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WO2024141641A2 - Signaux de sécrétion - Google Patents

Signaux de sécrétion Download PDF

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Publication number
WO2024141641A2
WO2024141641A2 PCT/EP2023/087985 EP2023087985W WO2024141641A2 WO 2024141641 A2 WO2024141641 A2 WO 2024141641A2 EP 2023087985 W EP2023087985 W EP 2023087985W WO 2024141641 A2 WO2024141641 A2 WO 2024141641A2
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WO
WIPO (PCT)
Prior art keywords
seq
sequence
signal peptide
encoding
copies
Prior art date
Application number
PCT/EP2023/087985
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English (en)
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WO2024141641A3 (fr
Inventor
Lucia Nancy COCONI LINARES
Yentil DE VLEESCHOUWER
Marie-Laure Juliette ERFFELINCK
Anton Alain An HEYMAN
Alrik Pieter Los
Deniz Güver MALAT
Original Assignee
Biotalys NV
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Publication date
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Publication of WO2024141641A2 publication Critical patent/WO2024141641A2/fr
Publication of WO2024141641A3 publication Critical patent/WO2024141641A3/fr

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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
    • 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
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • 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
    • 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
    • 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
    • C12R2001/80—Penicillium

Definitions

  • the present invention relates to the field of biotechnology, specifically to the field of recombinant protein expression. More specifically, the present invention relates to cells modified to express higher yields of recombinant protein or a protein encoded by a gene of interest.
  • the a-MF secretion signal could be suboptimal, and it may be preferable to use alternative secretion signals (Ng et al. The Journal of cell biology. 1996. 134 (2), 269-78).
  • the secretion signal a-MF has already been reported to cause a bottleneck in translocation (Fitzgerald & Glick. Microb Cell Fact. 2014. 13, 125; Zahri et al. Microbiology. 2018.).
  • the present invention further relates to the use of a microbial host cell, comprising one or more copies of an expression cassette comprising a promoter capable of promoting expression of a gene of interest, and the gene of interest, wherein the gene of interest is fused to a secretion signal sequence which comprises a signal peptide encoding sequence, and a terminator, and wherein the one or more copies of the expression cassette are integrated into the genome of the microbial host cell, for manufacturing a protein, where the protein is encoded by the gene of interest.
  • the present invention further relates to a nucleic acid comprising a secretion peptide-encoding sequence wherein the signal peptide-encoding sequence is the nucleotide sequence according to the nucleotide sequence of SEQ ID NO: 101 , a nucleotide sequence with at least 90% identity to SEQ ID NO:
  • the present invention further relates to a nucleic acid comprising a secretion peptide-encoding sequence wherein the signal peptide-encoding sequence is the nucleotide sequence according to the nucleotide sequence of SEQ ID NO: 102, a nucleotide sequence with at least 90% identity to SEQ ID NO:
  • nucleotide sequence encoding a signal peptide having the amino acid sequence of SEQ ID NO: 1 19 or a nucleotide sequence encoding a signal peptide having an amino acid sequence with at least 90% identity to SEQ ID NO: 1 19.
  • nucleotide sequence encoding a signal peptide having the amino acid sequence of SEQ ID NO: 120 or a nucleotide sequence encoding a signal peptide having an amino acid sequence with at least 90% identity to SEQ ID NO: 120.
  • the present invention further relates to a nucleic acid comprising a secretion peptide-encoding sequence wherein the signal peptide-encoding sequence is the nucleotide sequence according to the nucleotide sequence of SEQ ID NO: 106, a nucleotide sequence with at least 90% identity to SEQ ID NO: 106, a nucleotide sequence encoding a signal peptide having the amino acid sequence of SEQ ID NO: 123, or a nucleotide sequence encoding a signal peptide having an amino acid sequence with at least 90% identity to SEQ ID NO: 123.
  • the present invention further relates to an expression cassette comprising a nucleic acid of the invention, and a promoter operably linked to the nucleic acid, and optionally a gene of interest.
  • the present invention further relates to a vector comprising a nucleic acid of the invention or an expression cassette of the invention, or a vector comprising said nucleic acid and a promoter operable linked to said nucleic acid and optionally a gene of interest.
  • the present invention further relates to a method for producing a protein, the method comprising
  • the present invention further relates to a protein produced by said method.
  • the present invention further relates to a peptide comprising the amino acid sequence provided in SEQ ID NO: 1 18, or an amino acid sequence with at least 90% identity to SEQ ID NO: 1 18, a peptide comprising the amino acid sequence provided in SEQ ID NO: 1 19, or an amino acid sequence with at least 90% identity to SEQ ID NO: 1 19, a peptide comprising the amino acid sequence provided in SEQ ID NO: 120, or an amino acid sequence with at least 90% identity to SEQ ID NO: 120, or a peptide comprising the
  • the present invention provides a microbial host cell comprising: one or more copies of an expression cassette comprising a promoter capable of promoting expression of a gene of interest, and the gene of interest, wherein the gene of interest is fused to a secretion signal sequence which comprises a signal peptide encoding sequence, and where the expression cassette further comprises a terminator, and wherein the one or more copies of the expression cassette are integrated into the genome of the microbial host cell.
  • the one or more copies of the expression cassette are integrated into the chromosomal DNA of the microbial host cell.
  • expression cassette refers to a distinct functional unit of nucleotide sequence (i.e. DNA) comprising a regulatory sequence such as a promoter capable of expressing a gene of interest.
  • the expression cassette may comprise a gene of interest or alternatively may be provided in a form suitable for insertion of a gene of interest into the expression cassette (e.g., the expression cassette may comprise a multiple cloning site for insertion of the gene of interest).
  • translation of a gene of interest in an expression cassette according to this invention is initiated by a start codon, which initiates translation by the ribosomal translation machinery of the microbial cell.
  • the start codon is situated at the 5’-end of said secretion signal sequence.
  • a gene of interest in an expression cassette according to this invention is terminated with a stop-codon.
  • the expression cassette according to the invention may further include a terminator sequence, halting expressing progression at the end of the expression cassette.
  • An expression cassette may comprise additional regulatory and other sequences such as signal sequences, introns, IRES- sequences, ribosomal binding sites etc.
  • the expression cassettes may be provided as part of a vector.
  • One or more expression cassettes, and optionally further parts of said vectors, may be integrated into the genome of the microbial host cell.
  • the expression cassette may be first amplified by PCR using said vectors as a DNA template whereafter said PCR products can be used for transforming two or more different expression cassettes to the host cell.
  • the expression cassette is synthesized or assembled without the need for it to be included into a vector.
  • the microbial host cell of the present invention may thus have the PCR products containing the expression cassettes integrated into its genome (partially or completely). It might also be that some of the transformed vectors are integrated (partially or completely) into the genome of said host cells, whereas other of said transformed vectors are present as plasmids within the cytosol of said host cell.
  • a promoter that is “capable of promoting expression of a gene of interest” is a promoter that is operably linked to the gene of interest and which, under suitable conditions, promotes the expression of the gene of interest in and by the microbial host cell.
  • the gene of interest may be under the control of a constitutive promoter, or the gene of interest may be under the control of an inducible promoter.
  • methods of the invention may comprise a step of inducing expression of the gene of interest by the microbial host cell. It is said that the promoter and the gene of interest are operably linked.
  • the phrase “fused to” as used herein, means that two or more distinct nucleic acid sequences are so organized so that the amino acid sequences encoded by the two or more distinct nucleic acid sequences are expressed as one single polypeptide chain.
  • expression of a secretion signal sequence fused to a gene of interest will results in a single polypeptide chain comprising the secretion signal and the protein of interest (encoded by the gene of interest) linked in a single polypeptide chain.
  • the single polypeptide chain can thereafter be further processed and for example cleaved, such as the cleavage of a secretion signal from the protein of interest during secretion.
  • the single polypeptide chain may further comprise an additional amino acid sequence between the secretion signal and the protein of interest.
  • the secretion signal sequence may be fused to the gene of interest directly (no additional sequence in between) or indirectly (an additional amino acid sequence in between), provided that the protein of interest and the secretion signal are expressed in a single polypeptide chain.
  • a protein or “fusion protein” means that two or more distinct proteins or peptides (such as a signal sequence) are produced as one single polypeptide chain. It is said the two proteins may be fused.
  • a signal sequence may be fused to a protein of interest.
  • a fused protein results from the expression of two or more nucleic acids sequences that are expressed as a single polypeptide chain as described above.
  • An example of a fusion protein is a precursor protein where the signal sequence is fused to the N-terminus of the protein of interest.
  • the expression cassette comprises, in a 5' to 3' order, a promoter, a start codon, a secretion signal sequence, optionally a N-terminal tag, a gene of interest encoding a protein of interest, optionally a C-terminal tag, a stop codon and a terminator sequence.
  • a “start codon” as used herein refers to the first codon of a messenger RNA (mRNA) transcript translated by a ribosome or the first codon of a DNA sequence encoding the mRNA. The most common start codon is AUG (i.e ., ATG in the corresponding DNA sequence). Optionally, the start codon is preceded by a 5' untranslated region (5' UTR).
  • gene of interest refers to a sequence of nucleotides that encodes a protein of interest, e.g., a recombinant protein (e.g., a VHH) to be produced in a microbial host cell.
  • the gene of interest my comprise intron and exon sequence or sequences where the genetic information in the intron sequence or sequences may not necessarily be present in the final protein product.
  • this does not include additional sequences such as secretion signal sequences or tags which although fused to the gene of interest and may be present in the single amino acid sequence of the corresponding protein, these additional sequences are not construed as being part of the gene of interest.
  • the protein encoded by the gene of interest is the protein of interest defined by the amino acid sequence without any additional tags or secretion signals.
  • the inventors have found that the choice of a secretion signal sequence for the expression and secretion of a protein encoded by a gene of interest and produced by a microbial host cell is critical to increasing the production and/or yield of said protein.
  • the inventors have found that the production and/or yield of a protein can be improved when using a microbial host cell comprising one or more copies of an expression cassette comprising a promoter capable of promoting expression of a gene of interest, and the gene of interest, wherein the gene of interest is fused to a secretion signal sequence which comprises a signal peptide encoding sequence, and a terminator, and wherein the one or more copies of the expression cassette are integrated into the genome of the microbial host cell.
  • the current invention therefore relates to said microbial host cell and the use of the microbial host cell for manufacturing a protein, wherein the protein is encoded by the gene of interest.
  • the invention further relates to a nucleic acid comprising a signal peptide encoding sequence comprised in a secretion signal sequence, the use of said nucleic acid as a secretion signal sequence, an expression cassette comprising said nucleic acid and a vector comprising said nucleic acid or said expression cassette.
  • the invention further relates to a method for producing a protein and the protein produced by said method.
  • the present invention further relates to a peptide (i.e., a signal peptide) and a protein comprising said peptide and the use of said peptide as a secretion signal.
  • a peptide i.e., a signal peptide
  • the invention finally relates to a microbial host cell comprising said nucleic acid, said expression cassette, said vector, or said peptide.
  • a “secretion signal sequence” refers to a nucleic acid sequence that comprises a signal peptide encoding sequence.
  • a secretion signal sequence may also further comprise a “pro-sequence”, which is a nucleic acid sequence encoding a “pro-peptide” (also known as a “carrier peptide”).
  • the pro-sequence is preferably fused to the 3’ end of the signal peptide encoding sequence.
  • the pro-peptide is preferably fused to the C-terminus of the signal peptide.
  • a secretion signal sequence may be fused to a gene of interest.
  • a secretion signal sequence is preferably fused to the 5’-end of the gene of interest.
  • the secretion signal sequence is fused to the 5’-end of the gene of interest
  • the secretion signal will be fused to the N-terminal end of the protein encoded by the gene of interest.
  • the secretion signal when fused to a protein encoded by a gene of interest will signal the internal protein expression and secretion pathways of the microbial cell to secrete the protein encoded by the gene of interest into the surrounding environment.
  • the surrounding environment may be a fermentation broth or culture media.
  • the protein encoded by the gene of interest may be isolated or purified from the fermentation broth or culture media.
  • the secretion signal sequence comprises a signal peptide encoding sequence, also referred to as a “pre-sequence”, which encodes for a signal peptide.
  • a signal peptide encoding sequence may be sufficient (i.e., without a pro-sequence) for secretion of a protein of interest when expressed in a microbial host cell as a fusion protein.
  • a typical example of a signal peptide encoding sequence is the pre-sequence of the Saccharomyces a-mating factor (a-MF) of SEQ ID NO: 114, where the corresponding signal peptide is identified by the amino acid sequence according to SEQ ID NO: 131 .
  • the signal peptide-encoding sequence is from a Myceliophthora species. In other more preferred embodiments of the invention the signal peptide-encoding sequence is from an Aspergillus species. In some more preferred embodiments of the invention the signal peptide-encoding sequence is from Komagataella phaffii. In other more preferred embodiments of the invention the signal peptide-encoding sequence is from Saccharomyces cerevisiae. In other more preferred embodiments of the invention the signal peptide-encoding sequence is from Hansenula polymorpha. In other more preferred embodiments of the invention the signal peptide-encoding sequence is from Fusarium solani.
  • the signal peptide-encoding sequence is from Trichoderma reesei. In other more preferred embodiments of the invention the signal peptide-encoding sequence is from Myceliophthora thermophila or Myceliophthora heterothallica. In other more preferred embodiments of the invention the signal peptide-encoding sequence is from Aspergillus niger.
  • the signal peptide-encoding sequence is selected from any of (a) the nucleotide sequence of any one of SEQ ID Nos: 99 to 1 13 or 134, (b) a nucleotide sequence with at least 90% identity to any one of SEQ ID Nos: 99 to 1 13 or 134, (c) a nucleotide sequence encoding a signal peptide having the amino acid sequence of any one of SEQ ID Nos: 1 16 to 130 or 135, or (d) a nucleotide sequence encoding a signal peptide having an amino acid sequence with at least 90% identity to any one of SEQ ID Nos: 1 16 to 130 or 135.
  • the signal peptide-encoding sequence is an artificial signal peptide.
  • the signal peptide-encoding sequence is any of (a) the nucleotide sequence of SEQ ID NO: 101 , (b) a nucleotide sequence with at least 90% identity to SEQ ID NO: 101 , (c) a nucleotide sequence encoding a signal peptide having the amino acid sequence of SEQ ID NO: 1 18, or (d) a nucleotide sequence encoding a signal peptide having an amino acid sequence with at least 90% identity to SEQ ID NO: 1 18.
  • the signal peptide-encoding sequence is from the Saccharomyces cerevisiae Scw10 gene.
  • the signal peptide- encoding sequence is any of (a) the nucleotide sequence of SEQ ID NO: 105, (b) a nucleotide sequence with at least 90% identity to SEQ ID NO: 105, (c) a nucleotide sequence encoding a signal peptide having the amino acid sequence of SEQ ID NO: 122, or (d) a nucleotide sequence encoding a signal peptide having an amino acid sequence with at least 90% identity to SEQ ID NO: 122.
  • the signal peptide-encoding sequence is from the Komagataella phaffii Gcw14 gene.
  • the signal peptide-encoding sequence is any of (a) the nucleotide sequence of SEQ ID NO: 106, (b) a nucleotide sequence with at least 90% identity to SEQ ID NO: 106, (c) a nucleotide sequence encoding a signal peptide having the amino acid sequence of SEQ ID NO: 123, or (d) a nucleotide sequence encoding a signal peptide having an amino acid sequence with at least 90% identity to SEQ ID NO: 123.
  • the signal peptide-encoding sequence is from the Komagataella phaffii Cwp11 gene.
  • the signal peptide-encoding sequence is any of (a) the nucleotide sequence of SEQ ID NO: 107, (b) a nucleotide sequence with at least 90% identity to SEQ ID NO: 107, (c) a nucleotide sequence encoding a signal peptide having the amino acid sequence of SEQ ID NO: 124, or (d) a nucleotide sequence encoding a signal peptide having an amino acid sequence with at least 90% identity to SEQ ID NO: 124.
  • the signal peptide-encoding sequence is from the Pichia pastoris Flo10 gene.
  • the signal peptide-encoding sequence is any of (a) the nucleotide sequence of SEQ ID NO: 1 13, (b) a nucleotide sequence with at least 90% identity to SEQ ID NO: 1 13, (c) a nucleotide sequence encoding a signal peptide having the amino acid sequence of SEQ ID NO: 130, or (d) a nucleotide sequence encoding a signal peptide having an amino acid sequence with at least 90% identity to SEQ ID NO: 130.
  • the secretion signal sequence comprises only a signal peptide- encoding sequence (i.e. , no pro-sequence)
  • the secretion signal sequence and the signal peptide-encoding sequence may be identical.
  • the secretion signal and the signal peptide may be identical.
  • pro-sequence is the pro-sequence from the a-MF from Saccharomyces cerevisiae as identified in SEQ ID NO: 1 15 encoding the pro-peptide as identified in SEQ ID NO: 132.
  • An example of a pre-pro protein i.e., secretion signal
  • SEQ ID NO: 137 is given in SEQ ID NO: 137 and the corresponding nucleotide sequence is given in SEQ ID NO: 136.
  • the secretion signal is cleaved from the protein encoded by the gene of interest before, at or during the secretion process of the microbial host cell.
  • the secreted protein encoded by the gene of interest may not contain any residual secretion signal amino acids at its N-terminus.
  • a fraction of proteins encoded by the gene of interest that are secreted still contain the secretion signal fused to the N-terminus.
  • a fraction of proteins encoded by the gene of interest that are secreted still contain one or more amino acids of the secretion signal fused to the N-terminus.
  • the nucleic acid may further comprise a pro-sequence, such as a Saccharomyces a-mating factor prosequence according to the nucleotide sequence of SEQ ID NO: 1 15, encoding pro-peptide such as the Saccharomyces a-mating factor pro peptide according to the amino acid sequence of SEQ ID NO: 132.
  • a pro-sequence such as a Saccharomyces a-mating factor prosequence according to the nucleotide sequence of SEQ ID NO: 1 15, encoding pro-peptide such as the Saccharomyces a-mating factor pro peptide according to the amino acid sequence of SEQ ID NO: 132.
  • a nucleic acid of the invention may be used as a secretion signal sequence by fusing it to a gene of interest, for example, but not limited to, a VHH. Therefore, the nucleic acid of the invention can be further comprised in an expression cassette, wherein the expression cassette further comprises a promoter operably linked to the nucleic acid. In some embodiments, the expression cassette further comprises a gene of interest wherein the signal peptide-encoding sequence is fused to the gene of interest.
  • the expression cassette can be used in a microbial host cell to express the gene of interest, whereby the signal peptide encoded by the nucleic acid directs secretion of the protein encoded by the gene of interest into the surrounding environment (e.g., culture broth).
  • the expression cassette may further comprise a start codon, a stop-codon, a terminator sequence, or additional regulatory and other sequences such as signal sequences, introns, IRES- sequences, ribosomal binding sites etc.
  • the inventors In their efforts to search for an optimal peptides that may serve as a secretion signal, the inventors have furthermore identified unique peptides that may serve as a secretion signal. Therefore, the invention further relates to use of said peptide as or in a secretion signal.
  • the peptide of the current invention may comprise the amino acid sequence of SEQ ID NO: 1 18, or an amino acid sequence with at least 90% identity to SEQ ID NO: 1 18; or the amino acid sequence of SEQ ID NO: 1 19, or an amino acid sequence with at least 90% identity to SEQ ID NO: 1 19; or the amino acid sequence of SEQ ID NO: 120, or an amino acid sequence with at least 90% identity to SEQ ID NO: 120; or amino acid sequence of SEQ ID NO: 123, or an amino acid sequence with at least 90% identity to SEQ ID NO: 123.
  • the peptide may further comprise a pro-peptide, such as a Saccharomyces a-mating factor pro-peptide such as the Saccharomyces a-mating factor pro-peptide according to the amino acid sequence of SEQ ID NO: 132.
  • the peptides may be isolated and/or recombinant.
  • the current invention further relates to a protein comprising said peptide.
  • the protein may be a recombinant protein, such as a recombinant fusion protein comprising the peptide fused to a protein encoded by a gene of interest.
  • the recombinant protein may comprise the peptide of the invention at its N-terminus.
  • the invention also provides a composition comprising the peptide of the invention or a protein comprising the peptide of the invention.
  • the invention further relates to precursor proteins where the precursor protein comprises a signal sequence and a protein of interest, where the signal sequence is fused to the N-terminus of the protein of interest.
  • a precursor protein may be a recombinant protein.
  • precursor protein refers to the preliminary or temporary nature of the precursor protein, that is to say the fusion of the signal sequence to the N-terminus of the protein of interest is present mainly and often only present during the production and secretion of the precursor protein in the microbial host cell since the signal sequence will become cleaved from the protein of interest. That is to say, the precursor protein exists as a precursor protein until the N- terminal signal sequence is cleaved, releasing the protein of interest. In some cases the signal sequence will be incorrectly cleaved or not cleaved at all whereby in a final product comprising the protein of interest, remnants of the precursor protein may be still be present.
  • the precursor polypeptides comprises a signal sequence fused to a protein of interest.
  • the signal sequence comprises a signal peptide having an amino acid sequence according to any one of SEQ ID NOs: 1 16 to 130 or 135 or a signal peptide having an amino acid sequence with at least 90% identity to any one of SEQ ID Nos: 1 16 to 130 or 135.
  • thee precursor polypeptide comprises a signal sequence fused to a protein of interest and where the signal sequence further comprises a pro-sequence, preferably the Saccharomyces alpha mating factor pro-sequence.
  • the precursor polypeptide comprises a signal sequence fused to a VHH, and where the signal sequence comprises a signal peptide having an amino acid sequence according to any one of SEQ ID NOs: 1 16 to 130 or 135 or a signal peptide having an amino acid sequence with at least 90% identity to any one of SEQ ID Nos: 1 16 to 130 or 135.
  • the precursor polypeptide comprises a signal sequence fused to a VHH and where the signal sequence further comprises a prosequence, preferably the Saccharomyces alpha mating factor pro-sequence.
  • the inventors have surprisingly found that by increasing the copies of the expression cassette of the invention integrated into the genome of the microbial host cell the production and/or yield of the protein expressed by the gene of interest may be significantly improved.
  • the inventors have observed that this improvement in production and/or yield is surprisingly larger for certain secretion signal sequences (i.e. , the nucleic acids of the invention).
  • the inventors have found that, when multiple copies of an expression cassette are integrated in the genome of a host cell, significant increases in the production and/or yield of a protein encoded by a gene of interest can be achieved when using the secretion signal sequences of the invention, as opposed to the canonically used a mating factor (aMF) of Saccharomyces cerevisiae.
  • aMF mating factor
  • the current invention relates to a microbial host cell comprising one or more copies of an expression cassette.
  • the expression cassette comprises a promoter capable of promoting expression of a gene of interest, and the gene of interest, wherein the gene of interest is fused to a secretion signal sequence which comprises a signal peptide encoding sequence, and wherein the expression cassette further comprises a terminator and wherein the one or more copies of the expression cassette are integrated into the genome of the microbial host cell.
  • the microbial host cell comprises two or more copies of the expression cassette.
  • the microbial host cell comprises at least 3 or more copies of the expression cassette.
  • the microbial host cell comprises at least 4 or more copies of the expression cassette.
  • the microbial host cell comprises at least 5 or more copies of the expression cassette. In another preferred embodiment, the microbial host cell comprises at least 6 or more copies of the expression cassette. In another preferred embodiment, the microbial host cell comprises at least 7 or more copies of the expression cassette. In another preferred embodiment, the microbial host cell comprises at least 8 or more copies of the expression cassette. In another preferred embodiment, the microbial host cell comprises at least 9 or more copies of the expression cassette. In another preferred embodiment, the microbial host cell comprises at least 10 or more copies of the expression cassette. In another preferred embodiment, the microbial host cell comprises at least 1 1 or more copies of the expression cassette. In another preferred embodiment, the microbial host cell comprises at least 12 or more copies of the expression cassette.
  • the microbial host cell comprises at least 13 or more copies of the expression cassette. In another preferred embodiment, the microbial host cell comprises at least 14 or more copies of the expression cassette. In another preferred embodiment, the microbial host cell comprises at least 15 or more copies of the expression cassette. In another preferred embodiment, the microbial host cell comprises at least 16 or more copies of the expression cassette. In another preferred embodiment, the microbial host cell comprises at least 17 or more copies of the expression cassette. In another preferred embodiment, the microbial host cell comprises at least 18 or more copies of the expression cassette. In another preferred embodiment, the microbial host cell comprises at least 19 or more copies of the expression cassette. In another preferred embodiment, the microbial host cell comprises at least 20 or more copies of the expression cassette.
  • the microbial host cell comprises at least 30 or more copies of the expression cassette. In another preferred embodiment, the microbial host cell comprises at least 40 or more copies of the expression cassette. In another preferred embodiment, the microbial host cell comprises at least 50 or more copies of the expression cassette. In another preferred embodiment, the microbial host cell comprises at least 60 or more copies of the expression cassette. In another preferred embodiment, the microbial host cell comprises at least 70 or more copies of the expression cassette.
  • the expression cassette integrated into the genome of the cell in one or more copies comprises a secretion signal sequence which comprises a signal peptide-encoding sequence.
  • a secretion signal sequence which comprises a signal peptide-encoding sequence.
  • Suitable secretion signal sequences including where two, or three, or four, or five, or 10 or more copies of the expression cassette are integrated, are discussed herein above in the section entitled “Secretion signal sequences”.
  • the method may comprise the steps of providing a microbial host cell; and integrating into the genome of the microbial host cell one or more copies of an expression cassette, where the expression cassette comprises a promoter capable of promoting expression of a gene of interest, and the gene of interest, wherein the gene of interest is fused to a secretion signal sequence which comprises a signal peptide encoding sequence, and a terminator.
  • the expression cassette may be introduced into the microbial host cell according to any suitable method known to the skilled person.
  • the nucleic acid constructs may be introduced by transformation, for example chemical transformation, heat-shock based transformation, electroporation, biolistic transformation, or particle-based transformation.
  • the transformation is chemical-based transformation, for example comprises the use of lithium, calcium phosphate, cationic polymers, liposomes (lipofection) or dendrimers.
  • the transformation is nonchemical-based transformation, for example electroporation, sonoporation, optical transformation, or protoplast fusion transformation.
  • the transformation is particle-based transformation, for example, comprising the use of a gene gun or using glass beads, magnetofection (or magnet-assisted transformation), impalefection (comprising the use of elongated nanostructures that are used to impale the cell to be transformation), or particle bombardment.
  • Other methods of transformation include nucleofection or viral-based transformation, also referred to as transduction.
  • the expression cassette may be provided for transformation in the form a double stranded DNA product derived from a PCR.
  • the expression cassette may be provided on an integrative plasmid.
  • the expression cassette may be present on a vector that is linearized prior to being transformed.
  • the expression cassette may be in the form of a double stranded DNA product derived from a PCR and where the double stranded DNA product further comprises a selectable marker.
  • the expression cassette may be comprised on an integrative plasmid where the integrative plasmid further comprises a selectable marker.
  • the expression cassette may be present on a vector that is linearized prior to being transformed and where the linearized vector further comprises a selectable marker.
  • the selectable marker may be flanked by a site specific recombination sites (such as FRT sites) and where the corresponding recombinase (such as the FLP flippase recombinase) is also included next to the selectable marker to allow for the selectable marker, together with the gene encoding the recombinase to be removed from the cell by inducing the flippase gene, for example by inducing an inducible promoter driving the flippase recombinase.
  • a site specific recombination sites such as FRT sites
  • the corresponding recombinase such as the FLP flippase recombinase
  • a “selectable marker” or “selection marker” or “selection cassette” is a gene introduced into a cell that confers a trait suitable for artificial selection i.e. the cell receiving the selectable marker is capable of growing on or in a growth media containing or lacking a substance preventing cells without the selectable marker from growing or killing the cells lacking the selectable marker.
  • Selectable markers are often antibiotic-resistance genes.
  • Examples include the bleoR gene encoding the phleomycin resistance protein conferring resistance against the antibiotic phleomycin or zeocin, the hygB gene encoding the Hygromycin B resistance protein conferring resistance against the antibiotic Hygromycin B, the bsr gene conferring resistance against the antibiotic blasticidin, or the nat gene conferring resistance against the antibiotic nourseothricin.
  • the protein of interest may be formulated, for example into an agrochemical or pharmaceutical composition.
  • Fermentation broth, culture media or cell culture media as used herein can mean the entirety of liquid or solid material of a fermentation or culture at any time during or after that fermentation or culture, including the liquid or solid material that results after optional steps taken to isolate the protein.
  • the fermentation broth or culture media as defined herein includes the surroundings of the protein after isolation of the protein, during storage and/or during use as an agrochemical or pharmaceutical composition. Fermentation broth is also referred to herein as a culture medium or cell culture medium.
  • Agrochemical means suitable for use in the agrochemical industry (including agriculture, horticulture, floriculture and home and garden uses), but also products intended for non-crop related uses such as public health/pest control operator uses to control undesirable insects and rodents, household uses, such as household fungicides and insecticides and agents, for protecting plants or parts of plants, crops, bulbs, tubers, fruits (e.g. from harmful organisms, diseases or pests); for controlling, preferably promoting or increasing, the growth of plants; and/or for promoting the yield of plants, crops or the parts of plants that are harvested (e.g. its fruits, flowers, seeds etc.).
  • an agrochemical composition as used herein includes compositions comprising at least one biological molecule as an active ingredient, substance or principle for controlling pests in plants or in other agro-related settings (such for example in soil).
  • biological molecules being used as active principles in the agrochemical compositions disclosed herein are proteins (including antibodies and fragments thereof, such as but not limited to heavy chain variable domain fragments of antibodies, including VHH’s), nucleic acid sequences, (poly-) saccharides, lipids, vitamins, hormones glycolipids, sterols, and glycerolipids.
  • the protein encoded by the gene of interest may therefore be a recombinant or heterologous protein, since it may not be encoded by the wild-type genome of the microbial host cell.
  • the nucleic acid sequences may be integrated into the genome of the microbial host cell. This may result in the constitutive expression of the protein of interest. In other embodiments, the nucleic acid sequence may be transiently expressed by the microbial host cell.
  • Biostatic (effect) or “biostatic use”, as used herein, includes any effect or use of an active substance (optionally comprised in a biostatic, biocidal, fungicidal or fungistatic composition as defined herein) for controlling, modulating or interfering with the harmful activity of a pest, such as a plant pest or a plant pathogen, including but not limited to inhibiting the growth or activity of the pest, altering the behaviour of the pest, and repelling the pest in or on plants, plant parts or in other agro-related settings, such as for example for household uses or in soil.
  • a pest such as a plant pest or a plant pathogen
  • Pesticidal, biocidal, or biostatic activity of an active ingredient, substance or principle or a composition or agent comprising a pesticidal, biocidal, or biostatic active ingredient, substance or principle can be expressed as the minimum inhibitory activity (MIC) of an agent (expressed in units of concentration such as e.g. mg/mL), without however being restricted thereto.
  • MIC minimum inhibitory activity
  • “Fungicidal activity”, as used herein, means to interfere with the harmful activity of a fungus, including but not limited to killing the fungus.
  • the current invention provides microbial host cells, use of said microbial host cells for the manufacturing of a protein encoded by a gene of interest, expression cassettes comprising a gene of interest, and methods for manufacturing a protein encoded by a gene of interest.
  • the protein encoded by the gene of interest is a bioactive protein.
  • plant derived AMPs with antimicrobial or antiviral activities such as peptides composed of at least two helical domains connected by a linker/turn such as plant-derived amphipathic helix or two helices engineered into a helix-tum-helix (HTH) format in which homologous or heterogeneous helices are connected by a peptide linker.
  • peptides composed of at least two helical domains connected by a linker/turn
  • HTH helix-tum-helix
  • the bioactive protein that can be produced in a microbial fermentation reaction and are suitable for being formulated in an agrochemical or pharmaceutical composition is an antibody or a functional fragment thereof, a carbohydrate-binding domain, a heavy chain antibody or a functional fragment thereof, a single domain antibody, a heavy chain variable domain of an antibody or a functional fragment thereof, a heavy chain variable domain of a heavy chain antibody or a functional fragment thereof, a variable domain of camelid heavy chain antibody (VHH) or a functional fragment thereof, a variable domain of a new antigen receptor, a variable domain of shark new antigen receptor (vNAR) or a functional fragment thereof, a minibody, a nanobody, a nanoantibody, an affibody, an alphabody, a designed ankyrin-repeat domain, an anticalins, a knottins or an engineered CH2 domain.
  • the VHH may be a VHH that binds a specific lipid fraction of the cell membrane of a fungal spore.
  • VHHs may exhibit fungicidal activity through retardation of growth and/or lysis and explosion of spores, thus preventing mycelium formation.
  • the VHH may therefore have fungicidal or fungistatic activity.
  • the VHH may be a VHH that is capable of binding to a lipid-containing fraction of the plasma membrane of a fungus (for example Botrytis cinerea or other fungus).
  • Said lipid-containing fraction may be obtainable by chromatography.
  • said lipid-containing fraction may be obtainable by a method comprising: fractionating hyphae of a fungus (for example Botrytis cinerea or other fungus) by total lipid extract thin-layer chromatography and selecting the fraction with a Retention Factor (Rf) higher than the ceramide fraction and lower than the non-polar phospholipids fraction.
  • Rf Retention Factor
  • the VHHs may (specifically) bind to a membrane of a fungus or a component of a membrane of a fugus. In some embodiments, the VHHs do not (specifically) bind to a cell wall or a component of a cell wall of a fungus. For example, in some embodiments, the VHHs do not (specifically) bind to a glucosylceramide of a fungus.
  • said lipid-containing fraction may be obtainable by a method comprising: fractionating hyphae and/or conidia of a fungus (for example Botrytis cinerea or other fungus) by total lipid extract thin-layer chromatography and selecting the fraction with a Retention Factor (Rf) higher than the ceramide fraction and lower than the non-polar phospholipids fraction.
  • a fungus for example Botrytis cinerea or other fungus
  • Rf Retention Factor
  • the fraction may be obtained using normal-phase flash chromatography.
  • the method may comprise: fractionating hyphae and/or conidia of a fungus (for example Botrytis cinerea or other fungus) by total lipid extract normal-phase flash chromatography, and selecting the fraction with a Retention Factor (Rf) higher than the ceramide fraction and lower than the non-polar phospholipids fraction.
  • Rf Retention Factor
  • the lipid-containing fraction may be obtainable by a method comprising: fractionating hyphae and/or conidia of a fungus (for example Botrytis cinerea or other fungus) by total lipid extract normal-phase flash chromatography comprising dissolving the TLE in dichloromethane (CH2CI2) and MeOH and using CH2CI2/MeOH (for example 85/15%, v/v) as the eluent, followed by filtration of the fractions through a filter.
  • a fungus for example Botrytis cinerea or other fungus
  • CH2CI2/MeOH for example 85/15%, v/v
  • the lipid-containing fraction may be obtainable by a method comprising: fractionating hyphae and/or conidia of a fungus (for example Botrytis cinerea or other fungus) by total lipid extract normal-phase flash chromatography comprising dissolving the TLE in dichloromethane (CH2CI2) and MeOH loading the TLE on to a phase flash cartridge (for example a flash cartridge with 15 pm particles), running the column with CH2CI2/MeOH (85/15%, v/v) as the eluent, and filtering the fractions through a filter (for example a 0.45 pm syringe filter with a nylon membrane) and drying the fractions.
  • a fungus for example Botrytis cinerea or other fungus
  • CH2CI2 dichloromethane
  • MeOH MeOH
  • the VHH may (specifically) bind to a lipid-containing chromatographic fraction of the plasma membrane of a fungus, optionally wherein the lipid-containing chromatographic fraction is prepared into liposomes prior to testing the binding of the polypeptide thereto.
  • the chromatography may comprise the steps of: fractionating hyphae and/or conidia of a fungus (for example Botrytis cinerea or other fungus) by total lipid extract normal-phase flash chromatography, and selecting the fraction with a Retention Factor (Rf) higher than the ceramide fraction and lower than the non-polar phospholipids fraction.
  • a fungus for example Botrytis cinerea or other fungus
  • Rf Retention Factor
  • the chromatography may comprise the steps of: fractionating hyphae and/or conidia of a fungus (for example Botrytis cinerea or other fungus)by total lipid extract normal-phase flash chromatography comprising dissolving the TLE in dichloromethane (CH2CI2) and MeOH loading the TLE on to a phase flash cartridge (for example a flash cartridge with 15 pm particles), running the column with CH2CI2/MeOH (85/15%, v/v) as the eluent, and filtering the fractions through a filter (for example a 0.45 pm syringe filter with a nylon membrane) and drying the fractions.
  • a filter for example a 0.45 pm syringe filter with a nylon membrane
  • the protein encoded by the gene of interest is VHH-1 , VHH-2 or VHH-3.
  • the protein encoded by the gene of interest is a VHH comprising or consisting of a sequence selected from the group consisting of SEQ ID NOs: 1 , 2, 6, 10, 14 and 15.
  • the protein encoded by the gene of interest is a VHH comprising: a CDR1 comprising or consisting of a sequence selected from the group consisting of SEQ ID NOs
  • a CDR2 comprising or consisting of a sequence selected from the group consisting of SEQ ID NOs:
  • CDR3 comprising or consisting of a sequence selected from the group consisting of SEQ ID NOs:
  • the protein encoded by the gene of interest is a VHH comprising: a CDR1 comprising or consisting of the sequence of SEQ ID NO: 3, a CDR2 comprising or consisting of the sequence of SEQ ID NO: 4 and a CDR3 comprising or consisting of the sequence of SEQ ID NO: 5; a CDR1 comprising or consisting of the sequence of SEQ ID NO: 7, a CDR2 comprising or consisting of the sequence of SEQ ID NO: 8 and a CDR3 comprising or consisting of the sequence of SEQ ID NO: 9 or a CDR1 comprising or consisting of the sequence of SEQ ID NO: 1 1 , a CDR2 comprising or consisting of the sequence of SEQ ID NO: 12 and a CDR3 comprising or consisting of the sequence of SEQ ID NO: 13.
  • the protein encoded by the gene of interest is a VHH comprising SEQ ID NO:
  • “Heavy chain variable domain of an antibody or a functional fragment thereof” means (i) the variable domain of the heavy chain of a heavy chain antibody, which is naturally devoid of light chains, including but not limited to the variable domain of the heavy chain of heavy chain antibodies of camelids or sharks or (ii) the variable domain of the heavy chain of a conventional four-chain antibody (also indicated hereafter as VH), including but not limited to a camelized (as further defined herein) variable domain of the heavy chain of a conventional four-chain antibody (also indicated hereafter as camelized VH).
  • a method for numbering the amino acid residues of heavy chain variable domains is the method described by Chothia et al. (Nature 342, 877-883 (1989)), the so-called “AbM definition” and the so-called “contact definition”. Herein, this is the numbering system adopted.
  • the protein encoded by the gene of interest may be a heavy chain single variable domain.
  • the term “heavy chain single variable domain” as used herein in its broadest sense is not limited to a specific biological source or to a specific method of preparation.
  • a heavy chain single variable domain can be obtained (1 ) by isolating the VHH domain of a naturally occurring heavy chain antibody; (2) by isolating the VH domain of a naturally occurring four-chain antibody (3) by expression of a nucleotide sequence encoding a naturally occurring VHH domain; (4) by expression of a nucleotide sequence encoding a naturally occurring VH domain (5) by “camelization” (as described below) of a naturally occurring VH domain from any animal species, in particular a species of mammal, such as from a human being, or by expression of a nucleic acid encoding such a camelized VH domain; (6) by “camelisation” of a “domain antibody” or “Dab” as described by Ward et al (supra), or by expression of a nucleic acid encoding such a camelized VH domain (7) using synthetic or semi-synthetic techniques for preparing proteins, polypeptides or other amino acid sequences; (8) by “camel
  • Example 1 cloning of gene of interest into Komaqataella phaff ii and expression screening
  • the obtained plasmids were then used as a template for a standard PCR reaction using high fidelity polymerase such as used here Q5® High- Fidelity DNA Polymerase (New England Biolabs) followed by a PCR purification using for example the GeneJET PCR Purification Kit (Thermo Fisher Scientific) in order to produce double stranded DNA expression constructs for transformation into electrocompetent Komagataella phaffii cells. Electrocompetent cells were prepared according to the protocol as set out by Joan Lin-Cereghino et al. (2005) Condensed protocol for competent cell preparation and transformation of the methylotrophic yeast Pichia pastoris. Biotechniques: 38, 1:44-48.
  • Transformation of expression constructs to electrocompetent cells was done in a 2 mm electroporation cuvette using an electroporator (MicroPulser Electroporator, Biorad, cat n° 1652100) according to the manufacturer’s instructions for Komagataella phaffii. Note that due to the high frequency of random integration events of expression constructs in Komagataella phaffii, multiple copies of the expression cassette can be integrated randomly into the genome (Jan-Philipp Schwarzhans et al. (2016). Non-canonical integration events in Pichia pastoris encountered during standard transformation analysed with genome sequencing. Scientific Reports. 6: 38952).
  • Signal peptides 3 (SEQ ID NO: 118) and 4 (SEQ ID NO: 119) are artificial constructs designed by the inventors to find alternative or improved signal peptides and were shown to provide improved expression over a-MF as secretion signal (Table 1).
  • Table 1 VHH expression levels of strains transformed with expression constructs varying in signal peptide sequences relative to constructs comprising the a-MF signal peptide, in 96-deep well plates.
  • VHH concentration was performed by protein A affinity high performance liquid chromatography (PA-HPLC) or reverse phase high performance liquid chromatography (RP-HPLC).
  • PA-HPLC protein A affinity high performance liquid chromatography
  • RP-HPLC reverse phase high performance liquid chromatography
  • Example 4 Influence of signal peptides and VHH copy numbers on protein production.
  • the gene of interest is fused to a secretion signal sequence which comprises a signal peptide encoding sequence, and iii. a terminator, and b. wherein the one or more copies of the expression cassette are integrated into the genome of the microbial host cell.
  • the microbial host cell of claim 1 wherein the microbial host cell comprises two or more copies of the expression cassette.
  • the microbial host cell of statement 1 wherein the microbial host cell comprises at least 3 or more copies, at least 4 or more copies, at least 5 or more copies, at least 6 or more copies, at least or more 7 copies, at least 8 or more copies, at least 9 or more copies, at least 10 or more copies, at least 1 1 or more copies, at least 12 or more copies, at least 13 or more copies, at least 14 or more copies, at least 15 or more copies, at least 16 or more copies, at least 17 or more copies, at least 18 or more copies, at least 19 or more copies, at least 20 or more copies, at least 30 or more copies, at least 40 or more copies, at least 50 or more copies, at least 60 or more copies or at least 70 or more copies of the expression cassette.
  • the signal peptide-encoding sequence is selected from any of a. the nucleotide sequence provided in SEQ ID NO: 108, b. a nucleotide sequence with at least 90% identity to SEQ ID NO: 108, c. a nucleotide sequence encoding a signal peptide having the amino acid sequence provided in SEQ ID NO: 125, or d. a nucleotide sequence encoding a signal peptide having an amino acid sequence with at least 90% identity to SEQ ID NO: 125.
  • nucleotide sequence provided in SEQ ID NO: 110 b. a nucleotide sequence with at least 90% identity to SEQ ID NO: 110, c. a nucleotide sequence encoding a signal peptide having the amino acid sequence provided in SEQ ID NO: 127, or d. a nucleotide sequence encoding a signal peptide having an amino acid sequence with at least 90% identity to SEQ ID NO: 127.
  • nucleotide sequence with at least 90% identity to SEQ ID NO: 111 a nucleotide sequence with at least 90% identity to SEQ ID NO: 111
  • c. a nucleotide sequence encoding a signal peptide having the amino acid sequence provided in SEQ ID NO: 128, or d. a nucleotide sequence encoding a signal peptide having an amino acid sequence with at least 90% identity to SEQ ID NO: 128.
  • nucleotide sequence encoding a signal peptide having an amino acid sequence with at least 90% identity to SEQ ID NO: 130 The microbial host cell of any of statements 1 to 4, wherein the signal peptide-encoding sequence is selected from any of a. the nucleotide sequence provided in SEQ ID NO: 134, b. a nucleotide sequence with at least 90% identity to SEQ ID NO: 134, c. a nucleotide sequence encoding a signal peptide having the amino acid sequence provided in SEQ ID NO: 135, or d. a nucleotide sequence encoding a signal peptide having an amino acid sequence with at least 90% identity to SEQ ID NO: 135.
  • a nucleic acid comprising a signal peptide-encoding sequence, and wherein the signal peptide-encoding sequence is a. the nucleotide sequence of SEQ ID NO: 101 , b. a nucleotide sequence with at least 90% identity to SEQ ID NO: 101 , c. a nucleotide sequence encoding a signal peptide having the amino acid sequence of SEQ ID NO: 1 18, or d. a nucleotide sequence encoding a signal peptide having an amino acid sequence with at least 90% identity to SEQ ID NO: 118.
  • a nucleic acid comprising a signal peptide-encoding sequence, and wherein the signal peptide-encoding sequence is a. the nucleotide sequence of SEQ ID NO: 102, b. a nucleotide sequence with at least 90% identity to SEQ ID NO: 102, c. a nucleotide sequence encoding a signal peptide having the amino acid sequence of SEQ ID NO: 119, or d. a nucleotide sequence encoding a signal peptide having an amino acid sequence with at least 90% identity to SEQ ID NO: 119.
  • a nucleic acid comprising a signal peptide-encoding sequence, and wherein the signal peptide-encoding sequence is a. the nucleotide sequence of SEQ ID NO: 106, b. a nucleotide sequence with at least 90% identity to SEQ ID NO: 106, c. a nucleotide sequence encoding a signal peptide having the amino acid sequence of SEQ ID NO: 123, or d. a nucleotide sequence encoding a signal peptide having an amino acid sequence with at least 90% identity to SEQ ID NO: 123.
  • nucleic acid of any of the statements 32 to 35 where the nucleic acid further comprises a pro-sequence is provided.
  • An expression cassette comprising the nucleic acid of any of statements 32 to 38 and a promoter operably linked to the nucleic acid.
  • a method for producing a protein comprising a. culturing the microbial host cell of any one of statements 1 to 30, or a microbial host cell comprising the expression cassette any one of statements 40 to 47 or the vector of statement 48, under conditions to express the gene of interest, wherein the gene of interest encodes the protein, b. optionally isolating the protein, c. optionally purifying the protein, d. optionally modifying the protein, and e. optionally formulating the protein. 50.
  • a peptide comprising the amino acid sequence provided in SEQ ID NO: 1 18, or an amino acid sequence with at least 90% identity to SEQ ID NO: 1 18.
  • a peptide comprising the amino acid sequence provided in SEQ ID NO: 123, or an amino acid sequence with at least 90% identity to SEQ ID NO: 123.
  • a precursor protein comprising a secretion signal fused to a protein of interest, where the secretion signal comprises a signal peptide having the amino acid sequence of any one of SEQ ID NOs: 1 16 to 130 or 135, or a signal peptide having an amino acid sequence with at least 90% identity to any one of SEQ ID NOs: 1 16 to 130 or 135.
  • a nucleic acid comprising a gene of interest and a secretion signal sequence fused to the 5’ end of the gene of interest, wherein i. the secretion signal sequence comprises a signal peptide encoding sequence and a pro-sequence, ii. the signal peptide encoding sequence encodes a signal peptide having the amino acid sequence provided in any one of SEQ ID NOs: 1 16 to 130 or 135, or an amino acid sequence which is at least 90% identical to an amino acid sequence provided in any one of SEQ ID NOs: 1 16 to 130 or 135,
  • the pro-sequence encodes a pro-peptide having the amino acid sequence provided in SEQ ID NO: 1 15, or an amino acid sequence which is at least 90% identical to the amino acid sequence provided in SEQ ID NO: 1 15, and iv. the pro-sequence is fused to the 3’ end of the signal peptide encoding sequence.
  • the nucleic acid of statement 93 wherein the signal peptide encoding sequence encodes a signal peptide having the amino acid sequence provided in any one of SEQ ID NOs: 1 16, 121 , 125 or 135, or an amino acid sequence which is at least 90% identical to an amino acid sequence provided in any one of SEQ ID NOs: 1 16, 121 , 125 or 135.
  • An expression cassette comprising the nucleic acid of any one of statements 93 to 99.
  • the microbial host cell of statement 102 wherein 3 or more copies, 4 or more copies, 5 or more copies, 6 or more copies, or more 7 copies, 8 or more copies, 9 or more copies, 10 or more copies, 1 1 or more copies, 12 or more copies, 13 or more copies, 14 or more copies, 15 or more copies, 16 or more copies, 17 or more copies, 18 or more copies, 19 or more copies, 20 or more copies, 30 or more copies, 40 or more copies, 50 or more copies, 60 or more copies or 70 or more copies of the expression cassette of statement 100 are integrated into the genome of the microbial host cell.

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Abstract

La présente invention concerne une cellule hôte microbienne pour exprimer un gène d'intérêt, par exemple un VHH, le gène d'intérêt étant fusionné avec une séquence de signal de sécrétion comprenant une séquence de codage de peptide signal. Le gène d'intérêt fusionné avec la séquence de signaux de sécrétion est en outre compris dans une cassette d'expression dont un ou plusieurs éléments sont intégrés dans la cellule hôte microbienne. La présente invention propose en outre des acides nucléiques codant pour des séquences de signaux de sécrétion et des peptides codés par lesdits acides nucléiques. La présente invention propose en outre des cassettes d'expression comprenant lesdits acides nucléiques et un promoteur lié de manière fonctionnelle à celles-ci et des vecteurs comprenant lesdits acides nucléiques ou lesdites cassettes d'expression. Enfin, la présente invention concerne une cellule hôte microbienne comprenant lesdits acides nucléiques, des cassettes d'expression ou des vecteurs, un procédé de production d'une protéine et une protéine comprenant le peptide codé par lesdits acides nucléiques.
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