Patent Publication Number: US-2015064765-A1

Title: Polypeptide expression method

Description:
FIELD OF THE INVENTION 
     The present invention relates to a method for the production of a polypeptide of interest in a host cell. The invention also relates to a modified polypeptide. Also, the invention relates to a method for improving the expression level of a polypeptide and to the use of a polypeptide of interest which is modified so as to increase the expression level of that polypeptide in a host cell. 
     BACKGROUND OF THE INVENTION 
     The production of recombinant polypeptides in bacterial, yeast and fungal host cells is known in the art. Current production of polypeptides is performed in various ways. 
     The state of the art process for the production of recombinant polypeptides is by means of fermentation of a host cell comprising an expression construct, said expression construct comprising inter alia a promoter operably linked to a polynucleotide encoding the polypeptide of interest. The resulting polypeptide might accumulate intracellularly or may be further secreted via the secretory pathway of the host cell. In the latter case, the polypeptide of interest typically comprises a signal sequence. In Broekhuijsen et al (Journal of Biotechnology, 31 (1993) 135-145, Broekhuijsen et al; Secretion of heterologous proteins by  Aspergillus niger : Production of active human interleukin-6 in a protease deficient mutant by KEX2-like processing of a glucoamylase-hIL6 fusion protein), a recombinant protein is expressed in  Aspergillus niger  using the signal sequence of the secreted polypeptide glucoamylase. 
     The yield of production of the recombinant polypeptide of interest may be enhanced by increasing the production and secretion efficiency, for example by using modified Kozak sequences (WO2008000632), codon-pair optimization (WO2008000632), introduction of improved signal sequences for secretion (WO2010/121933). These methods do not change the amino acid composition of the mature polypeptide. 
     Recently, protein feature optimization (PFO) was introduced as a novel method for improving the secretion of a polypeptide of interest by a eukaryotic host cell by modifying the value of a set of relevant protein features in the amino acid backbone of the polypeptide to fall within an optimal range or to become more close to an optimal value for one or more protein features in the eukaryotic host. The protein features are properties that can be computationally derived from the protein amino acid sequence and DNA sequence (WO2010/102982). 
     In an industrial context, high yields of polypeptides produced are required. Consequently, to enhance the yield of production of a polypeptide of interest still further, there is a need to improve secretion efficiency. It is an object of the invention to provide an improved process for the production of a recombinant polypeptide. 
     SUMMARY OF THE INVENTION 
     Knowledge of factors influencing protein production may be employed to improve enzyme production rates in industrial settings. High production yields can in some cases be obtained for homologous gene expression, but yields are often limited for heterologous gene expression. We have applied sequence-based machine learning techniques to identify relevant protein sequence features. The amino-acid composition of the protein sequence was found to be most predictive and interpretation revealed that, for both homologous and heterologous gene expression, the same features are important. In particular, methionine (M) and Lysine (K) were found to have a negative contribution to high-level production. 
     Accordingly, the present invention relates to a method for improving the secretion of a polypeptide of interest by a host cell, such as a eukaryotic host cell, by reducing the number of methionine, or lysine, or methionine and lysine amino acids in the amino acid backbone of the mature polypeptide. 
     One advantage is that reduction of the lysine (K), methionine (M), or lysine (L) and methionine (M) content contributes to improved protein production and leads to the development of lower cost processes. 
     According to the invention, there is thus provided a method for the production of a polypeptide of interest in a host cell, which method comprises:
         a. providing a host cell which harbours a nucleic acid encoding a polypeptide of interest, wherein the polypeptide of interest is modified so that it comprises fewer methionine and/or lysine residues than a reference polypeptide, excluding any initial methionine amino acid located at the N-terminal end of the polypeptide sequence,   b. cultivating the host cell under conditions suitable for production of the polypeptide; and, optionally,   c. recovering the compound of interest.       

     Typically, the polypeptide may be modified with respect to the coding sequence, including or excluding one or more or all control sequences (such as a signal sequence). Typically, modification excludes the N-terminal amino acid. 
     The invention also relates to:
         a modified polypeptide which comprises fewer methionine and/or lysine residues than a reference polypeptide, excluding any initial methionine amino acid located at the N-terminal end of the polypeptide sequence;   method for improving the expression level of a polypeptide in a host cell, which method comprises reducing the number of methionine and/or lysine amino acids in the polypeptide as compared with a reference polypeptide, excluding any initial methionine amino acid located at the N-terminal end of the polypeptide sequence;   use of a polypeptide of interest which is modified so that it comprises fewer methionine and/or lysine residues than a reference polypeptide, excluding any initial methionine amino acid located at the N-terminal end of the polypeptide sequence, to increase the expression level of the polypeptide of interest in a host cell; and
 
a polypeptide having the amino acid sequence set out in SEQ ID NOs: 16, 17, 18, 19, 20, 8, 9, 10, 11, 12, 26, 27, 43, 44, 45, 28 or 29.
       

    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         FIG. 1  shows the weights of the classifier. For each (negatively correlated) amino acid (x-axis), the bar indicates the importance/weight of the frequency of occurrence of this amino acid in a protein for obtaining a high production after overexpression. 
         FIG. 2  shows a comparison of hom and het classifiers. Amino acid contributions obtained from hom and het classifiers are the x- and y-values, respectively. Contributions are normalized per classifier (axis): each contribution is divided by the maximum absolute contribution. The plot shows the contributions obtained from classifiers trained using the protein amino acid composition. 
         FIG. 3  depicts a plasmid map of expression vector pGBFINEBA205 (construction described in Example 1). Indicated are the glaA flanking regions relative to the amdS selection marker cassette. In addition are indicated sequences of the glaA promoter and the EBA205 sequences encoding variant enzymes according a method of the invention. The  E. coli  DNA can be removed by digestion with restriction enzyme NotI, prior to transformation of the  A. niger  strains. 
         FIG. 4  depicts a plasmid map of expression vector pGBFINFUA (construction described in Example 1). Indicated are the glaA flanking regions relative to the amdS selection marker cassette. In addition are indicated sequences of the glaA promoter and the EBA205 sequences encoding variant enzymes according a method of the invention. The  E. coli  DNA can be removed by digestion with restriction enzyme NotI, prior to transformation of the  A. niger  strains. 
         FIG. 5  sets out EBA205 protein M-variant expression in shake-flask fermentation measured as FUA activity in supernatant; normalized to M-content 100% and protein concentartion (mg/ml) WT at 100%; data-points are average 1-3 independently selected strains and 1× standard deviation is shown. 
         FIG. 6  sets out EBA205 protein K-variant expression in shake-flask fermentation measured as FUA activity in supernatant; normalized to K-content 100% and protein concentration (mg/ml) WT at 100%; data-points are average 1-3 independently selected strains and 1× standard deviation is shown. 
         FIG. 7  sets out FUA protein M-variant expression in shake-flask fermentation measured as fungal alpha-amylase activity in supernatant; normalized to M-content 100% and activity WT at 100%; data-points are average 1-3 independently selected strains and 1× standard deviation is shown. 
         FIG. 8  sets out FUA protein K-variants expression in shake-flask fermentation measured as fungal alpha-amylase activity in supernatant; normalized to K-content 100% and activity WT at 100%; data-points are average 1-3 independently selected strains and 1× standard deviation is shown. 
     
    
    
     DESCRIPTION OF THE SEQUENCE LISTING 
     SEQ ID NO: 1: PEPTIDE: SS_EBA205 signal sequence EBA205; 18 amino acids. 
     SEQ ID NO: 2: PEPTIDE: SS_FUA signal sequence FUA; 20 amino acids. 
     SEQ ID NO: 3: PEPTIDE: SS_PmeA signal sequence; 17 amino acids. 
     SEQ ID NO: 4: DNA: SS_PmeA signal sequence; 51 base pairs: codon-pair optimized. 
     SEQ ID NO: 5: PROTEIN: EBA205 wild-type sequence, including signal sequence (SEQ ID NO: 1). 
     SEQ ID NO: 6: PROTEIN: FUA wild-type sequence, including signal sequence (SEQ ID NO: 2). 
     SEQ ID NO: 7: PROTEIN: FUA wild-type sequence, where signal sequence (SEQ ID NO: 2) is replaced by (SEQ ID NO: 3). 
     SEQ ID NO: 8-27: PROTEIN: EBA205 variant sequences based on SEQ ID NO: 5. 
     SEQ ID NO: 28-52: PROTEIN: FUA variant sequences based on SEQ ID NO: 7 SEQ ID NO: 53: DNA: codon-pair optimized (CPO): EBA205 sequence for SEQ ID NO: 5. 
     SEQ ID NO: 54: DNA: codon-pair optimized (CPO): FUA sequence for SEQ ID NO: 7. 
     SEQ ID NO: 55-74: DNA EBA205 sequence variants for SEQ ID NO: 8-27 based on template SEQ ID NO: 53. 
     SEQ ID NO: 75-99: DNA FUA sequence variants for SEQ ID NO: 28-52 based on template SEQ ID NO: 54. 
     DETAILED DESCRIPTION 
     Throughout the present specification and the accompanying claims, the words “comprise”, “include” and “having” and variations such as “comprises”, “comprising”, “includes” and “including” are to be interpreted inclusively. That is, these words are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context allows. 
     The articles “a” and “an” are used herein to refer to one or to more than one (i.e. to one or at least one) of the grammatical object of the article. By way of example, “an element” may mean one element or more than one element 
     In the current invention, a polypeptide redesign method is applied to alter sequence properties without affecting enzymatic function. The rationale behind this is the observed characteristic amino acid compositions for proteins that provide high yields after over-expression compared to those that do not; thereby indicating that alteration of this characteristic can be used to improve secretion yields. However, the enzymatic activity should remain similar to the original activity to make the method useful for the enzyme industry. A characteristic difference in amino acid composition can be obtained by comparing individual amino acids composition of successful with less successful examples. 
     Protein engineering, using both rationalized and randomized approaches, has been used to e.g. increase thermostability, change by-product spectrum, let enzymes work in organic solvent, etc., avoid inclusion bodies, or form inclusion bodies, etc. Recently one approach focused on protein-feature optimization for increasing the expression of proteins (WO2010/102982). Recently, others (Goltermann et al (2010) approached protein evolution via amino acid and codon elimination and showed that it was possible to prepare a GFP free of phenylalanine. Others have looked at reduced amino-acid alphabets that will sustain life. Natural evolution produces complex protein folds with a 20-amino acid alphabet. Primordial protein synthesis, however, is believed to have involved only a handful of amino acids. Several studies have demonstrated that considerably reduced amino acid alphabets may be sufficient to encode native-like proteins (Tanaka et al. 2011, v6(3), e18034; Walter et al., 2005, The Journal of Biological Chemistry vol. 280, no. 45, pp. 37742-37746). for example Walter et al. 2005 (The Journal of Biological Chemistry vol. 280, NO. 45, pp. 37742-37746) constructed an active enzyme from a 9-amino acid alphabet, basically aiming at novel structures and functions. The simplified 9-amino acid alphabet (Asp, Glu, Asn, Lys/Phe, Ile, Leu, Met plus Arg) contains methionine and lysine. Tanaka et al. (Protein Science 2010 Vol. 19:786-795) evaluated comparative characterization of random sequence proteins consisting of 5, 12, and 20 kinds of amino acids. The 12-amino acid table contained lysine and methionine; the 5-amino acid alphabet (Ala, Gly, Val, Asp, and Glu) did not yield proteins exhibiting extensively well-folded structure. 
     We have observed, however, that, the Methionine (M) and Lysine (K) compositional fraction of a mature polypeptide is negatively correlated with expressivity. Accordingly, the invention relates to polypeptides and polypeptide expression methods, wherein the number of methionine and/or lysine amino acids is modified in a polypeptide, in particular reduced in comparison with a reference/starting polypeptide. 
     That is to say, the current invention takes the approach of modification, in particular reduction, of the amino acids methionine or lysine, or methionine and lysine (taken together) to increase expression of a protein, and, in the case of a catalytic enzyme, without losing its catalytic activity. This may lead to improved protein production levels and to consequent lower cost processes. The method according to the invention may be referred to a Protein Sequence Optimisation (PSO). 
     The reduction in the number of methionine and/or lysine amino acids in a polypeptide of interest may be carried out on the basis of the polypeptide including all of its control sequences (such as a signal sequence) or on the basis of the polypeptide excluding one or more or all of its control sequences (such as a signal sequence). Typically, the N-terminal amino acid is excluded. 
     According to the invention, there is thus provided a method for the production of a polypeptide of interest in a host cell, which method comprises:
         a. providing a host cell which harbours a nucleic acid encoding a polypeptide of interest, wherein the polypeptide of interest is modified so that it comprises fewer methionine and/or lysine residues than a reference polypeptide, excluding any initial methionine amino acid located at the N-terminal end of the polypeptide sequence;   b. cultivating the host cell under conditions suitable for production of the polypeptide; and, optionally,   c. recovering the compound of interest.       

     Thus, in the method of the invention, a polypeptide is selected and modified so that its expression level in a host cell may be increased. Typically, the nucleic acid sequence encoding the polypeptide is modified so that the resulting polypeptide is modified as desired. The nucleic acid that is harboured by the host cell may be in the form of a nucleic acid construct. 
     The term “nucleic acid construct” is herein referred to as a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or which has been modified to contain segments of nucleic acid which are combined and juxtaposed in a manner which would not otherwise exist in nature. The term nucleic acid construct is synonymous with the term “expression cassette” when the nucleic acid construct contains all the control sequences required for expression of a coding sequence, wherein said control sequences are operably linked to said coding sequence. 
     The term “operably linked” is defined herein as a configuration in which a control sequence is appropriately placed at a position relative to the coding sequence of the DNA sequence such that the control sequence directs the production of a polypeptide. 
     The term “control sequences” is defined herein to include all components, which are necessary or advantageous for the expression of mRNA and/or a polypeptide, either in vitro or in a host cell. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader, Shine-Delgarno sequence, optimal translation initiation sequences (as described in Kozak, 1991, J. Biol. Chem. 266:19867-19870), a polyadenylation sequence, a pro-peptide sequence, a pre-pro-peptide sequence, a promoter, a signal sequence, and a transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. Control sequences may be optimized to their specific purpose. Preferred optimized control sequences used in the present invention are those described in WO2006/077258, which is herein incorporated by reference. 
     A signal sequence (sometimes referred to as a signal peptide) may be a short (for example from about 5 to about 30 amino acids long) peptide present at the N-terminus of the majority of newly synthesized proteins that are destined towards the secretory pathway. These proteins include those that reside either inside certain organelles (the endoplasmic reticulum, golgi or endosomes), secreted from the cell, or inserted into most cellular membranes. Although most type I membrane-bound proteins have signal peptides, the majority of type II and multi-spanning membrane-bound proteins are targeted to the secretory pathway by their first transmembrane domain, which biochemically resembles a signal sequence except that it is not cleaved. 
     The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleic acid sequence encoding a polypeptide. 
     The control sequence may be an appropriate promoter sequence (promoter). 
     The control sequence may also be a suitable transcription terminator (terminator) sequence, a sequence recognized by a filamentous fungal cell to terminate transcription. The terminator sequence is operably linked to the 3′-terminus of the nucleic acid sequence encoding the polypeptide. Any terminator, which is functional in the cell, may be used in the present invention. 
     Preferred terminator sequences for filamentous fungal cells are obtained from the polynucleotides encoding  A. oryzae  TAKA amylase,  A. niger  glucoamylase (glaA),  A. nidulans  anthranilate synthase,  A. niger  alpha-glucosidase, trpC and  Fusarium oxysporum  trypsin-like protease. 
     The control sequence may also be a suitable leader sequence (leaders), a non-translated region of a mRNA which is important for translation by the filamentous fungal cell. The leader sequence is operably linked to the 5′-terminus of the nucleic acid sequence encoding the polypeptide. Any leader sequence, which is functional in the cell, may be used in the present invention. 
     Preferred leaders for filamentous fungal cells are obtained from the polynucleotides encoding  A. oryzae  TAKA amylase and  A. nidulans  triose phosphate isomerase and  A. niger  glaA and phytase. 
     Other control sequences may be isolated from the  Penicillium  IPNS gene, or pcbC gene, the beta tubulin gene. All the control sequences cited in WO 01/21779 are herewith incorporated by reference. 
     The control sequence may also be a polyadenylation sequence, a sequence which is operably linked to the 3′-terminus of the nucleic acid sequence and which, when transcribed, is recognized by the filamentous fungal cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence, which is functional in the cell, may be used in the present invention. 
     Preferred polyadenylation sequences for filamentous fungal cells are obtained from the polynucleotides encoding  A. oryzae  TAKA amylase,  A. niger  glucoamylase,  A. nidulans  anthranilate synthase,  Fusarium oxysporum  trypsin-like protease and  A. niger  alpha-glucosidase. 
     The term “promoter” is defined herein as a DNA sequence that binds RNA polymerase and directs the polymerase to the correct downstream transcriptional start site of a nucleic acid sequence encoding a biological compound to initiate transcription. RNA polymerase effectively catalyzes the assembly of messenger RNA complementary to the appropriate DNA strand of a coding region. The term “promoter” will also be understood to include the 5′-non-coding region (between promoter and translation start) for translation after transcription into mRNA, cis-acting transcription control elements such as enhancers, and other nucleotide sequences capable of interacting with transcription factors. The promoter may be any appropriate promoter sequence suitable for a eukaryotic or prokaryotic host cell, which shows transcriptional activity, including mutant, truncated, and hybrid promoters, and may be obtained from polynucleotides encoding extra-cellular or intracellular polypeptides either homologous (native) or heterologous (foreign) to the cell. The promoter may be a constitutive or inducible promoter. 
     Modification of a polypeptide is herein intended to encompass any event resulting in a change in the amino acid sequence of the polypeptide. A modification is construed as one or more modifications. Modification may be accomplished by the introduction (insertion), substitution or removal (deletion) of one or more amino acids in the polypeptide backbone. Clearly, such modification may readily be effected by modifying the sequence coding for the polypeptide of interest such that the desired modifications are present. 
     Typically, modification in the invention is reduction of methionine and/or lysine residues. The number of methionine residues or lysine residues or methionine and lysine residues (taken together) may be reduced. 
     Modification according to the invention may be carried out on the basis of the polypeptide of interest including control sequences (such as a signal sequence) or may be carried out on the basis of the polypeptide of interest minus one or more or all of its control sequences (such as a signal sequence). 
     In the method of the invention, the methionine and/or lysine amino acids in the reference polypeptide may be substituted with non-methionine and/or non-lysine amino acids and/or deleted. That is to say, all or some of the methionine and/or lysine amino acids in the reference polypeptide may be substituted and/or deleted. 
     In the present invention, a polypeptide may be modified in order to increase its expression level in a host cell. An increase in expression level encompasses any way in which the amount of resulting polypeptide may be increased. Typically, an increase in secretion will be preferred. The term ‘secretion’ refers to the appearance of a polypeptide in the extracellular medium, typically the growth medium or production medium. The polypeptide which is secreted is free from the biomass. The level of secretion may be measured by methods known in the art, including by activity assays (units of activity), specific activity (units per weight protein), quantitative PAGE analysis, quantitative mass spectrometry and antibody assays. 
     Thus, the method of the invention may be used to increase/improve secretion of a polypeptide. Accordingly, in a method of the invention, the polypeptide of interest may be a secreted protein. 
     The expression ‘improvement of the secretion of a polypeptide’ refers to an increase in the amount of polypeptide which is secreted in the extracellular medium of a cell. The improvement may be reflected by the fact that a polypeptide which is normally not secreted, such as for example an intracellular polypeptide, is now secreted. The improvement may also reside in the fact that a polypeptide which is expected to be secreted, for example because it contains a signal sequence, and which is not secreted, is now secreted. Improvement is of course always measured with reference to identical host genetic background and identical culture or fermentation conditions. In these cases, improved secretion may be clear from, for example, the appearance of a protein band in a polyacrylamide gel, where there was no band visible before improvement. 
     Alternatively, the improvement may be reflected by the fact that a polypeptide which is secreted in very low amounts, shows increased levels of secretion. 
     The amount of polypeptide secreted may be determined by measuring the activity of the polypeptide in the extracellular medium. In comparison to the situation before improvement, the activity in the extracellular medium may be increased by at least 5%, at least 10%, at least 15% or at least 20%. Preferably the activity is increased by at least 25%, at least 30%, at least 35% or at least 40%. In a more preferred embodiment, the activity is at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500% or at least 1000% increased. The activity may be increased from no activity to some activity in the extracellular medium. “Activity” in this context typically refers to the total activity of the polypeptide in the extracellular medium which is used as a measure of an increased amount of protein in the extracellular medium. “Activity” is not intended to be a measure of specific activity—that is to say, “activity” is used to identify proteins which are secreted better than non-modified polypeptides (the intrinsic enzymatic activity of the polypeptide itself may be unaffected). 
     According to the method of the invention, the number of a set of relevant amino acids to be chosen from methionine and/or lysine in the amino acid backbone is modified to be reduced by at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99% or more. These percentages refer to the amount of modification with respect to the total number of methionine or lysine residues (depending on which is being modified). If methionine and lysine residues are modified, these percentages typically refer to the total modifications of both types of residue taken together. However, they can refer to the total modifications made with respect to one of the residues. 
     According to the method of the invention, the number of a set of relevant amino acids to be chosen from methionine and/or lysine in the amino acid backbone is modified to be reduced by at two, three, four, five, six, seven, eight, nine, ten, about fifteen or about twenty, or about twenty five, or about 30 or more. That is to say, in the method of the invention a reference polypeptide may be modified by carrying out modifications to two, three, four, five, six, seven, eight, nine, ten, about fifteen or about twenty, or about twenty five, or about 30 or more methionine residues and/or by carrying out modifications to two, three, four, five, six, seven, eight, nine, ten, about fifteen or about twenty, or about twenty five, or about 30 or more lysine residues. 
     Such modifications may be, for example, substitutions, replacements or deletions. A substitution will typically be with an amino acid other than methionine or lysine. A preferred substitution may be with an amino acid other than methionine or lysine which occurs at a corresponding position in a related polypeptide (for example the corresponding polypeptide from another species from which the reference polypeptide is derived). 
     Typically, any initial methionine amino acid located at the N-terminal end of the polypeptide sequence is excluded from modification. Also, any methionines and/or lysine amino acids in one or more control sequences, in particular one or more signal sequences, may be excluded from modification. 
     Also, one or more methionine and/or lysine residues may be essential for the functionality of a polypeptide of interest. Typically, such an amino acid will not be modified in the invention. Such an amino acid may be indicated if it is shared between a set of related polypeptides (for example the cognate polypeptide from different species), i.e. appears at the same corresponding position within that set of polypeptides. 
     On the other hand, a polypeptide which may be preferentially modified may be one where there appears to be variation in nature, i.e. different amino acids appear at the same corresponding position within a related set of polypeptides. 
     The number of modifications is determined in relation to a reference polypeptide. The reference polypeptide may be any polypeptide the expression of which it is desired to increase in a host cell. In particular, a reference polypeptide may be a wild-type polypeptide. Wild-type herein may refer to the typical form of the polypeptide as it appears in nature. It is of course the case that gene loci may exist in a variety of allelic forms. Typically, the most prevalent allele may be considered the wild-type. However, for the purposes of the invention, any naturally-occurring allele may be the wild-type. However, the reference polypeptide may be any polypeptide, such as a modified wild-type or a hybrid polypeptide composed of two or more wild-type sequences (e.g. a consensus sequence) or a mixture of wild-type and non-wild-type sequences. Also, the reference polypeptide may be a non-wild type sequence. Such a polypeptide may be modified according to the invention so that its expression level in a host cell in increased. 
     The reference polypeptide could be a polypeptide which has been modified according to the invention. That is to say, the method according to the invention may be an iterative process. Such an iterative process could be carried out by first modifying the number of one or methionine or lysine and then carrying out a second modification based on the other of methionine or lysine. One round of modification of methionine and/or lysine could be followed by one or more additional rounds of modification of methionine and/or lysine. 
     Accordingly, in the invention, a polypeptide, that is to say a reference polypeptide, is provided. That polypeptide is then modified according to the invention, that is to say, the number of methionine and/or lysine residues in the polypeptide is reduced. In this way, the expression level of the polypeptide may be increased. 
     A reference polypeptide may be a polypeptide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% identity with one or more corresponding wild-type polypeptides. 
     Further, the number of methionine and/or lysine amino acids in the reference polypeptide may be determined as the average number of methionine and/or lysine amino acids in two or more reference polypeptides. Thus, the number of methionine and/or lysine residues could be averaged over two or more corresponding wild-type sequences obtained from two or more different species. That is to say, the reference polypeptide could be a consensus sequence. The polypeptide of interest would be modified in that it would contain fewer methionine and/or lysine residues than this average. 
     In the method of the invention, the reduction in methionine and/or lysine amino acids in polypeptide of interest may be determined over a length of at least about 80 or more contiguous amino acids. The reduction in methionine and/or lysine amino acids may be determined within one functional domain of the polypeptide, in particular if the polypeptide of interest is a fusion protein or a chimeric protein. 
     Typically, the reduction of methionine and/or lysine amino acids in the polypeptide of interest is determined without taking into account signal sequences that may be processed in the host cell. That is to say, the reduction of methionine and/or lysine amino acids in the polypeptide of interest may typically be determined with reference to a mature polypeptide sequence, for example after processing of pre- and/or pro-sequences, for example, has occurred. 
     The invention also relates to a polypeptide modified as set out herein. That is to say, the invention concerns a modified polypeptide which comprises fewer methionine and/or lysine residues than a reference polypeptide, excluding any initial methionine amino acid located at the N-terminal end of the polypeptide sequence. The reference polypeptide may be a corresponding wild-type polypeptide. 
     Accordingly, the invention relates to a polypeptide having the amino acid sequence set out in SEQ ID NOs: 16, 17, 18, 19, 20, 8, 9, 10, 11, 12, 26, 27, 43, 44, 45, 28 or 29. 
     The invention relates to variants of these polypeptides in which one or more amino acids other than lysine or methionine residues are modified and which has at least about 35% identity, more preferably at least 40% identity, more preferably at least 45% identity, more preferably at least 50% identity, even more preferably at least 55% identity, even more preferably at least 60% identity, even more preferably at least 65% identity, even more preferably at least 70% identity, even more preferably at least 75% identity, even more preferably at least 80% identity, even more preferably at least 85% identity, even more preferably at least 90% identity, for example at least 91% identity, for example at least 92% identity, for example at least 93% identity, for example at least 94% identity, for example at least 95% identity, for example at least 96% identity, for example at least 97% identity, for example at least 98% identity, for example at least 99% identity, for example 100% identity with a sequence as set out in any one of SEQ ID NOs: 16, 17, 18, 19, 20, 8, 9, 10, 11, 12, 26, 27, 43, 44, 45, 28 or 29. 
     The invention also relates to a method for improving the expression level of a polypeptide in a host cell, which method comprises reducing the number of methionine and/or lysine amino acids in the polypeptide as compared with a reference polypeptide, excluding any initial methionine amino acid located at the N-terminal end of the polypeptide sequence. 
     Also, the invention relates to use of a polypeptide of interest which is modified so that it comprises fewer methionine and/or lysine residues than a reference polypeptide, typically excluding any initial methionine amino acid located at the N-terminal end of the polypeptide sequence, and/or optionally excluding one or more control sequences (such as) to increase the expression level of the polypeptide of interest in a host cell. 
     According to the method of the invention, modifications, in particular reductions of the numbers of methionine and/or lysine residues, are made to the polypeptide backbone. In this context, the term “backbone” refers to the regular structure which is formed when amino acids are linked together through peptide bonds and form a sequence of covalently linked amino acids. In the present invention, preferably the backbone of the mature polypeptide is modified. In the context of the present invention “mature polypeptide” is defined herein as a polypeptide that is in its final functional form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. 
     The polypeptide before modification is referred to as the parent or reference (for example a wild-type polypeptide) to distinguish it from the modified polypeptide which results from it, which may be referred to as the polypeptide of interest. The terms “parent-”, and “reference-polypeptide” are used interchangeably herein. When the polypeptide is a chimeric polypeptide, i.e. a translational fusion with an efficiently secreted polypeptide, preferably a polypeptide native to the host cell, the entire chimeric polypeptide may be modified according to the invention. When the chimeric polypeptide comprises an efficiently secreted polypeptide as a leader polypeptide fused to polypeptide of interest, the polypeptide of interest is preferably modified. A reference polypeptide may be a variant of a different parent polypeptide. 
     As is known to the person skilled in the art it is possible that the N-termini of the mature polypeptide might be heterogeneous as well as the C-terminus of the mature polypeptide due to processing errors during maturation. In particular such processing errors might occur upon overexpression of the polypeptide. In addition, exo-protease activity might give rise to heterogeneity. The extent to which heterogeneity occurs depends also on the host and fermentation protocols that are used. Such N-terminal and C-terminal processing artefacts might lead to shorter polypeptides or longer polypeptides compared with the expected mature polypeptide. 
     Methods to identify amino acids crucial for essential functional properties of interest are known in the art. Suitable tools include using a 3D structure or a 3D model of the protein of interest, mutagenesis studies of the protein of interest or of homologous proteins, the use of site saturated libraries to establish functionally neutral substitutions versus functional substitutions. 
     When introducing amino acid sequence features, such as methionine and/or lysine substitutions/deletions according to the invention, substitutions may preferably be chosen in such a way that at the given position the required amino acid sequence characteristic are selected from the group of amino acids which is observed in homologous sequences. State of the art modeling techniques may be applied to identify allowable substitutions which are not observed in natural homologues. Preferred references for modelling techniques which allow the generation of new sequences adopting a given fold are: 
     Kuhlman B, Dantas G, Ireton G C, Varani G, Stoddard B L, Baker D (2003). Design of a novel globular protein fold with atomic-level accuracy Science 302, 1364-8. 
     Baker D (2006). Prediction and design of macromolecular structures and interactions.  Philos. Trans. R. Soc. Lond., B, Biol. Sci.  361, 459-63 De Novo protein design: towards fully automated sequence selection  Journal of Molecular Biology , Volume 273, Issue 4, 7 Nov. 1997, Pages 789-796 Bassil I. Dahiyat, Catherine A. Sarisky, Stephen L. Mayo 
     State of the art computational method allow for the generation of numerous potential sequences which may adopt a given protein fold. By introducing protein sequence optimisation (PSO) into the scoring functions which are used to filter out the most optimal sequences the most optimal sequences for a given production host might be selected in a computational way. 
     The amino-acid fraction computed from the entire polypeptide or DNA sequence is an average value for the entire mature protein, which may not reveal local protein properties. For example, a protein could be on average hydrophilic but still contain a large internal hydrophobic region. Local protein properties can be computed using a sliding window method of 30, 50, and preferably 80 amino acids or more. 
     In order to modulate the hydrophilicity of the protein accessible surface a 3D structure or a 3D structural model may be required. The 3D structure of protein can be determined by X-ray crystallography and by NMR. In addition comparative modelling or template based modelling can be applied to construct reliable 3D models for a given sequences based on 3D structures of homologous proteins (http://en.wikipedia.org/wiki/Homology_modeling). Various servers and software packages for comparative modelling be found at: http://en.wikipedia.org/wiki/Protein_structure_prediction_software 
     For a recent review on protein structure prediction and modelling see Yang Zhang, Current Opinion in Structural Biology 2008, 18:342-348. 
     Given the atomic coordinates of a 3D structure or 3D model the accessible surface can be calculated by methods known in the art. A well known method is the calculation via a rolling-ball algorithm developed by Frederic Richards (1977, “Areas, volumes, packing and protein structure.” Annu Rev Biophys Bioeng, 6:151-176). See also http://en.wikipedia.org/wiki/Accessible_surface_area 
     For determination of the accessible surface the quaternary structure of the final mature protein should be considered in order to avoid that substitutions will disturb the interaction between the individual polypeptides (the monomers) in the multimer (e.g. dimer, trimer, tetramer etc) 
     According to the invention, there is provided a process for the production of a compound of interest. The process comprises cultivating cells capable of producing a polypeptide of interest, because they comprise a polynucleotide encoding the polypeptide of interest, in a reaction medium under conditions which allow for production of the polypeptide of interest. 
     The compound of interest may then be recovered from the medium (or from the cells in the event that the compound of interest is not secreted). 
     It is to be understood that the methods according to the present invention can conveniently be combined with a state of the art technique to increase levels of protein production or with combinations of one or more of these techniques. These include but are not limited to application of strong promoters, increase of copy number, optimal Kozak sequence, mRNA stabilizing elements and optimized codon usage (WO2008/000632). 
     The host cell used in the invention may be a prokaryotic cell or a eukaryotic cell. 
     A host cell suitable for use in the method according to the invention may be a prokaryotic cell. Preferably, the prokaryotic host cell is a bacterial cell. The term “bacterial cell” includes both Gram-negative and Gram-positive microorganisms. Suitable bacteria may be selected from e.g.  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.    
     Any eukaryotic cell may be used in the method of the invention. 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, PerC6 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&#39;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, Geosmithia, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Phanerochaete, Pleurotus, Rasamsonia, Schizophyllum, Talaromyces, Thermoascus, Thermomyces, Thielavia, Tolypocladium , and  Trichoderma.    
     Preferred filamentous fungal cells belong to a species of an  Acremonium, Aspergillus, Chrysosporium, Myceliophthora, Penicillium, Rasamsonia, Talaromyces, Thielavia, Fusarium  or  Trichoderma  genus, and most preferably a species of  Aspergillus niger, Acremonium alabamense, Aspergillus awamori, Aspergillus foetidus, Aspergillus sojae, Aspergillus fumigatus, Talaromyces emersonii, Talaromyces thermophilus, Thermomyces lanuginosus, Thermoascus thermophilus, Thermoascus aurantiacus, Thermoascus crustaceus, Rasamsonia emersonii, Rasamsonia byssochlamyoides, Rasamsonia argillacea, Rasamsonia brevistipitata, Rasamsonia cylindrospora, Aspergillus oryzae, Chrysosporium lucknowense, Fusarium oxysporum, Myceliophthora thermophila, Trichoderma reesei, Thielavia terrestris  or  Penicillium chrysogenum . A more preferred host cell belongs to the genus  Aspergillus , more preferably the host cell belongs to the species  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. A more preferred host cell belongs to the genus  Penicillium , more preferably the host cell belongs to the species  Penicillium chrysogenum . When the host cell according to the invention is a  Penicillium chrysogenum  host cell, the host cell preferably is Wisconsin 54-1255 or a derivative thereof. A more preferred host cell belongs to the genus  Rasamsonia  also known as  Talaromyces , more preferably the host cell belongs to the species  Talaromyces emersonii  also known as  Rasamsonia emersonii.    
     Several strains of filamentous fungi are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL), and All-Russian Collection of Microorganisms of Russian Academy of Sciences, (abbreviation in Russian—VKM, abbreviation in English—RCM), Moscow, Russia. Useful strains in the context of the present invention may be  Aspergillus niger  CBS 513.88, CBS124.903,  Aspergillus oryzae  ATCC 20423, IFO 4177, ATCC 1011, CBS205.89, ATCC 9576, ATCC14488-14491, ATCC 11601, ATCC12892,  P. chrysogenum  CBS 455.95,  P. chrysogenum  Wisconsin 54-1255 (ATCC28089),  Penicillium citrinum  ATCC 38065,  Penicillium chrysogenum  P2,  Thielavia terrestris  NRRL8126,  Talaromyces emersonii  CBS 124.902,  Acremonium chrysogenum  ATCC 36225 or ATCC 48272,  Trichoderma reesei  ATCC 26921 or ATCC 56765 or ATCC 26921,  Aspergillus sojae  ATCC11906,  Myceliophthora thermophila  Cl, Garg 27K, VKM-F 3500 D,  Chrysosporium lucknowense  Cl, Garg 27K, VKM-F 3500 D, ATCC44006 and derivatives thereof. 
     In one embodiment, the eukaryotic cell is a host cell in which the polypeptide is produced by recombinant technology. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. ( Molecular Cloning: A Laboratory Manual,  2 nd , ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), Davis et al.,  Basic Methods in Molecular Biology  (1986) and other laboratory manuals. Accordingly, the present invention also relates to a method for the production of a polypeptide of interest by applying a method according to the invention to improve the expression, for example secretion, of the polypeptide to the polypeptide of interest and producing the polypeptide modified according to the invention by recombinant technology. The present invention also relates to said recombinantly produced polypeptide. The present invention also relates to a polypeptide obtainable by a method according to the invention to improve the expression, for example secretion, of the polypeptide; preferably said polypeptide is obtained by a method according to the invention to improve the secretion of the polypeptide. 
     The polypeptide of interest of which the expression level is to be improved is improved according to a method of the invention may be any polypeptide having a biological activity of interest. The polypeptide may be a collagen or gelatin, or a variant or hybrid thereof. The polypeptide 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 such as serum albumin, e.g. Bovine Serum Albumin and Human Serum Albumin, or such as a transferrin, e.g. lactoferrin, a protein involved in secretion process, a protein involved in folding process, a chaperone, a peptide amino acid transporter, a glycosylation factor, a transcription factor, a synthetic peptide or oligopeptide, a protein which in its native form is an intracellular protein and is secreted by methods known in the art such as fusion to a signal peptide and fusion to a polypeptide that is already secreted in its native form. Such intracellular protein may be an enzyme such as a protease, ceramidases, epoxide hydrolase, aminopeptidase, acylases, aldolase, hydroxylase, aminopeptidase, lipase. The polypeptide may be an enzyme secreted extracellularly in its native form. 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, 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, endo polygalacturonases, 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, 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. The polypeptide of which the secretion is improved may be homologous or heterologous to the host cell. A suitable example of a homologous polypeptide is an  Aspergillus niger  protein which is cloned into and produced by an  Aspergillus niger . Suitable examples of heterologous expression include a bacterial polypeptide, for example from  E. coli  or  Bacillus , cloned into and produced by a filamentous fungus or a yeast, or a mammalian protein, for example from bovine or goat, which is cloned into and produced by a filamentous fungus or a yeast, or a filamentous fungal polypeptide which is cloned and produced by a yeast, or a filamentous fungal protein which is cloned into and produced by another fungus. Preferably, the nucleic acids encoding the polypeptides are optimized, for example by codon pair optimization, for expression in the relevant host cell. Codon-pair optimization is a method wherein the nucleotide sequences encoding a polypeptide have been modified with respect to their codon-usage, in particular the codon-pairs that are used, to obtain improved expression of the nucleotide sequence encoding the polypeptide and/or improved production of the encoded polypeptide. Codon pairs are defined as a set of two subsequent triplets (codons) in a coding sequence. Codon-pair optimization is preferably performed as described in WO2008/000632. 
     Preferably, the specificity of the modified polypeptide is substantially the same as before the improvement in expression level. This means for example that substrate specificity or binding specificity is substantially maintained. In this context, the term “substantially maintained” means that more than 60%, more than 65%, more than 70% or more than 75% of the specificity is maintained. Preferably more than 80%, 85% or 90% of the specificity is maintained. Most preferably, more than 95%, 96%, 97%, 98% or 99% of the specificity is maintained. 
     According to the method of the invention, the level of activity in the extracellular medium may be increased, which is an indication of improved secretion. However, specific activity of the modified polypeptide does not have to be increased, as long as it is not decreased. Therefore, specific activity is preferably substantially the same as or higher than before the improvement of secretion. In a preferred embodiment, specific activity is substantially the same as before improvement. In this context the phrase ‘substantially the same level of activity’ refers to a level of activity which differs less than 15%, preferably less than 12% or less than 10%, more preferably less than 8%, less than 6% or less than 4% from the level of activity of the parent polypeptide. 
     In the present context, the terms ‘polypeptide’ and protein′ are used interchangeably. Any type of polypeptide may have its secretion improved by the method of the invention. In a preferred embodiment, the polypeptide is one of the list cited earlier herein. For the purpose of this invention, it is defined here that in order to determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the complete sequences are aligned for optimal comparison purposes. In order to optimize the alignment between the two sequences gaps may be introduced in any of the two sequences that are compared. Such alignment may be carried out over the full length of the sequences being compared or on the basis of longest identity. The identity is the percentage of identical matches between the two sequences over the reported aligned region. 
     A comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. The homology or identity between two aligned sequences may be calculated as follows: Number of corresponding positions in the alignment showing an identical amino acid in both sequences divided by the total length of the alignment after subtraction of the total number of gaps in the alignment. The skilled person will be aware of the fact that several different computer programs are available to align two sequences and determine the homology between two sequences (Kruskal, J. B. (1983) An overview of sequence comparison In D. Sankoff and J. B. Kruskal, (ed.), Time warps, string edits and macromolecules: the theory and practice of sequence comparison, pp. 1-44 Addison Wesley). The percent identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm for the alignment of two sequences. (Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453). The algorithm aligns amino acid sequences as well as nucleotide sequences. The Needleman-Wunsch algorithm has been implemented in the computer program NEEDLE. For the purpose of this invention the NEEDLE program from the EMBOSS package was used (version 2.8.0 or higher,  EMBOSS: The European Molecular Biology Open Software Suite  (2000) Rice, P. Longden, I. and Bleasby, A. Trends in Genetics 16, (6) pp 276-277, http://emboss.bioinformatics.nl/). For protein sequences, EBLOSUM62 is used for the substitution matrix. For nucleotide sequences, EDNAFULL is used. Other matrices can be specified. For purpose of the invention, No brief is yes and the parameters used for alignment of amino acid sequences are a gap-open penalty of 10 and a gap extension penalty of 0.5. The skilled person will appreciate that all these different parameters will yield slightly different results but that the overall percentage identity of two sequences is not significantly altered when using different algorithms. The identity defined as herein may be obtained from NEEDLE by using the NOBRIEF option and is labelled in the output of the program as “longest-identity”. For purposes of the invention the level of identity (homology) between two sequences (amino acid or nucleotide) may be calculated according to the definition of “longest-identity” as can be carried out by using the program NEEDLE. 
     The protein sequences mentioned herein can further be used as a “query sequence” to perform a search against sequence databases, for example to identify other family members or related sequences. Such searches can be performed using the BLAST programs. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov). BLASTP is used for amino acid sequences and BLASTN for nucleotide sequences. In the BLAST program, the default settings may be used:
         Cost to open gap: default=5 for nucleotides/11 for proteins   Cost to extend gap: default=2 for nucleotides/1 for proteins   Penalty for nucleotide mismatch: default=−3   Reward for nucleotide match: default=1   Expect value: default=10   Wordsize: default=11 for nucleotides/28 for megablast/3 for proteins       

     The nucleic acid sequences as mentioned herein can further be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score=100, word-length=12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. 
     The sequence information as provided herein should not be so narrowly construed as to require inclusion of erroneously identified bases. The specific sequences disclosed herein can be readily used to isolate the complete gene from filamentous fungi, in particular  A. niger  which in turn can easily be subjected to further sequence analyses thereby identifying sequencing errors. 
     Unless otherwise indicated, all nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer and all amino acid sequences of polypeptides encoded by DNA molecules determined herein were predicted by translation of a nucleic acid sequence determined as above. Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art. As is also known in the art, a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion. 
     The person skilled in the art is capable of identifying such erroneously identified bases and knows how to correct for such errors. 
     A reference herein to a patent document or other matter which is given as prior art is not to be taken as an admission that that document or matter was known or that the information it contains was part of the common general knowledge as at the priority date of any of the claims. 
     The disclosure of each reference set forth herein is incorporated herein by reference in its entirety. 
     EXAMPLES 
     It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. 
     Strains 
       A. niger  Strains: 
     WT 1: This  A. niger  strain is used as a wild-type strain. This strain was deposited on 10 Aug. 1988 at the Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands. 
     WT 2: This  A. niger  strain is a WT 1 strain comprising a deletion of the gene encoding glucoamylase (glaA). WT 2 was constructed by using the “MARKER-GENE FREE” approach as described in EP 0 635 574 B1. In this patent it is extensively described how to delete glaA specific DNA sequences in the genome of CBS 513.88. The procedure resulted in a MARKER-GENE FREE ΔglaA recombinant  A. niger  CBS 513.88 strain, possessing finally no foreign DNA sequences at all. 
     WT 3: To disrupt the pepA gene encoding the major extracellular aspartic protease PepA in WT 2, pepA specific DNA sequences in the genome of WT 2 were deleted, as described by van den Hombergh et al. (van den Hombergh J P, Sollewijn Gelpke M D, van de Vondervoort P J, Buxton F P, Visser J. (1997)—Disruption of three acid proteases in  Aspergillus niger —effects on protease spectrum, intracellular proteolysis, and degradation of target proteins—Eur J Biochem. 247(2): 605-13). The procedure resulted in a MARKER-GENE FREE WT 3 strain, with the pepA gene inactivated in the WT 2 strain background. 
     WT 4: To delete the hdfA gene in WT 3, the method as earlier described in detail in WO05/095624 was used to generate  Aspergillus niger  WT 4 (ΔglaA, ΔpepA, ΔhdfA). 
     WT 5: This  A. niger  strain is a WT 4 strain comprising a deletion which results in an oxalate deficient  A. niger  strain. WT 5 was constructed by using the method as described in EP1157100 and U.S. Pat. No. 6,936,438, in which an oxalate deficient strain was obtained by deletion of the oahA gene, encoding oxaloacetate hydrolase, Strain WT 5 was selected as a representative strain with the oahA gene inactivated in the WT 4 strain background. 
     WT 6: This  A. niger  strain is a WT 5 strain comprising the deletion of three genes encoding alpha-amylases (amyB, amyBI and amyBII) in three subsequent steps. The construction of deletion vectors and genomic deletion of these three genes has been described in detail in WO2005095624. The vectors pDEL-AMYA, pDEL-AMYBI and pDEL-AMYBII, described in WO2005095624, have been used according the “MARKER-GENE FREE” approach as described in EP 0 635 574 B1. The procedure described above resulted in WT 6, an oxalate deficient, MARKER-GENE FREE ΔglaA, ΔpepA, ΔhdfA, ΔamyA, ΔamyBI and ΔamyBII amylase-negative recombinant  A. niger  CBS 513.88 strain, possessing finally no foreign DNA sequences at all. As such, strain WT 6 has a low amylase background, has a higher HR/NHR ratio for more efficient targeting of sequences and is more optimized for extracellular protein expression and detection compared to WT 1. 
     Fungal Alpha-Amylase Activity Assay 
     Example 2 
     To determine the alpha-amylase activity in  A. niger  culture broth, the Megazyme cereal alpha-amylase kit is used (Megazyme, CERALPHA alpha amylase assay kit, catalogue, ref. K-CERA, year 2000-2001), according protocol of the supplier. The measured activity is based on hydrolysis of non-reducing-end blocked p-nitrophenyl maltoheptaoside in the presence of excess glucoamylase and [alpha]-glucosidase. The amount of formed p-nitrophenol is a measure for alpha-amylase activity present in a sample. 
     Fungal Alpha-Amylase Activity Assay 
     Example 3 
     The assay to determine the alpha-amylase activity in  A. niger  culture broth was based on the Megazyme cereal alpha-amylase kit (Megazyme, CERALPHA alpha amylase assay kit, catalogue, ref. K-CERA, year 2000-2001), with some minor modifications to make it suitable for running on an auto-analyzer (Konelab Arena 30). The assay is executed at pH 5.20 taking into account the pH optima for a-glucosidase and amyloglucosidase (pH range 5-6). The measured activity is based on hydrolysis of non-reducing-end blocked p-nitrophenyl maltoheptaoside in the presence of excess glucoamylase and [alpha]-glucosidase. The amount of formed p-nitrophenol is a measure for alpha-amylase activity present in a sample. The change in absorption per minute of incubation in the linear part of the time curve is used as a measure for the activity of the enzyme. 
     Cellobiohydrolase Activity Assay 
     25 μL of enzyme solution was incubated with 10 mM para-nitrophenyl-8-D-cellobioside and 10 mM gluconolactone in 0.2 M sodium acetate buffer of pH 4.5 at 40° C. Samples were taken at time intervals of 10 min up to 30 min. The reaction was stopped by addition of cold 1M sodium carbonate solution at a ratio of 1:1. The absorbance of the final solutions is measured at 405 nm. The change in absorption per minute of incubation in the linear part of the time curve is used as a measure for the activity of the enzyme. 
     LC-90 
     The LabChip 90 system (Caliper) was used to perform 1D electrophoretic separation of supernatant (protein) samples. Sample preparation was performed according to supplier&#39;s manual (HT Protein express reagent kit Catno. 760328) using DTT as a denaturing agent. HT protein express LabChip (Catno. 760499) that was prepared according to supplier&#39;s manual was used in combination with the LabChip HT protein express 200 software program, for sizing of protein ranging from 14 kDa to 200 kDa, to analyze the samples. 
     SDS-PAGE Electrophoresis 
     Example 2 
     Sample Pre-Treatment: 
     30 μl sample was added to 35 μl water and 25 μl NuPAGE™ LDS sample buffer (4×) Invitrogen and 10 μl NuPAGE™ Sample Reducing agent (10×) Invitrogen. Samples were heated for ten minutes at 70° C. in a thermo mixer. 
     SDS-PAGE was performed in duplicate according to the supplier&#39;s instructions (Invitrogen: Gel: 4-12% Bis-Tris gel, Buffer: MES SDS running buffer, Runtime: 35 minutes). One of the two gels was used for blotting, 10 μl of the sample solutions and 1 μl marker M12 (Invitrogen) were applied on the gels (NuPAGE™ BisTris, Invitrogen). 
     The gels were run at 200V, using the XCELL Surelock, with 600 ml 20 times diluted MES-SDS buffer in the outer buffer chamber and 200 ml 20 times diluted MES-SDS buffer, containing 0.5 ml of antioxidant (NuPAGE™ Invitrogen) in the inner buffer chamber. After running, the gels were fixed for one hour with 50% Methanol/7% Acetic acid (50 ml), rinsed twice with demineralised water and stained with Sypro Ruby (50 ml, Invitrogen) overnight. 
     Images were made using the Typhoon 9200 (610 BP 30, Green (532 nm), PMT 600V, 100 micron) after washing the gel for ten minutes with demineralised water. 
     Quantitative SDS-PAGE 
     Example 3 
     To determine the concentration of the specific protein of interest in the presence of one or more other proteins with a different molecular weight on SDS-PAGE, a technique called quantitative SDS-PAGE (qPAGE) was used. For qPAGE a Bovine Serum Albumin protein standard from Sigma-Aldrich (P0914, 1 mg/ml) was used to create a calibration curve. The five calibration BSA samples ranged from 0.02 up to 0.10 mg/ml. The BSA calibration samples were applied together with the fermentation samples containing the protein of interest for which the protein concentration should be determined. From the calibration BSA standard samples, 65 μl of was added to 25 μl NuPAGE™ LDS sample buffer (4×) Invitrogen and 10 μl NuPAGE™ Sample Reducing Agent (10×) Invitrogen. The mixtures were heated in a thermomixer for 10 minutes at 70° C. 
     Every fermentation sample of the protein of interest was diluted in order to fit the calibration curve generated with the BSA standard samples. After this dilution, 65 μl of the diluted sample was added to 25 μl NuPAGE™ LDS sample buffer (4×) Invitrogen and 10 μl NuPAGE™ Sample Reducing Agent (10×) Invitrogen. The mixtures were heated in a thermomixer for 10 minutes at 70° C. Subsequently, 5 μl of the sample solutions; 5 ul of each of the BSA standards and 3 μl marker M12 (Invitrogen) were applied on the 17 wells gels (4-12% Bis-Tris NuPAGE™, Invitrogen). The gels were run for 55 minutes at 200V using the XCELL Surelock, with 600 ml 20× diluted MOPS SDS buffer in the outer buffer chamber and 200 ml 20× diluted SDS buffer, containing 0.5 ml of anti-oxidant (NuPAGE™ Invitrogen) in the inner buffer chamber. After running, the gels were fixed for one hour with 50% Methanol/7% Acetic acid (50 ml), rinsed twice with demineralized water and stained with Sypro Ruby (50 ml, Invitrogen) overnight. An image was made using the Typhoon 9200 (Amersham/GE-Healthcare; 610 BP 30, Green (532 nm), PMT 530V, 100 micron) after washing the gels with demineralized water. 
     Using the Typhoon with the program Image Quant Tools version 2003.02 (Amersham/GE-Healthcare), grey values were measured for the BSA and the band(s) corresponding to the protein of interest. Calibration lines were made with the five BSA dilutions (0.02, 0.04, 0.06, 0.08 and 0.10 mg/ml) and from these calibration lines the protein concentrations of the protein of interest (on the same gel) were calculated. It should be noted that a calibration curve was accepted if 1) it was constituted of at least 4 standards, 2) if for each standard, the bias with respect to the nominal concentration was within +/−15.0%. Only the (diluted) sample concentrations that felt between the lowest and the highest standards of an accepted calibration curve were calculated. 
     Molecular Biology Techniques 
     In the examples herein, using molecular biology techniques known to the skilled person (see: Sambrook &amp; Russell,  Molecular Cloning: A Laboratory Manual,  3rd Ed., CSHL Press, Cold Spring Harbor, N.Y., 2001), several genes were over expressed and others were down regulated as described below. 
     All gene replacement vectors described and used, were designed according to known principles and constructed according to routine cloning procedures. In essence, these vectors comprise approximately 1-2 kb flanking regions of the respective ORF sequences, to target for homologous recombination at the predestined genomic loci. In addition, they contain the  A. nidulans  bi-directional amdS selection marker for transformation, in-between direct repeats. The method applied for gene deletion in all examples herein uses linear DNA, which integrates into the genome at the homologous locus of the flanking sequences by a double cross-over, thus substituting the gene to be deleted by the amdS gene. After transformation, the direct repeats allow for the removal of the selection marker by a (second) homologous recombination event. The removal of the amdS marker can be done by plating on fluoro-acetamide media, resulting in the selection of marker-gene-free strains. Using this strategy of transformation and subsequent counter-selection, which is also described as the “MARKER-GENE FREE” approach in EP 0 635 574, the amdS marker can be used indefinitely in strain modification programs. The general procedure for gene disruption is depicted in FIG. 6 of WO2006040312. The general design of deletion vectors was previously described in EP635574B and WO 98/46772 and the use of general cloning vector pGBDEL for constructing deletion vectors and the counter-selection procedure were a.o. described in WO06/040312. 
     Examples of the general design of expression vectors and specifically pGBFIN-expression vectors for gene over expression, transformation, use of markers and selective media can be found in W0199846772, W0199932617, WO2001121779, WO2005095624, EP 635574B and WO2005100573. 
     Shake Flask Fermentations 
     Example 2 
       A. niger  strains were pre-cultured in 20 ml CSL pre-culture medium (100 ml flask, baffle) as described in the Examples: “ Aspergillus niger  shake flask fermentations” section of WO 99/32617. After growth for 18-24 hours at 34° C. and 170 rpm, 10 ml of this culture is transferred to Fermentation Medium (FM). Fermentation in FM is performed in 500 ml flasks with baffle with 100 ml fermentation broth at 34° C. and 170 rpm for the number of days indicated, generally as described in WO99/32617. 
     The CSL medium consisted of (in amount per litre): 100 g Corn Steep Solids (Roquette), 1 g NaH 2 PO 4 *H 2 0, 0.5 g MgSO 4 *7H 2 O, 10 g glucose*H 2 O and 0.25 g Basildon (antifoam). The ingredients were dissolved in demi-water and the pH was adjusted to pH 5.8 with NaOH or H 2 SO 4 ; 100 ml flasks with baffle and foam ball were filled with 20 ml fermentation medium and sterilized for 20 minutes at 120° C. 
     The fermentation medium (FM) consisted of (in amount per liter): 150 g maltose*H 2 O, 60 g Soytone (peptone), 1 g NaH 2 PO 4 *H2O, 15 g MgSO 4 *7H2O, 0.08 g Tween 80, 0.02 g Basildon (antifoam), 20 g MES, 1 g L-arginine. The ingredients were dissolved in demi-water and the pH was adjusted to pH 6.2 with NaOH or H 2 504; 500 ml flasks with baffle and foam ball were filled with 100 ml fermentation broth and sterilized for 20 minutes at 120° C. 
     Shake flask fermentations 
     Example 3 
       A. niger  strains were pre-cultured in 20 ml START pre-culture medium (100 ml flask, baffle) as described in the Examples: “ Aspergillus niger  shake flask fermentations” section of WO 99/32617. After growth for 18-24 hours at 34° C. and 170 rpm, 10 ml of this culture is transferred to Fermentation Medium (FM). Fermentation in FM is performed in 500 ml flasks with baffle with 100 ml fermentation broth at 34° C. and 170 rpm for the number of days indicated, generally as described in WO99/32617. 
     The START medium consisted of (in amount per litre): 30 g maltose, 10 g pepton aus casein (Merck 2239), 5 g yeast extract, 0.5 g MgSO4*7H2O, 1 g KH2PO4, 30 mg ZnCl2, 20 mg CaCl2, 10 mg MnSO4*4H2O, 0.3 g FeSO4*7H2O, 3 g Tween-80. The ingredients were dissolved in demi-water and the pH was adjusted to pH 5.5 with H2SO4; 100 ml flasks with baffle and foam ball were filled with 20 ml fermentation medium and sterilized for 15 minutes at 110° C. 
     The fermentation medium (FM) consisted of (in amount per liter): 60 g Glucose.H2O, 10 g maltose, 1 g KH2PO4, 12.5 g Yeast extract (Difco), 25 g Pepton aus casein (Merck 2239), 2 g K2SO4, 0.5 g MgSO4.7H2O, 3 ml ZnCl2 (10 mg/ml), 8 g CaCl2, 0.9 ml MnSO4.1H2O (10 mg/ml), 0.3 ml FeSO4.7H2O (10 mg/ml), 48.8 g MES. The ingredients were dissolved in demi-water and pH was adjusted to pH 5.6 with 6N KOH/H2SO4; 500 ml flasks with baffle and foam ball were filled with 100 ml fermentation medium and sterilized for 15 minutes at 110° C. Pre-culturing was done in START medium 
     Alternatively for the production of the EBA205 cellobiohydrolases the  A. niger  strains were pre-cultured using CSL medium. The CSL medium consisted of (in amount per litre): 100 g Corn Steep Solids (Roquette), 1 g NaH 2 PO 4 *H 2 O, 0.5 g MgSO 4 *7H 2 O, 10 g glucose*H 2 O and 0.25 g Basildon (antifoam). The ingredients were dissolved in demi-water and the pH was adjusted to pH 5.8 with NaOH or H 2 SO 4 ; 100 ml flasks with baffle and foam ball were filled with 20 ml fermentation medium and sterilized for 20 minutes at 120° C. 
     The fermentation was performed in CSM/MES medium. The CSM/MES medium consisted of (in amount per litre): 150 g Maltose.H2O, 60 g Soytone, 1 g NaH2PO4.H2O, 15 g (NH4)2SO4.H2O, 1 g MgSO4.7H2O. 0.08 g Tween80, 0.02 g Basildon, 20 g MES, 1 g L-arginine. The ingredients were dissolved in demi-water and pH was adjusted to pH 6.2 with NaOH/H2SO4; 500 ml flasks with baffle and foam ball were filled with 100 ml fermentation medium and sterilized for 15 minutes at 110° C. 
     Example 1 
     Relating Sequence Characteristics to High-Level Protein Production in  Aspergillus niger    
     Knowledge on factors influencing protein production could be employed to improve enzyme production rates in industrial settings. High production yields can be obtained for homologous gene expression, but yields are often limited for heterologous gene expression. To learn about sequence properties that might influence production rates, we applied sequence-based machine learning techniques to identify relevant protein sequence features. The composition of the protein sequence was found to be most predictive and interpretation revealed that, for both homologous and heterologous gene expression, the same features are important. Methionine (M) and Lysine (K) were found to have a negative contribution to high-level production. 
     Data 
     Two protein data sets were experimentally tested for high-level production and secretion, one for homologous gene expression and one for heterologous gene expression. Binary success scores were obtained by over-expression of the gene, introduced randomly into the  A. niger  genome behind the strong constitutive glucoamylase promoter. After growing in a shake flask, the filtered broth was put on gel. A positive success score was given when a clear visible band was present, negative otherwise. Proteins in the heterologous data set originated from 14 different fungal donor organisms (see Table 1). The resulting data from both hom and het data sets is set out in Table 2. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 The names and abbreviations of the 14 fungal donor organisms for 
               
               
                 which there are proteins in the heterologous data set (het) 
               
            
           
           
               
               
               
            
               
                   
                 abbreviation 
                 organism 
               
               
                   
                   
               
               
                   
                 Apul 
                 
                   Aureobasidium pullulans 
                 
               
               
                   
                 Ares 
                 
                   Amorphotheca resinae 
                 
               
               
                   
                 Ccin 
                 
                   Coprinus cinereus 
                 
               
               
                   
                 Cele 
                 
                   Cunninghamella elegans 
                 
               
               
                   
                 Clau 
                 
                   Cryptococcus laurentii 
                 
               
               
                   
                 Gpan 
                 
                   Geomyces pannorum 
                 
               
               
                   
                 Gtra 
                 
                   Gloeophyllum trabeum 
                 
               
               
                   
                 Ledo 
                 
                   Lentinula edodes 
                 
               
               
                   
                 Lsco 
                 
                   Leucosporidium scottii 
                 
               
               
                   
                 Opil 
                 
                   Ophiostoma piliferum 
                 
               
               
                   
                 Pchr 
                 
                   Phanerochaete chrysosporium 
                 
               
               
                   
                 Sthe 
                 
                   Sporotrichum thermophile 
                 
               
               
                   
                 Tlan 
                   Thermomyces  ( Humicola )  lanuginosa   
               
               
                   
                 Tver 
                 
                   Trametes versicolor 
                 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE S2 
               
             
            
               
                   
               
               
                 The total number of proteins and the number of successfully (pos) and unsuccessfully 
               
               
                 (neg) high-level produced proteins in both hom and het, and per organism in het 
               
            
           
           
               
               
            
               
                   
                 Organism 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                 hom 
                 het 
                 Apul 
                 Ares 
                 Ccin 
                 Cele 
                 Clau 
                 Gpan 
                 Gtra 
                 Ledo 
                 Lsco 
                 Opil 
                 Pchr 
                 Sthe 
                 Tlan 
                 Tver 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 # proteins 
                 345 
                 991 
                 87 
                 68 
                 1.40 
                 39 
                 14 
                 83 
                 81 
                 169 
                 60 
                 39 
                 55 
                 65 
                 16 
                 78 
               
               
                 % 
                 1.0 
                 1.0 
                 0.09 
                 0.07 
                 0.14 
                 0.04 
                 0.01 
                 0.08 
                 0.08 
                 0.17 
                 0.06 
                 0.04 
                 0.06 
                 0.07 
                 0.02 
                 0.08 
               
               
                 # pos 
                 178 
                 163 
                 13 
                 7 
                 29 
                 2 
                 2 
                 12 
                 20 
                 9 
                 4 
                 9 
                 18 
                 17 
                 6 
                 15 
               
               
                 % pos 
                 0.52 
                 0.16 
                 0.15 
                 0.1 
                 0.21 
                 0.05 
                 0.14 
                 0.14 
                 0.25 
                 0.05 
                 0.07 
                 0.23 
                 0.33 
                 0.26 
                 0.38 
                 0.2 
               
               
                 # neg 
                 167 
                 828 
                 74 
                 61 
                 111 
                 37 
                 12 
                 71 
                 61 
                 160 
                 56 
                 30 
                 37 
                 46 
                 10 
                 60 
               
               
                 % neg 
                 0.48 
                 0.84 
                 0.85 
                 0.9 
                 0.79 
                 0.95 
                 0.86 
                 0.86 
                 0.75 
                 0.95 
                 0.93 
                 0.77 
                 0.67 
                 0.74 
                 0.62 
                 0.8 
               
               
                   
               
            
           
         
       
     
     All proteins have a signal peptide (length &gt;10 amino acids) as predicted by SignalP 3.0 [Dyrlov Bendtsen J, Nielsen H, von Heijne G, Brunak S (2004) Improved prediction of signal peptides: SignalP 3.0. Journal of molecular biology 340: 783-795] and a total sequence length longer than 100 amino acids. Proteins containing an ER retention signal (Cterminal KDEL) and proteins predicted to be transmembrane by both TMHMM [Krogh A, Larsson B, Von Heijne G, Sonnhammer E (2001) Predicting transmembrane protein topology with a hidden markov model: application to complete genomes. Journal of molecular biology 305: 567-580] and Phobius [. K″all L, Krogh A, Sonnhammer E (2004) A combined transmembrane topology and signal peptide prediction method. Journal of molecular biology 338: 1027-1036] were filtered out of the data set. 
     To avoid biasing subsequent analyses, sequence redundancy was reduced using BLASTCLUST [Dondoshansky I (2002) Blastclust (NCBI Software Development Toolkit). NCBI, Bethesda, Md]. Two sequences were considered redundant when the aligned sequences shared &gt;40% identity over a length of minimal 90% for at least one of the sequences. From the obtained protein clusters, we selected a representative protein, with the shortest average distance to all other proteins in the cluster, and removed the remainder. If a cluster contained proteins with both positive and negative labels, one positive and one negative protein was selected. This resulted in data sets horn and het containing 345 proteins (187 positives, 167 negatives) and 991 proteins (163 positives, 828 negatives), respectively. 
     To train a classifier on horn en test it on het, a data set het hom  was constructed that contains the het data set without proteins that share &gt;40% identity with any protein in horn. This data set contained 906 (128 positives, 778 negatives) proteins. 
     Classification 
     A linear support vector machine (LIBSVM [Chang C, Lin C (2011) LIBSVM: a library for support vector machines. ACM Transactions on Intelligent Systems and Technology (TIST) 2: 27]) was used for classification [Ben-Hur A, Ong C, Sonnenburg S, Scholkopf B, Ratsch G (2008) Support vector machines and kernels for computational biology. PLoS computational biology 4: e1000173]. Parameter C was optimized using a simple grid search. In case of multiple kernels, both C and the kernel weights were optimized using a covariance matrix adaptation evolutionary algorithm (CMA) [Hulsman M, Reinders M, de Ridder D (2009) Evolutionary optimization of kernel weights improves protein complex comembership prediction. IEEE/ACM Transactions on Computational Biology and Bioinformatics (TCBB) 6: 427-437; and Hansen N (2006) The CMA evolution strategy: a comparing review. Towards a new evolutionary computation: 75-102]. Classifier performance on a data set was obtained by running a double 10-fold cross-validation (CV) loop, in which C and the kernel weights were optimized in an inner CV-loop on the training set. As performance measure we used the area under the receiver-operator characteristic curve (auroc) [Fawcett T (2006) An introduction to ROC analysis. Pattern recognition letters 27: 861-874]. Classifier performance is defined as the average auroc over the CV-loops. When separate training and test sets are used, a classifier was trained on the first data set, optimizing C and kernel weights in a 10-fold CV-loop, and tested on the second data set, again using the auroc as performance measure. 
     Results 
     We exploited a dataset of proteins to predict successful high-level secretion, showing that the frequency of occurrence of a limited number amino acids are most predictive for high-yield secretion (see  FIG. 1 ). Lysine and methione show the highest negative correlation. 
       FIG. 2  shows the correlation between weights for both het and hom classifiers. The correlation shows that both classifiers are indeed similar. For both horn and het, a remarkable negative negative contribution of methionine (M) and lysine (K) was found. Considering amino acid properties, it was observed that the basic and the sulphur-containing amino acids have a negative contribution. 
     Prediction performance scores (AUROC) for amino acid composition were very good. Score 0.83 and 0.70 respectively for the mature protein AA composition, based on horn training and evaluation for horn, het training and evaluation on het, respectively. Similar characteristics are important for both data sets, as derived from ROC-curves of the composition-based classifiers. Remarkably, similar weights resulted as the classifiers trained on het, suggesting that the homologous classifier generalizes well to predict high-level production for het. The good generalization suggests that classifiers trained on hom and het are similar, i.e. perform their predictions based on the same sequence characteristics. 
     Example 2 
     Construction  A. niger  Expression Vectors for Wild-Type Enzymes and Enzyme Variants According a Method of the Invention 
     In this example a number of expression vectors are constructed for variants of the enzymes of the invention. All variants for expression in  Aspergillus  are cloned in a pGBFIN-5 or a pGBTOP-expression vector. The construction, general layout and use of these vectors is described in detail in WO1999/32617. 
       A. niger  Constructs 
     For expression of EBA205 and FUA (SEQ ID No. 5 and 7) in  A. niger , the cDNA sequence was codon pair optimized using the method described in WO2008/000632 (SEQ ID No. 54 and 55) and is prepared synthetically (e.g. DNA2.0, USA, GeneArt, Germany). 
     The DNA sequence of the amyB gene encoding the alpha-amylase protein (FUA) was disclosed in J. Biochem. Mol. Biol. 37(4):429-438(2004) (Matsubara T., Ammar Y. B., Anindyawati T., Yamamoto S., Ito K., lizuka M., Minamiura N. “Molecular cloning and determination of the nucleotide sequence of raw starch digesting alpha-amylase from  Aspergillus awamori  KT-I I.”) and also can be retrieved from EMBL Nucleotide Sequence Database (http://www.ebi.ac.uk/embl/index.html) under accession number AB083159.  A. niger  WT 6 is a strain where both the native 2 copies amyBI and amyBII were removed from the genome [An12g06930 and An05g02100] ( A. niger  genome sequence (EMBL: AM269948-AM270415; Pel et al., “Genome sequencing and analysis of the versatile cell factory  Aspergillus niger  CBS 513.88”). Nat Biotechnol. 2007 February; 25 (2):221-231). 
     The  T. emersonii  EBA205, cellobiohydrolase 1 is identical to the one in Genbank [AAL33603]. In the case of FUA, the signal sequence for secretion (SEQ ID NO: 2) is replaced by an optimal signal sequence (SEQ ID NO: 3) (as detailed in WO2010121933). 
     For both proteins, cDNA sequences including the signal sequences were designed by codon-pair optimization directly from the amino acid sequence (as detailed in WO2008000632). The translational initiation sequence of the glucoamylase glaA promoter is modified into 5′-CACCGTCAAA ATG-3′ in all expression constructs generated (as also detailed in WO2006/077258). In addition, an optimal translational termination sequence is used: 5′-TAAA-3′ (as detailed in WO2006/077258) in all expression constructs. 
     The  Talaromyces emersonii  EBA205 was codon pair optimized (as detailed in WO2008000632) and with all appropriate control elements is synthesized completely as PacI-AscI fragment, subcloned and sequence verified. The PacI-AscI restriction sites at the ends of the synthesized fragments are used to allow cloning in the large vector fragment of a PacI-AscI digested pGBFIN-5 expression vector, generating an expression vector pGBFINEBA205 (see  FIG. 3 ). In addition and in a similar way as for the EBA205, an optimized FUA construct was codon pair optimized (as detailed in WO2008000632) and with all appropriate control elements is cloned as PacI-AscI fragment in pGBFIN-5, generating pGBFINFUA ( FIG. 4 ). 
     Protein sequence optimization (PSO) is applied to the FUA,  A. niger  fungal amylase protein sequence [An12g06930; An05g02100], and the  T. emersonii  EBA205, cellobiohydrolase 1 [Genbank: AAL33603]. All proteins designed are obtained via gene-synthesis providers (e.g. DNA2.0, USA, GeneArt, Germany). The obtained fragments are cloned in a pGBFIN vector using the method essentially as described above, generating various pGBFIN-expression constructs. All relevant protein modification details for  A. niger  FUA and EBA205 constructs can be found in Tables 3-10. The complete overview with decrease/increase in methionine and lysine content for the mature protein is given in Table 11 and 12. 
     The experimental design is set-up in such a way that either M-residues, or K-residues, or both M and K residues are replaced in order to study the generic effect of reducing the compositional percentage of these amino-acids. For providing additional evidence on the specific effect of M or K, additional constructs with increased M or K content are used. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 EBA205: decreased M content 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 gene 
                 164 
                 228 
                 252 
                 283 
                 374 
                 378 
                 382 
                 386 
                 396 
                 M 
               
               
                   
               
               
                 EBA205 
                 M 
                 M 
                 M 
                 M 
                 M 
                 M 
                 M 
                 M 
                 M 
                 reduction 
               
               
                 mature 
                 146 
                 210 
                 234 
                 265 
                 356 
                 360 
                 364 
                 368 
                 378 
               
               
                 substitute 
                 L 
                 L 
                 R 
                 L 
                   
                 L 
                 H 
                 L 
                 A 
                 % 
               
               
                 EBA205-M01 
                   
                   
                 R 
                 L 
                   
                   
                   
                 L 
                   
                 33% 
               
               
                 EBA205-M02 
                   
                   
                 R 
                   
                 I 
                 L 
                   
                 L 
                   
                 44% 
               
               
                 EBA205-M03 
                   
                   
                 R 
                 L 
                 I 
                 L 
                   
                   
                   
                 44% 
               
               
                 EBA205-M04 
                   
                   
                 R 
                 L 
                 I 
                 L 
                   
                 L 
                   
                 56% 
               
               
                 EBA205-M05 
                   
                 L 
                 R 
                 L 
                 V 
                 L 
                   
                 L 
                   
                 67% 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
               
                   
               
               
                 EBA205: increased M content 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 gene 
                 32 
                 123 
                 126 
                 134 
                 148 
                 170 
                 195 
                 201 
                 236 
                 293 
               
               
                   
               
               
                 EBA205 wt 
                 L 
                 L 
                 L 
                 I 
                 V 
                 V 
                 L 
                 E 
                 I 
                 K 
               
               
                 mature 
                 14 
                 105 
                 108 
                 116 
                 130 
                 152 
                 177 
                 183 
                 218 
                 275 
               
               
                 substitute 
                 M 
                 M 
                 M 
                 M 
                 M 
                 M 
                 M 
                 M 
                 M 
                 M 
               
               
                 EBA205-M06 
                   
                   
                 M 
                 M 
                   
                 M 
               
               
                 EBA205-M07 
                 M 
                 M 
                 M 
                 M 
                   
                 M 
               
               
                 EBA205-M08 
                 M 
                 M 
                 M 
                 M 
                   
                 M 
                   
                   
                   
                 M 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 gene 
                 301 
                 371 
                 M 
               
               
                   
                   
               
               
                   
                 EBA205 wt 
                 F 
                 L 
                 increase 
               
               
                   
                 mature 
                 283 
                 353 
               
               
                   
                 substitute 
                 M 
                 M 
                 % 
               
               
                   
                 EBA205-M06 
                   
                 M 
                 44% 
               
               
                   
                 EBA205-M07 
                   
                   
                 56% 
               
               
                   
                 EBA205-M08 
                   
                 M 
                 78% 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 EBA205: decreased K content 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 gene 
                 136 
                 172 
                 177 
                 181 
                 196 
                 293 
                 322 
                 330 
                 356 
                 373 
                 444 
               
               
                   
               
               
                 EBA205 
                 K 
                 K 
                 K 
                 K 
                 K 
                 K 
                 K 
                 K 
                 K 
                 K 
                 K 
                 % 
               
               
                 mature 
                 118 
                 154 
                 159 
                 163 
                 178 
                 275 
                 304 
                 312 
                 338 
                 355 
                 426 
               
               
                 substitute 
                 N 
                 R 
                 T 
                 Q 
                   
                 L 
                 R 
                 N 
                   
                 G 
                 R 
               
               
                 EBA205-K01 
                 N 
                   
                   
                   
                   
                   
                 R 
                 N 
                   
                 G 
                   
                 36% 
               
               
                 EBA205-K02 
                 N 
                 R 
                   
                   
                   
                   
                 R 
                 N 
                   
                 G 
                 R 
                 55% 
               
               
                 EBA205-K03 
                 N 
                   
                   
                   
                   
                 L 
                 R 
                 N 
                   
                 G 
                   
                 45% 
               
               
                 EBA205-K04 
                 N 
                 R 
                 T 
                 Q 
                   
                   
                 R 
                 N 
                   
                 G 
                 R 
                 73% 
               
               
                 EBA205-K05 
                 N 
                 R 
                 T 
                 Q 
                   
                 L 
                 R 
                 N 
                   
                 G 
                 R 
                 82% 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6 
               
               
                   
               
               
                 EBA205: increased K content 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 gene 
                 36 
                 49 
                 83 
                   
                 112 
                 117 
                 131 
                 140 
                 208 
                 286 
                 298 
                 299 
               
               
                   
               
               
                 EBA205 wt 
                 E 
                 A 
                 V 
                 R 
                 N 
                 S 
                 T 
                 R 
                 Q 
                 T 
                 T 
                 Q 
               
               
                 mature 
                 18 
                 31 
                 65 
                 92 
                 94 
                 99 
                 113 
                 122 
                 190 
                 268 
                 280 
                 281 
               
               
                 substitute 
                 K 
                 K 
                 K 
                 K 
                 K 
                 K 
                 K 
                 K 
                 K 
                 K 
                 K 
                 K 
               
               
                 EBA205-K06 
                 K 
                 K 
                   
                 K 
                   
                   
                   
                   
                   
                   
                   
                 K 
               
               
                 EBA205-K07 
                   
                 K 
                   
                 K 
                   
                   
                   
                 K 
                   
                   
                   
                 K 
               
               
                 EBA205-K08 
                 K 
                   
                   
                 K 
                   
                   
                   
                   
                 K 
                 K 
                   
                 K 
               
               
                 EBA205-K09 
                 K 
                 K 
                 K 
                 K 
                   
                   
                   
                   
                   
                   
                   
                 K 
               
               
                 EBA205-K10 
                 K 
                 K 
                   
                 K 
                   
                   
                   
                 K 
                 K 
                 K 
                   
                 K 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 gene 
                 300 
                 319 
                 338 
                 357 
                 367 
                 368 
                 376 
                 380 
                 407 
                 411 
               
               
                   
               
               
                 EBA205 wt 
                 P 
                 S 
                 D 
                 Q 
                 Q 
                 H 
                 A 
                 Q 
                 A 
                 T 
               
               
                 mature 
                 282 
                 301 
                 320 
                 339 
                 349 
                 350 
                 358 
                 362 
                 389 
                 393 
               
               
                 substitute 
                 K 
                 K 
                 K 
                 K 
                 K 
                 K 
                 K 
                 K 
                 K 
                 K 
               
               
                 EBA205-K06 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 36% 
               
               
                 EBA205-K07 
                   
                 K 
                   
                   
                   
                 K 
                   
                   
                   
                   
                 55% 
               
               
                 EBA205-K08 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 45% 
               
               
                 EBA205-K09 
                   
                   
                   
                 K 
                   
                   
                 K 
                   
                   
                   
                 64% 
               
               
                 EBA205-K10 
                   
                 K 
                   
                   
                   
                 K 
                   
                   
                   
                   
                 82% 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 FUA: decreased M content 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 gene 
                 75 
                 132 
                 135 
                 143 
                 266 
                 289 
                 295 
                 416 
                 475 
               
               
                   
               
               
                 wt 
                 M 
                 M 
                 M 
                 M 
                 M 
                 M 
                 M 
                 M 
                 M 
                 reduction 
               
               
                 mature 
                 55 
                 112 
                 115 
                 123 
                 246 
                 269 
                 275 
                 396 
                 455 
                 M 
               
               
                 substitute 
                 L 
                 I 
                 L 
                 F 
                 L 
                 I 
                 E 
                 L 
                 L 
                 % 
               
               
                 FUA-M01 
                   
                   
                   
                   
                 x 
                   
                   
                 x 
                 x 
                 33% 
               
               
                 FUA-M02 
                   
                   
                   
                   
                 x 
                 x 
                   
                   
                 x 
                 33% 
               
               
                 FUA-M03 
                   
                   
                   
                   
                 x 
                   
                 x 
                 x 
                   
                 33% 
               
               
                 FUA-M04 
                   
                   
                   
                   
                 x 
                 x 
                 x 
                 x 
                 x 
                 56% 
               
               
                 FUA-M05 
                   
                   
                   
                 x 
                   
                 x 
                 x 
                 x 
                 x 
                 56% 
               
               
                 FUA-M06 
                 x 
                   
                   
                 x 
                 x 
                   
                   
                 x 
                 x 
                 56% 
               
               
                 FUA-M07 
                 x 
                   
                   
                 x 
                 x 
                 x 
                 x 
                   
                   
                 56% 
               
               
                 FUA-M08 
                 x 
                 x 
                 x 
                 x 
                 x 
                 x 
                 x 
                 x 
                 x 
                 100%  
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 FUA: increased M content 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 gene 
                 180 
                 206 
                 214 
                 247 
                 274 
                 299 
                 313 
                 328 
                 455 
               
               
                   
               
               
                 wt 
                 Q 
                 E 
                 L 
                 C 
                 I 
                 V 
                 V 
                 I 
                 T 
                 increase 
               
               
                 mature 
                 160 
                 186 
                 194 
                 227 
                 254 
                 279 
                 293 
                 308 
                 435 
                 M 
               
               
                 substitute 
                 M 
                 M 
                 M 
                 M 
                 M 
                 M 
                 M 
                 M 
                 M 
                 % 
               
               
                 FUA-M09 
                   
                   
                   
                   
                 x 
                 x 
                   
                 x 
                   
                 33% 
               
               
                 FUA-M10 
                   
                 x 
                   
                   
                   
                   
                 x 
                   
                 x 
                 33% 
               
               
                 FUA-M11 
                   
                 x 
                   
                   
                 x 
                 x 
                 x 
                 x 
                 x 
                 67% 
               
               
                 FUA-M12 
                 x 
                 x 
                 x 
                 x 
                 x 
                 x 
                 x 
                 x 
                 x 
                 100%  
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 9 
               
               
                   
               
               
                 FUA: decreased K content 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 gene 
                 56 
                 68 
                 121 
                 158 
                 200 
                 204 
                 229 
                 233 
                 241 
                 241 
                 283 
                 300 
               
               
                   
               
               
                 wt 
                 K 
                 K 
                 K 
                 K 
                 K 
                 K 
                 K 
                 K 
                 K 
                 K 
                 K 
                 K 
               
               
                 mature 
                 36 
                 48 
                 101 
                 138 
                 180 
                 184 
                 209 
                 213 
                 221 
                 221 
                 263 
                 280 
               
               
                 substitute 
                 I 
                 H 
                 R 
                 N 
                 S 
                 R 
                 R 
                 P 
                 D 
                 S 
                 Q 
                 A 
               
               
                 FUA-K01 
                   
                   
                   
                 x 
                   
                 x 
                   
                   
                 x 
                   
                 x 
               
               
                 FUA-K02 
                   
                 x 
                   
                 x 
                   
                   
                   
                   
                   
                 x 
                   
                 x 
               
               
                 FUA-K03 
                   
                   
                 x 
                   
                   
                   
                   
                   
                   
                   
                 x 
               
               
                 FUA-K04 
                   
                 x 
                   
                 x 
                   
                 x 
                   
                   
                 x 
                   
                 x 
               
               
                 FUA-K05 
                   
                   
                 x 
                 x 
                   
                   
                   
                   
                   
                 x 
                 x 
                 x 
               
               
                 FUA-K06 
                   
                 x 
                 x 
                 x 
                   
                 x 
                   
                   
                   
                 x 
                 x 
                 x 
               
               
                 FUA-K07 
                   
                 x 
                 x 
                 x 
                   
                 x 
                   
                 x 
                   
                 x 
                 x 
                 x 
               
               
                 FUA-K08 
                 x 
                 x 
                 x 
                 x 
                 x 
                 x 
                 x 
                 x 
                   
                 x 
                 x 
                 x 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 gene 
                 332 
                 380 
                 395 
                 395 
                 403 
                 409 
                 418 
                 432 
                 488 
                 493 
               
               
                   
               
               
                 wt 
                 K 
                 K 
                 K 
                 K 
                 K 
                 K 
                 K 
                 K 
                 K 
                 K 
                 reduction 
               
               
                 mature 
                 312 
                 360 
                 375 
                 375 
                 383 
                 389 
                 398 
                 412 
                 468 
                 473 
                 K 
               
               
                 substitute 
                 Q 
                 Q 
                 Q 
                 A 
                 Q 
                 Q 
                 R 
                 L 
                 R 
                 G 
                 % 
               
               
                 FUA-K01 
                   
                   
                   
                   
                   
                 x 
                   
                   
                   
                 x 
                 30% 
               
               
                 FUA-K02 
                   
                 x 
                   
                   
                   
                   
                   
                   
                   
                 x 
                 30% 
               
               
                 FUA-K03 
                   
                   
                 x 
                   
                 x 
                 x 
                   
                 x 
                   
                   
                 30% 
               
               
                 FUA-K04 
                   
                 x 
                   
                 x 
                   
                 x 
                   
                 x 
                   
                 x 
                 50% 
               
               
                 FUA-K05 
                   
                 x 
                 x 
                   
                 x 
                 x 
                   
                   
                   
                 x 
                 50% 
               
               
                 FUA-K06 
                   
                 x 
                 x 
                   
                 x 
                 x 
                   
                 x 
                   
                 x 
                 65% 
               
               
                 FUA-K07 
                   
                 x 
                 x 
                   
                 x 
                 x 
                   
                 x 
                 x 
                 x 
                 75% 
               
               
                 FUA-K08 
                 x 
                 x 
                 x 
                   
                 x 
                 x 
                 x 
                 x 
                 x 
                 x 
                 100%  
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 10 
               
               
                   
               
               
                 FUA: increased K content 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 gene 
                 27 
                 63 
                 87 
                 212 
                 304 
                 384 
                 387 
                 390 
                 417 
                 453 
               
               
                   
               
               
                 wt 
                 R 
                 Q 
                 A 
                 G 
                 P 
                 S 
                 A 
                 N 
                 R 
                 Q 
               
               
                 mature 
                 7 
                 43 
                 67 
                 192 
                 284 
                 364 
                 367 
                 370 
                 397 
                 433 
               
               
                 substitute 
                 K 
                 K 
                 K 
                 K 
                 K 
                 K 
                 K 
                 K 
                 K 
                 K 
               
               
                 FUA-K09 
                   
                   
                 x 
                 x 
                 x 
                   
                 x 
                   
                   
                 x 
               
               
                 FUA-K10 
                   
                   
                 x 
                 x 
                 x 
                   
                 x 
                   
                 x 
                 x 
               
               
                 FUA-K11 
                 x 
                 x 
                 x 
                 x 
                 x 
                 x 
                 x 
                 x 
                 x 
                 x 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 gene 
                 470 
                 481 
               
               
                   
                   
               
               
                   
                 wt 
                 N 
                 R 
                 increase 
               
               
                   
                 mature 
                 450 
                 461 
                 K 
               
               
                   
                 substitute 
                 K 
                 K 
                 % 
               
               
                   
                 FUA-K09 
                   
                   
                 25% 
               
               
                   
                 FUA-K10 
                   
                 x 
                 35% 
               
               
                   
                 FUA-K11 
                 x 
                 x 
                 60% 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                 Overview EBA-205 experiment (21 constructs) 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 Protein 
                 M− 
                 M+ 
                 K− 
                 K+ 
                 KM− 
               
               
                   
                   
               
               
                   
                 EBA-205 
                   
                   
                   
                   
                   
               
               
                   
                 EBA205-M01 
                 33% 
               
               
                   
                 EBA205-M02 
                 44% 
               
               
                   
                 EBA205-M03 
                 44% 
               
               
                   
                 EBA205-M04 
                 56% 
               
               
                   
                 EBA205-M05 
                 67% 
               
               
                   
                 EBA205-M06 
                   
                 44% 
               
               
                   
                 EBA205-M07 
                   
                 56% 
               
               
                   
                 EBA205-M08 
                   
                 78% 
               
               
                   
                 EBA205-K01 
                   
                   
                 33% 
               
               
                   
                 EBA205-K02 
                   
                   
                 44% 
               
               
                   
                 EBA205-K03 
                   
                   
                 44% 
               
               
                   
                 EBA205-K04 
                   
                   
                 56% 
               
               
                   
                 EBA205-K05 
                   
                   
                 67% 
               
               
                   
                 EBA205-K06 
                   
                   
                   
                 36% 
               
               
                   
                 EBA205-K07 
                   
                   
                   
                 55% 
               
               
                   
                 EBA205-K08 
                   
                   
                   
                 45% 
               
               
                   
                 EBA205-K09 
                   
                   
                   
                 64% 
               
               
                   
                 EBA205-K10 
                   
                   
                   
                 82% 
               
               
                   
                 EBA205-KM01 
                 33% 
                   
                 45% 
                   
                 35% 
               
               
                   
                 EBA205-KM02 
                 44% 
                   
                 64% 
                   
                 60% 
               
               
                   
                   
               
            
           
         
       
     
                     TABLE 12                  Overview FUA experiment (26 constructs)                                     Protein   M−   M+   K−   K+   KM−               FUA                           FUA-M01   33%       FUA-M02   33%       FUA-M03   33%       FUA-M04   56%       FUA-M05   56%       FUA-M06   56%       FUA-M07   56%       FUA-M08   100%        FUA-M09       33%       FUA-M10       33%       FUA-M11       67%       FUA-M12       100%        FUA-K01           30%       FUA-K02           30%       FUA-K03           30%       FUA-K04           50%       FUA-K05           50%       FUA-K06           65%       FUA-K07           75%       FUA-K08           100%        FUA-K09               25%       FUA-K10               35%       FUA-K11               60%       FUA-KM01   33%       56%       31%       FUA-KM02   30%       60%       62%                    
Expression of Wild-Type and PSO Optimized Fungal Enzymes in  A. niger  
 
     The pGBFINFUA- and pGBFINEBA205-expression constructs are introduced by transformation using  A. niger . In order to introduce the different pGBFINFUA- and pGBFINEBA205-vectors (see Tables 3 to 10) in WT 6, a transformation and subsequent selection of transformants is carried out as described in WO1998/46772 and WO1999/32617. In brief, linear DNA of all the pGBFIN-constructs was isolated and used to transform  A. niger  WT 6. Transformants were selected on acetamide media and colony purified according standard procedures. Colonies were diagnosed for integration at the glaA locus and for copy number using PCR. Three independent transformants for each pGBFINFUA- and pGBFINEBA205-construct with similar estimated copy numbers (putative single copy) were selected and named using the number of the transforming plasmid, as for example FUA-M01-1, FUA-M01-2, FUA-M01-3, EBA205-M01-1, EBA205-M01-2, EBA205-M01-3, etc. . . . , respectively. 
     EBA-205 Constructs and Expression 
     Transformants of the  A. niger  EBA205-transformants containing the different constructs are picked, resulting about 80% successful overexpression strains, including the wild-type EA205 (wild-type protein). For the subset, the production of CBHI expressed by each of the transformants of the  A. niger  EBA205-transformants containing the different constructs, is measured in the culture supernatant at day 3. In addition, the culture supernatants sampled at day 4 are analyzed by SDS gel electrophoresis and staining. 
     Protein sequence optimization by reducing M, K or both M and K amino acid has a positive impact on protein secretion and results in increased protein expression levels and increased activity levels for the cellobiohydrolase I enzyme. A clear positive trend may be seen when plotting from left to right the percentage {increase-wild type-percentage decrease} of methionine versus expression level, and in a similar way for activity. The same effect may be seen for lysine. 
     FUA Constructs and Expression 
     Transformants of the  A. niger  FUA-transformants containing the different constructs are picked, resulting about 80% successful overexpression strains, including the wild-type FUA (wild-type protein). For the subset, the production of alpha-amylase expressed by each of the transformants of the  A. niger  FUA-transformants containing the different constructs, is measured in the culture supernatant at day 3. In addition, the culture supernatants sampled at day 4 are analyzed by SDS gel electrophoresis and staining. 
     Protein sequence optimization by reducing M, K or both M and K amino acid has a positive impact on protein secretion and results in detectable and thus increased protein expression levels and increased activity levels for the alpha-amylase enzyme. A clear positive trend is observed when plotting from left to right the percentage {increase-wild type-percentage decrease of methionine versus expression level, and in a similar way for activity. The same effect may be seen for lysine. 
     Example 3 
     Construction  A. niger  Expression Vectors for Wild-Type Enzymes and Enzyme Variants According a Method of the Invention 
     In this example a number of expression vectors were constructed for variants of the enzymes of the invention. All variants for expression in  Aspergillus  were cloned in a pGBFIN-5 or a pGBTOP-expression vector. The construction, general layout and use of these vectors is described in detail in WO1999/32617. 
       A. niger  Constructs 
     For expression of EBA205 and FUA (SEQ ID No. 5 and 7) in  A. niger , the cDNA sequence was codon pair optimized using the method described in WO2008/000632 (SEQ ID No. 54 and 55) and is prepared synthetically (e.g. DNA2.0, USA, GeneArt, Germany). 
     The DNA sequence of the amyB gene encoding the alpha-amylase protein (FUA) was disclosed in J. Biochem. Mol. Biol. 37(4):429-438(2004) (Matsubara T., Ammar Y. B., Anindyawati T., Yamamoto S., Ito K., lizuka M., Minamiura N. “Molecular cloning and determination of the nucleotide sequence of raw starch digesting alpha-amylase from  Aspergillus awamori  KT-I I.”) and also can be retrieved from EMBL Nucleotide Sequence Database (http://www.ebi.ac.uk/embl/index.html) under accession number AB083159.  A. niger  WT 6 is a strain where both the native 2 copies amyBI and amyBII were removed from the genome [An12g06930 and An05g02100] ( A. niger  genome sequence (EMBL: AM269948-AM270415; Pel et al., “Genome sequencing and analysis of the versatile cell factory  Aspergillus niger  CBS 513.88”). Nat Biotechnol. 2007 February; 25 (2):221-231). 
     The  T. emersonii  EBA205, cellobiohydrolase 1 is identical to the one in Genbank [AAL33603]. In the case of FUA, the signal sequence for secretion (SEQ ID NO: 2) is replaced by an optimal signal sequence (SEQ ID NO: 3) (as detailed in WO2010121933). 
     For both proteins, cDNA sequences including the signal sequences were designed by codon-pair optimization directly from the amino acid sequence (as detailed in WO2008000632). The translational initiation sequence of the glucoamylase glaA promoter was modified into 5′-CACCGTCAAA ATG-3′ in all expression constructs generated (as also detailed in WO2006/077258). In addition, an optimal translational termination sequence was used: 5′-TAAA-3′ (as detailed in WO2006/077258) in all expression constructs. 
     The  Talaromyces emersonii  EBA205 was codon pair optimized (as detailed in WO2008000632) and with all appropriate control elements is synthesized completely as PacI-AscI fragment, subcloned and sequence verified. The PacI-AscI restriction sites at the ends of the synthesized fragments are used to allow cloning in the large vector fragment of a PacI-AscI digested pGBFIN-5 expression vector, generating an expression vector pGBFINEBA205 (see  FIG. 3 ). In addition and in a similar way as for the EBA205, an optimized FUA construct was codon pair optimized (as detailed in WO2008000632) and with all appropriate control elements was cloned as PacI-AscI fragment in pGBFIN-5, generating pGBFINFUA ( FIG. 4 ). 
     Protein sequence optimization (PSO) was applied to the FUA,  A. niger  fungal amylase protein sequence [An12g06930; An05g02100], and the  T. emersonii  EBA205, cellobiohydrolase 1 [Genbank: AAL33603]. All proteins designed were obtained via gene-synthesis providers (e.g. DNA2.0, USA, GeneArt, Germany). The obtained fragments were cloned in a pGBFIN vector using the method essentially as described above, generating various pGBFIN-expression constructs. All relevant protein modification details for  A. niger  FUA and EBA205 constructs can be found in Tables 3-10. The complete overview with decrease/increase in methionine and lysine content for the mature protein is given in Table 11 and 12. 
     The experimental design was set-up in such a way that either M-residues, or K-residues, or both M and K residues were replaced in order to study the generic effect of reducing the compositional percentage of these amino-acids. For providing additional evidence on the specific effect of M or K, additional constructs with increased M or K content were used. 
     Expression of Wild-Type and PSO Optimized Fungal Enzymes in  A. niger    
     The pGBFINFUA- and pGBFINEBA205-expression constructs were introduced by transformation using  A. niger . In order to introduce the different pGBFINFUA- and pGBFINEBA205-vectors (see Tables 3 to 10) in WT 6, a transformation and subsequent selection of transformants was carried out as described in WO01998/46772 and WO01999/32617. In brief, linear DNA of all the pGBFIN-constructs was isolated and used to transform  A. niger  WT 6. Transformants were selected on acetamide media and colony purified according standard procedures. Colonies were diagnosed for integration at the glaA locus and for copy number using PCR. Three independent transformants for each pGBFINFUA- and pGBFINEBA205-construct with similar estimated copy numbers (putative single copy) were selected and named using the number of the transforming plasmid, as for example FUA-M01-1, FUA-M01-2, FUA-M01-3, EBA205-M01-1, EBA205-M01-2, EBA205-M01-3, etc. . . . , respectively. 
     EBA-205 Constructs and Expression 
     Transformants of the  A. niger  EBA205-transformants containing the different constructs were picked, resulting about 80% successful overexpression strains, including the wild-type EA205 (wild-type protein). For the subset, the production of GBHI expressed by each of the transformants of the  A. niger  EBA205-transformants containing the different constructs, was measured in the culture supernatant at day 5. In addition, the culture supernatants sampled at day 5 were analyzed by quantitative SDS PAGE. 
     
       
         
           
               
             
               
                 TABLE 13 
               
             
            
               
                   
               
               
                 Methionine variants versus WT; expression level in % of WT; 
               
               
                 measurements italics left out corresponding to FIG. 5. 
               
            
           
           
               
               
               
            
               
                   
                 Strain 
                 % WT 
               
               
                   
                   
               
               
                   
                 EBA-205-1 
                  92% 
               
               
                   
                 EBA-205-2 
                  0% 
               
               
                   
                 EBA-205-3 
                 108% 
               
               
                   
                 EBA-205-M01-1 
                 130% 
               
               
                   
                 EBA-205-M01-2 
                  78% 
               
               
                   
                 EBA-205-M01-3 
                 114% 
               
               
                   
                 EBA-205-M02-1 
                 117% 
               
               
                   
                 EBA-205-M02-2 
                 123% 
               
               
                   
                 EBA-205-M02-3 
                  0% 
               
               
                   
                 EBA-205-M03-1 
                 145% 
               
               
                   
                 EBA-205-M03-2 
                 136% 
               
               
                   
                 EBA-205-M03-3 
                  79% 
               
               
                   
                 EBA-205-M04-1 
                 129% 
               
               
                   
                 EBA-205-M04-2 
                 115% 
               
               
                   
                 EBA-205-M04-3 
                 134% 
               
               
                   
                 EBA-205-M05-1 
                 108% 
               
               
                   
                 EBA-205-M05-2 
                 128% 
               
               
                   
                 EBA-205-M05-3 
                 136% 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 14 
               
             
            
               
                   
               
               
                 Methionine variants versus WT; expression level in % of WT; 
               
               
                 measurements italics left out corresponding to FIG. 6. 
               
            
           
           
               
               
               
            
               
                   
                 Strain 
                 % WT 
               
               
                   
                   
               
               
                   
                 EBA-205-1 
                  92% 
               
               
                   
                 EBA-205-2 
                  0% 
               
               
                   
                 EBA-205-3 
                 108% 
               
               
                   
                 EBA-205-K01-1 
                 108% 
               
               
                   
                 EBA-205-K01-2 
                 198% 
               
               
                   
                 EBA-205-K01-3 
                 107% 
               
               
                   
                 EBA-205-K02-1 
                 108% 
               
               
                   
                 EBA-205-K02-2 
                 168% 
               
               
                   
                 EBA-205-K02-3 
                 142% 
               
               
                   
                 EBA-205-K03-1 
                 121% 
               
               
                   
                 EBA-205-K03-2 
                  0% 
               
               
                   
                 EBA-205-K03-3 
                  0% 
               
               
                   
                 EBA-205-K04-1 
                 664% 
               
               
                   
                 EBA-205-K04-2 
                 132% 
               
               
                   
                 EBA-205-K04-3 
                 120% 
               
               
                   
                 EBA-205-K05-1 
                 135% 
               
               
                   
                 EBA-205-K05-2 
                  59% 
               
               
                   
                 EBA-205-K05-3 
                  0% 
               
               
                   
                 EBA-205-K06-1 
                 120% 
               
               
                   
                 EBA-205-K06-2 
                  93% 
               
               
                   
                 EBA-205-K06-3 
                 120% 
               
               
                   
                 EBA-205-K07-1 
                 150% 
               
               
                   
                 EBA-205-K07-2 
                 983% 
               
               
                   
                 EBA-205-K07-3 
                 152% 
               
               
                   
                 EBA-205-K08-1 
                  57% 
               
               
                   
                 EBA-205-K08-2 
                  88% 
               
               
                   
                 EBA-205-K08-3 
                  63% 
               
               
                   
                 EBA-205-K09-1 
                  98% 
               
               
                   
                 EBA-205-K09-2 
                  86% 
               
               
                   
                 EBA-205-K09-3 
                  47% 
               
               
                   
                 EBA-205-K10-1 
                  64% 
               
               
                   
                 EBA-205-K10-2 
                  86% 
               
               
                   
                 EBA-205-K10-3 
                  0% 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 15 
               
             
            
               
                   
               
               
                 Combined methionine and lysine variants 
               
               
                 versus WT; expression level in % of WT; 
               
            
           
           
               
               
               
            
               
                   
                 Strain 
                 % WT 
               
               
                   
                   
               
               
                   
                 FUA-1 
                  92% 
               
               
                   
                 FUA-2 
                  0% 
               
               
                   
                 FUA-3 
                 108% 
               
               
                   
                 EBA205-KM01-1 
                  86% 
               
               
                   
                 EBA205-KM01-2 
                 136% 
               
               
                   
                 EBA205-KM01-3 
                 101% 
               
               
                   
                 EBA205-KM02-1 
                 150% 
               
               
                   
                   
               
            
           
         
       
     
     From the tables and figures, it is clear that protein sequence optimization (PSO) by reducing M, K or both M and K amino acid has a positive impact on protein secretion and results in increased protein expression levels for the cellobiohydrolase I enzyme. Although not all constructs resulted in triplicate producing strains and expression data, still a clear correlation is observed when plotting from left to right the percentage {decrease-wild type-percentage increase} of methionine versus expression level. 
     A similar effect is shown for lysine reduction versus expression level. 
     FUA Constructs and Expression 
     Transformants of the  A. niger  FUA-transformants containing the different constructs were picked, resulting about 80% successful overexpression strains, including the wild-type FUA (wild-type protein). For the subset, the production of alpha-amylase expressed by each of the transformants of the  A. niger  FUA-transformants containing the different constructs, was measured with the fungal alpha-amylase activity assay in the culture supernatant at day 4. The activity is a measure for expression level. 
     
       
         
           
               
             
               
                 TABLE 16 
               
             
            
               
                   
               
               
                 Methionine variants versus WT; activity level in % of 
               
               
                 WT; measurements italics left out corresponding FIG. 7. 
               
            
           
           
               
               
               
            
               
                   
                 Strain 
                 % WT 
               
               
                   
                   
               
               
                   
                 FUA-1 
                 105%  
               
               
                   
                 FUA-2 
                 89%  
               
               
                   
                 FUA-3 
                 107%  
               
               
                   
                 FUA-M01-1 
                 111%  
               
               
                   
                 FUA-M01-2 
                 124%  
               
               
                   
                 FUA-M01-3 
                 196%  
               
               
                   
                 FUA-M02-1 
                 151%  
               
               
                   
                 FUA-M02-2 
                 135%  
               
               
                   
                 FUA-M02-3 
                 126%  
               
               
                   
                 FUA-M03-1 
                 18%  
               
               
                   
                 FUA-M03-2 
                 15%  
               
               
                   
                 FUA-M03-3 
                 27%  
               
               
                   
                 FUA-M04-1 
                 1% 
               
               
                   
                 FUA-M04-2 
                 1% 
               
               
                   
                 FUA-M04-3 
                 2% 
               
               
                   
                 FUA-M05-1 
                 0% 
               
               
                   
                 FUA-M05-2 
                 0% 
               
               
                   
                 FUA-M05-3 
                 0% 
               
               
                   
                 FUA-M06-1 
                 26%  
               
               
                   
                 FUA-M06-2 
                 18%  
               
               
                   
                 FUA-M06-3 
                 14%  
               
               
                   
                 FUA-M07-1 
                 0% 
               
               
                   
                 FUA-M07-2 
                 1% 
               
               
                   
                 FUA-M07-3 
                 0% 
               
               
                   
                 FUA-M08-1 
                 0% 
               
               
                   
                 FUA-M08-2 
                 0% 
               
               
                   
                 FUA-M08-3 
                 0% 
               
               
                   
                 FUA-M09-1 
                 224%  
               
               
                   
                 FUA-M09-2 
                 137%  
               
               
                   
                 FUA-M09-3 
                 0% 
               
               
                   
                 FUA-M10-1 
                 57%  
               
               
                   
                 FUA-M10-2 
                 45%  
               
               
                   
                 FUA-M10-3 
                 70%  
               
               
                   
                 FUA-M11-1 
                 35%  
               
               
                   
                 FUA-M11-2 
                 69%  
               
               
                   
                 FUA-M11-3 
                 35%  
               
               
                   
                 FUA-M12-1 
                 4% 
               
               
                   
                 FUA-M12-2 
                 5% 
               
               
                   
                 FUA-M12-3 
                 5% 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 17 
               
             
            
               
                   
               
               
                 Methionine variants versus WT; activity level in % of 
               
               
                 WT; measurements italics left out corresponding FIG. 8. 
               
            
           
           
               
               
               
            
               
                   
                 Strain 
                 % WT 
               
               
                   
                   
               
               
                   
                 FUA-1 
                 106%  
               
               
                   
                 FUA-2 
                 101%  
               
               
                   
                 FUA-3 
                 93% 
               
               
                   
                 FUA-K01-1 
                 45% 
               
               
                   
                 FUA-K01-2 
                 50% 
               
               
                   
                 FUA-K01-3 
                 85% 
               
               
                   
                 FUA-K02-1 
                  0% 
               
               
                   
                 FUA-K02-2 
                  0% 
               
               
                   
                 FUA-K02-3 
                 134%  
               
               
                   
                 FUA-K03-1 
                 109%  
               
               
                   
                 FUA-K03-2 
                 44% 
               
               
                   
                 FUA-K03-3 
                 142%  
               
               
                   
                 FUA-K10-1 
                 65% 
               
               
                   
                 FUA-K10-2 
                 76% 
               
               
                   
                 FUA-K10-3 
                 73% 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 18 
               
             
            
               
                   
               
               
                 Combined methionine and lysine variants 
               
               
                 versus WT; activity level in % of WT; 
               
            
           
           
               
               
               
            
               
                   
                 Strain 
                 % WT 
               
               
                   
                   
               
               
                   
                 FUA-1 
                 105%  
               
               
                   
                 FUA-2 
                 89% 
               
               
                   
                 FUA-3 
                 107%  
               
               
                   
                 FUA-KM01-1 
                  0% 
               
               
                   
                 FUA-KM01-2 
                 17% 
               
               
                   
                 FUA-KM01-3 
                 18% 
               
               
                   
                 FUA-KM02-1 
                  2% 
               
               
                   
                 FUA-KM02-2 
                 11% 
               
               
                   
                 FUA-KM02-3 
                  1% 
               
               
                   
                   
               
            
           
         
       
     
     Note that some of the PSO engineered variants with reduced M-content show no or reduced activity levels. Typically those proteins likely are too much affected in their folding and no longer fold correctly nor express well. In addition, the culture supernatants sampled at day 4 were analyzed by LC90, which show a similar trend for expression versus no expression of picked strains, although is not quantitative for expression levels. Table 18 shows extremely engineered combined M/K variants, which have not survived the engineering approach and are no longer active. 
     For a majority of the shown FUA example, protein sequence optimization by reducing M or K K amino acid has a positive impact on protein secretion and results in detectable and thus increased protein expression levels and increased activity levels for the alpha-amylase enzyme. A clear positive trend is observed when plotting from left to right the percentage {decrease-wild type-percentage increase of methionine versus a activity level, and thus in a similar way for expression assuming that the specific activity is not substantially changed for those variants. For lysine also a clear correlation is observed, although less strains and thereby less data-points are available.