WO2013007820A1 - Screening method - Google Patents
Screening method Download PDFInfo
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- WO2013007820A1 WO2013007820A1 PCT/EP2012/063802 EP2012063802W WO2013007820A1 WO 2013007820 A1 WO2013007820 A1 WO 2013007820A1 EP 2012063802 W EP2012063802 W EP 2012063802W WO 2013007820 A1 WO2013007820 A1 WO 2013007820A1
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- amino acid
- acid sequence
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- signal peptide
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
- C12N15/625—DNA sequences coding for fusion proteins containing a sequence coding for a signal sequence
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- 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)
- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
- C12N9/2411—Amylases
- C12N9/2414—Alpha-amylase (3.2.1.1.)
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/036—Fusion polypeptide containing a localisation/targetting motif targeting to the medium outside of the cell, e.g. type III secretion
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/50—Fusion polypeptide containing protease site
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01133—Glucan 1,4-alpha-maltohydrolase (3.2.1.133), i.e. maltogenic alpha-amylase
Definitions
- the present invention relates to a screening method for screening protein constructs for optimal protein secretion.
- it relates to a method for efficient screening for optimal combinations between a signal peptide and a protein of interest.
- Secretion can be improved by modifying the protein's amino acid sequence, but this may negatively affect protein activity.
- proteins may be fused to a foreign signal sequence.
- the foreign signal sequence is typically from a protein which is well secreted.
- a disadvantage of current methods used to recombine signal sequences in front of proteins to be secreted is that often additional amino acids are introduced at the N-terminus of the protein to be secreted (Brockmeier et al. J. Mol. Biol. (2006) 362, p. 393-402) This is undesirable as that could have unpredictable effects on the characteristics of the secreted protein.
- Another disadvantage is that there seems to be an optimal combination between a protein to be secreted and a signal peptide, which cannot be predicted in advance and differs from protein to protein (Brockmeier et al. (2006).
- Fig. 1 Schematic representation of an embodiment in which a first polynucleotide comprises a nucleotide sequence A which encodes for an amino acid sequence of interest.
- the amino acid sequence of interest comprises a unique restriction site in the nucleotides encoding the first few amino acids of the N-terminus of the amino acid sequence of interest;
- a second polynucleotide comprises a nucleotide sequence B encoding a signal peptide having at its carboxyterminus the first few amino acids of the N-terminus of the amino acid sequence of interest.
- Combination of A and B results in a nucleotide fusion product C which allows for secretion of an unmodified amino acid sequence of interest (D).
- White nucleotides encoding a signal peptide
- grey nucleotides encoding (part of) an amino acid sequence of interest
- black amino acid sequence of interest.
- Fig. 2 Schematic representation of an embodiment in which a first polynucleotide comprises a nucleotide sequence A which encodes the part of an amino acid sequence of interest which includes the unique restriction site and the nucleotides encoding the amino acid sequence of interest downstream of this site; a second polynucleotide comprises a nucleotide sequence B encoding a signal peptide having at its carboxyterminus the part of the amino acid sequence of interest which is encoded by the nucleotides forming the unique restriction site and the nucleotides encoding the amino acid sequence of interest upstream of the unique restriction site.
- Combination of A and B results in a nucleotide fusion product C which allows for secretion of an unmodified amino acid sequence of interest (D).
- White nucleotides encoding a signal peptide
- grey nucleotides encoding (part of) an amino acid sequence of interest
- black amino acid sequence of interest.
- Fig. 3 Plasmid pBAH1 MAM2 containing the modified amyM sequence with the silent mutation which resulted in a unique Nhe ⁇ restriction site (underlined). Grey: nucleotides encoding an amino acid sequence of interest; White: nucleotides encoding a signal peptide.
- Fig. 4 Schematic representation of a combination of PCR fragment (A) and digested plasmid pBAH1 MAM2 (B).
- the PCR fragment encodes a signal peptide having at its carboxyterminus the first few amino acids of amyM.
- Combination results in a nucleotide fusion product (C) which after cloning and expression yields unmodified maltogenic alpha-amylase.
- Fig. 5 Maltogenic alpha-amylase activity in shake flasks at 44 hours in SMM medium.
- Fig. 6 Examples of amino acid sequences encoded by nucleotide fusion products according to the invention. The N-terminus of the protein to be secreted is not changed. 6A: SacC fusion product; 6B: ybfO fusion product; 6C: yjcM fusion product; 6D: yurl fusion product, 6F: ynfF fusion product. For comparison, wild type (wt) maltogenic alpha-amylase with its native signal peptide is also shown (6E); Signal peptides sequences are underlined.
- 6A SacC fusion product
- 6B ybfO fusion product
- 6C yjcM fusion product
- 6D yurl fusion product
- 6F ynfF fusion product.
- wild type (wt) maltogenic alpha-amylase with its native signal peptide is also shown (6E); Signal peptides sequences are underlined.
- the present invention relates to a method for constructing a nucleotide fusion product which encodes a fusion between a signal peptide and an amino acid sequence of interest, wherein the method comprises combining
- a first polynucleotide which comprises a nucleotide sequence encoding the amino acid sequence of interest, wherein the nucleotide sequence encoding the amino acid sequence of interest contains a unique restriction site in the nucleotides encoding the first few amino acids of the N-terminus of the amino acid sequence of interest;
- a second polynucleotide which comprises a nucleotide sequence encoding the signal peptide having at its carboxy terminus the first few amino acids of the N-terminus of the amino acid sequence of interest, wherein the nucleotide sequence comprises a unique restriction site in the nucleotides encoding the first few amino acids of the N- terminus of the amino acid sequence of interest; whereby a nucleotide fusion product is obtained which encodes a fusion between the signal peptide and the amino acid sequence of interest, which fusion allows for the secretion of the amino acid sequence of interest without modifications to the amino acid sequence of the amino acid sequence of interest.
- Fig.1 One embodiment of this method is schematically represented in Fig.1 .
- nucleotide fusion product obtained encodes a clean fusion between signal sequence and amino acid sequence of interest, i.e. the fusion is made without introducing amino acid modifications to the amino acid sequence of the amino acid sequence of interest.
- the fusion is made without introducing amino acid modifications to the amino acid sequence of the amino acid sequence of interest.
- no amino acids are deleted from, inserted in, added to or changed in the N- terminus of the amino acid of interest, neither in the fusion with the signal peptide, nor in the secreted amino acid sequence. In this way, the introduction of undesired and/or unexpected properties to the amino acid sequence produced is prevented.
- the method may conveniently be used to combine one amino acid sequence of interest with a great number of signal peptides, e.g. a whole library, which are to be tested in order to select from this collection of signal peptide-amino acid sequence of interest fusions, those fusions which are the most interesting or optimal fusions for a certain purpose or application. Therefore, the present invention also encompasses a method for constructing and optionally screening a library for selecting optimal combinations between an amino acid sequence of interest and a variety of signal peptides, which method comprises
- the method according to the present invention allows for fast screening and identification of improved polypeptide-signal peptide combinations.
- the method of the invention may be used to obtain secretion of an intracellular amino acid sequence of interest. Therefore, in another aspect, the present invention relates to a method for obtaining or improving the secretion of an amino acid sequence of interest, wherein the method comprises:
- a first polynucleotide which comprises a nucleotide sequence encoding the amino acid sequence of interest, wherein the nucleotide sequence encoding the amino acid sequence of interest contains a unique restriction site in the nucleotides encoding the first few amino acids of the N-terminus of the amino acid sequence of interest; and (ii) a second polynucleotide which comprises a nucleotide sequence encoding the signal peptide having at its carboxy terminus the first few amino acids of the N-terminus of the amino acid sequence of interest, wherein the nucleotide sequence comprises a unique restriction site in the nucleotides encoding the first few amino acids of the N- terminus of the amino acid sequence of interest; whereby a nucleotide fusion product is obtained which encodes a fusion between the signal peptide and the amino acid sequence of interest, which fusion allows for the secretion of the amino acid sequence of interest without modifications to its amino acid sequence;
- the invention may also be used to improve the secretion of an amino acid sequence of interest which is already secreted after production, for example an extracellular protein, if higher levels of secretion are desired.
- Extracellular proteins are produced with a signal peptide (unprocessed protein).
- the signal peptide is typically spliced off upon secretion, yielding a processed protein.
- the secretion of an extracellular protein may be improved by replacing the signal peptide which is present with another signal peptide. Therefore, in another aspect the invention relates to a method for improving the secretion of an amino acid sequence of interest comprising:
- a first polynucleotide sequence which comprises a nucleotide sequence encoding the amino acid sequence of interest and a signal peptide to be replaced, wherein the sequence part encoding the amino acid sequence of interest contains a unique restriction site in the nucleotides encoding the first few amino acids of the N-terminus of the amino acid sequence of interest;
- a second polynucleotide sequence which comprises a nucleotide sequence encoding a signal peptide to be tested having at its carboxy terminus the first few amino acids of the N-terminus of the amino acid sequence of interest, wherein the nucleotide sequence comprises a unique restriction site in the nucleotides encoding the first few amino acids of the N-terminus of the amino acid sequence of interest; whereby a nucleotide fusion product is obtained which encode a fusion between the signal peptide to be tested and the amino acid sequence of interest, which fusion allows for the secretion of the amino acid sequence of interest without modifications to its amino acid sequence;
- first and second polynucleotides of the methods of the invention are not necessary to use overlapping parts in the first and second polynucleotides of the methods of the invention. All or some of the nucleotides encoding the amino acid sequence of interest upstream of the unique restriction site may be omitted from the first polynucleotide and all or some of the nucleotides encoding the amino acid sequence of interest downstream of the unique restriction site may be omitted from the second polynucleotide.
- the present invention also relates to a method for constructing a nucleotide fusion product which comprises a fusion between a nucleotide sequence encoding a signal peptide and a nucleotide sequence encoding an amino acid sequence of interest, wherein the nucleotide sequence encoding the amino acid sequence of interest comprises a unique restriction site, the method comprising combining
- a first polynucleotide which comprises a nucleotide sequence which encodes a part of the amino acid sequence of interest, which nucleotide sequence comprises the unique restriction site and the nucleotides encoding the amino acid sequence of interest downstream of the unique restriction site;
- a second polynucleotide which comprises a nucleotide sequence which encodes the signal peptide having at its carboxyterminus part of the amino acid sequence of interest, and which nucleotide sequence comprises the nucleotides forming the unique restriction site and the nucleotides encoding the amino acid sequence of interest upstream of the unique restriction site,
- nucleotide fusion product which encodes a fusion product which allows for the secretion of the amino acid sequence of interest without modifications to the amino acid sequence of the amino acid sequence of interest.
- Fig. 2 One embodiment of this method is schematically represented in Fig. 2.
- 'upstream' and 'downstream' refer to the region at or towards the 5' end and at or towards the 3'end, respectively.
- the unique restriction sites are used for combining the nucleotide sequence encoding all or part the amino acid sequence of interest with the nucleotide sequence encoding the signal peptide.
- the use of a restriction enzyme will yield compatible ends, which may be sticky or blunt and which may be combined using any suitable method, for example by ligation.
- the term 'combining' refers to any method which can be used to combine two or more nucleotide sequences, including ligation, fusion PCR, PCR, cloning with the use of a ligase, in vivo recombination and in vitro recombination.
- the signal peptide to be replaced may be a wild type signal peptide, i.e. a signal peptide which the protein is usually associated with when produced in the cell before secretion from the cell. However, it may also be a signal peptide which the amino acid sequence of interest is not usually associated with, but to which the amino acid sequence of interest has become associated by manipulation, e.g. by genetic engineering. In any case, the signal peptide to be replaced is a signal peptide to which an alternative is sought in order to optimise the secretion of the amino acid sequence of interest. The secretion of the amino acid sequence of interest is typically increased if the method of the invention is used.
- it increases with at least 1 %, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100%. More preferably, with at least 1 10%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190% or at least 200%.
- the secretion is increased while the amino acid sequence of the amino acid of interest remains unchanged, i.e. is identical to the sequence which the amino acid of interest had when it was processed from the signal peptide to be replaced.
- the terms wild type signal peptide, wild type signal sequence, native signal sequence, native signal peptide, natural signal peptide are used interchangeably herein. Increased secretion may be measured using any available technique in the art.
- increased secretion is measured by analysing enzyme activity or productivity in the external medium, and optionally comparing it to the enzyme activity before replacing, modifying or introducing a signal peptide. If secretion is increased, typically enzyme activity or productivity in the external medium is increased.
- enzyme activity or productivity in the external medium increases with at least 1 %, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100%. More preferably, with at least 1 10%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190% or at least 200%.
- the signal peptide and the signal peptide to be tested may be any signal peptide. It may be from the same species as the signal peptide to be replaced, or from a different species. It may be from the same species as the amino acid sequence of interest, or from a different species. It may be obtained from various organisms, such as for example from plants, animals or microorganisms. In one embodiment, it is obtained from a prokaryotic cell, e.g. a Gram-negative or Gram-positive bacterium.
- Suitable bacteria include Escherichia, Anabaena, Caulobactert, Gluconobacter, Rhodobacter, Pseudomonas, Paracoccus, Bacillus, Brevibacterium, Corynebacterium, Rhizobium (Sinorhizobium), Flavobacterium, Klebsiella, Enterobacter, Lactobacillus, Lactococcus, Methylobacterium, Staphylococcus or Streptomyces.
- the bacterial cell is selected from the group consisting of B. subtilis, B. amyloliquefaciens, B. licheniformis, B. puntis, B. megaterium, B. halodurans, B.
- pumilus pumilus, G. oxydans, Caulobactert crescentus CB 15, Methylobacterium extorquens, Rhodobacter sphaeroides, Pseudomonas zeaxanthinifaciens, Paracoccus denitrificans, E. coli, C. glutamicum, Staphylococcus carnosus, Streptomyces lividans, Sinorhizobium melioti and Rhizobium radiobacter.
- the signal peptide or the signal peptide to be tested is obtained from a eukaryotic cell.
- the eukaryotic cell is a mammalian, insect, plant, fungal, or algal cell.
- Preferred mammalian cells include e.g. Chinese hamster ovary (CHO) cells, COS cells, 293 cells, Per.C6® cells, and hybridomas.
- Preferred insect cells include e.g. Sf9 and Sf21 cells and derivatives thereof. More preferably, the eukaryotic cell is a fungal cell, i.e.
- yeast cell such as Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia strain. More preferably from Kluyveromyces lactis, S. cerevisiae, Hansenula polymorpha, Yarrowia lipolytica and Pichia pastoris, or a filamentous fungal cell. Most preferably, the eukaryotic cell is a filamentous fungal cell.
- Filamentous fungi include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, U K).
- the filamentous fungi are 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.
- Filamentous fungal strains include, but are not limited to, strains of Acremonium, Agaricus, Aspergillus, Aureobasidium, Chrysosporium, Coprinus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Panerochaete, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, and Trichoderma.
- Preferred filamentous fungal cells belong to a species of an Aspergillus, Chrysosporium, Penicillium, Talaromyces, Fusarium or Trichoderma genus, and most preferably a species of Aspergillus niger, Aspergillus awamori, Aspergillus foetidus, Aspergillus sojae, Aspergillus fumigatus, Talaromyces emersonii, Aspergillus oryzae, Chrysosporium lucknowense, Myceliophtora thermophila, Fusarium oxysporum, Trichoderma reesei or Penicillium chrysogenum. Alternatively, it may be produced synthetically.
- the signal peptide or the signal peptide to be tested may or may not improve the secretion of the amino acid sequence of interest. Typically, only those alternative signal peptides which improve the secretion of the amino acid sequence of interest will be selected. In certain embodiments, the signal peptide is selected from the signal peptides listed in Table 1 .
- the term 'signal peptide' refers to a protein which direct the secretion of a protein to a specific location in the cell e.g. chloroplast, endoplasmic reticulum or outside the cell. In industrial practice, extracellular secretion is of particular interest, because it enables the convenient recovery of an amino acid sequences of interest.
- Signal peptides consist of short stretches of amino acids which, after protein delivery to the correct subcellular compartment, are frequently removed by specialized signal peptidases. Before removal they are typically situated at the N-terminus of the protein which they direct. Signal peptides typically have a start codon at their 5' end. The nucleotide sequence of the start codon is ususally AUG (or ATG), but there are also alternative start codons, such as for example GUG (or GTG) and UUG (or TTG), which are used in prokaryotes.
- the signal peptide is typically rich in hydrophobic amino acids which helps to transport the entire polypeptide through intracellular or cellular membranes.
- the amino acid sequence of interest may be a peptide of interest, a polypeptide of interest or a protein of interest. In the present context, these terms are used interchangeably.
- the amino acid sequence of interest may be a sequence which is isolated from an organism, such as an original, native or wild type sequence, but it may also be a synthetic sequence. It may be an existing sequence , but it may also be part of an existing sequence or a modified version of an existing sequence, such as a variant or a codon optimised version of an original, native or wild type sequence.
- the amino acid sequence may be any amino acid sequence of interest. It may be an extracellular protein or an intracellular protein. It may be native or heterologous to the host cell.
- It may be an antibody or parts thereof, an antigen, a clotting factor, an enzyme, a hormone or a hormone variant, a receptor or parts thereof, a regulatory protein, a structural protein, a reporter, or a transport protein, protein involved in secretion process, protein involved in folding process, chaperone, peptide amino acid transporter, glycosylation factor, transcription factor, synthetic peptide or oligopeptide.
- enzymes include proteases, ceramidases, epoxide hydrolases, aminopeptidases, acylases, aldolases, hydroxylases, lipolytic enzymes.
- the polypeptide may be an enzyme secreted extracel lu larly.
- Such enzymes may belong to the grou ps of oxidored uctase, transferase, hydrolase, lyase, isomerase, ligase, catalase, cellulase, chitinase, cutinase, deoxyribonuclease, dextranase, esterase.
- the enzyme may be a carbohydrase, such as a cell wall degrading enzyme, e.g. cellulases such as endoglucanases, ⁇ -glucanases, cellobiohydrolases or ⁇ -glucosidases, hemicellulases or pectinolytic enzymes such as xylanases, xylosidases, mannanases, galactanases, galactosidases, pectin methyl esterases, pectin lyases, pectate lyases, endopolygalacturonases, exopolygalacturonases rhamnogalacturonases, arabanases, arabinofuranosidases, arabinoxylan hydrolases, galacturonases, lyases, or amylolytic enzymes; hydrolase, isomerase, or ligase, phosphatases such as phytases, esterase
- the enzyme may be a phytase.
- the enzyme may be an aminopeptidase, asparaginase, amylase, a maltogenic alpha-amylase, carbohydrase, carboxypeptidase, endo-protease, metallo-protease, serine-protease catalase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, beta- glucosidase, haloperoxidase, protein deaminase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phospholipase, polyphenoloxida
- the amino acid sequence of interest is an enzyme, in particular a maltogenic alpha-amylase (EC 3.2.1 .133). More in particular, a maltogenic alpha-amylase with an amino acid sequence according to amino acids 34-719 of SEQ ID No. 1 1 . Also included are naturally occurring allelic, codon optimised and engineered variations of the above-mentioned polypeptides and parts of the above-mentioned polypeptides. Codon optimisation may be performed using methods known in the art, preferably as described in WO2006/077258 or
- a library constructed using a method according to the invention is also part of the invention.
- the amino acid sequence of interest is fused to various different signal sequences to be tested, in order to be able to quickly screen for the optimal combination(s) of amino acid sequence and signal peptide.
- the library may contain an unlimited amount of combinations.
- the library contains more than 100, more than 150, more than 200 different combinations of clean fusions which can be screened within 6 months.
- the libary contains nucleotide fusion constructs which encode fusions of a signal peptides fused to the mature or active form of the amino acid sequence of interest, so that no further processing or engineering of the protein is required for measuring its activity after secretion of the protein.
- the libary contains nucleotide fusion constructs which encode fusions of the signal peptides and an inactive form of the amino acid sequence of interest which can readily be converted to its active form by the action of another compound, typically by the action of an enzyme.
- the library would contain prochymosin-signal peptide fusions, wherein the prochymosin after secretion would be converted to chymosin.
- the unique restriction may be naturally present or may have been introduced by mutation of the nucleotide sequences encoding the amino acid sequence of interest. If it is introduced by mutation , the mutation must be a silent mutation in the sequence which encodes the amino acid sequence of interest, i.e. any convenient site may be introduced in the nucleotide sequence as long as it does not change the amino acid sequence of the amino acid sequence of interest. The skilled person will be able to determine suitable restriction sites for a given amino acid sequence. In one embodiment, a silent mutation is introduced in a sequence encoding a maltogenic alpha-amylase, resulting in a unique Nhe ⁇ restriction site. The amino acid sequence is not changed by the introduction of the Nhe ⁇ restriction site.
- the second polynucleotide sequence comprises a unique restriction site which is different from the unique restriction site in the first polynucleotide, unique to the second polynucleotide and which is compatible with the unique restriction site in the first polynucleotide sequence.
- two restriction sites are 'compatible' with each other when the ends or overhangs of the two restriction sites can be combined with each other. Lists of compatible restriction sites are available in the prior art, see for example New England Biolabs Inc. 2007- 2008 Catalog & Technical Reference, Ipswich, MA USA.
- restriction sites which are compatible with Nhe ⁇ restriction sites include sites for ⁇ and Bfal.
- the unique restriction site is located in the nucleotides encoding the first few amino acids of the N-terminus of the amino acid sequence of interest. Preferably, it is located within the nucleotides encoding the first 25 amino acids of the N-terminus sequence of the amino acid sequence of interest. More preferably, it is located within the nucleotides encoding the first 20, 15 or 10 amino acids of the N-terminus of the amino acid sequence of interest. Most preferably, it is located within the nucleotides encoding the first 9, 8, 7, 6, 5, 4, 3, 2 amino acids or the first amino acid of the N- terminus of the amino acid sequence of interest.
- the second polynucleotide sequence comprises a nucleotide sequence encoding a signal peptide having at its carboxyterminus the first few amino acids of the N-terminus of the amino acid sequence of interest.
- the nucleotide sequence encodes a signal peptide having at its carboxyterminus the first 25 amino acids of the N-terminus of the amino acid sequence of interest, or less. More preferably, the nucleotide sequence encodes a signal peptide having at its carboxyterminus the first 20, 15 or 10 amino acids of the N-terminus of the amino acid sequence of interest, or less.
- the nucleotide sequence encodes a signal peptide having at its carboxyterminus the first 9, 8, 7, 6, 5, 4, 3, 2 amino acids of the N-terminus of the amino acid sequence of interest, or less, or the first amino acid of N-terminus of the amino acid sequence of interest.
- a suitable host cell is transformed with the nucleotide fusion products according to the invention in order to bring the amino acid sequence of interest to expression.
- the host may be any suitable host. It may be a host which is known to produce the signal peptide to be tested under certain conditions, but it does not have to be. It may be a host which is known to produce the amino acid sequence of interest under certain conditions, but it does not have to be.
- the host cell is a prokaryotic host cell, e.g. a Gram-negative or Gram-positive bacterium.
- Suitable bacteria include Escherichia, Anabaena, Caulobactert, Gluconobacter, Rhodobacter, Pseudomonas, Paracoccus, Bacillus, Brevibacterium, Corynebacterium, Rhizobium (Sinorhizobium), Flavobacterium, Klebsiella, Enterobacter, Lactobacillus, Lactococcus, Methylobacterium, Staphylococcus or Streptomyces.
- the bacterial cell is selected from the group consisting of B. subtilis, B. amyloliquefaciens, B. licheniformis, B. puntis, B. megaterium, B. halodurans, B.
- pumilus pumilus, G. oxydans, Caulobactert crescentus CB 15, Methylobacterium extorquens, Rhodobacter sphaeroides, Pseudomonas zeaxanthinifaciens, Paracoccus denitrificans, E. coli, C. glutamicum, Staphylococcus carnosus, Streptomyces lividans, Sinorhizobium melioti and Rhizobium radiobacter.
- the host cell is a eukaryotic host cell.
- the eukaryotic cell is a mammalian, insect, plant, fungal, or algal cell.
- Preferred mammalian cells include e.g. Chinese hamster ovary (CHO) cells, COS cells, 293 cells, Per.C6® cells, and hybridomas.
- Preferred insect cells include e.g. Sf9 and Sf21 cells and derivatives thereof.
- the eukaryotic cell is a fungal cell, i.e. a yeast cell, such as Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia strain.
- the eukaryotic cell is a filamentous fungal cell.
- Filamentous fungi include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of
- filamentous fungi are 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. Filamentous fungal strains include, but are not limited to, strains of Acremonium,
- Preferred filamentous fungal cells belong to a species of an Aspergillus, Chrysosporium, Penicillium, Rasamsonia, Talaromyces, Fusarium or Trichoderma genus, and most preferably a species of Aspergillus niger, Aspergillus awamori, Aspergillus foetidus, Aspergillus sojae, Aspergillus fumigatus, Rasamsonia emersonii, Talaromyces emersonii, Aspergillus oryzae, Chrysosporium lucknowense, Myceliophthora thermophila, Fusarium oxysporum, Trichoderma reesei or Penicillium chrysogenum.
- a more preferred host cell is Aspergillus niger.
- the host cell according to the invention is an Aspergillus niger host cell
- the host cell preferably is CBS 513.88, CBS124.903 or a derivative thereof.
- the suitable host cell is cultured to bring the amino acid sequence of interest to expression.
- culturing of the host cell may be at any suitable scale, such as in a chemostat or in Eschweiler, for high throughput screening library, culturing is preferably at microtiter scale, e.g. in microtiter plates.
- the library constructed in this way is also part of the present invention.
- the library contains an amino acid sequence of interest fused to a signal peptide to be tested, whereby the N-terminus of the amino acid sequence of interest remains the same once the signal peptide is removed by a signal peptidase.
- the optimal protein-signal peptide combination or combinations can be easily selected from the library.
- the method of the invention can be applied to improve protein expression in a host, although the expression construct and host has already several other optimizations, such as for example a strong promoter, an improved translation initiation sequence, an improved translational termination sequence, an optimized codon and codon pair usage and / or an improved host for protein expression.
- the present invention relates to a polynucleotide which comprises a nucleotide sequence encoding a signal peptide having at its carboxy terminus the first few amino acids of the N-terminus of an amino acid sequence of interest.
- the nucleotide sequence includes a unique restriction site which allows for fusion with a nucleotide sequence which encodes the whole or part of the amino acid sequence of interest in such a way that a nucleotide fusion product is obtained which encodes a fusion between the signal peptide and the amino acid sequence of interest which allows for the secretion of the amino acid sequence of interest without modifications to its amino acid sequence.
- This polynucleotide sequence may be part of a vector and form a recombinant nucleotide which may be introduced into a host cell.
- the vector, recombinant nucleotide sequence and host cell comprising the recombinant nucleotide are also part of the present invention.
- the vector may be any vector, including a plasmid and an expression vector.
- Suitable recombinant host cells may be selected by the skilled person. Suitable examples are mentioned above and include Aspergillus and Bacillus, Escherichia, Kluyveromyces, Penicillium, Pseudomonas, Saccharomyces and Streptomyces species.
- 'polynucleotide sequence' and 'nucleotide sequence' may refer to forms of DNA and RNA of any length and any origin, i.e. it includes both natural, isolated and manipulated and synthetically produced nucleotides.
- the present invention relates to the use of a library according to the invention in a screening method for increasing the secretion of amino acids of interest.
- the present invention relates to a nucleotide fusion product obtainable by the method according to the present invention.
- This nucleotide fusion product comprises a polynucleotide encoding a signal sequence which polynucleotide is at its 3' site and in reading frame linked to a polynucleotide encoding an amino acid sequence of interest, whereby the nucleotide fusion product encodes a fusion between the signal peptide and the amino acid sequence of interest, which fusion allows for the secretion of the amino acid sequence of interest without modifications to its amino acid sequence.
- Polynucleotides encoding fusions between signal peptides and their corresponding natural polypeptides are typically excluded from the present invention.
- polynucleotides encoding a fusion between maltogenic alpha-amylase and its natural signal peptide are excluded from this invention.
- the polynucleotides of the invention encode fusions between a signal peptide and an amino acid sequence of interest wherein the signal peptide and amino acid of interest are obtained from different strains, subspecies, species, genera, families, orders, classes or kingdoms.
- nucleotide fusion products may be obtained by a method according to the invention.
- Suitable examples of such nucleotide fusion products include nucleotide fusion products which encode amino acid sequences as given in Fig. 6A-6D or which include the signal peptides given in Table 1 .
- Such a nucleotide fusion product encodes a clean fusion between the signal peptide and the amino acid sequence of interest, i.e. fusions of an amino acid sequence of interest and a signal peptide, without modifications to its amino acid sequence, in particular, without modifications to the N- terminus of the amino acid of interest.
- nucleotide fusion products encoding optimal amino acid of interest-signal peptide combinations are also encompassed by the present invention. Screening among these nucleotide fusion products will reveal nucleotide fusions products with optimised secretion of the amino acid sequence of interest in comparison to the situation before the fusion.
- the method of the invention is used to find a signal peptide to optimize secretion of maltogenic alpha-amylase from Bacillus stearothermophilus later typed as Geobacillus stearothermophilus
- a library was constructed, containing about 168 signal sequences to be tested fused to the sequence encoding the processed/coding sequence of maltogenic alpha-amylase. Screening revealed that the yurl fliL, vpr, glpQ, phy, lytC, ywsB, ybbD, ybxl, yolA, ylqB, ybbC, pel, yckD, ywaD, ywmD, yweA, yraJ, dacF, yfjS, yybN, yrpD, yvcE, wprA, yxaL, ykwD, yncM2, sacB, phrC, SacC, yoqM, ykoJ, lip, yfkN, yurl, ybfO, yfkD, yoaJ
- the present invention relates to an amino acid sequence produced by a method according to the invention in industrial applications, in particular in the cosmetic industry, detergent industry, feed or food industry, paper and pulp industry, more in particularly in baking, brewing, dairy processing, sport drinks, grain processing, fruit juice and the nutraceutical industry.
- the present invention includes the following aspects.
- a method for constructing a nucleotide fusion product which comprises a fusion between a nucleotide sequence encoding a signal peptide and a nucleotide sequence encoding an amino acid sequence of interest, wherein the nucleotide sequence encoding the amino acid sequence of interest comprises a unique restriction site, the method comprising combining
- a first polynucleotide which comprises a nucleotide sequence which encodes a part of the amino acid sequence of interest, which nucleotide sequence comprises the unique restriction site and the nucleotides encoding the amino acid sequence of interest downstream of the unique restriction site;
- a second polynucleotide which comprises a nucleotide sequence which encodes the signal peptide having at its carboxyterminus part of the amino acid sequence of interest, and which nucleotide sequence comprises the nucleotides forming the unique restriction site and the nucleotides encoding the amino acid sequence of interest upstream of the unique restriction site,
- nucleotide fusion product which encodes a fusion product which allows for the secretion of the amino acid sequence of interest without modifications to the amino acid sequence of the amino acid sequence of interest.
- Method according aspect 1 wherein the first polynucleotide also comprises all or some of the nucleotides encoding the amino acid sequence of interest which are upstream of the unique restriction site.
- Method according aspect 1 or 2 wherein the second polynucleotide also comprises all or some of the nucleotides encoding the amino acid sequence of interest which are downstream of the unique restriction site.
- Method according to aspects 1 -3 wherein the unique restriction site is a silent mutation in the nucleotide sequence which encodes the amino acid sequence of interest.
- the second polynucleotide comprises a nucleotide sequence encoding a signal peptide having at its carboxyterminus not more than the first 25 nucleotides encoding the N-terminal amino acids of the amino acid sequence of interest.
- amino acid sequence of interest is an antibody or parts thereof, an antigen, a clotting factor, an enzyme, a hormone or a hormone variant, a receptor or parts thereof, a regulatory protein, a structural protein, a reporter, or a transport protein, protein involved in secretion process, protein involved in folding process, chaperone, peptide amino acid transporter, glycosylation factor, transcription factor, synthetic peptide or oligopeptide.
- amino acid sequence of interest is an enzyme, in particular an aminopeptidase, amylase, maltogenic alpha-amylase, carbohydrase, carboxypeptidase, endo-protease, metallo-protease, serine-protease, catalase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, dextranase, esterase, alpha-galactosidase, beta-galactosidase, cell wall degrading enzyme, glucoamylase, alpha-glucosidase, beta-glucosidase, haloperoxidase, hydrolase, isomerase, protein deaminase, invertase, laccase, ligase, lipolytic enzyme, lyase, mannosidase, mutanase, oxidase
- an enzyme in particular an aminopeptida
- Method according to aspects 1 -8 wherein the secretion, productivity or activity of the amino acid sequence of interest in an external medium is measured and found to be improved by at least 50% compared to the secretion, productivity or activity before combination.
- a method for constructing and optionally screening a library for selecting optimal combinations between an amino acid sequence of interest and a variety of signal peptides comprising:
- a library for selecting optimal combinations between an amino acid sequence of interest and a variety of signal peptides obtainable by the method according to aspects 10. 12.
- a polynucleotide sequence which comprises a nucleotide sequence encoding a signal peptide having at its carboxy terminus the first few amino acids of the N-terminus of an amino acid sequence of interest including a unique restriction site, which unique restriction site allows for fusion to the amino acid of interest and allows for expression of the amino acid of interest without modifications to its amino acid sequence.
- a recombinant host cell comprising a polynucleotide sequence according to aspect 12 or 13.
- a method according to aspect 10 a library according to aspect 1 1 or a recombinant host cell according to aspect 14, wherein the host cell is an Aspergillus, Bacillus,
- Bacillus subtilis strain BS154 (CBS 363.94) ⁇ AaprE, AnprE, AamyE, spo) is described in Quax and Broekhuizen 1994 Appl Microbiol Biotechnol. 41 : 425-431 .
- Bacillus subtilus 168 (trpC2) is described in Anagnostopoulos C and Spizizen J. (1961 ) J Bacteriol. 1961 81 (5)741 -746.
- NCIMB1 1873 Bacillus stearothermophilus C599 (NCIMB1 1873) is described in WO91/04669.
- plasmid pNAPBH27 is contained in Bacillus subtilis strain BS154
- Plasmid pNAPHB27 contains a Bacillus expression vector with a bleomycine and neomcyin resistance marker and is described in Quax and Broekhuizen 1994 Appl Microbiol Biotechnol. 41 : 425-431 .
- the maltogenic alpha-amylase productivity in the culture broth of B. subtilis was quantified by measuring activity using a Megazyme CERALPHA alpha-amylase assay kit (Megazyme International Ireland Ltd., Co. Wicklow, Ireland) according to the manufacturer's instruction.
- the amyM gene including its native terminator sequence was amplified by polymerase chain reaction (PCR) with the following primers.
- a forward primer to amplify the amyM gene and introduce a Nde ⁇ site A forward primer to amplify the amyM gene and introduce a Nde ⁇ site:
- PCR was performed on a thermocycler with Phusion High-Fidelity DNA polymerase (Finnzymes OY, Aspoo, Finland) according to the instructions of the manufacturer.
- the resulting PCR fragment was digested with the restriction enzymes Ndel and Hind ⁇ and ligated with T4 DNA ligase into Ndel and Hind ⁇ digested pNAPHB27 plasmid.
- the ligation mixture was transformed into B. subtilis strain BS154.
- a clone was selected and the amyM expression plasmid was named pBHA1 MAM1 .
- the sequence of the plasmid was confirmed by DNA sequencing.
- a silent mutation was introduced on pBHA1 MAM1 at the wobble position of the 37 th amino acid of the unprocessed maltogenic alpha-amylase protein to create a unique Nhe ⁇ restriction site.
- the wild type signal peptide of maltogenic alpha-amylase used in this example and as described in SEQ ID NO 1 1 contains amino acids 1 -33 of SEQ ID NO: 1 1.
- the silent mutation in this example was therefore introduced within the nucleotides of the fourth amino acid of the amino acid of interest, in this example the fourth amino acid of the maltogenic alpha-amylase with the amino acid sequence according to amino acids 34-719 of SEQ ID No. 1 1 .
- a 483 bp synthetic DNA fragment containing Nhe ⁇ , Nde ⁇ and Acc ⁇ restriction sites was synthesized (SEQ ID NO.3):
- the ligation mixture was transformed into B. subtilis strain BS154.
- a clone was selected and the modified amyM expression plasmid was named pBHA1 MAM2 (Fig. 3). The seq uence of the plasm id was confirmed by DNA sequencing and Nhe ⁇ digestion.
- the pBHA1 MAM2 plasmid was digested with Nhe ⁇ and Nde ⁇ to remove the native signal sequence.
- the linear vector fragment without the signal sequence was gel- purified.
- the plasmid fragment was excised from the gel and the DNA was recovered using the NucleoSpin Extract II kit (Macherey-Nagel, GmbH & Co. Dijren, Germany) according to the manufacturer's instructions. This fragment was used to introduce the signal sequence encoding DNA fragments.
- Genomic DNA from B. subtilis 168 was isolated by FastD NA S P I N Kit (M P Biomedicals, Solon, OH, USA) and used as DNA template in these PCR reactions.
- the Nde ⁇ restriction site in the forward primers allowed for cloning at the ATG start codon.
- the reverse primers contained the first four DNA codons of the N-terminal amino acids of the predicted mature maltogenic alpha-amylase protein and the Nhe ⁇ restriction site at the wobble position of the 37 th amino acid of the unprocessed maltogenic alpha-amylase protein.
- the generated DNA fragments were purified by Nucleospin 96 extract II (Macherey-Nagel, GmbH & Co. Dijren, Germany) according to instructions of the manufacturer.
- the purified PCR fragments were digested with Nde ⁇ and Nhe ⁇ .
- the restriction enzymes were heat inactivated by incubating 20 minutes at 65°C.
- the digested vector and signal sequence fragments were ligated using the Quick ligation Kit (New England Biolabs, Inc., USA) according the protocol of the manufacturer. See Figure 4 for a schematic representation of this ligation.
- the ligation products were transformed to competent B. subtilis BS154 cells.
- the correct clones were identified by PCR with the signal sequence specific forward primers used to amplify the signal sequences and the reverse amyM primer annealing 184 bp down stream of the amyM ATG start codon.
- B. subtilis BS154 strains containing the pBHA1 MAM2 plasmid (Fig. 3) with the various signal sequences fused to the mature maltogenic alpha-amylase protein were grown in 0.5 ml U96 microWell Plates (Nunc A/S, Roskilde, Denmark).
- a 200 ⁇ pre-culture was made in 2xTY medium composed of 1 .6% (w/w) Bacto tryptone, 1 % (w/w) yeast extract and 0.5% (w/w) NaCI. Kanamycin was added to the culture medium in a final concentration of 25 ⁇ 9/ ⁇ and the MicroWell plates were covered by a Breathseal (Greiner bio-one, Frickenhausen, Germany).
- SMM pre-medium contains 1 .25% (w/w) yeast extract, 00.5% (w/w) CaCI 2 , 0.075% (w/w) MgCI 2 .6H 2 0, 15 pg/l MnS0 4 .4H 2 0 , 1 0 pg/l CoCI 2 .6H 2 0 , 0.05% (w/w) citric acid, 0.025% (w/w) antifoam 86/013 (Basildon Chemicals, Abingdon, UK).
- SMM medium 20 ml of 5% (w/v) maltose and 20 ml of a 200 mM Na-phosphate buffer stock solution (pH 6.8), both prepared and sterilized separately, were added to 60 ml SMM pre-medium. These cultures were incubated in a Microton incubator shaker (Infors AG, Bottmingen, Switzerland) for 48 hours at 37 ° C, 550 rpm and 80% humidity. The supernatants were harvested by centrifugation for 30 min at 4000g and analysed for enzyme productivity.
- a Microton incubator shaker Infors AG, Bottmingen, Switzerland
- B. subtilis strains containing the pBHA1 MAM2 plasmid with the phy, sacC, ybfO, yddT, yjcM, yojL, ynfF, yomL or yurl signal sequences were also tested in a shake flask fermentation experiments. These shake flaks contained 20 ml 2xTY medium composed of 1 .6% (w/w) Bacto tryptone, 1 % (w/w) yeast extract and 0.5% (w/w) NaCI. Kanamycin was added to the culture medium to reach a final concentration of 25 ⁇ g/ ⁇ l.
- the cultures were shaken vigorously at 37 ° C and 250 rpm for 16 hours and 0.2 ml culture medium was used to inoculate 20 ml SMM medium. These cultures were incubated for 44 hours at 37 ° C and 250 rpm. The supernatants were harvested and analysed for enzyme productivity. The maltogenic alpha-amylase productivity of the signal sequence fusion constructs was compared to the strain containing the native amyM signal sequence. Samples were assayed for maltogenic alpha-amylase activity. As shown in Fig. 5, a clear positive effect for the use of different signal sequences from Bacillus subtilis is also observed in shake flaks fermentation.
- the polypeptide containing growth medium was used to determine the amino acid composition of the N-terminus of the maltogenic alpha-amylase polypeptide through LC-MS/MS.
- the predicted sequence for some illustrative fusion products is shown in Fig 6A-D and SEQ ID NO. 12-15 and 82.
- the corresponding fusion with the wild type signal sequence is shown as comparison in Fig. 6E and SEQ ID NO.1 1 (the wild type signal peptide is represented by amino acids 1 -33, the maltogenic alpha-amylase is represented by amino acids 34-719 in SEQ ID NO. 1 1 ).
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Abstract
The present invention relates to a method for constructing a nucleotide fusion construct encoding a fusion between a signal peptide and an amino acid sequence of interest, which allows for secretion of the amino acid sequence of interest without modifications to its amino acid sequence.
Description
SCREENING METHOD
Field of the invention
The present invention relates to a screening method for screening protein constructs for optimal protein secretion. In particular, it relates to a method for efficient screening for optimal combinations between a signal peptide and a protein of interest.
Background of the invention
In industrial enzyme production, secretion of enzymes of interest into the extracellular medium is of the utmost importance. Therefore, much research has been performed into the optimisation of protein secretion, i.e. to obtain secretion of an intracellular protein which is normally not secreted or to increase the secretion of a protein which is normally secreted.
Secretion can be improved by modifying the protein's amino acid sequence, but this may negatively affect protein activity.
Alternatively, proteins may be fused to a foreign signal sequence. The foreign signal sequence is typically from a protein which is well secreted. A disadvantage of current methods used to recombine signal sequences in front of proteins to be secreted is that often additional amino acids are introduced at the N-terminus of the protein to be secreted (Brockmeier et al. J. Mol. Biol. (2006) 362, p. 393-402) This is undesirable as that could have unpredictable effects on the characteristics of the secreted protein. Another disadvantage is that there seems to be an optimal combination between a protein to be secreted and a signal peptide, which cannot be predicted in advance and differs from protein to protein (Brockmeier et al. (2006).
Short description of the figures
Fig. 1 Schematic representation of an embodiment in which a first polynucleotide comprises a nucleotide sequence A which encodes for an amino acid sequence of interest. The amino acid sequence of interest comprises a unique restriction site in the nucleotides encoding the first few amino acids of the N-terminus of the amino acid
sequence of interest; a second polynucleotide comprises a nucleotide sequence B encoding a signal peptide having at its carboxyterminus the first few amino acids of the N-terminus of the amino acid sequence of interest. Combination of A and B results in a nucleotide fusion product C which allows for secretion of an unmodified amino acid sequence of interest (D). White: nucleotides encoding a signal peptide; grey: nucleotides encoding (part of) an amino acid sequence of interest; black: amino acid sequence of interest.
Fig. 2 Schematic representation of an embodiment in which a first polynucleotide comprises a nucleotide sequence A which encodes the part of an amino acid sequence of interest which includes the unique restriction site and the nucleotides encoding the amino acid sequence of interest downstream of this site; a second polynucleotide comprises a nucleotide sequence B encoding a signal peptide having at its carboxyterminus the part of the amino acid sequence of interest which is encoded by the nucleotides forming the unique restriction site and the nucleotides encoding the amino acid sequence of interest upstream of the unique restriction site. Combination of A and B results in a nucleotide fusion product C which allows for secretion of an unmodified amino acid sequence of interest (D). White: nucleotides encoding a signal peptide; grey: nucleotides encoding (part of) an amino acid sequence of interest; black: amino acid sequence of interest.
Fig. 3 Plasmid pBAH1 MAM2 containing the modified amyM sequence with the silent mutation which resulted in a unique Nhe\ restriction site (underlined). Grey: nucleotides encoding an amino acid sequence of interest; White: nucleotides encoding a signal peptide.
Fig. 4 Schematic representation of a combination of PCR fragment (A) and digested plasmid pBAH1 MAM2 (B). The PCR fragment encodes a signal peptide having at its carboxyterminus the first few amino acids of amyM. Combination results in a nucleotide fusion product (C) which after cloning and expression yields unmodified maltogenic alpha-amylase.
Fig. 5 Maltogenic alpha-amylase activity in shake flasks at 44 hours in SMM medium.
Fig. 6 Examples of amino acid sequences encoded by nucleotide fusion products according to the invention. The N-terminus of the protein to be secreted is not changed. 6A: SacC fusion product; 6B: ybfO fusion product; 6C: yjcM fusion product; 6D: yurl fusion product, 6F: ynfF fusion product. For comparison, wild type (wt)
maltogenic alpha-amylase with its native signal peptide is also shown (6E); Signal peptides sequences are underlined.
Detailed description
The present invention relates to a method for constructing a nucleotide fusion product which encodes a fusion between a signal peptide and an amino acid sequence of interest, wherein the method comprises combining
(i) a first polynucleotide which comprises a nucleotide sequence encoding the amino acid sequence of interest, wherein the nucleotide sequence encoding the amino acid sequence of interest contains a unique restriction site in the nucleotides encoding the first few amino acids of the N-terminus of the amino acid sequence of interest; and
(ii) a second polynucleotide which comprises a nucleotide sequence encoding the signal peptide having at its carboxy terminus the first few amino acids of the N-terminus of the amino acid sequence of interest, wherein the nucleotide sequence comprises a unique restriction site in the nucleotides encoding the first few amino acids of the N- terminus of the amino acid sequence of interest; whereby a nucleotide fusion product is obtained which encodes a fusion between the signal peptide and the amino acid sequence of interest, which fusion allows for the secretion of the amino acid sequence of interest without modifications to the amino acid sequence of the amino acid sequence of interest. One embodiment of this method is schematically represented in Fig.1 .
The terms signal sequence and signal peptide are used interchangeably herein.
The great advantage of this method is that the nucleotide fusion product obtained encodes a clean fusion between signal sequence and amino acid sequence of interest, i.e. the fusion is made without introducing amino acid modifications to the amino acid sequence of the amino acid sequence of interest. In particular, there are no modifications to the N-terminus of the amino acid sequence of interest. This means that no amino acids are deleted from, inserted in, added to or changed in the N- terminus of the amino acid of interest, neither in the fusion with the signal peptide, nor
in the secreted amino acid sequence. In this way, the introduction of undesired and/or unexpected properties to the amino acid sequence produced is prevented.
The method may conveniently be used to combine one amino acid sequence of interest with a great number of signal peptides, e.g. a whole library, which are to be tested in order to select from this collection of signal peptide-amino acid sequence of interest fusions, those fusions which are the most interesting or optimal fusions for a certain purpose or application. Therefore, the present invention also encompasses a method for constructing and optionally screening a library for selecting optimal combinations between an amino acid sequence of interest and a variety of signal peptides, which method comprises
(a) transforming a collection of fusion products obtainable by a method according to the invention coding for diverse combinations of a signal peptide with the amino acid sequence of interest into a population of suitable host cells; and optionally
(b) culturing the population of transformed host cells to bring the amino acid sequence of interest to expression, and optionally
(c) screening the population of transformed host cells for the optimal combination of amino acid sequence of interest and signal peptide for a specific purpose or application.
The method according to the present invention allows for fast screening and identification of improved polypeptide-signal peptide combinations.
The method of the invention may be used to obtain secretion of an intracellular amino acid sequence of interest. Therefore, in another aspect, the present invention relates to a method for obtaining or improving the secretion of an amino acid sequence of interest, wherein the method comprises:
(a) combining
(i) a first polynucleotide which comprises a nucleotide sequence encoding the amino acid sequence of interest, wherein the nucleotide sequence encoding the amino acid sequence of interest contains a unique restriction site in the nucleotides encoding the first few amino acids of the N-terminus of the amino acid sequence of interest; and
(ii) a second polynucleotide which comprises a nucleotide sequence encoding the signal peptide having at its carboxy terminus the first few amino acids of the N-terminus of the amino acid sequence of interest, wherein the nucleotide sequence comprises a unique restriction site in the nucleotides encoding the first few amino acids of the N- terminus of the amino acid sequence of interest; whereby a nucleotide fusion product is obtained which encodes a fusion between the signal peptide and the amino acid sequence of interest, which fusion allows for the secretion of the amino acid sequence of interest without modifications to its amino acid sequence;
(b) transforming the resulting nucleotide fusion product into a suitable host cell; and optionally
(c) culturing the host cell to bring the polypeptide represented by the amino acid sequence of interest to expression.
However, it may also be used to improve the secretion of an amino acid sequence of interest which is already secreted after production, for example an extracellular protein, if higher levels of secretion are desired. Extracellular proteins are produced with a signal peptide (unprocessed protein). The signal peptide is typically spliced off upon secretion, yielding a processed protein. The secretion of an extracellular protein may be improved by replacing the signal peptide which is present with another signal peptide. Therefore, in another aspect the invention relates to a method for improving the secretion of an amino acid sequence of interest comprising:
(a) combining
(i) a first polynucleotide sequence which comprises a nucleotide sequence encoding the amino acid sequence of interest and a signal peptide to be replaced, wherein the sequence part encoding the amino acid sequence of interest contains a unique restriction site in the nucleotides encoding the first few amino acids of the N-terminus of the amino acid sequence of interest; and
(ii) a second polynucleotide sequence which comprises a nucleotide sequence encoding a signal peptide to be tested having at its carboxy terminus the first few amino acids of the N-terminus of the amino acid sequence of interest, wherein the nucleotide sequence comprises a unique restriction site in the nucleotides encoding the first few amino acids of the N-terminus of the amino acid sequence of interest;
whereby a nucleotide fusion product is obtained which encode a fusion between the signal peptide to be tested and the amino acid sequence of interest, which fusion allows for the secretion of the amino acid sequence of interest without modifications to its amino acid sequence;
(b) transforming the resulting nucleotide fusion product into a suitable host cell; and
(c) culturing the host cell to bring the amino acid sequence of interest to expression, and optionally
(d) screening for the optimal combination of amino acid sequence of interest and signal peptide.
The skilled person will understand that it is not necessary to use overlapping parts in the first and second polynucleotides of the methods of the invention. All or some of the nucleotides encoding the amino acid sequence of interest upstream of the unique restriction site may be omitted from the first polynucleotide and all or some of the nucleotides encoding the amino acid sequence of interest downstream of the unique restriction site may be omitted from the second polynucleotide. Therefore, the present invention also relates to a method for constructing a nucleotide fusion product which comprises a fusion between a nucleotide sequence encoding a signal peptide and a nucleotide sequence encoding an amino acid sequence of interest, wherein the nucleotide sequence encoding the amino acid sequence of interest comprises a unique restriction site, the method comprising combining
(a) a first polynucleotide which comprises a nucleotide sequence which encodes a part of the amino acid sequence of interest, which nucleotide sequence comprises the unique restriction site and the nucleotides encoding the amino acid sequence of interest downstream of the unique restriction site; and
(b) a second polynucleotide which comprises a nucleotide sequence which encodes the signal peptide having at its carboxyterminus part of the amino acid sequence of interest, and which nucleotide sequence comprises the nucleotides forming the unique restriction site and the nucleotides encoding the amino acid sequence of interest upstream of the unique restriction site,
whereby a nucleotide fusion product is obtained which encodes a fusion product which allows for the secretion of the amino acid sequence of interest without modifications to
the amino acid sequence of the amino acid sequence of interest. One embodiment of this method is schematically represented in Fig. 2.
The terms 'upstream' and 'downstream' refer to the region at or towards the 5' end and at or towards the 3'end, respectively.
The unique restriction sites are used for combining the nucleotide sequence encoding all or part the amino acid sequence of interest with the nucleotide sequence encoding the signal peptide. The use of a restriction enzyme will yield compatible ends, which may be sticky or blunt and which may be combined using any suitable method, for example by ligation. In the present context, the term 'combining' refers to any method which can be used to combine two or more nucleotide sequences, including ligation, fusion PCR, PCR, cloning with the use of a ligase, in vivo recombination and in vitro recombination.
The signal peptide to be replaced may be a wild type signal peptide, i.e. a signal peptide which the protein is usually associated with when produced in the cell before secretion from the cell. However, it may also be a signal peptide which the amino acid sequence of interest is not usually associated with, but to which the amino acid sequence of interest has become associated by manipulation, e.g. by genetic engineering. In any case, the signal peptide to be replaced is a signal peptide to which an alternative is sought in order to optimise the secretion of the amino acid sequence of interest. The secretion of the amino acid sequence of interest is typically increased if the method of the invention is used. Preferably, it increases with at least 1 %, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100%. More preferably, with at least 1 10%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190% or at least 200%. Even more preferably with at least 210%, at least 220%, at least 230%, at least 240%, at least 250%, at least 260%, at least 270%, at least 280%, at least 290% or at least 300%. The secretion is increased while the amino acid sequence of the amino acid of interest remains unchanged, i.e. is identical to the sequence which the amino acid of interest had when it was processed from the signal peptide to be replaced.
The terms wild type signal peptide, wild type signal sequence, native signal sequence, native signal peptide, natural signal peptide are used interchangeably herein. Increased secretion may be measured using any available technique in the art. In one embodiment, increased secretion is measured by analysing enzyme activity or productivity in the external medium, and optionally comparing it to the enzyme activity before replacing, modifying or introducing a signal peptide. If secretion is increased, typically enzyme activity or productivity in the external medium is increased. Preferably, enzyme activity or productivity in the external medium increases with at least 1 %, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100%. More preferably, with at least 1 10%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190% or at least 200%.
Even more preferably with at least 210%, at least 220%, at least 230%, at least 240%, at least 250%, at least 260%, at least 270%, at least 280%, at least 290% or at least 300%. The signal peptide and the signal peptide to be tested may be any signal peptide. It may be from the same species as the signal peptide to be replaced, or from a different species. It may be from the same species as the amino acid sequence of interest, or from a different species. It may be obtained from various organisms, such as for example from plants, animals or microorganisms. In one embodiment, it is obtained from a prokaryotic cell, e.g. a Gram-negative or Gram-positive bacterium. Suitable bacteria include Escherichia, Anabaena, Caulobactert, Gluconobacter, Rhodobacter, Pseudomonas, Paracoccus, Bacillus, Brevibacterium, Corynebacterium, Rhizobium (Sinorhizobium), Flavobacterium, Klebsiella, Enterobacter, Lactobacillus, Lactococcus, Methylobacterium, Staphylococcus or Streptomyces. Preferably, the bacterial cell is selected from the group consisting of B. subtilis, B. amyloliquefaciens, B. licheniformis, B. puntis, B. megaterium, B. halodurans, B. pumilus, G. oxydans, Caulobactert crescentus CB 15, Methylobacterium extorquens, Rhodobacter sphaeroides, Pseudomonas zeaxanthinifaciens, Paracoccus denitrificans, E. coli, C. glutamicum,
Staphylococcus carnosus, Streptomyces lividans, Sinorhizobium melioti and Rhizobium radiobacter.
According to another embodiment, the signal peptide or the signal peptide to be tested is obtained from a eukaryotic cell. Preferably, the eukaryotic cell is a mammalian, insect, plant, fungal, or algal cell. Preferred mammalian cells include e.g. Chinese hamster ovary (CHO) cells, COS cells, 293 cells, Per.C6® cells, and hybridomas. Preferred insect cells include e.g. Sf9 and Sf21 cells and derivatives thereof. More preferably, the eukaryotic cell is a fungal cell, i.e. a yeast cell, such as Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia strain. More preferably from Kluyveromyces lactis, S. cerevisiae, Hansenula polymorpha, Yarrowia lipolytica and Pichia pastoris, or a filamentous fungal cell. Most preferably, the eukaryotic cell is a filamentous fungal cell. Filamentous fungi include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, U K). The filamentous fungi are 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. Filamentous fungal strains include, but are not limited to, strains of Acremonium, Agaricus, Aspergillus, Aureobasidium, Chrysosporium, Coprinus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Panerochaete, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, and Trichoderma. Preferred filamentous fungal cells belong to a species of an Aspergillus, Chrysosporium, Penicillium, Talaromyces, Fusarium or Trichoderma genus, and most preferably a species of Aspergillus niger, Aspergillus awamori, Aspergillus foetidus, Aspergillus sojae, Aspergillus fumigatus, Talaromyces emersonii, Aspergillus oryzae, Chrysosporium lucknowense, Myceliophtora thermophila, Fusarium oxysporum, Trichoderma reesei or Penicillium chrysogenum. Alternatively, it may be produced synthetically. The signal peptide or the signal peptide to be tested may or may not improve the secretion of the amino acid sequence of interest. Typically, only those alternative signal peptides which improve the secretion of the amino acid sequence of interest will be selected. In certain embodiments, the signal peptide is selected from the signal peptides listed in Table 1 .
In the present context, the term 'signal peptide' refers to a protein which direct the secretion of a protein to a specific location in the cell e.g. chloroplast, endoplasmic reticulum or outside the cell. In industrial practice, extracellular secretion is of particular interest, because it enables the convenient recovery of an amino acid sequences of interest.
Signal peptides consist of short stretches of amino acids which, after protein delivery to the correct subcellular compartment, are frequently removed by specialized signal peptidases. Before removal they are typically situated at the N-terminus of the protein which they direct. Signal peptides typically have a start codon at their 5' end. The nucleotide sequence of the start codon is ususally AUG (or ATG), but there are also alternative start codons, such as for example GUG (or GTG) and UUG (or TTG), which are used in prokaryotes. The signal peptide is typically rich in hydrophobic amino acids which helps to transport the entire polypeptide through intracellular or cellular membranes.
The amino acid sequence of interest may be a peptide of interest, a polypeptide of interest or a protein of interest. In the present context, these terms are used interchangeably. The amino acid sequence of interest may be a sequence which is isolated from an organism, such as an original, native or wild type sequence, but it may also be a synthetic sequence. It may be an existing sequence , but it may also be part of an existing sequence or a modified version of an existing sequence, such as a variant or a codon optimised version of an original, native or wild type sequence. The amino acid sequence may be any amino acid sequence of interest. It may be an extracellular protein or an intracellular protein. It may be native or heterologous to the host cell. It may be an antibody or parts thereof, an antigen, a clotting factor, an enzyme, a hormone or a hormone variant, a receptor or parts thereof, a regulatory protein, a structural protein, a reporter, or a transport protein, protein involved in secretion process, protein involved in folding process, chaperone, peptide amino acid transporter, glycosylation factor, transcription factor, synthetic peptide or oligopeptide.
Preferably it is an enzyme. Suitable examples of enzymes include proteases, ceramidases, epoxide hydrolases, aminopeptidases, acylases, aldolases, hydroxylases, lipolytic enzymes. The polypeptide may be an enzyme secreted extracel lu larly. Such enzymes may belong to the grou ps of oxidored uctase,
transferase, hydrolase, lyase, isomerase, ligase, catalase, cellulase, chitinase, cutinase, deoxyribonuclease, dextranase, esterase. The enzyme may be a carbohydrase, such as a cell wall degrading enzyme, e.g. cellulases such as endoglucanases, β-glucanases, cellobiohydrolases or β-glucosidases, hemicellulases or pectinolytic enzymes such as xylanases, xylosidases, mannanases, galactanases, galactosidases, pectin methyl esterases, pectin lyases, pectate lyases, endopolygalacturonases, exopolygalacturonases rhamnogalacturonases, arabanases, arabinofuranosidases, arabinoxylan hydrolases, galacturonases, lyases, or amylolytic enzymes; hydrolase, isomerase, or ligase, phosphatases such as phytases, esterases such as lipases, proteolytic enzymes, oxidoreductases such as oxidases, transferases, or isomerases. The enzyme may be a phytase. The enzyme may be an aminopeptidase, asparaginase, amylase, a maltogenic alpha-amylase, carbohydrase, carboxypeptidase, endo-protease, metallo-protease, serine-protease catalase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, beta- glucosidase, haloperoxidase, protein deaminase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phospholipase, polyphenoloxidase, ribonuclease, transglutaminase, or glucose oxidase, hexose oxidase, monooxygenase. In a preferred embodiment, the amino acid sequence of interest is an enzyme, in particular a maltogenic alpha-amylase (EC 3.2.1 .133). More in particular, a maltogenic alpha-amylase with an amino acid sequence according to amino acids 34-719 of SEQ ID No. 1 1 . Also included are naturally occurring allelic, codon optimised and engineered variations of the above-mentioned polypeptides and parts of the above-mentioned polypeptides. Codon optimisation may be performed using methods known in the art, preferably as described in WO2006/077258 or
WO2008/000632.
A library constructed using a method according to the invention is also part of the invention. In the library, the amino acid sequence of interest is fused to various different signal sequences to be tested, in order to be able to quickly screen for the optimal combination(s) of amino acid sequence and signal peptide. The skilled person will understand that the library may contain an unlimited amount of combinations. Preferably, the library contains more than 100, more than 150, more than 200 different combinations of clean fusions which can be screened within 6 months.
In one embodiment, the libary contains nucleotide fusion constructs which encode fusions of a signal peptides fused to the mature or active form of the amino acid sequence of interest, so that no further processing or engineering of the protein is required for measuring its activity after secretion of the protein. In another embodiment, the libary contains nucleotide fusion constructs which encode fusions of the signal peptides and an inactive form of the amino acid sequence of interest which can readily be converted to its active form by the action of another compound, typically by the action of an enzyme. For example, the library would contain prochymosin-signal peptide fusions, wherein the prochymosin after secretion would be converted to chymosin.
In all methods according to the invention, the unique restriction may be naturally present or may have been introduced by mutation of the nucleotide sequences encoding the amino acid sequence of interest. If it is introduced by mutation , the mutation must be a silent mutation in the sequence which encodes the amino acid sequence of interest, i.e. any convenient site may be introduced in the nucleotide sequence as long as it does not change the amino acid sequence of the amino acid sequence of interest. The skilled person will be able to determine suitable restriction sites for a given amino acid sequence. In one embodiment, a silent mutation is introduced in a sequence encoding a maltogenic alpha-amylase, resulting in a unique Nhe\ restriction site. The amino acid sequence is not changed by the introduction of the Nhe\ restriction site. In one embodiment, the second polynucleotide sequence comprises a unique restriction site which is different from the unique restriction site in the first polynucleotide, unique to the second polynucleotide and which is compatible with the unique restriction site in the first polynucleotide sequence. In the present context, two restriction sites are 'compatible' with each other when the ends or overhangs of the two restriction sites can be combined with each other. Lists of compatible restriction sites are available in the prior art, see for example New England Biolabs Inc. 2007- 2008 Catalog & Technical Reference, Ipswich, MA USA. For example, restriction sites which are compatible with Nhe\ restriction sites include sites for Ανή\ and Bfal.
The unique restriction site is located in the nucleotides encoding the first few amino acids of the N-terminus of the amino acid sequence of interest. Preferably, it is located within the nucleotides encoding the first 25 amino acids of the N-terminus sequence of the amino acid sequence of interest. More preferably, it is located within the nucleotides encoding the first 20, 15 or 10 amino acids of the N-terminus of the amino acid sequence of interest. Most preferably, it is located within the nucleotides encoding the first 9, 8, 7, 6, 5, 4, 3, 2 amino acids or the first amino acid of the N- terminus of the amino acid sequence of interest. The second polynucleotide sequence comprises a nucleotide sequence encoding a signal peptide having at its carboxyterminus the first few amino acids of the N-terminus of the amino acid sequence of interest. Preferably, the nucleotide sequence encodes a signal peptide having at its carboxyterminus the first 25 amino acids of the N-terminus of the amino acid sequence of interest, or less. More preferably, the nucleotide sequence encodes a signal peptide having at its carboxyterminus the first 20, 15 or 10 amino acids of the N-terminus of the amino acid sequence of interest, or less. Most preferably, the nucleotide sequence encodes a signal peptide having at its carboxyterminus the first 9, 8, 7, 6, 5, 4, 3, 2 amino acids of the N-terminus of the amino acid sequence of interest, or less, or the first amino acid of N-terminus of the amino acid sequence of interest.
A suitable host cell is transformed with the nucleotide fusion products according to the invention in order to bring the amino acid sequence of interest to expression. The host may be any suitable host. It may be a host which is known to produce the signal peptide to be tested under certain conditions, but it does not have to be. It may be a host which is known to produce the amino acid sequence of interest under certain conditions, but it does not have to be. In one embodiment, the host cell is a prokaryotic host cell, e.g. a Gram-negative or Gram-positive bacterium. Suitable bacteria include Escherichia, Anabaena, Caulobactert, Gluconobacter, Rhodobacter, Pseudomonas, Paracoccus, Bacillus, Brevibacterium, Corynebacterium, Rhizobium (Sinorhizobium), Flavobacterium, Klebsiella, Enterobacter, Lactobacillus, Lactococcus, Methylobacterium, Staphylococcus or Streptomyces. Preferably, the bacterial cell is selected from the group consisting of B. subtilis, B. amyloliquefaciens, B. licheniformis, B. puntis, B. megaterium, B. halodurans, B. pumilus, G. oxydans, Caulobactert
crescentus CB 15, Methylobacterium extorquens, Rhodobacter sphaeroides, Pseudomonas zeaxanthinifaciens, Paracoccus denitrificans, E. coli, C. glutamicum, Staphylococcus carnosus, Streptomyces lividans, Sinorhizobium melioti and Rhizobium radiobacter.
According to another embodiment, the host cell is a eukaryotic host cell. Preferably, the eukaryotic cell is a mammalian, insect, plant, fungal, or algal cell. Preferred mammalian cells include e.g. Chinese hamster ovary (CHO) cells, COS cells, 293 cells, Per.C6® cells, and hybridomas. Preferred insect cells include e.g. Sf9 and Sf21 cells and derivatives thereof. More preferably, the eukaryotic cell is a fungal cell, i.e. a yeast cell, such as Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia strain. More preferably from Kluyveromyces lactis, S. cerevisiae, Hansenula polymorpha, Yarrowia lipolytica and Pichia pastoris, or a filamentous fungal cell. Most preferably, the eukaryotic cell is a filamentous fungal cell. Filamentous fungi include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of
The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK). The filamentous fungi are 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. Filamentous fungal strains include, but are not limited to, strains of Acremonium,
Agaricus, Aspergillus, Aureobasidium, Chrysosporium, Coprinus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Panerochaete, Pleurotus, Schizophyllum, Rasamsonia Talaromyces, Thermoascus, Thielavia, Tolypocladium, and Trichoderma. Preferred filamentous fungal cells belong to a species of an Aspergillus, Chrysosporium, Penicillium, Rasamsonia, Talaromyces, Fusarium or Trichoderma genus, and most preferably a species of Aspergillus niger, Aspergillus awamori, Aspergillus foetidus, Aspergillus sojae, Aspergillus fumigatus, Rasamsonia emersonii, Talaromyces emersonii, Aspergillus oryzae, Chrysosporium lucknowense, Myceliophthora thermophila, Fusarium oxysporum, Trichoderma reesei or Penicillium chrysogenum. A more preferred host cell is Aspergillus niger. When the host cell according to the invention is an Aspergillus niger host cell, the host cell preferably is CBS 513.88, CBS124.903 or a derivative thereof.
The suitable host cell is cultured to bring the amino acid sequence of interest to expression. Although culturing of the host cell may be at any suitable scale, such as in a chemostat or in Eschweiler, for high throughput screening library, culturing is preferably at microtiter scale, e.g. in microtiter plates.
In this way a library can be constructed which allows for fast and efficient screening of multiple protein-signal peptide combinations. The library constructed in this way is also part of the present invention. The library contains an amino acid sequence of interest fused to a signal peptide to be tested, whereby the N-terminus of the amino acid sequence of interest remains the same once the signal peptide is removed by a signal peptidase. The optimal protein-signal peptide combination or combinations can be easily selected from the library.
The method of the invention can be applied to improve protein expression in a host, although the expression construct and host has already several other optimizations, such as for example a strong promoter, an improved translation initiation sequence, an improved translational termination sequence, an optimized codon and codon pair usage and / or an improved host for protein expression.
In another aspect, the present invention relates to a polynucleotide which comprises a nucleotide sequence encoding a signal peptide having at its carboxy terminus the first few amino acids of the N-terminus of an amino acid sequence of interest. The nucleotide sequence includes a unique restriction site which allows for fusion with a nucleotide sequence which encodes the whole or part of the amino acid sequence of interest in such a way that a nucleotide fusion product is obtained which encodes a fusion between the signal peptide and the amino acid sequence of interest which allows for the secretion of the amino acid sequence of interest without modifications to its amino acid sequence. This polynucleotide sequence may be part of a vector and form a recombinant nucleotide which may be introduced into a host cell. The vector, recombinant nucleotide sequence and host cell comprising the recombinant nucleotide are also part of the present invention. The vector may be any vector, including a plasmid and an expression vector. Suitable recombinant host cells may be selected by the skilled person. Suitable examples are mentioned above and include Aspergillus and Bacillus, Escherichia, Kluyveromyces, Penicillium, Pseudomonas, Saccharomyces and Streptomyces species.
In the present context, 'polynucleotide sequence' and 'nucleotide sequence' may refer to forms of DNA and RNA of any length and any origin, i.e. it includes both natural, isolated and manipulated and synthetically produced nucleotides.
In yet another aspect, the present invention relates to the use of a library according to the invention in a screening method for increasing the secretion of amino acids of interest. In yet another aspect, the present invention relates to a nucleotide fusion product obtainable by the method according to the present invention. This nucleotide fusion product comprises a polynucleotide encoding a signal sequence which polynucleotide is at its 3' site and in reading frame linked to a polynucleotide encoding an amino acid sequence of interest, whereby the nucleotide fusion product encodes a fusion between the signal peptide and the amino acid sequence of interest, which fusion allows for the secretion of the amino acid sequence of interest without modifications to its amino acid sequence. Polynucleotides encoding fusions between signal peptides and their corresponding natural polypeptides are typically excluded from the present invention. For example, polynucleotides encoding a fusion between maltogenic alpha-amylase and its natural signal peptide are excluded from this invention. Preferably, the polynucleotides of the invention encode fusions between a signal peptide and an amino acid sequence of interest wherein the signal peptide and amino acid of interest are obtained from different strains, subspecies, species, genera, families, orders, classes or kingdoms.
Such nucleotide fusion products may be obtained by a method according to the invention. Suitable examples of such nucleotide fusion products include nucleotide fusion products which encode amino acid sequences as given in Fig. 6A-6D or which include the signal peptides given in Table 1 . Such a nucleotide fusion product encodes a clean fusion between the signal peptide and the amino acid sequence of interest, i.e. fusions of an amino acid sequence of interest and a signal peptide, without modifications to its amino acid sequence, in particular, without modifications to the N- terminus of the amino acid of interest. This means that no amino acids are deleted from, inserted in, added to or changed in the N-terminus of the amino acid of interest, neither in the fusion with the signal peptide, nor in the secreted amino acid sequence.
Nucleotide fusion products encoding optimal amino acid of interest-signal peptide combinations are also encompassed by the present invention. Screening among these nucleotide fusion products will reveal nucleotide fusions products with optimised secretion of the amino acid sequence of interest in comparison to the situation before the fusion. Once these combinations have been identified using the method of the invention, the skilled person will be able to produce, synthesize, use and/or apply the corresponding nucleotide fusion products almost without any effort for optimized secretion and production of the amino acid of interest. In one embodiment, the method of the invention is used to find a signal peptide to optimize secretion of maltogenic alpha-amylase from Bacillus stearothermophilus later typed as Geobacillus stearothermophilus
Using the method according to the invention, a library was constructed, containing about 168 signal sequences to be tested fused to the sequence encoding the processed/coding sequence of maltogenic alpha-amylase. Screening revealed that the yurl fliL, vpr, glpQ, phy, lytC, ywsB, ybbD, ybxl, yolA, ylqB, ybbC, pel, yckD, ywaD, ywmD, yweA, yraJ, dacF, yfjS, yybN, yrpD, yvcE, wprA, yxaL, ykwD, yncM2, sacB, phrC, SacC, yoqM, ykoJ, lip, yfkN, yurl, ybfO, yfkD, yoaJ, xynA, penP, ydjM, yddT, yojL, yomL, yqxl, yrvJ, yvpA, yjcM, yjfA, ypjP, ggt, yoqH, ywtD, y/aE, yraJ, lytB, lytD, nprB, nucB, rpIR, yfhK, yjdB, ykv\l, ybbE, yuiC, ylbL, yacD, yvpB and ynfF signal sequences in combination with amyM increased the secretion of maltogenic alpha- amylase from Bacillus subtilis. In many cases, the increase was more than 50% compared to the wild type signal sequence (Table 2). Since these combinations have been identified, the skilled person will be able to produce or synthesize the nucleotide sequences and corresponding amino acid sequences for the fusion products in any other convenient way. Amino acid sequences which are particularly suitable for facilitating secretion of maltogenic alpha-amylase are given in SEQ ID NO. 12 (SacC- maltogenic alpha-amylase fusion product), SEQ ID NO. 13 (ybfO-maltogenic alpha- amylase fusion product), SEQ ID. NO. 14 (yjcM-maltogenic alpha-amylase fusion product) and SEQ ID NO. 15 (yurl-maltogenic alpha-amylase fusion product), SEQ ID
NO. 82 (ynfF-maltogenic alpha-amylase fusion product) and these amino acid sequences are also encompassed by the present invention. Other suitable fusions may be made with nucleotide sequences encoding the signal peptides given in Table 1 .
In yet another aspect, the present invention relates to an amino acid sequence produced by a method according to the invention in industrial applications, in particular in the cosmetic industry, detergent industry, feed or food industry, paper and pulp industry, more in particularly in baking, brewing, dairy processing, sport drinks, grain processing, fruit juice and the nutraceutical industry.
The present invention includes the following aspects.
A method for constructing a nucleotide fusion product which comprises a fusion between a nucleotide sequence encoding a signal peptide and a nucleotide sequence encoding an amino acid sequence of interest, wherein the nucleotide sequence encoding the amino acid sequence of interest comprises a unique restriction site, the method comprising combining
(a) a first polynucleotide which comprises a nucleotide sequence which encodes a part of the amino acid sequence of interest, which nucleotide sequence comprises the unique restriction site and the nucleotides encoding the amino acid sequence of interest downstream of the unique restriction site; and
(b) a second polynucleotide which comprises a nucleotide sequence which encodes the signal peptide having at its carboxyterminus part of the amino acid sequence of interest, and which nucleotide sequence comprises the nucleotides forming the unique restriction site and the nucleotides encoding the amino acid sequence of interest upstream of the unique restriction site,
whereby a nucleotide fusion product is obtained which encodes a fusion product which allows for the secretion of the amino acid sequence of interest without modifications to the amino acid sequence of the amino acid sequence of interest.
Method according aspect 1 , wherein the first polynucleotide also comprises all or some of the nucleotides encoding the amino acid sequence of interest which are upstream of the unique restriction site.
Method according aspect 1 or 2, wherein the second polynucleotide also comprises all or some of the nucleotides encoding the amino acid sequence of interest which are downstream of the unique restriction site.
Method according to aspects 1 -3, wherein the unique restriction site is a silent mutation in the nucleotide sequence which encodes the amino acid sequence of interest.
Method according to aspects 1 -4, wherein the unique restriction site is located within the nucleotides encoding the first 25 amino acids of the N-terminus of the amino acid sequence of interest
Method according to aspects 1 -5, wherein the second polynucleotide comprises a nucleotide sequence encoding a signal peptide having at its carboxyterminus not more than the first 25 nucleotides encoding the N-terminal amino acids of the amino acid sequence of interest.
Method according to aspects 1 -6, wherein the amino acid sequence of interest is an antibody or parts thereof, an antigen, a clotting factor, an enzyme, a hormone or a hormone variant, a receptor or parts thereof, a regulatory protein, a structural protein, a reporter, or a transport protein, protein involved in secretion process, protein involved in folding process, chaperone, peptide amino acid transporter, glycosylation factor, transcription factor, synthetic peptide or oligopeptide.
Method according to aspects 1 -7, wherein the amino acid sequence of interest is an enzyme, in particular an aminopeptidase, amylase, maltogenic alpha-amylase, carbohydrase, carboxypeptidase, endo-protease, metallo-protease, serine-protease, catalase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, dextranase, esterase, alpha-galactosidase, beta-galactosidase, cell wall degrading enzyme, glucoamylase, alpha-glucosidase, beta-glucosidase, haloperoxidase, hydrolase, isomerase, protein deaminase, invertase, laccase, ligase, lipolytic enzyme, lyase, mannosidase, mutanase, oxidase, oxidoreductase, pectinolytic enzyme, peroxidase, phosphatase, phospholipase, polyphenoloxidase, ribonuclease, transferase, transglutaminase, cellulase, xylanase, asparaginase or glucose oxidase,.
Method according to aspects 1 -8, wherein the secretion, productivity or activity of the amino acid sequence of interest in an external medium is measured and found to be
improved by at least 50% compared to the secretion, productivity or activity before combination.
10. A method for constructing and optionally screening a library for selecting optimal combinations between an amino acid sequence of interest and a variety of signal peptides, wherein the method comprises:
(a) transforming a collection of the fusion products obtainable by the methods according to aspects 1 -9 coding for diverse combinations of a signal peptide with the amino acid sequence of interest into a population of suitable host cells; and optionally (b) culturing the population of transformed host cells to bring the amino acid sequence of interest to expression, and optionally
(c) screening the population of transformed host cells for the optimal combination between amino acid sequence of interest and signal peptide for a specific purpose or application.
1 1 . A library for selecting optimal combinations between an amino acid sequence of interest and a variety of signal peptides obtainable by the method according to aspects 10. 12. A polynucleotide sequence which comprises a nucleotide sequence encoding a signal peptide having at its carboxy terminus the first few amino acids of the N-terminus of an amino acid sequence of interest including a unique restriction site, which unique restriction site allows for fusion to the amino acid of interest and allows for expression of the amino acid of interest without modifications to its amino acid sequence.
13. A polynucleotide sequence according to aspect 12, wherein the nucleotide sequence encodes an amino acid sequence according to SEQ ID NO. 12, 13, 14, 15, 82, or a fusion of an amino acid sequence selected from SEQ ID NO. 4, 7, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 31 , 32, 33, 34, 35, 37, 39, 40, 41 , 42, 43, 44, 45, 46, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 71 ,
72, 73, 74, 75, 76, 77, 78, 79,80 and 81 directly fused to the N-terminal sequence of maltogenic alpha-amylase (SEQ ID NO. 1 1 , amino acids 34-719).
A recombinant host cell comprising a polynucleotide sequence according to aspect 12 or 13. A method according to aspect 10, a library according to aspect 1 1 or a recombinant host cell according to aspect 14, wherein the host cell is an Aspergillus, Bacillus,
Chrysosporium, Escherichia, Kluyveromyces, Penicillium, Pseudomonas, Saccharomyces, Streptomyces or Talaromyces species, in particular a Bacillus subtilis. The present invention is further illustrated by the following examples.
EXAMPLES
Strains and plasmids
Bacillus subtilis strain BS154 (CBS 363.94) {AaprE, AnprE, AamyE, spo) is described in Quax and Broekhuizen 1994 Appl Microbiol Biotechnol. 41 : 425-431 .
Bacillus subtilus 168 (trpC2) is described in Anagnostopoulos C and Spizizen J. (1961 ) J Bacteriol. 1961 81 (5)741 -746.
Bacillus stearothermophilus C599 (NCIMB1 1873) is described in WO91/04669.
plasmid pNAPBH27 is contained in Bacillus subtilis strain BS154
Molecular biology techniques
In this strain, using molecular biology techniques known to the skilled person (see: Sambrook & Russell, Molecular Cloning: A Laboratory Manual, 3rd Ed., CSHL Press, Cold Spring Harbor, NY, 2001 ). B. subtilis transformations were performed as described by Anagnostopolous and Spizizen. 1961 J. Bacteriol. 81 : 741 -746.
Plasmid pNAPHB27 contains a Bacillus expression vector with a bleomycine and neomcyin resistance marker and is described in Quax and Broekhuizen 1994 Appl Microbiol Biotechnol. 41 : 425-431 .
Maltogenic alpha-amylase productivity
The maltogenic alpha-amylase productivity in the culture broth of B. subtilis was quantified by measuring activity using a Megazyme CERALPHA alpha-amylase assay
kit (Megazyme International Ireland Ltd., Co. Wicklow, Ireland) according to the manufacturer's instruction.
LC-MS analyses
The relative abundance of the identified peptides in the culture medium was estimated based on their LC-MS peak areas, using the accurate mass determined in the Orbitrap. The percentage of each N-terminal maltogenic alpha-amylase variant was estimated based on LC-MS peak areas. Example 1
Construction of modified Bacillus expression plasmids for maltogenic alpha- amylase (amvM)
To express the maltogenic alpha-amylase protein from Geobacillus stearothermophilus, which was formerly known as Bacillus stearothermophilus C599, the amyM gene including its native terminator sequence was amplified by polymerase chain reaction (PCR) with the following primers.
A forward primer to amplify the amyM gene and introduce a Nde\ site:
5'-GTGATAATCATATGAAAAAGAAAACGCTTTCTTTATTTGTGG-3' (SEQ ID NO. 1 ) A reverse primer to amplify the amyM gene and introduce a Hind\\\ site
5'-TAAATATAAAAGCTTGCAAGCAAGTGCATATCCTG-3' (SEQ ID NO. 2)
PCR was performed on a thermocycler with Phusion High-Fidelity DNA polymerase (Finnzymes OY, Aspoo, Finland) according to the instructions of the manufacturer. The resulting PCR fragment was digested with the restriction enzymes Ndel and Hind\\\ and ligated with T4 DNA ligase into Ndel and Hind\\\ digested pNAPHB27 plasmid. The ligation mixture was transformed into B. subtilis strain BS154. A clone was selected and the amyM expression plasmid was named pBHA1 MAM1 . The sequence of the plasmid was confirmed by DNA sequencing.
A silent mutation was introduced on pBHA1 MAM1 at the wobble position of the 37th amino acid of the unprocessed maltogenic alpha-amylase protein to create a unique Nhe\ restriction site. The wild type signal peptide of maltogenic alpha-amylase used in this example and as described in SEQ ID NO 1 1 contains amino acids 1 -33 of SEQ ID NO: 1 1. The silent mutation in this example was therefore introduced within the nucleotides of the fourth amino acid of the amino acid of interest, in this example the fourth amino acid of the maltogenic alpha-amylase with the amino acid sequence
according to amino acids 34-719 of SEQ ID No. 1 1 . A 483 bp synthetic DNA fragment containing Nhe\, Nde\ and Acc\ restriction sites was synthesized (SEQ ID NO.3):
5 - CATATGAAAAAGAAAACGCTTTCTTTATTTGTGGGACTGATGCTCCTCATCGGTCT TCTGTTCAGCGGTTCTCTTCCGTACAATCCAAACGCCGCTGAAGCCAGCAGTTCC G CTAG CGTCAAAG GG G ACGTG ATT A CCAG ATTATCATTG/\ CCG GTTTTACG ATG G G G ACAC G AC G AAC AACAATC CTG C CAAAAGTTATG G ACTTTAC G ATC C GA CCAA ATCGAAGTGGAAAATGTATTGGGGCGGGGATCTGGAGGGGGTTCGTCAAAAACT TCCTTATCTTAAACAGCTGGGCGTAACGACAATCTGGTTGTCCCCGGTTTTGGAC AATCTG GATACACTG G CG G G CA CCG ATAACACG G GCTATCACG G ATACTG GACG C G C G ATTTTAAACAG ATTG AG G AAC ATTTC G G G AATTG GA CCAC ATTTG AC AC GTT G GTCAATG ATG CJCA CCAAAACG GAATCAAGGTG ATTGTCG AC-3 ' (SEQ ID NO.3) The synthetic DNA fragment (SEQ ID NO.3) was digested with the restriction enzymes
Nde\ and Acc\ and ligated with T4 DNA ligase into Nde\ and Acc\ digested pBHA1 MAM1 . The ligation mixture was transformed into B. subtilis strain BS154. A clone was selected and the modified amyM expression plasmid was named pBHA1 MAM2 (Fig. 3). The seq uence of the plasm id was confirmed by DNA sequencing and Nhe\ digestion.
Example 2
Amplification of the various signal sequences
The pBHA1 MAM2 plasmid was digested with Nhe\ and Nde\ to remove the native signal sequence. The linear vector fragment without the signal sequence was gel- purified. The plasmid fragment was excised from the gel and the DNA was recovered using the NucleoSpin Extract II kit (Macherey-Nagel, GmbH & Co. Dijren, Germany) according to the manufacturer's instructions. This fragment was used to introduce the signal sequence encoding DNA fragments.
In order to obtain a signal sequence library, 163 DNA fragments encoding signal sequences from B. subtilis (see Table 1 for the amino acid sequences of the signal sequences which were the best performers in this experiment) were amplified by PCR using primer pairs designed on the basis of amino acid sequences of the signal sequences. The forward primers were extended by the Ndel restriction site and the
reverse primers were extended by three serine residues and the Nhel restriction site. For example, for dacF with amino acid sequence:
Met Lys Arg Leu Leu Ser Thr Leu Leu lie Gly lie Met Leu Leu Thr Phe Ala Pro Ser Ala Phe Ala
(SEQ ID NO. 4)
the following forward primer was used:
5'- TTTTTCATATGAAACGTCTTTTATCCACTTT-3' (SEQ ID NO.5)
and the following reverse primer was used:
5'-TTTTG CTAG CGG AACTG CTTG CAAATG CAGACG GTG CAAAT-3 ' (SEQ ID NO.6); for fliL with amino acid sequence:
Met Lys Lys Lys Leu Met lie lie Leu Leu lie lie Leu lie Val lie Gly Ala Leu Gly Ala Ala Ala
(SEQ ID NO.7)
the following forward primer was used:
5'-TTTTTCATATGAAGAAAAAGTTAATGATCAT-3' (SEQ ID NO.8)
and the following reverse primer was used:
5'- TTTTG CTAG CG G AACTG CTG CTTCACTTTTTTCG GACTTTC-3 ' (SEQ ID NO.9) Genomic DNA from B. subtilis 168 was isolated by FastD NA S P I N Kit (M P Biomedicals, Solon, OH, USA) and used as DNA template in these PCR reactions. The Nde\ restriction site in the forward primers allowed for cloning at the ATG start codon. The reverse primers contained the first four DNA codons of the N-terminal amino acids of the predicted mature maltogenic alpha-amylase protein and the Nhe\ restriction site at the wobble position of the 37th amino acid of the unprocessed maltogenic alpha-amylase protein. The generated DNA fragments were purified by Nucleospin 96 extract II (Macherey-Nagel, GmbH & Co. Dijren, Germany) according to instructions of the manufacturer. The purified PCR fragments were digested with Nde\ and Nhe\. The restriction enzymes were heat inactivated by incubating 20 minutes at 65°C. The digested vector and signal sequence fragments were ligated using the Quick ligation Kit (New England Biolabs, Inc., USA) according the protocol of the manufacturer. See Figure 4 for a schematic representation of this ligation. The ligation products were transformed to competent B. subtilis BS154 cells. The correct clones were identified by PCR with the signal sequence specific forward primers used to amplify the signal sequences and the reverse amyM primer annealing 184 bp down stream of the amyM ATG start codon.
5'-GATTGTTGTTCGTCGTGTCC-3' (SEQ ID NO.10)
In addition positive colonies were identified by halo formation on soluble starch containing agar plates that were overlaid with Lugol solution (SIGMA-ALDRICH, St. Louis, MO, USA).
Table 1
Signal SEQ peptide ID
NO. dacF MKRLLSTLLIGIMLLTFAPSAFA 4 fliL MKKKLMIILLIILIVIGALGAAA 7 ggt MKRTWNVCLTALLSVLLVAGSVPFHA 16 gipQ MRKNRILALFVLSLGLLSFMVTPVSA 17 lip MKKVLMAFIICLSLILSVLA 18 lytB MKSCKQLIVCSLAAILLLIPSVSFA 19 lytC MRSYIKVLTMCFLGLILFVPTALA 20 lytD MKKRLIAPMLLSAASLAFFAMSGSAQA 21 nprB MRNLTKTSLLLAGL CTAAQ M V F VT H AS A 22 nucB MKKWMAGLFLAAAVLLCLMVPQQIQG 23 pel MKKVMLATALFLGLTPAGANA 24 penP MKLKTKASIKFGICVGLLCLSITGFTPFFNSTHA 25 phrC MKLKSKLFVICLAAAAIFTAAGVSANA 26 phy MKVPKTMLLSTAAGLLLSLTATSVSA 27 rpIR MITKTSKNAARLKRHARVRAKLSGTA 28 sacB MNIKKFAKQATVLTFTTALLAGGATQAFA 29
SacC MKKRLIQVMIMFTLLLTMAFSADA 30 vpr MKKGIIRFLLVSFVLFFALSTGITGVQA 31 wprA MKRRKFSSVVAAVLIFALIFSLFSPGTKA 32 xynA MFKFKKNFLVGLSAALMSISLFSATASA 33 yacD MKSRTIWTIILGALLVCCIAVA 34 ybbC MRKTIFAFLTGLMMFGTITAASA 35 ybbD MRPVFPLILSAVLFLSCFFGARQTEA 36 ybbE MKTKTLFIFSAILTLSIFAPNETFA 37 ybfO MKRMIVRMTLPLLIVCLAFSSFSASARA 38 ybxl MKKWIYVVLVLSIAGIGGFSVHA 39 yckD MKRITINIITMFIAAAVISLTGTAEA 40 yddT MRKKRVITCVMAASLTLGSLLPAGYASA 41 ydjM MLKKVILAAFILVGSTLGAFSFSSDA 42 yfjS MKW MCS I CCAAVLLAGGAAQA 43 yfhK MKKKQVMLALTAAAGLGLTALHSAPAAKA 44 yfkD MMKKLFHSTLIVLLFFSFFGVQPIHA 45 yfkN MRIQKRRTHVENILRILLPPIMILSLILPTPPIHA 46 yjcM MKKELLASLVLCLSLSPLVSTNEVFA 47 yjdB M N F K KTVVS ALS I S AL ALS VS G VAS A 48 yjfA MKRLFMKASLVLFAWFVFAVKG 49 ykoJ MLKKKWMVGLLAGCLAAGGFSYNAFA 50 ykvV MLTKRLLTIYIMLLGLIAWFPGAAQA 51 ykwD MKKAFILSAAAAVGLFTFGGVQQASA 52 ylaE MKKTFVKKAMLTTAAMTSAALLTFGPDAASA 53 ylbL MLRKKHFSWMLVILILIAVLSFIKLPYYITKPGEA 54 ylqB MKKIGLLFMLCLAALFTIGFPAQQADA 55 yncM2 MAKPLSKGGILVKKVLIAGAVGTAVLFGTLSSGIP 56
GLPAADAQVAKA
yoaJ MKKIMSAFVGMVLLTIFCFSPQASA 57
yojL MKKKI VAG LAVSAWGSS MAAAPAEA 58 yolA M K K R 1 T YS L L AL LAWAF AFT DS S KAKA 59
SEQ
signal ID peptide NO. yomL MRKKRVITCVMAASLTLGSLLPAGYATA 60 yoqH MKRFILVLSFLSIIVAYPIQTNA 61 yoqM MKLRKVLTGSVLSLGLLVSASPAFA 62 ypjP MKRKLTICLLIALIFYNGNAKA 63 yqxl MFKKLLLATSALTFSLSLVLPLDGHA 64 yral MVSESKSLTGCKKVKRTAFIRGGYKVNKLKRLS 65
MLTVMIASVFIFSSHALA
yraJ MTLTKLKMLSMLTVMIASLFIFSSQALA 66 yrpD MMKKGLLAGALTATVLFGTCA 67 yrvJ MNKKYFVLIVCIIFTSALFPTFSSVTA 68 yuiC MMLNMIRRLLMTCLFLLAFGTTFLSVS 69 yurl MTKKAWFLPLVCVLLISGWLAPAASASA 70 yvcE MRKSLITLGLASVIGTSSFLIPFTSKTASA 71 yvpA MKKIVSILFMFGLVMGFSQFQPSTVFA 72 yvpB MKTLRTLCVLMILSGVIFFGLKIDA 73 ywaD MKKLLTVMTMAVLTAGTLLLPAQSVTPAAHA 74 yweA MLKRTSFVSSLFISSAVLLSILLPSGQAHA 75 ywmD MKKLLAAGIIGLLTVSIASPSFA 76 ywsB MN KPTKLFSTLALAAGMTAAAAGGAGTI HA 77 ywtD MNTLANWKKFLLVAVIICFLVPIMTKA 78 yxaL MVKSFRMKALIAGAAVAAAVSAGA 79 yybN MNKFLKSNFRFLLAAALGISLLASSNFIKA 80 ynfF MIPRIKKTICVLLVCFTMLSVMLGPGATEVLA 81
Example 3
Expression of wild-type and modified expression constructs for maltogenic alpha-amylase in B. subtilis
B. subtilis BS154 strains containing the pBHA1 MAM2 plasmid (Fig. 3) with the various signal sequences fused to the mature maltogenic alpha-amylase protein were grown in 0.5 ml U96 microWell Plates (Nunc A/S, Roskilde, Denmark). A 200 μΙ pre-culture was made in 2xTY medium composed of 1 .6% (w/w) Bacto tryptone, 1 % (w/w) yeast extract and 0.5% (w/w) NaCI. Kanamycin was added to the culture medium in a final concentration of 25 μ9/μΙ and the MicroWell plates were covered by a Breathseal (Greiner bio-one, Frickenhausen, Germany). After overnight growth at 37°C, 550 rpm and 80% humidity in a Microton incubator shaker (Infors AG, Bottmingen, Switzerland), 1 % (v/v) is inoculated in 0.5 ml U96 microWell Plates (Nunc A/S, Roskilde, Denmark) with 200 μΙ SMM medium. SMM pre-medium contains 1 .25% (w/w) yeast extract, 00.5% (w/w) CaCI2, 0.075% (w/w) MgCI2.6H20, 15 pg/l MnS04 .4H20 , 1 0 pg/l
CoCI2.6H20 , 0.05% (w/w) citric acid, 0.025% (w/w) antifoam 86/013 (Basildon Chemicals, Abingdon, UK). To complete SMM medium, 20 ml of 5% (w/v) maltose and 20 ml of a 200 mM Na-phosphate buffer stock solution (pH 6.8), both prepared and sterilized separately, were added to 60 ml SMM pre-medium. These cultures were incubated in a Microton incubator shaker (Infors AG, Bottmingen, Switzerland) for 48 hours at 37°C, 550 rpm and 80% humidity. The supernatants were harvested by centrifugation for 30 min at 4000g and analysed for enzyme productivity.
Surprisingly, a clear positive effect of the use of other signal sequences on maltogenic alpha-amylase production was observed. The results can be found in Table 2. Thus, a clear positive effect for the use of different signal sequences from Bacillus subtilis and more specifically the signal sequences of fliL, vpr, glpQ, lytC, ywsB, ybbD, ybxl, yolA, ylqB, ybbC, pel, yckD, ywaD, ywmD, yweA, yraJ, dacF, yfjS, yybN, yrpD, yvcE, wprA, yxaL, ykwD, yncM2, sacB, phrC, yoqM, ykoJ, lip, yral, yfkN, yfkD, yoaJ, xynA, penP, ydjM, yqxl, yrvJ, yvpA, yjfA, ypjP, ggt, yoqH, and ywtD on amyM expression was observed.
Table 2 Effect of the use of other signal sequences on maltogenic alpha-amylase production compared to the construct with the native signal peptide.
Relative
Signal Relative Signal productivity sequence productivity (%) sequence (%)
amyM
(native) 100 yxaL 134
flil 100 ykwD 137
vpr 102 ync 2 139
glpQ 103 sacB 139
lytC 104 phr 140
yws 105 yoq 143
ybbD 105 yko 146
ybx\ 106 lip 146
yolk 107 yra\ 147
ylqB 107 y Z N 151
ybbC 108 yfkD 163
pel 109 yoaJ 165
yckD 1 1 1 xynA 165
ywaD 1 14 penP 165
ywmD 120 ydjM 168
yweA 122 yqx\ 169
yraJ 122 yrvJ 172
dacF 125 yvpA 176
yfjS 125 yjfA 180
yybN 130 ypjP 188
yrpD 132 ggt 193
yvcE 134 yoqH 194
wprA 134 ywtD 202
Example 4
Expression of wild-type and modified expression constructs for maltogenic alpha-amylase in B. subtilis in shake flasks.
B. subtilis strains containing the pBHA1 MAM2 plasmid with the phy, sacC, ybfO, yddT, yjcM, yojL, ynfF, yomL or yurl signal sequences were also tested in a shake flask fermentation experiments. These shake flaks contained 20 ml 2xTY medium composed of 1 .6% (w/w) Bacto tryptone, 1 % (w/w) yeast extract and 0.5% (w/w) NaCI. Kanamycin was added to the culture medium to reach a final concentration of 25 μg/μl. The cultures were shaken vigorously at 37°C and 250 rpm for 16 hours and 0.2 ml culture medium was used to inoculate 20 ml SMM medium. These cultures were incubated for 44 hours at 37°C and 250 rpm. The supernatants were harvested and analysed for enzyme productivity. The maltogenic alpha-amylase productivity of the signal sequence fusion constructs was compared to the strain containing the native amyM signal sequence. Samples were assayed for maltogenic alpha-amylase activity. As shown in Fig. 5, a clear positive effect for the use of different signal sequences from Bacillus subtilis is also observed in shake flaks fermentation.
Example 5
Determination of the N-terminus of the maltogenic alpha-amylase produced.
The polypeptide containing growth medium was used to determine the amino acid composition of the N-terminus of the maltogenic alpha-amylase polypeptide through LC-MS/MS. The predicted sequence for some illustrative fusion products is shown in Fig 6A-D and SEQ ID NO. 12-15 and 82. The corresponding fusion with the wild type signal sequence is shown as comparison in Fig. 6E and SEQ ID NO.1 1 (the wild type signal peptide is represented by amino acids 1 -33, the maltogenic alpha-amylase is represented by amino acids 34-719 in SEQ ID NO. 1 1 ).
When the native maltogenic alpha-amylase polypeptide construct including its own signal peptide was used (amyM) more than 90% of the mature protein had the following amino acid sequence at the N-terminus; SSSASVKGDVIYQIIIDR (SEQ ID NO. 1 1 , amino acids 34-51 and Fig. 6E). When the yjcM or sacC signal sequence was fused with the sequence of the amyM gene also more than 90% mature maltogenic alpha-amylase present in the growth medium contained the same N-terminus (Table 3). This demonstrates that the signal peptide of a secreted protein can be exchanged
and that other signal peptides can increase its productivity without changing the amino acid composition of the mature protein.
Clearly, these examples show how the method of the invention can be used for improved secretion and production of enzymes.
Table 3 N-terminus of maltogenic alpha-amylase in 44 hour shake flask samples N-term ID after Arg-C digest
Signal sequence N-terminus of the mature %
protein amyM (native) SSSASVKGDVIYQIIIDR >90
yjc SSSASVKGDVIYQIIIDR >90
SacC SSSASVKGDVIYQIIIDR >90
Claims
1 . A polynucleotide sequence which comprises a nucleotide sequence encoding a signal peptide having at its carboxyterminus the first few amino acids of the N- terminus of an amino acid sequence of interest including a unique restriction site, which unique restriction site allows for fusion to the amino acid of interest and allows for expression of the amino acid sequence of interest without modifications to its amino acid sequence.
2. A polynucleotide sequence according to claim 1 , wherein the nucleotide sequence encodes an amino acid sequence according to SEQ ID NO. 82, 12, 13, 14 or 15, or a fusion of an amino acid sequence selected from SEQ ID NO. 81 , 4, 7, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 31 , 32, 33, 34, 35, 37, 39, 40, 41 , 42, 43, 44, 45, 46, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 71 , 72, 73, 74, 75, 76, 77, 78, 79, and 80 directly fused to the N- terminal sequence of maltogenic alpha-amylase (SEQ ID NO. 1 1 , amino acids 34- 719).
3. A method for constructing a nucleotide fusion product which comprises a fusion between a nucleotide sequence encoding a signal peptide and a nucleotide sequence encoding an amino acid sequence of interest, wherein the nucleotide sequence encoding the amino acid sequence of interest comprises a unique restriction site, the method comprising combining
(a) a first polynucleotide which comprises a nucleotide sequence which encodes a part of the amino acid sequence of interest, which nucleotide sequence comprises the unique restriction site and the nucleotides encoding the amino acid sequence of interest downstream of the unique restriction site; and
(b) a second polynucleotide which comprises a nucleotide sequence which encodes the signal peptide having at its carboxyterminus part of the amino acid sequence of interest, and which nucleotide sequence comprises the nucleotides forming the unique restriction site and the nucleotides encoding the amino acid sequence of interest upstream of the unique restriction site, whereby a nucleotide fusion product is obtained which encodes a fusion product which allows for the secretion of the amino acid sequence of interest without modifications to the amino acid sequence of the amino acid sequence of interest.
4. Method according claim 3, wherein the first polynucleotide also comprises all or some of the nucleotides encoding the amino acid sequence of interest which are upstream of the unique restriction site.
5. Method according claim 3 or 4, wherein the second polynucleotide also comprises all or some of the nucleotides encoding the amino acid sequence of interest which are downstream of the unique restriction site.
6. Method according to claims 3-5, wherein the unique restriction site is a silent mutation in the nucleotide sequence which encodes the amino acid sequence of interest.
7. Method according to claims 3-6, wherein the unique restriction site is located within the nucleotides encoding the first 25 amino acids of the N-terminus of the amino acid sequence of interest
8. Method according to claims 3-7, wherein the second polynucleotide comprises a nucleotide sequence encoding a signal peptide having at its carboxyterminus not more than the first 25 nucleotides encoding the N-terminal amino acids of the amino acid sequence of interest.
9. Method according to claims 3-8, wherein the amino acid sequence of interest is an antibody or parts thereof, an antigen, a clotting factor, an enzyme, a hormone or a hormone variant, a receptor or parts thereof, a regulatory protein, a structural protein, a reporter, or a transport protein, protein involved in secretion process, protein involved in folding process, chaperone, peptide amino acid transporter, glycosylation factor, transcription factor, synthetic peptide or oligopeptide.
10. Method according to claims 3-9, wherein the amino acid sequence of interest is an enzyme, in particular an aminopeptidase, amylase, maltogenic alpha-amylase, carbohydrase, carboxypeptidase, endo-protease, metallo-protease, serine- protease, catalase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, dextranase, esterase, alpha-galactosidase, beta- galactosidase, cell wall degrading enzyme, glucoamylase, alpha-glucosidase, beta- glucosidase, haloperoxidase, hydrolase, isomerase, protein deaminase, invertase, laccase, ligase, lipolytic enzyme, lyase, mannosidase, mutanase, oxidase, oxidoreductase, pectinolytic enzyme, peroxidase, phosphatase, phospholipase, polyphenoloxidase, ribonuclease, transferase, transglutaminase, cellulase, xylanase, asparaginase or glucose oxidase,.
1 1 . Method according to claims 3-10, wherein the secretion, productivity or activity of the amino acid sequence of interest in an external medium is measured and found to be improved by at least 50% compared to the secretion, productivity or activity before combination.
12. A method for constructing and optionally screening a library for selecting optimal combinations between an amino acid sequence of interest and a variety of signal peptides, wherein the method comprises:
(a) transforming a collection of the fusion products obtainable by the methods according to claims 3-1 1 coding for diverse combinations of a signal peptide with the amino acid sequence of interest into a population of suitable host cells; and optionally
(b) culturing the population of transformed host cells to bring the amino acid sequence of interest to expression, and optionally
(c) screening the population of transformed host cells for the optimal combination between amino acid sequence of interest and signal peptide for a specific purpose or application.
13. A library for selecting optimal combinations between an amino acid sequence of interest and a variety of signal peptides obtainable by the method according to claim 12.
14. A recombinant host cell comprising a polynucleotide sequence according to claim 1 or 2.
15. A method according to claim 12, a library according to claim 13 or a recombinant host cell according to claim 14, wherein the host cell is an Aspergillus, Bacillus, Chrysosporium, Escherichia, Kluyveromyces, Penicill iu m , Pseudomonas, Saccharomyces, Streptomyces or Talaromyces species, in particular a Bacillus subtilis.
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