the heterologous promoter is a tandem promoter comprising the P3 promoter or is a tandem promoter derived from the P3 promoter.
  • the signal peptide is obtained from a b-transglycosidase polypeptide (EC 2.4.1.-).
  • the signal peptide is obtained from a chitin b-1 ,3/1 ,6-glucanosyltransferase polypeptide (EC 2.4.1 .-) polypeptide.
  • the signal peptide is obtained from an endo-b-1 ,3-glucanase polypeptide or laminarinase polypeptide (EC 3.2.1.39). In one embodiment, the signal peptide is obtained from a polypeptide, such as a mannanase, transglycosidase, glycosyltransferase, laminarinase, or glucanase, expressed by a filamentous fungal host cell.
  • a polypeptide such as a mannanase, transglycosidase, glycosyltransferase, laminarinase, or glucanase
  • the signal peptide is obtained from a polypeptide expressed by a Aspergillus host cell, such as an Aspergillus luchuensis.
  • the first polynucleotide encoding the signal peptide has a sequence identity of at least 80%, e.g. at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to the mature polypeptide coding sequence of SEQ ID NO:1 or SEQ ID NO:3; most preferably the polynucleotide comprises, consists essentially of, or consists of the mature polypeptide coding sequence of SEQ ID NO:1 , SEQ ID NO:3, SEQ ID NO:44, SEQ ID NO:46, or SEQ ID NO:48;.
  • the first polynucleotide encoding the signal peptide has a sequence identity of at least 80%, e.g. at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to the mature polypeptide coding sequence of SEQ ID NO:1 ; most preferably the polynucleotide comprises, consists essentially of, or consists of the mature polypeptide coding sequence of SEQ ID NO:1.
  • the first polynucleotide encoding the signal peptide has a sequence identity of at least 80%, e.g. at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to the mature polypeptide coding sequence of SEQ ID NO:3; most preferably the polynucleotide comprises, consists essentially of, or consists of the mature polypeptide coding sequence of SEQ ID NO:3.
  • the first polynucleotide encoding the signal peptide has a sequence identity of at least 80%, e.g. at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to the mature polypeptide coding sequence of SEQ ID NO:44; most preferably the polynucleotide comprises, consists essentially of, or consists of the mature polypeptide coding sequence of SEQ ID NO:44.
  • the first polynucleotide encoding the signal peptide has a sequence identity of at least 80%, e.g. at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to the mature polypeptide coding sequence of SEQ ID NO:46; most preferably the polynucleotide comprises, consists essentially of, or consists of the mature polypeptide coding sequence of SEQ ID NO:46.
  • the first polynucleotide encoding the signal peptide has a sequence identity of at least 80%, e.g. at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to the mature polypeptide coding sequence of SEQ ID NO:48; most preferably the polynucleotide comprises, consists essentially of, or consists of the mature polypeptide coding sequence of SEQ ID NO:48.
  • the signal peptide is obtained from a glycosidase expressed by an Aspergillus species selected from the group consisting of Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus luchuensis, or Aspergillus oryzae.
  • an Aspergillus species selected from the group consisting of Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus luchuensis, or Aspergillus oryzae.
  • the signal peptide comprises, consists essentially of, or consists of SEQ ID NO:2.
  • the signal peptide has a sequence identity of at least 85%, e.g. at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to SEQ ID NO:4.
  • the signal peptide has a sequence identity of at least 85%, e.g. at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to SEQ ID NO:45.
  • the signal peptide has a sequence identity of at least 85%, e.g. at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to SEQ ID NO:49.
  • the wherein the signal peptide comprises, consists essentially of, or consists of SEQ ID NO:49.
  • the alpha-lactalbumin polypeptide is a human alpha-lactalbumin polypeptide.
  • the alpha-lactalbumin polypeptide comprises, consists essentially of, or consists of the mature polypeptide of SEQ ID NO:6.
  • the polypeptide having phytase activity has a sequence identity of at least 80%, e.g. at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to the mature polypeptide of SEQ ID NO:12.
  • the N- and/or C-terminal end of the polypeptide having phytase activity has been extended by addition of one or more amino acids. It is expected that the invention will be just as effective when employing a signal peptide that is highly similar to the signal peptide disclosed in SEQ ID NO:45, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO: 47, and SEQ ID NO: 49, encoded by SEQ ID NO:1 ,SEQ ID NO:3, SEQ ID NO:44, SEQ ID NO: 46, and SEQ ID NO:48, respectively.
  • One or more non-essential amino acids may, for example, be altered.
  • Non- essential amino acids in a signal peptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081 -1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant molecules are tested for signal peptide activity to identify amino acid residues that are critical to the activity of the molecule and residues that are non-essential. See also, Hilton et al., 1996, J. Biol. Chem. 271 : 4699-4708. The identity of essential and non-essential amino acids can also be inferred from an alignment with one or more related signal peptide.
  • Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241 : 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625.
  • Other methods that can be used include error-prone PCR, phage display (e.g. Lowman et al., 1991 , Biochemistry 30: 10832-10837; U.S. Patent No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner ef a/., 1988, DNA 7: 127).
  • Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide.
  • the signal peptide is a variant of the mature polypeptide of SEQ ID NO:45, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO: 47, or SEQ ID NO: 49comprising 1-10 alterations, e.g., 1-5, such as 1 , 2, 3, 4, or 5 alterations, compared to SEQ ID NO:45, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO: 47, or SEQ ID NO: 49 respectively.
  • the first and second polynucleotide are operably linked in translational fusion.
  • the term “operably linked in translation fusion” means that the signal peptide encoded by the first polynucleotide and the polypeptide encoded by the second polynucleotide are encoded in frame and translated together as a single polypeptide.
  • the signal peptide is removed to provide the mature phytase polypeptide or the mature ALAB polypeptide.
  • the signal peptide is not removed, or only removed partly to provide the mature ALAB polypeptide orthe mature polypeptide having phytase activity and comprising at least a fragment of the signal peptide.
  • the first and second polynucleotide may be manipulated in a variety of ways to provide for expression of a variant. Manipulation of the polynucleotide priorto its insertion into a nucleic acid construct or expression vector may be desirable or necessary depending on the construct or vector. The techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.
  • nucleic acid constructs of the invention may be operably linked to one or more further control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.
  • the control sequence may be a promoter, a polynucleotide that is recognized by a host cell for expression of a polynucleotide encoding a polypeptide of the present invention.
  • the promoter contains transcriptional control sequences that mediate the expression of the polypeptide.
  • the promoter may be any polynucleotide that shows transcriptional activity in the host cell including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.
  • the nucleic acid construct further comprises a heterologous promoter, and wherein said promoter, the first polynucleotide, and the second polynucleotide are operably linked.
  • the promoter is orientated upstream of the first polynucleotide.
  • the promoter is a heterologous promoter.
  • the promoter is a tandem promoter. More preferably, the promoter is a P3 promoter or a P3-based promoter.
  • promoters for directing transcription of the polynucleotide of the present invention in a filamentous fungal host cell are promoters obtained from Aspergillus, Fusarium, Rhizomucor and Trichoderma cells, such as the promoters described in Mukherjee et al., 2013, “Trichoderma-. Biology and Applications”, and by Schmoll and Dattenbock, 2016, “Gene Expression Systems in Fungi: Advancements and Applications”, Fungal Biology.
  • the promoter is a promoter, such as a P3 promoter, operably linked to an mRNA stabilizer region.
  • the mRNA stabilizer region is the cry 11 IA mRNA stabilizer region.
  • control sequence may also be an mRNA stabilizer region downstream of a promoter and upstream of the coding sequence of a gene which increases expression of the gene.
  • mRNA stabilizer regions are obtained from a Bacillus thuringiensis crylllA gene (WO 94/25612) and a Bacillus subtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177: 3465-3471).
  • mRNA stabilizer regions for fungal cells are described in Geisberg et al., 2014, Cell 156(4): 812-824, and in Morozov et al., 2006, Eukaryotic Cell 5(11): 1838-1846.
  • the control sequence may also be a transcription terminator, which is recognized by a host cell to terminate transcription.
  • the terminator is operably linked to the 3’-terminus of the polynucleotide encoding the polypeptide. Any terminator that is functional in the host cell may be used in the present invention.
  • Preferred terminators for filamentous fungal host cells may be obtained from Aspergillus or Trichoderma species, such as obtained from the genes for Aspergillus niger glucoamylase, Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, and Trichoderma reesei endoglucanase I, such as the terminators described in Mukherjee et al., 2013, “Trichoderma-. Biology and Applications”, and by Schmoll and Dattenbock, 2016, “Gene Expression Systems in Fungi: Advancements and Applications”, Fungal Biology.
  • Preferred terminators for yeast host cells may be obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase.
  • Other useful terminators for yeast host cells are described by Romanos et al., 1992, Yeast 8: 423-488.
  • the control sequence may also be a propeptide coding sequence that encodes a propeptide positioned at the N-terminus of a polypeptide.
  • the resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases).
  • a propolypeptide is generally inactive and can be converted to an active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide.
  • the propeptide coding sequence may be obtained from the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor miehei aspartic proteinase, and Saccharomyces cerevisiae alpha-factor.
  • the propeptide sequence is positioned next to the N-terminus of a polypeptide and the signal peptide sequence is positioned next to the N-terminus of the propeptide sequence.
  • the polypeptide may comprise only a part of the signal peptide sequence and/or only a part of the propeptide sequence.
  • the final or isolated polypeptide may comprise a mixture of mature polypeptides and polypeptides which comprise, either partly or in full length, a propeptide sequence and/or a signal peptide sequence.
  • regulatory sequences that regulate expression of the polypeptide relative to the growth of the host cell.
  • regulatory sequences are those that cause expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound.
  • yeast the ADH2 system or GAL1 system may be used.
  • filamentous fungi the Aspergillus niger glucoamylase promoter, Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzae glucoamylase promoter, Trichoderma reesei cellobiohydrolase I promoter, and Trichoderma reesei cellobiohydrolase II promoter may be used.
  • the recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the polynucleotide.
  • the choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced.
  • the vector may be a linear or closed circular plasmid.
  • a construct or vector comprising a polynucleotide is introduced into a host cell so that the construct or vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector as described earlier.
  • the choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source.
  • the polypeptide encoded by the introduced polynucleotide can be native or heterologous to the recombinant host cell.
  • at least one of the one or more control sequences can be heterologous to the polynucleotide encoding the polypeptide.
  • the recombinant host cell may comprise a single copy, or at least two copies, e.g. three, four, five or more copies of the polynucleotide of the present invention.
  • the host cell comprises two or more copies of the nucleic acid construct and/or the expression vector.
  • the fungal host cell may be a yeast cell.
  • yeast as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). For purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, Passmore, and Davenport, editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).
  • the yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cell.
  • the yeast host cell is a Pichia or Komagataella cell, e.g., a Pichia pastoris cell ( Komagataella phaffii).
  • the fungal host cell may be a filamentous fungal cell.
  • “Filamentous fungi” include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra).
  • the filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides.
  • Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic.
  • vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative.
  • the filamentous fungal host cell may be an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell.
  • the filamentous fungal host cell is an Aspergillus, Trichoderma or Fusarium cell. In a further preferred embodiment, the filamentous fungal host cell is an Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, or Fusarium venenatum cell.
  • the filamentous fungal host cell may be an Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zona
  • the host cell is an Aspergillus cell.
  • the host cell is an Aspergillus niger cell.
  • the host cell is an Aspergillus oryzae cell.
  • the host cell is isolated.
  • the host cell comprises at least two copies of the nucleic acid construct and/or the expression vector, such as two copies, three copies, four copies or more than four copies.
  • the present invention also relates methods of producing an alpha-lactalbumin (ALAB) polypeptide, the method comprising: a) cultivating a host cell according to the third aspect under conditions conducive for production of the ALAB polypeptide; and optionally b) recovering the ALAB polypeptide.
  • ALAB alpha-lactalbumin
  • the present invention also relates to methods of producing a polypeptide having phytase activity, the method comprising: a) cultivating a host cell according to the third aspect under conditions conducive for production of the polypeptide having phytase activity; and optionally b) recovering the polypeptide having phytase activity.
  • the present invention also relates to a fermentation broth formulation or a cell composition comprising a polypeptide having phytase activity.
  • the fermentation broth product further comprises additional ingredients used in the fermentation process, such as, for example, cells (including, the host cells containing the nucleic acid constructs of the present invention which are used to produce the polypeptide having phytase activity), cell debris, biomass, fermentation media and/or fermentation products.
  • the composition is a cell-killed whole broth containing organic acid(s), killed cells and/or cell debris, and culture medium.
  • the composition contains an organic acid(s), and optionally further contains killed cells and/or cell debris.
  • the killed cells and/or cell debris are removed from a cell- killed whole broth to provide a composition that is free of these components.
  • a whole broth or cell composition as described herein is typically a liquid, but may contain insoluble components, such as killed cells, cell debris, culture media components, and/or insoluble enzyme(s). In some embodiments, insoluble components may be removed to provide a clarified liquid composition.
  • SP cutinase with SEQ ID NO:27 is derived from Humicola insolens cutinase.
  • Transformants constructed as in example 1 were fermented in 96-well multi-titer-plate (MTP) containing % YPG Ac, and 1% SBP at 30C for 3 days.
  • the yield is detected by measuring phytase activity in culture supernatants using artificial substrate (described under section “pNP assay”).
  • pNP assay As can be seen from Table 3, increased phytase activities were measured using SP GH26 and SP GH16, compared to phytase activities using SP cutinase.
  • SP GH26 showed 187% yield increase compared to SP cutinase
  • SP GH16 showed 12% yield increase compared to SP cutinase.
  • GH16 signal peptide was compared to SP GH13 by lab-tank fermented under the current standard conditions.
  • the strains have genetically the same background, but integrated gene copy number of phytase signal variants are different as indicated in Table 4. Results are shown in Table 4, where JSP010 signal peptide GH16 showed significantly higher yield compared to SP GH13, i.e. 2.3-fold increase. The yield is measured by phytase activity using phytase as substrate (FYT(B) assay).
  • Signal peptide variants (shown in table 5) for expression of bovine alpha-lactalbumin (ALAB, SEQ ID NO:6) was constructed by 2 transformation steps.
  • the first transformation (host C2552) was done to integrate an expression cassette of bovine alpha-lactalbumin, but an intron of an amylase containing PAM sequence is inserted in place of signal sequence.
  • second transformation was performed.
  • the target signal sequence fragments were amplified by PCR and integrated in between the promoter and mature sequence of bovine alpha-lactalbumin by CRISPR system. Obtained strains were bovine alpha-lactalbumin expressing strains under six different signal peptides.
  • SP GH16 with SEQ ID NO:4 is derived from A. luchuensis endo-b-1 ,3-glucanase GH16 (SEQ ID NO:23).
  • SP GH26 with SEQ ID NO:2 is derived from A. luchuensis endo-1 ,4-beta-mannanase GH26 (SEQ ID NO:21).
  • SP pepesin A with SEQ ID NO:47.
  • Example 5 Increased yield of alpha-lactalbumin using SP GH26
  • Transformants JSP002 and JSP031 constructed as in example 4 were fermented by MTP or by buffled shake flasks (SF). The culture broth was centrifuged (3,500 x g, 15 min) and the supernatant was used for yield evaluation by MALDI-TOF MS semi-quantification method.
  • Signal peptide variants for expression of bovine ALAB was constructed as described in Example 4.
  • E.coli DH5a (Toyobo) is used for plasmid construction and amplification. Amplified plasmids are recovered with Qiagen Plasmid Kit (Qiagen). Ligation is done with either Rapid DNA Dephos & Ligation Kit (Roche) or Gibson assembly kit (NEB) according to the manufactory instructions. Polymerase Chain Reaction (PCR) is carried out with KOD-Plus system (TOYOBO) or PrimeSTAR MAX DNA Polymerase (Takara). QIAquickTM Gel Extraction Kit (Qiagen) is used for the purification of PCR fragments and extraction of DNA fragment from agarose gel.
  • Qiagen Plasmid Kit Qiagen Plasmid Kit
  • Ligation is done with either Rapid DNA Dephos & Ligation Kit (Roche) or Gibson assembly kit (NEB) according to the manufactory instructions.
  • Polymerase Chain Reaction (PCR) is carried out with KOD-Plus system (TOYOBO) or PrimeSTAR MA
  • Enzymes for DNA manipulations e.g. restriction endonucleases, ligases etc. are obtainable from New England Biolabs, Inc. and were used according to the manufacturer’s instructions.
  • SEQ ID NO:11 coding sequence
  • SEQ ID NO:12 amino acid sequence
  • the expression host strain Aspergillus niger C2552 was isolated by Novozymes and is a derivative of Aspergillus niger NN049184 which was isolated from soil described in example 14 in WQ2012/160093.
  • C3085, C5553, C6242 are strains which can produce the glucoamylase (1 ,4-alpha-D-glucan glucohydrolase, EC 3.2.1 .3) from Gloeophyllum sepiarium (Gs AMG).
  • COVE trace metals solution was composed of 0.04 g of NaB4O7 «10H2Q, 0.4 g of CuSO4 «5H2O, 1 .2 g of FeSO4 «7H2O, 0.7 g of MnSO4 «H2O, 0.8 g of Na2MoQ2 «2H20, 10 g of ZnSO4 «7H2O, and deionized water to 1 liter.
  • 50X COVE salts solution was composed of 26 g of KCI, 26 g of MgSO4 «7H2O, 76 g of KH2PO4, 50 ml of COVE trace metals solution, and deionized water to 1 liter.
  • COVE medium was composed of 342.3 g of sucrose, 20 ml of 50X COVE salts solution, 10 ml of 1 M acetamide, 10 ml of 1 .5 M CsCI2, 25 g of Noble agar, and deionized water to 1 liter.
  • COVEII plus 5-Fluorocytosine top agarose was composed of 34 g of sucrose, 20 ml of 50X COVE salts solution, 10 ml of 1 M acetamide, 2 ml of 5 g/L 5-Fluorocytosine, 10 g of low melt agarose, and deionized water to 1 liter.
  • STC buffer was composed of 0.8 M sorbitol, 25 mM Tris pH 8, and 25 mM CaCI2.
  • LB medium was composed of 10 g of tryptone, 5 g of yeast extract, 5 g of sodium chloride, and deionized water to 1 liter.
  • LB plus ampicillin plates were composed of 10 g of tryptone, 5 g of yeast extract, 5 g of sodium chloride, 15 g of Bacto agar, ampicillin at 100 pg per ml, and deionized water to 1 liter.
  • YPG medium was composed of 10 g of yeast extract, 20 g of Bacto peptone, 20 g of glucose, and deionized water to 1 liter.
  • SOC medium was composed of 20 g of tryptone, 5 g of yeast extract, 0.5 g of NaCI, 10 ml of 250 mM KCI, and deionized water to 1 liter.
  • TAE buffer was composed of 4.84 g of Tris Base, 1.14 ml of Glacial acetic acid, 2 ml of 0.5 M EDTA pH 8.0, and deionized water to 1 liter.
  • 1/4YPG Ac,1 % SBP was composed of 5.0 g/L glucose, 2.5 g/L yeast extract, 5 g/L peptone, 10g/L soy bean powder, 5ml/L 2M sodium acetate buffer, pH4.5
  • Amyloglycosidase trace metal solution is 13.9 g/L of FeSO4 7H2O, 13.56 g/L of MnSO4 5H2O, 6.8 g/L of ZnCI2, 2.5 g/L of CuSO4-5H2O, 0.24 g/L of NiCI2-6H2O, 3 g/L of Citric acid H2O.
  • Transformation of Aspergillus species can be achieved using the general methods for yeast transformation.
  • Aspergillus niger host strain was inoculated to 100 ml of YPG medium supplemented with 10 mM uridine and incubated for 16 h at 32°C at 80 rpm. Pellets were collected and washed with 0.6 M KCI, and resuspended 20 ml 0.6 M KCI containing a commercial beta-glucanase product (GLUCANEXTM, Novozymes A/S, Bagsvaerd, Denmark) at a final concentration of 20 mg per ml. The suspension was incubated at 32°C at 80 rpm until protoplasts were formed, and then washed twice with STC buffer.
  • GLUCANEXTM commercial beta-glucanase product
  • PCR Polymerase Chain Reaction
  • KOD plus reaction mix is shown in table 10.
  • KOD plus PCR cycle is shown in table 11 .
  • PrimeSTAR reaction mix is shown in table 12, and corresponding PCR cycle is shown in table 13.
  • Spores are inoculated to 100 ml of MSG media in buffled flask and fermented on a rotary shaking table at 220 rpm, 30°C.
  • Ten-milli liters of cultured broth was transferred tol OOml MU1 -glu with 4ml 50% urea in 500 ml baffled flasks and further fermented for 3 to 5 days.
  • Fermentation was done as fed-batch fermentation (H. Pedersen 2000, Appl Microbiol Biotechnol, 53: 272-277). Selected strains were pre-cultured in liquid media then grown mycelia were transferred to the tanks for further cultivation of protein production. Cultivation was done at pH 4 to 7, 30 to 34°C, for 6 ⁇ 8 days with the feeding of glucose and ammonium without over-dosing. Culture supernatant after centrifugation was used for yield evaluation. pNP assay
  • Phytase activity was measured as FYT(B) (Phytase (Braakii) Units), relative to an enzyme standard of a declared strength.
  • the samples and standard phytase reacts with sodium phytase (phytic acid dodeca sodium salt C e H e O24P6Nai2) and releases inorganic phosphate.
  • Catalytic reaction parameters and conditions are shown in table 14. This phosphate is determined spectrophometically from a yellow complex formed by an acidic complex reagent containing molybdate/vanadate. The yellow complex is measured spectrophotometrically at a wavelength of 405 nm. The rate of phosphate release can be observed by Konelab (Thermo Fisher Scientific). Colorimetric reaction parameters and conditions are shown in Table 15. Table 16 shows reaction buffers and reagent compositions.
  • the activity of the enzyme samples is determined relative to the standard curve.
  • a nucleic acid construct comprising: a first polynucleotide encoding a signal peptide having a sequence identity of at least 80% to SEQ ID NO:45, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO: 47, or SEQ ID NO: 49; and a) a second polynucleotide encoding an alpha-lactalbumin (ALAB) polypeptide having a sequence identity of at least 70% to the polypeptide sequence of SEQ ID NO:6; or b) a second polynucleotide encoding a polypeptide having phytase activity; wherein the first polynucleotide and the second polynucleotide are operably linked in translational fusion.
  • ALAB alpha-lactalbumin
  • nucleic acid construct according to any of embodiment 1 or 2, wherein the signal peptide is a naturally occurring signal peptide, or a functional fragment or functional variant of a naturally occurring signal peptide.
  • nucleic acid construct according to any of embodiments 1 to 3, wherein the signal peptide is from a filamentous fungal glycosidase.
  • nucleic acid construct according to any of embodiments 1 to 4, further comprising a third polynucleotide downstream of the first polynucleotide and upstream of the second polynucleotide.
  • nucleic acid construct according to any previous embodiment, wherein the third polynucleotide has a sequence identity of at least 80%, e.g. at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to SEQ ID NO:17 (gtaagtaacatccactctgttctagtgccatgctgagattgtacag).
  • nucleic acid construct according to any previous embodiment, wherein the nucleic acid construct further comprises a heterologous promoter, and wherein said promoter, the first polynucleotide, the second polynucleotide, and optionally the third polynucleotide, are operably linked.
  • nucleic acid construct according to any of the preceding embodiments, wherein the signal peptide is obtained from a glycosidase expressed by an Aspergillus species selected from the group consisting of Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus luchuensis, or Aspergillus oryzae.
  • Aspergillus species selected from the group consisting of Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus luchuensis, or Aspergillus oryzae.
  • nucleic acid construct according to any of the preceding embodiments, wherein the signal peptide comprises, consists essentially of, or consists of SEQ ID NO:45.
  • nucleic acid construct according to any of the preceding embodiments, wherein the signal peptide comprises, consists essentially of, or consists of SEQ ID NO:49.
  • nucleic acid construct according to any of the preceding embodiments, wherein the signal peptide has a sequence identity of at least 80%, e.g. at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to SEQ ID NO:4.
  • nucleic acid construct according to any of the preceding embodiments, wherein the N- and/or C- terminal end of the signal peptide has been extended by addition of one or more amino acids.
  • nucleic acid construct according to any of embodiments 1 to 25, wherein the signal peptide is a fragment of the signal peptides of any of embodiments 1 to 25.
  • polynucleotide encoding the alpha-lactalbumin polypeptide has a sequence identity of at least 60%, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to the mature polypeptide coding sequence of SEQ ID NO:5; most preferably the polynucleotide comprises, consists essentially of, or consists of the mature polypeptide coding sequence of SEQ ID NO:5.
  • nucleic acid construct according to any of the preceding embodiments, wherein the alphalactalbumin is a human alpha-lactalbumin.
  • nucleic acid construct according to any preceding embodiments, wherein the alpha-lactalbumin polypeptide has a sequence identity of at least 60%, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to the mature polypeptide of SEQ ID NO:6.
  • the alpha-lactalbumin polypeptide comprises, consists essentially of, or consists of the mature polypeptide of SEQ ID NO:6.
  • nucleic acid construct according to any of the preceding embodiments, wherein polynucleotide encoding the polypeptide having phytase activity has a sequence identity of at least 80%, e.g. at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to the mature polypeptide coding sequence of SEQ ID NO: 11 ; most preferably the polynucleotide comprises, consists essentially of, or consists of the mature polypeptide coding sequence of SEQ ID NO:11 .

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Abstract

La présente invention concerne des constructions d'acide nucléique comprenant un premier polynucléotide codant pour un peptide signal, par exemple, à partir d'une glycosidase fongique, et un second polynucléotide codant pour un polypeptide alpha-lactalbumine (ALAB) ; des vecteurs d'expression et des cellules hôtes comprenant lesdites constructions d'acide nucléique ; des procédés de production de polypeptides ALAB ; et des protéines de fusion comprenant un polypeptide ALAB et un peptide signal.
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US5571697A (en) * 1989-05-05 1996-11-05 Baylor College Of Medicine Texas Medical Center Expression of processed recombinant lactoferrin and lactoferrin polypeptide fragments from a fusion product in Aspergillus
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