WO2013007821A1

WO2013007821A1 - Screening method

Info

Publication number: WO2013007821A1
Application number: PCT/EP2012/063807
Authority: WO
Inventors: Evert Tjeerd VAN RIJ; Antonius Cornelis Henrica Maria RENNIERS; Zoltan Pragai; Noël Nicolaas Maria Elisabeth VAN PEIJ
Original assignee: Dsm Ip Assets B.V.
Priority date: 2011-07-14
Filing date: 2012-07-13
Publication date: 2013-01-17
Also published as: WO2013007820A1

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. The collection of signal peptide encoding nucleotide sequences has been designed in such a way that the collection is widely applicable.

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 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 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. (2006) 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 sequence comprises a nucleotide sequence A which encodes for an amino acid sequence of interest which comprises an extra alanine (Ala) at its 5' end; a second polynucleotide sequence comprises a nucleotide sequence B encoding a signal peptide lacking the last amino acid at its carboxyterminus. 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), i.e. without the extra Alanine. White: nucleotides encoding a signal peptide; grey: nucleotides encoding (part of) an amino acid sequence of interest; black: protein.

Fig. 2 Schematic representation of the modified xynA expression plasmid pBHA1XAS1 . Fig. 3 Schematic representation of the 5' end of the xynA gene and the location of the signal peptidase cleavage site (A). Mutations (bold) were introduced in xynA at nucleotide position 80 (C>G), 81 (T>C) and 84(T>A) to create a unique Fsp\ restriction site (B). The introduction of this site allows the removal of the signal peptide encoding DNA sequence with the exception of the last three nucleotides encoding an alanine (C).

Fig. 4 Schematic representation of the modified xynA expression plasmid pBHA1 XAS2 into which an Fsp\ site is introduced at the 3' end of the signal sequence part of xynA, in order to be able to remove all but the three last nucleotides of the native signal sequence, resulting in a coding sequence with an extra alanine at its 5' end.

Fig. 5 Schematic representation of the ligation of digested vector comprising an extra codon coding for alanine 5' of the coding sequence of xynA (A) and the PCR fragments encoding various different signal sequences lacking the last three nucleotides (B). Combination of A and B results in a nucleotide fusion product C which allows for secretion of the unmodified XynA protein.

White: nucleotides encoding a signal peptide or part there of; grey: nucleotides encoding the mature XynA protein.

Fig. 6 Schematic representation of the modified xynA expression plasmid pBHA1XAS3 to allow type two S restriction enzyme cloning with the restriction enzyme SsmBI. (A). A PCR fragment containing a signal sequence and two SsmBI sites (B). The SsmBI recognition sites are in italic and the unique four nucleotide cutting sites are underlined.

Fig. 7 Relative xylanase activity of strains expressing fusions between xynA and different signal sequences. The xynA control contains the native xynA signal sequence. Strains were grown for 48 hours in SMM medium. Fig. 8 Fusions products according to the invention. A) fusion of B. subtilis bgIC signal peptide and XynA; B) fusion of pelB and XynA; C) fusion of oligol 53 signal peptide and XynA; D) fusion of dbam17260 signal peptide and XynA; E) fusion of dbam20830 signal peptide and XynA; F) fusion of dbam35060 signal peptide and XynA.

Fig. 9 XynA wild type or native signal peptide and XynA. This fusion product is not part of the invention.

Detailed description of the invention

The present invention relates to a method for constructing a fusion of (a) a nucleotide sequence encoding an amino acid sequence of interest and (b) a nucleotide sequence encoding a signal peptide, the method comprises combining

(i) a first polynucleotide sequence comprising a nucleotide sequence encoding the amino acid sequence of interest having at its 5'end an extra codon coding for the amino acid alanine and optionally one extra nucleotide upstream to said extra codon; with

(ii) a second polynucleotide sequence comprising a nucleotide sequence encoding the signal peptide lacking on one or both of the nucleotide strands of the nucleotide sequence the last three or four nucleotides which encode the signal peptide's carboxyterminus;

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 .

In an embodiment of the method according to the invention the method for constructing a fusion of (a) a nucleotide sequence encoding an amino acid sequence of interest and (b) a nucleotide sequence encoding a signal peptide, the method comprises combining

(i) a first polynucleotide sequence comprising a nucleotide sequence encoding the amino acid sequence of interest having at its 5'end an extra codon coding for the amino acid alanine and one extra nucleotide upstream to said extra codon; with (ii) a second polynucleotide sequence comprising a nucleotide sequence encoding the signal peptide lacking on one or both of the nucleotide strands of the nucleotide sequence the last four nucleotides which encode the signal peptide carboxyterminus 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.

(i) a first polynucleotide sequence comprising a nucleotide sequence encoding the amino acid sequence of interest having at its 5'end an extra codon coding for the amino acid alanine; with

(ii) a second polynucleotide sequence comprising a nucleotide sequence encoding the signal peptide lacking on one or both of the nucleotide strands of the nucleotide sequence the last three nucleotides which encode the signal peptide carboxyterminus;

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 unexpected properties of the polypeptide produced is prevented.

In an embodiment of the method according to the invention the first and second polynucleotide sequences are prepared by cloning and digestion using restriction enzymes. The method may conveniently be used to combine one amino acid sequence of interest with a collection of signal peptides, thereby creating a library of combinations, and select from this library those amino acid sequence-signal peptide combinations which are the most interesting or optimal 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. The method comprises:

(a) transforming a collection of nucleotide fusion products 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 between 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 optimal and interesting amino acid sequence-signal peptide combinations. 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, in order to be able to quickly screen for the optimal combination between 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. Preferably the library containing more than 100, more than 150, more than 200 different combinations of clean fusions may be screened within six months, preferably in less than one month.

In one embodiment, the method of the invention is used to obtain secretion of an intracellular polypeptide of interest. Optimal combination between intracellular peptide and signal peptide can be screened for using a library constructed according to a method according to the invention. In this way, secretion of a normally intracellular polypeptide may be obtained. Therefore in one embodiment, in the method for constructing and optionally screening a library according to the invention, the amino acid sequence of interest is an intracellular polypeptide of interest and in step (c) the purpose is to find the optimal combination between intracellular polypeptide and signal peptide to obtain secretion of the intracellular polypeptide.

In another embodiment, the method of the invention is used to improve the secretion of a protein which is already secreted after production, i.e. an extracellular protein. For example, 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. It will be clear to those skilled in the art that in the context of the present invention the words protein, polypeptide, peptide are used interchangeably.

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 or further 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.

Therefore in one embodiment of the method for constructing and optionally screening a library according to the invention, the amino acid sequence of interest is an extracellular protein and in step (c) the purpose is to find the optimal combination between extracellular protein and signal peptide to improve the secretion of the extracellular protein. 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%.

In an embodiment of the method according to the invention the productivity or activity of the amino acid sequence of interest in an external medium is improved by at least 5% compared to the secretion, productivity or activity before combination.

In an embodiment of the method according to the invention the productivity or activity of the amino acid sequence of interest in an external medium is improved by at least 1 0% compared to the secretion, productivity or activity before combination.

In an embodiment of the method according to the invention the productivity or activity of the amino acid sequence of interest in an external medium is improved by at least 20%, preferably at least 50% compared to the secretion, productivity or activity before combination.

In an embodiment of the method according to the invention the productivity or activity of the amino acid sequence of interest in an external medium is improved by at least 100% compared to the secretion, productivity or activity before combination. The signal peptide which is combined with the amino acid sequence of interest may be any signal peptide. The term "signal peptide" is defined herein as a peptide linked (fused) in frame to the amino terminus of a polypeptide, and directs the polypeptide into the cell secretory pathway. 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, Gluconobacter oxydans, Caulobactert crescentus CB 15, Methylobacterium extorquens, Rhodobacter sphaeroides, Pseudomonas zeaxanthinifaciens, Paracoccus denitrificans, E. coli, Corynebacterium glutamicum, Staphylococcus carnosus, Streptomyces lividans, Sinorhizobium melioti and Rhizobium radiobacter.

According to another embodiment, the signal peptide which is combined with the amino acid sequence of interest 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, 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 obligatory 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, 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, Myceliophtora thermophila, Fusarium oxysporum, Trichoderma reesei or Penicillium chrysogenum. Alternatively, it may be produced synthetically. The signal peptide which is combined with the amino acid sequence of interest may or may not improve the secretion of the amino acid sequence of interest. DNA fragments encoding signal peptides may be identified in the genomic sequence of microorganisms using the methods described by Tjalsma and van Dijl, Proteomics (2005) 5: 4472-4482. 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 .

According to one embodiment of the invention the signal sequence or signal peptide to be combined with the amino acid sequence of interest may be derived from a prokaryote, preferably from a bacterium, more preferably a bacterium belonging to the genus Bacillus. It will be clear to those skilled in the art that the term signal sequence and signal peptide are used interchangeably in the context of the present invention.

The polynucleotide sequence comprising the nucleotides sequence encoding the amino acid sequence of interest having at its 5'-end an extra codon encoding an alanine and optionally an extra nucleotide upstream to said extra codon may be combined with the polynucleotide sequence comprising the nucleotide sequence encoding the signal peptide lacking on one or both of the nucleotide strands the last three or four nucleotides encoding the signal peptide's carboxyterminus by any method in the art which is suitable for combining two or more nucleotide sequences, including ligation, ligation independent cloning, fusion PCR, PCR, cloning with the use of a ligase, in vivo recombination and in vitro recombination. In one embodiment, the two polynucleotide sequences are combined by ligation after digestion with a restriction enzyme. 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 certain embodiments, these blunt or sticky compatible ends have been created not by the use of a restriction enzyme, but by adding nucleotides.

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, in particular to complete an alanine codon. Alanine codons are GCA, GCT, GCG and GCC. Lists of compatible restriction sites are available in the prior art, see for example page 31 1 of New England Biolabs Inc. 2007-2008 Catalog & Technical Reference, Ipswich, MA USA, which are hereby incorporated by reference. The polynucleotide sequence comprising the nucleotide sequence encoding the amino acid sequence of interest having at its 5'end an extra codon encoding an alanine and optionally an extra nucleotide upstream to said extra codon may be prepared by any method known in the art, be it by PCR, synthetically, cloning or isolation from a larger DNA sequence as a vector or genomic DNA.

In one embodiment, the polynucleotide sequence comprising the nucleotide sequence encoding the amino acid sequence of interest having at its 5'end an extra codon encoding an alanine is prepared from a plasmid isolated fragment that includes the nucleotides sequence encoding the amino acid sequence of interest having at its 5'end an extra codon encoding an alanine and a unique blunt restriction site. Upon digestion of the restriction site, a blunt DNA fragment is created where the 5'end encodes an alanine. The blunt 5' end can be ligated in frame to nucleotide sequences encoding signal peptides that lack the last three nucleotides of their carboxytermini. Suitable restriction enzymes include enzymes which create a blunt DNA fragment encoding for alanine, i.e. a GCA, GCG, GCC or GCT. Such enzymes are known in the art and include Afe\, Sto\ and Fsp\.

In another embodiment, the polynucleotide sequence comprising the nucleotides sequence encoding the amino acid sequence of interest having at its 5'end an extra codon encoding an alanine and an extra nucleotide upstream to said extra codon is prepared from a plasmid isolated fragment that includes the nucleotides sequence encoding the amino acid sequence of interest having at its 5'end an extra codon encoding an alanine and a unique restriction site generating a sticky end. For example, a unique type II restriction site may be present or introduced with the restriction enzyme's recognition site at the 5'end of the extra codon encoding an alanine and the restriction enzyme's digestion site at the extra codon encoding an alanine. Upon digestion of the restriction site a polynucleotide sequence is created that encodes the amino acid sequence of interest having at its 5'end a sticky-end that encodes the extra alanine and comprises an extra nucleotide upstream to the extra codon coding for alanine. The sticky 5'end can be ligated in frame with nucleotides sequences encoding signal peptides with a compatible 3' sticky end. The sticky end overhang has 4 nucleotides, most preferably it is 3 nucleotides. In one embodiment it is 3 nucleotides, in particular 3 nucleotides corresponding to GCA, GCG, GCT or GCC, which corresponds to the alanine codon. The use of type II restriction enzymes is preferred. Suitable type II restriction enzymes which can produce a GCA, GCG, GCC or GCT overhang are known in the art and include Sap\. Suitable type II restriction enzymes which can produce an overhang ending in a XGCA, XGCG, XGCC or XGCT are known in the art and include BsmB\ (X may be any one of A, C, G, T)

In yet another embodiment, the polynucleotide sequence comprising the nucleotides sequence encoding the amino acid sequence of interest having at its 5'end an extra codon encoding an alanine and an extra nucleotide upstream to said extra codon is prepared by PCR amplification using synthetic primers in combination with restriction enzyme digestion. These primers add a extra alanine encoding nucleotides at the 5'end of the nucleotide sequence.

In one preferred embodiment of the invention, a nucleotide fusion product was prepared using a polynucleotide sequence comprising the nucleotide sequence encoding the amino acid sequence of interest having at its 5'end an extra blunt ended alanine. In another preferred embodiment, a nucleotide fusion product was prepared using a polynucleotide sequence comprising the nucleotide sequence encoding the amino acid sequence of interest having at its 5'end a sticky end that encodes the extra alanine and has an overhang of four or three nucleotides.

The polynucleotide sequence comprising the nucleotide sequence encoding the signal peptide lacking on one or both of the nucleotide strands of the nucleotide sequence the nucleotides which encode the last three or four nucleotides encoding the signal peptide's carboxyterminus may be prepared by any method known in the art, be it by PCR, synthetically or cloning or isolation from a larger DNA sequence as a vector or genomic DNA. I n one embod iment, it is prepared by PCR amplification using synthetic primers in combination with restriction enzyme digestion which, after digestion, introduces a sticky 5' end and the polymerase generates a blunt 3'end in the PCR reaction. This restriction site may have been naturally present or may have been introduced by the primers used in the PCR reaction. In yet another embodiment, this polynucleotide sequence is prepared by PCR amplification using synthetic primers in combination with restriction enzyme digestion which introduces sticky ends which leave a GCA, GCT, GCC or GCG overhang at the 3'end. The restriction enzyme's recognition site(s) may have been naturally present or may have been introduced by the primers used in the PCR reaction. Upon digestion of the restriction site(s) a polynucleotide sequence is created which comprises a nucleotide sequence which encodes the signal peptide lacking on one nucleotide strand of the nucleotide sequence the last four nucleotides, or the last three nucleotides. This can now be ligated in frame with a polynucleotide sequence comprising a nucleotide sequence encoding the amino acid sequence of interest having compatible 5' sticky end. For example, this can be achieved by using primers that introduce type II restriction sites at the 3'end and 5'end of the polynucleotide. The use of type II restriction enzymes is preferred. Suitable type II restriction enzymes which produce an overhang ending in a XGCA, XGCG, XGCC or CGCT overhang (wherein X can be any of A, G. T. C) are known in the art and include BsmB\.

In one preferred embodiment of the invention, a nucleotide fusion product was prepared using a polynucleotide sequence comprising the nucleotide sequence encoding the signal peptide lacking on one of the nucleotide strands of the nucleotide sequence the last four nucleotides encoding the signal peptide's carboxyterminus. In another preferred embodiment, a nucleotide fusion product was prepared using a polynucleotide sequence comprising the nucleotide sequence encoding the signal peptide lacking on both of the nucleotide strands of the nucleotide sequence the last three nucleotides encoding the signal peptide's carboxyterminus.

In all methods according to the invention, the unique restriction site may be naturally present or may have been introduced by mutation. Any convenient site may be introduced at the 5'end as long as it does not change the amino acid sequence of the amino acid sequence of interest and the extra alanine codon. The skilled person will be able to determine suitable restriction sites for a given amino acid sequence. In one embodiment, mutations are introduced in a sequence encoding a xylanase, resulting in the unique blunt restriction site Fspl. Digestion of the DNA with Fsp\ results in a blunt 5'end of which the first codon encodes an alanine. Other blunt restriction sites leaving a blunt 5'end and an alanine encoding codon are Sfo\ and Afe\. In the present context, the phrase 'unique restriction sites' is used to indicate that this site is present only once in a certain sequence of interest.

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 to the extracellular. In industrial practice, extracellular secretion is of particular interest, because it enables the convenient recovery of proteins 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 usually 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 help to transport the entire polypeptide through intracellular or cellular membranes.

The amino acid sequence of interest may be a peptide, polypeptide or protein of interest. In the present context, these terms are used interchangeably. The amino acid sequence of interest may be a sequence which is identified in, isolated, amplified or copied 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 extracellularly. Such enzymes may belong to the groups of oxidoreductase, 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, an alpha amylase, a beta amylase, a maltogenic 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, triacyl glycerollipase, a galactolipase, 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 an aminopeptidase, amylase, alpha amylase, a beta amylase, maltogenic alpha-amylase, carbohydrase, carboxypeptidase, endo-protease, metallo-protease, serine-p rotea se , cata l a se , ch iti n a se , cu ti n ase , cyc l od extri n 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, more preferably an endoxylanase, in particular an endo-1 ,4-beta-xylanase (EC 3.2.1 .8). More in particular, an endo-1 ,4-beta-xylanase with an amino acid sequence according to SEQ ID NO: 47. 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 (poly)peptide- signal peptide combination. The skilled person will understand that the library may contain an unlimited amount of (poly)peptide-signal peptide combinations. Preferably, the library contains more than 100, more than 150, more than 200 different combinations of clean fusions. Preferably, the library 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.

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. Iicheniformis, 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, 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.

In an embodiment of the method according to the invention the host cell is an Aspergillus, Bacillus, Chrysosporium, Escherichia, Kluyveromyces, Penicillium, Pseudomonas, Saccharomyces, Streptomyces, Rasamsonia or Talaromyces species.

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 of a 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. In particular, there are no modifications to the N-terminus of the amino acid sequence of interest once secreted. This means that no amino acids are deleted from, inserted in, added to or changed in the N-terminus in the secreted amino acid sequence. In this way, the introduction of undesired and unexpected properties of the polypeptide produced is prevented. The optimal protein-signal peptide combination or combinations can be easily selected from the library. A library according to the invention may be presented in any suitable form, for example in a container, such as a microtiter plate, or kit.

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 sequence which comprises a nucleotide sequence encoding a signal peptide lacking on one or more of the nucleotide strands of the nucleotide sequence the last few nucleotides which encode the amino acids at the signal peptide's carboxyterminus.

In an embodiment of the polynucleotide sequence according to the invention, said polynucleotide sequence comprises a nucleotide sequence encoding a signal peptide lacking on one or both of the nucleotide strands of the nucleotide sequence the last four nucleotides which encode the amino acids at the signal peptide's carboxyterminus.

In an embodiment of the polynucleotide sequence according to the invention, said polynucleotide sequence comprises a nucleotide sequence encoding a signal peptide lacking on one or both of the nucleotide strands of the nucleotide sequence the last three nucleotides which encode the amino acids at the signal peptide's carboxy terminus.

The nucleotide sequence includes a unique restriction site or a compatible nucleotide 3'end sequence which allows for fusion with a nucleotide sequence which encodes the whole or part of the amino acid sequence of interest and an extra alanine codon at is 5'end 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.

A collection of polynucleotide sequences comprising nucleotide sequences encoding signal peptides which lack on one or both of the nucleotide strands of the nucleotide sequence the last nucleotides encoding the signal peptide's carboxyterminus as described herein is also part of the present invention. Preferably, at least 80%, at least 85 or at least 90% of the polynucleotide sequences in the collection comprise nucleotide sequences which encode signal peptides which lack on one or both of the nucleotide strands of the nucleotide sequence the last few nucleotides which encode the signal peptide's carboxyterminus. More preferably, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the polynucelotide sequences in the collection comprise nucleotide sequences which encode signal peptides which lack on one or both of the nucleotide strands of the nucleotide sequence the last nucleotides which encode the signal peptide's carboxyterminus. Most preferably, the collection consists of polynucleotide sequences which comprise nucleotide sequences which encode signal peptides that lack on one or both of the nucleotide strands of the nucleotide sequence the last few nucleotides which encode the signal peptide's carboxyterminus. Typically, the last six nucleotides or less on one or both strands are missing. Preferably, the last five nucleotides or less on one or both strands are missing. Most preferably, the last four nucleotides or less on one or both strands are missing. Even more preferably, the last three nucleotides on one or both strands, i.e. the last codon at the signal peptide carboxyterminus, is lacking. This codon may encode any amino acid

This collection of polynucleotide sequences is universal, i.e. it can be used for screening optimal combinations with various different amino acid sequence of interest, because it is independent of the amino acid sequence of interest for which an optimal signal peptide is sought. The use of this collection of polynucleotide sequences is much more convenient and time efficient for screening combinations than existing systems because it does not require adapting the signal sequence encoding nucleotide sequences to a specific amino acid sequence of interest. The collection can be conveniently combined with polynucleotide sequences comprising nucleotide sequences encoding an amino acid sequence of interest with an extra alanine at its 5'end. A collection according to the invention may be present in any suitable form, for example in a container, such as a microtiter plate, or kit .

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 sequence encoding a signal sequence which polynucleotide sequence is at its 3' end linked in reading frame to a polynucleotide sequence 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 endo-1 ,4-beta-xylanase 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 genes, 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 Table 2 in which the signal peptides given in Table 1 are directly fused to an amino acid sequence of interest. 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 optimized 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 endo-1 ,4-beta-xylanase from Bacillus subtilis. Using the method according to the invention, a library was constructed, containing 486 signal sequences to be tested fused to the sequence encoding the processed/coding sequence of endo-1 ,4-beta-xylanase. Screening revealed that about thirty signal sequences in combination with xynA increased the secretion of endo-1 ,4-beta-xylanase from Bacillus subtilis. In many cases, the increase was more than 10% compared to the wild type signal sequence (Figure 7). 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 endo-1 ,4-beta-xylanase are given in Table 2 and SEQ ID NO: 48, 49, 50, 51 , 52 and 53 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 the use of an amino acid sequence of interest prepared 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.

In aspect 1 the invention relates to a method for constructing a fusion of (a) a nucleotide sequence encoding an amino acid sequence of interest and (b) a nucleotide sequence encoding a signal peptide, the method comprises combining:

(i) a first polynucleotide sequence comprising the nucleotide sequence encoding the amino acid sequence of interest having at its 5'end an extra codon coding for the amino acid alanine and optionally one extra nucleotide upstream to said extra codon

(ii) a second polynucleotide sequence comprising the nucleotide sequence encoding the signal peptide lacking on one or both of the nucleotide strands of the nucleotide sequence the last three or four nucleotides which encode the signal peptide's carboxy terminus;

In aspect 2 the invention relates to the method according to aspect 1 , comprising combining:

(i) a first polynucleotide sequence comprising the nucleotide sequence encoding the amino acid sequence of interest having at its 5'end an extra codon coding for the amino acid alanine; with

(ii) a second polynucleotide sequence comprising a nucleotide sequence encoding the signal peptide lacking on one or both of the nucleotide strands of the nucleotide sequence the last three nucleotides which encode the signal peptide carboxy terminus;

wherein 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. In aspect 3 the invention relates to a method according to claim 1 , the method comprising combining:

(i) a first polynucleotide sequence comprising the nucleotide sequence encoding the amino acid sequence of interest having at its 5'end an extra codon coding for the amino acid alanine and one extra nucleotide upstream to said extra codon; with

(ii) a second polynucleotide sequence comprising a nucleotide sequence encoding the signal peptide lacking on one or both of the nucleotide strands of the nucleotide sequence the last four nucleotides which encode the signal peptide carboxyterminus;

In aspect 4 the invention relates to the method according to aspect 1 to 3, wherein the first and second polynucleotide sequence are prepared by cloning and digestion using a restriction enzymes.

In aspect 5 the invention relates to the method according to aspect 4, wherein the restriction enzymes are type II restriction enzymes.

In aspect 6 the invention relates to the method according to any one of aspects 1 to 5, 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.

In aspect 7 the invention relates to the method according to any one of aspects 1 to 6, wherein the host cell is an Aspergillus, Bacillus, Chrysosporium, Escherichia,

Kluyveromyces, Penicillium, Pseudomonas, Saccharomyces, Streptomyces, Rasamsonia or Talaromyces species.

In aspect 8 the invention relates to the method according to any one of aspects 1 to 7, wherein the secretion, productivity or activity of the amino acid sequence of interest in an external medium is improved by at least 1 % compared to the secretion, productivity or activity before combination.

In aspect 9 the invention relates to a nucleotide fusion product obtainable by the method according to any one of aspects 1 to 8.

In aspect 10 the invention relates to a nucleotide fusion product according to aspect 9, which encodes an amino acid sequence according to any one of SEQ ID NO: 48, 49, 50, 51 , 52 or 53.

In aspect 1 1 the invention relates to 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 nucleotide fusion products according to aspect 9 or 10 coding for diverse combinations of a signal peptide with the amino acid sequence of interest into a population of suitable host cells; and optionally

In aspect 12 the invention relates to a method according to aspect 1 1 wherein the amino acid sequence of interest is an intracellular polypeptide of interest and wherein in step (c) the purpose is to find the optimal combination between intracellular polypeptide and signal peptide to obtain secretion of the intracellular polypeptide.

In aspect 13 the invention relates to a method according to aspect 1 1 wherein the amino acid sequence of interest is an extracellular protein and wherein in step (c) the purpose is to find the optimal combination between extracellular protein and signal peptide to improve the secretion of the extracellular protein.

In aspect 14 the invention relates to a library for selecting optimal combinations between an amino acid sequence of interest and a variety of signal peptides, wherein the library is obtainable by the method according to any one of aspects 10 to 13.

In aspect 15 the invention relates to a collection of polynucleotide sequences comprising a nucleotide sequence encoding a signal peptide lacking on one or both of the nucleotide strands of the nucleotide sequence the last few nucleotides which encode the signal peptide's carboxyterminus.

In aspect 16 the invention relates to a collection according to aspect 15, wherein the nucleotide sequence encoding the signal peptide is lacking on one or both of the nucleotide strands of the nucleotide sequence the last six or less nucleotides, preferably the last five or less nucleotides, preferably the last four or less nucleotides, preferably the last three or less nucleotides, preferably the last two or less nucleotides, preferably the last nucleotide which encode the signal peptide's carboxyterminus. In aspect 17 the invention relates to a collection according to aspects 15 or 16, wherein the signal peptide has an amino acid sequence according to any one of SEQ ID NO: 4, 7, 12, 15, 18, 19, 20, 21 , 22, 23, 24, 25, 26 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45 or 46. In aspect 18 the invention relates to a recombinant host cell comprising a nucleotide fusion product according to aspect 9 or 10 or a polynucleotide sequence from a collection according to aspect 15 or 16.

The present invention is further illustrated by the following examples.

Examples

Strain construction

Bacillus subtilis strain BS154 (CBS 363.94) (AaprE, AnprE, AamyE, spo) is described in Quax and Broekhuizen 1994 Appl Microbiol Biotechnol. 41 : 425-431 . This strain also contains plasmid pNAPBH27 which is also described.

Bacillus subtilus 168 (ATCC 23857) (trpC2) in Anagnostopoulos C and Spizizen J. (1961 ) J Bacteriol. 1961 81 (5)741 -746, Bacillus licheniformis DSM13 (ATCC 14580) (Veith, 2004 Mol Microbiol Biotechnol. 2004;7(4):204-1 1 .) and Bacillus amyloliquefaciens BZ9 (CBS121452). Molecular biology techniques

Molecular biology techniques used are known to the skilled person and described in 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, C, and J. Spizizen. 1961 . Requirements for transformation in Bacillus subtilis. J. Bacteriol. 81 : 741 -746.

PCR was performed on a thermocycler with Phusion High-Fidelity DNA polymerase (Finnzymes OY, Aspoo, Finland) according to the instructions of the manufacturer.

Xylanase activity

The xylanase activity and their by the productivity of the xylanase producing strains was determined by measuring the xylanase activity in the culture broth of B. subtilis. This was quantified by EnzChek ® Ultra Xylanase Assay Kit E33650 (Invitrogen, Ltd. Paisley, UK), this kit was used according to the instructions of the supplier.

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 xylanase variant was estimated based on LC-MS peak areas.

Example 1

Construction of pBHA1XAS2, a modified an endo-1 ,4-beta-xylanase (xynA) expression plasmid

The DNA sequence of the xynA gene encoding the endo-1 ,4-beta-xylanase protein was retrieved from EMBL Nucleotide Sequence Database (http://www.ebi.ac.Uk/embl/i/Vc/ex.html) Accession numbers CDS:CAB13776. To express the endo-1 ,4-beta-xylanase gene from Bacillus subtilis 168 (Bacillus Genetic Stock Centre) the xynA gene was amplified from the B. subtilis 168 genome by PCR (Polymerase Chain Reaction) with the following primers. These primers introduced an Ale\ and Pad site up stream of xynA and a Hind\\\ site doen stream of the xynA gene. Forward primer for the 5' part of the xynA gene (Ale\, and Pad sites) was used: 5'-

CCAACACATTTGTGTTAATTAAAAAAGGAGCGATTTACATATGTTTAAGTTTAAAAAGAAT TTC-3' (SEQ ID NO: 1 )

Reverse primer for the 3' region of the xynA gene (Hind\\\) was used:.

5'-TAAATATAAAAGCTTCTCCAGCAATTCCAAGGCCGTTC-3' (SEQ ID NO: 2) The resulting PCR fragment was digested with the restriction enzymes Ale\ and Hind\\\ and ligated with T4 DNA ligase into Ale\ and Hind\\\ digested pNAPHB27 plasmid. The ligation mixture was transformed into B. subtilis strain BS154. A clone was selected and the xynA expression plasmid was named pBHAIXASI (Fig. 2). The sequence of the plasmid was confirmed by DNA sequencing.

The Fspl site was introduced in pBHAI XASI (Fig. 2) at the xynA nucleotide position 80 (C>G), 81 (T>C) and 84(T>A) (Fig. 3). The introduction of this site allows the removal of the signal peptide encoding DNA sequence with the exception of the last three nucleotides encoding an alanine. To introduce these mutations a synthetic DNA fragment was ordered that contained the three point mutations and the Pad and Nhe\ sites which are unique in pBHAI XASI .

A 1 16 bp synthetic DNA fragment with the following sequence

5 - TTAATTAAAAAAGGAGCGATTTACATATGTTTAAGTTTAAAAAGAATTTCTTAGTTGGATT ATCG G CAG CTTTAATG AGTATTAG CTTGTTTTCG G CAACCG CCTG CG CAG CTAG C-3 ' (SEQ ID NO: 3).

This synthetic DNA fragment was digested with the restriction enzymes Pad and Nhe\ and ligated with T4 DNA ligase into Pad and Nhe\ digested pBHAIXASI . The ligation mixture was transformed into B. subtilis strain BS154. A clone was selected and the modified xynA expression plasmid was named pBHA1XAS2 (Fig 4). The sequence of the plasmid was confirmed by DNA sequencing and Fspl digestion. Example 2

Preparation of the digested vector

The pBHA1 XAS2 plasmid was digested with Pad and the blunt restriction enzyme Fsp\ to remove all but the last three nucleotides of the native signal sequence. In this way, the alanine encoding codon remains in front of the coding sequence of endo-1 ,4-beta-xylanase. 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 vector fragment was used for ligation with the signal peptide encoding DNA fragments.

Example 3

Amplification of the various signal sequences and ligation into the vector

The signal sequence library, DNA fragments encoding signal peptides was identified in the genomic sequence of Bacillus subtilis 168, Bacillus licheniformis DSM13 and B. amylolqiuefaciens BZ9 using the methods as described by Tjalsma and van Dijl Proteomics (2005) 5:4472-4482. The identified sequences were used to design primers for the amplification of the signal sequences. The genomic template DNA of these strains was isolated by FastDNA SPIN Kit (MP Biomedicals, Solon, OH, USA). Primer pairs for PCR were based on the basis of nucleotide sequences of the specific signal sequences identified. The forward primers contained a Pad restriction site that allowed directional cloning. The reverse primers were designed to amplify the C-terminal region of the signal sequences with the exception of the three nucleotides, which encode the last amino acid of the signal peptide. For example, for csn from B. subtilis with amino acid sequence:

MKISMQKADFWKKAAISLLVFTMFFTLMMSETVFA (SEQ ID NO: 4)

the following forward primer was used:

5'- G G AAATTAATTAAAAAAG GAG CGATTTACATATGAAAATCAGTATG CAAAAAG CAG-3 ' (SEQ ID NO: 5)

and the following reverse primer was used:

5'-AAAAACCGTTTCGCTCATCATCAGG -3' (SEQ ID NO: 6);

for yhcC with amino acid sequence:

MAI I IAI IAAVIVIAALITFNVRNA (SEQ ID NO: 7)

the following forward primer was used: 5'- G G AAATTAATTAAAAAAGG AG CG ATTTACATATG G CCATTATTATAG CAATAATTG-3 ' (SEQ ID NO: 8)

and the following reverse primer was used:

5'- GTTGCGGACGTTAAATGTAATAAGGG -3' (SEQ ID NO: 9);

The primers were ordered in 96 well skirted reaction plates, SP-0037 (Abgene Limited, Epsom, United Kingdom). A mix of the forward and reverse primer pairs in the same 96 well skirted reaction plates was using in the PCR reactions. The signal sequence encoding DNA fragments were amplified by PCR using Phusion High-Fidelity DNA polymerase (Finnzymes OY, Aspoo, Finland) which creates blunt DNA fragments. The DNA fragments were generated in 96 well PCR plates, 4ti-0750 (BIOKE, Leiden, The Netherlands) and purified by Nucleospin 96 extract II (Macherey-Nagel, GmbH & Co. Dijren, Germany) according to instructions of the manufacturer. The purified PCR fragments were digested in 96 well PCR plates with Pad and this enzyme was heath inactivated by incubating 20 minutes at 65°C. The digested vector of Example 2 and the signal sequence fragments were ligated in in 96 well PCR plates using the compatible Pad sites and the blunt Fsp\ site of the vector and the blunt 3' PCR fragment. The Quick ligation Kit (New England Biolabs, Inc., USA) was used according the protocol of the manufacturer. See Figure 5 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 primer 5'- AGAGGTGATCTCGTCCAAC-3' (SEQ ID NO: 10) annealing 31 1 bp down stream of the xynA ATG start cod on

Example 4

Type two S restriction enzyme cloning

As an alternative to the classical restriction enzyme cloning, also the Stargate (IBA GmbH,Gottingen

Germany) or type two S restriction enzyme cloning method can be used to combine a signal sequence and the mature protein of interest. An example of a type two S restriction enzyme is SsmBI. Two SsmBI recognition sites can be introduced in pBHA1 XAS1 (Fig. 2) by synthesizing a 145 bp synthetic DNA fragment with the following sequence: 5'-

TTAATTAAAAAAGGAGCGATTTACATATGGG/\G/\CGCGC/AATGTTTAAGTTTAAAAAGAA TTTCTTAGTTG GATTATCG G CAG CTTTAATG AGTATTAG CTTGTTTTCG G CAACCG CCTC TGCA GCGCG7C7CCTGCAGCTAGC -3' (SEQ ID NO: 1 1 ) containing the Pad and Nhe\ restriction sites (in bold). The two BsmBI\ sites in italics with the unique four nucleotide cutting sites are under lined. This xynA signal sequence can now be digested with the restriction enzymes Pad and Nhe\ and ligated with T4 DNA ligase into Pad\ and Nhe\ digested pBHA1XAS1 . The ligation mixture is transformed into B. subtilis strain BS154. A clone that contains the modified xynA expression plasmid is depicted in Figure 4 as pBHA1XAS3. The sequence of the plasmid is subsequently confirmed by DNA sequencing and SsmBI digestion at 55°C.

The pBHA1XAS3 plasmid (Fig. 4) is digested with SsmBI which leaves a unique four nucleotide overhang TATG at the start of the gene and four unique nucleotide overhang encoding the last nucleotides of a signal sequence TGCA. 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 vector fragment is used for ligation with the signal peptide encoding DNA fragments which can be identified in the genomic sequence of Badllus subtilis 168, Badllus licheniformis DSM13 and B. amylolqiuefadens BZ9 using the methods as described by Tjalsma and van Dijl Proteomics (2005) 5: 4472-4482. The identified sequences are used to design primers for the amplification of the signal sequences. The genomic template DNA of these strains is isolated by FastDNA SPIN Kit (MP Biomedicals, Solon, OH, USA). The forward and reverse primers contain a SsmBI restriction site and the specific four nucleotide overhang that allowes directional cloning. The four specific nucleotides of the reverse primer dictate the last four nucleotides of all signal sequences to be TGCA, thereby forcing the last codon in into GCA, which encodes an alanine. For example, for the abnA signal sequence from S. subtilis with amino acid sequence

MKKKKTWKRFLHFSSAALAAGLIFTSAAPAEA (SEQ ID NO: 12)

the following forward primer is used (ATG in bold, four nucleotide overhang underlined): /AGCGCG7C7CCTATGAAAAAGAAAAAAACATGGAAACGCTTCT (SEQ ID NO: 13) and the following reverse primer is used (four nucleotide overhang underlined): /AGCGCG7C7CC7GCATCTGCGGGAGCAGCAGAAGTG (SEQ ID NO: 14)

for amyE from B. subtilis with amino acid sequence:

MFAKRFKTSLLPLFAGFLLLFHLVLA (SEQ ID NO: 15)

the following forward primer is used (ATG in bold, four nucleotide overhang underlined): /AGCGCG7C7CCTATGTTTGCAAAACGATTCAAAACCTC (SEQ ID NO: 16)

and the following reverse primer is used (four nucleotide overhang underlined):

A GCGCGTCTCC 7GCAAGAAC CAAATG AAACAG CAATAAAAATC (SEQ ID NO: 17) The signal sequence encoding DNA fragments is amplified by PCR using Phusion High- Fidelity DNA polymerase (Finnzymes OY, Aspoo, Finland). The generated DNA fragments are purified by Nucleospin 96 extract II (Macherey-Nagel, GmbH & Co. Dijren, Germany) according to instructions of the manufacturer. The purified PCR fragments are digested with Bsm \ and this enzyme is heat inactivated by incubating 20 minutes at 80°C. The digested vector and signal sequence fragments are ligated using the compatible three nucleotide overhanging fragments ( Fig. 6). The Quick ligation Kit (New England Biolabs, Inc., USA) is used according the protocol of the manufacturer. The ligation products are transformed to competent B. subtilis BS154 cells. The correct clones are identified by PCR with the signal sequence specific forward primers and the reverse primer

5'-AGAGGTGATCTCGTCCAAC-3' (SEQ ID NO: 10 ) annealing 31 1 bp down stream of the xynA ATG start codon.

Example 5

Expression of modified expression constructs for endo-1 ,4-beta-xylanase in B. subtilis

B. subtilis BS154 strains containing the pBHA1 XAS2 plasmid (Fig. 4) with the various signal sequences fused to the xynA gene encoding the mature endo-1 ,4-beta-xylanase protein (constructed in Example 3) 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 μg/μl and the MicroWell plates were covered by a Breathseal (Greiner bio-one, Frickenhausen, Germany). After over night 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, 10 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 positive effect of the use of other signal sequences on endo-1 ,4-beta- xylanase production was observed with different signal sequences from B. subtilis and more specifically the signal sequences of cwlD, bgIC, ylqB, yoaJ, ybfO, xynD. Also a positive effect was seen with the B. lichenifomis signal sequences ydhT, yfjS, BU01299, yvgO, bgIC, BU02101 , pelB, yqil, BL02555, yurl, BL03614, yvnB, vpr, BL00947 (oligol 53) and with B. amyloliquefaciens signal sequences dbam21030, dbam01880, dbam03020, dbam12980, dbam35060, dbam30360, dbam17260, dbam20830, dbam00430 (Fig. 7). Amino acid sequences of these signal peptides are presented in Table 1 .

Example 6 Determination of the N-terminus of the endo-1 ,4-beta-xylanase produced.

The polypeptide containing growth medium was used to determine the amino acid composition of the N-terminus of the endo-1 ,4-beta-xylanase polypeptide through LC- MS/MS. The predicted sequences for some fusion products are shown in Table 2, Fig. 8A-F and SEQ ID NO: 48-53. The corresponding fusion with the wild type signal sequence is shown as comparison in Fig. 9 and SEQ ID NO: 47 (the wild type signal peptide is represented by amino acids 1 -28, the endo-1 ,4-beta-xylanase is represented by amino acids 29-213 in SEQ ID NO: 47). When the native endo-1 ,4-beta-xylanase polypeptide construct including its own signal peptide was used {xynA, native) more than 90% of the mature protein had ASTDYWQNWTDGGGIVNAVNGSGGNYSVNWSNTGNFWGK (SEQ ID NO: 47 and Fig. 9, amino acids 29-70) as the N-terminus. When the bgIC, pelB, oligo l 53, dbam17260, dbam20830, or dbam35060 signal sequence was fused with the sequence of the xynA gene also more than 90% mature endo-1 ,4-beta-xylanase 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 1.

Signal peptides Amino acid sequence SEQ β. subtilis ID NO. bgIC MKRSISIFITCLLITLLTMGGMIASPASA 18 cwlD MRKKLKWLSFLLGFIILLFLFKYQFS 19 xynD MRKKCSVCLWILVLLLSCLSGKSAYA 20 ybfO MKRMIVRMTLPLLIVCLAFSSFSASARA 21 ylqB MKKIGLLFMLCLAALFTIGFPAQQADA 22 yoaJ MKKIMSAFVGMVLLTIFCFSPQASA 23

Signal peptides

β. licheniformis

ydhT MKKNIVCSIFALLLAFAVSQPSYA 24 yfjS M M KR I CAI CCG F LLTLAFSG N AE A 25

BU01299 MMVMKWLGKSLVASSLLAGILAVSGSPMHVQA 26 yvgO M KKMFAFFTALM LG LTCLP YLSEA 27 bgIC MAAEKVFSKNKIIGGKRMSYMKRSISVFIACFMVAALGISGIIAPKAAA 28

BU02101 MENMYTDNIWLEVDNMNIGKLSKYFLSLAIALATILSFHAVSANA 29 pelB MKLIKNASFIISF LAAAG I YF LLGTVAASA 30 yqil MKLLIKSFVLLLFSFMAAFPAAFA 31

BL02555 MKKLVLLMLVLLLVYPHVSKA 32 yurl MNRKCVIPFILMLSAMCAPAQN 33

BL03614 MKKLLFMVILSVLTLVFGSSSVSFA 34 yvnB MKKYRNRQTLLLLAAVLLFALQIPISSASG 35 vpr MRKSIVRYFVMAFILLFALSTFLTGVQA 36

BL00947 MKKLISIIFIFVLGWGS LTAAVS AE A 37 (oligo153)

Signal peptides

a

amyloliquefaciens

dbam21030 MFKKILLATSALTFSLSLVLPLDGHA 38 d bam 01880 MAIIIAVIAAVIVIALLITFNVRSTA 39 dbam03020 VRGKKVWISLLFALALIFTMAFGSTSSAQA 40 dbam 12980 MKRRRLTALKKMWIGLLAAAVVLCTIPKISLA 41 dbam35060 MKKLFLALLILAGSTLGAISYSSDASA 42 dbam30360 MKTLAAAILTMVISFSFLPQHKAFA 43 dbam 17260 MKNRFKRLIRFSCAAVTAGLLLMSSSPASA 44 dbam20830 MTKKLWFLPIVCLYFIFGWAAPSASA 45 dbam00430 MRKLFHATMILLLSFSIFATPSAGA 46

Table 2. Amino acid sequence of the nucleotide fusion products of different signal sequences and endo-1 ,4-beta-xylanase.

Signal Amino acid sequence of the fusion product of the signal peptides SEQ ID and the mature endo-1 ,4-beta-xylanase protein

peptides NO. bgIC MKRSISIFITCLLITLLTMGGMIASPASAASTDYWQNWTDGGGIVN 48

AVNGSGGNYSVNWSNTGNFWGKGWTTGSPFRTINYNAGVWAP NGNGYLTLYGWTRSPLIEYYVVDSWGTYRPTGTYKGTVKSDGGT YDIY I I I RYNAPSIDGDRTTFTQYWSVRQSKRPTGSNATITFSNHV NAWKSHGMNLGSNWAYQVMATEGYQSSGSSNVTVW

pelB MKLIKNASFIISFLAAAGIYFLLGTVAASAASTDYWQNWTDGGGIV 49

NAVNGSGGNYSVNWSNTGNFWGKGWTTGSPFRTINYNAGVWA PNGNGYLTLYGWTRSPLIEYYVVDSWGTYRPTGTYKGTVKSDGG TYDIY I I I RYNAPSIDGDRTTFTQYWSVRQSKRPTGSNATITFSNH VNAWKSHGMNLGSNWAYQVMATEGYQSSGSSNVTVW

BL00947 MKKLISIIFIFVLGVVGSLTAAVSAEAASTDYWQNWTDGGGIVNAV 50 (oligo153) NGSGGNYSVNWSNTGNFWGKGWTTGSPFRTINYNAGVWAPNG

NGYLTLYGWTRSPLIEYYVVDSWGTYRPTGTYKGTVKSDGGTYDI Y I N RYNAPSIDGDRTTFTQYWSVRQSKRPTGSNATITFSNHVNA WKSHGMNLGSNWAYQVMATEGYQSSGSSNVTVW

dbam172 MKNRFKRLIRFSCAAVTAGLLLMSSSPASAASTDYWQNWTDGG 51 60 GIVNAVNGSGGNYSVNWSNTGNFWGKGWTTGSPFRTINYNAGV

WAP NGN G YLTLYG WTRS P L I E YYVVDSWGTYRPTGTYKGTVKS D GGTYDIY I I I RYNAPSIDGDRTTFTQYWSVRQSKRPTGSNATITFS NHVNAWKSHGMNLGSNWAYQVMATEGYQSSGSSNVTVW

dbam208 MTKKLWFLPIVCLYFIFGWAAPSASAASTDYWQNWTDGGGIVNA 52 30 VNGSGGNYSVNWSNTGNFVVGKGWTTGSPFRTINYNAGVWAPN

GNGYLTLYGWTRSPLIEYYVVDSWGTYRPTGTYKGTVKSDGGTY DIY I I I RYNAPSIDGDRTTFTQYWSVRQSKRPTGSNATITFSNHVN AWKSHGMNLGSNWAYQVMATEGYQSSGSSNVTVW

dbam350 MKKLFLALLILAGSTLGAISYSSDASAASTDYWQNWTDGGGIVNA 53 60 VNGSGGNYSVNWSNTGNFVVGKGWTTGSPFRTINYNAGVWAPN

GNGYLTLYGWTRSPLIEYYVVDSWGTYRPTGTYKGTVKSDGGTY DIY I I I RYNAPSIDGDRTTFTQYWSVRQSKRPTGSNATITFSNHVN AWKSHGMNLGSNWAYQVMATEGYQSSGSSNVTVW Table 3. N-terminus of endo-1 ,4-beta-xylanase after trypsin digestion

Signal N-terminus of the mature protein after trypsin digestion % sequence

xynA (native) ASTDYWQNWTDGGGIVNAVNGSGGNYSVNWSNTGNFVVGK >90 bgIC ASTDYWQNWTDGGGIVNAVNGSGGNYSVNWSNTGNFVVGK >90 pelB ASTDYWQNWTDGGGIVNAVNGSGGNYSVNWSNTGNFVVGK >90

BL00947 ASTDYWQNWTDGGGIVNAVNGSGGNYSVNWSNTGNFVVGK >90 (oligol 53)

dbam17260 ASTDYWQNWTDGGGIVNAVNGSGGNYSVNWSNTGNFVVGK >90 dbam20830 ASTDYWQNWTDGGGIVNAVNGSGGNYSVNWSNTGNFVVGK >90 dbam35060 ASTDYWQNWTDGGGIVNAVNGSGGNYSVNWSNTGNFVVGK >90

Claims

1 . A method for constructing a fusion of (a) a nucleotide sequence encoding an amino acid sequence of interest and (b) a nucleotide sequence encoding a signal peptide, the method comprises combining:

(i) a first polynucleotide sequence comprising the nucleotide sequence encoding the amino acid sequence of interest having at its 5'end an extra codon coding for the amino acid alanine and optionally one extra nucleotide upstream to said extra codon ; with

2. A method according to claim 1 , the method comprising combining:

(ii) a second polynucleotide sequence comprising a nucleotide sequence encoding the signal peptide lacking on one or both of the nucleotide strands of the nucleotide seq u en ce th e l a st th ree n u c l eoti d es wh i ch e n cod e th e s i g n a l pe pti d e carboxyterminus;

3. A method according to claim 1 , the method comprising combining:

(i) a first polynucleotide sequence comprising the nucleotide sequence encoding the amino acid sequence of interest having at its 5'end an extra codon coding for the amino acid alanine and one extra nucleotide upstream to said extra codon; with (ii) a second polynucleotide sequence comprising a nucleotide sequence encoding the signal peptide lacking on one or both of the nucleotide strands of the nucleotide sequence the last four nucleotides which encode the signal peptide carboxyterminus; 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.

A method according to any one of claims 1 to 3, wherein the first and second polynucleotide sequence are prepared by cloning and digestion using a restriction enzyme.

A method according to claim 4, wherein the restriction enzyme is a type I I restriction enzyme.

A method according to any one of claims 1 to 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.

A method according to any one of claims 1 to 6, wherein the host cell is an Aspergillus, Bacillus, Chrysosporium, Escherichia, Kluyveromyces, Penicillium, Pseudomonas, Saccharomyces, Streptomyces, Rasamsonia or Talaromyces species.

A method according to any one of claims 1 to 7, wherein the secretion, productivity or activity of the amino acid sequence of interest in an external medium is improved by at least 1 % compared to the secretion, productivity or activity before combination.

9. A nucleotide fusion product obtainable by the method according to any one of claims 1 to 8.

10. A nucleotide fusion product according to claim 9, which encodes an amino acid sequence according to any one of SEQ ID NO:. 48, 49, 50, 51 , 52 or 53.

1 1 . 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 nucleotide fusion products according to claim 9 or

10 coding for diverse combinations of a signal peptide with the amino acid sequence of interest into a population of suitable host cells; and optionally

12. The method according to claim 1 1 wherein the amino acid sequence of interest is an intracellular polypeptide of interest and wherein in step (c) the purpose is to find the optimal combination between intracellular polypeptide and signal peptide to obtain secretion of the intracellular polypeptide.

13. The method according to claim 1 1 wherein the amino acid sequence of interest is an extracellular protein and wherein in step (c) the purpose is to find the optimal combination between extracellular protein and signal peptide to improve the secretion of the extracellular protein.

14. A library for selecting optimal combinations between an amino acid sequence of interest and a variety of signal peptides, wherein the library is obtainable by the method according to any one of claims 1 1 to 13.

15. A collection of polynucleotide sequences comprising a nucleotide sequence encoding a signal peptide lacking on one or both of the nucleotide strands of the nucleotide sequence the last three or four nucleotides which encode the signal peptide carboxyterminus.

16. A collection according to claim 15 , wherein the signal peptide has an amino acid sequence according to any one of SEQ ID NO: 4, 7, 12, 15, 18, 19, 20, 21 , 22, 23, 24, 25, 26 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45 or 46.

17. A recombinant host cell comprising a nucleotide fusion product according to claim 9 or 10 or a polynucleotide sequence from a collection according to any one of claims 15 or 16.