# Patent

## Title
Amylase-deficient strain

## Abstract

The present invention relates to a mutant microbial host cell which has been modified, preferably in its genome, to result in a deficiency in the production of a polypeptide having α-amylase activity AmyC or an homologous thereof. It has been surprisingly found that when the mutant microbial host cell according to the invention is used in a method to produce a compound of interest, for example an enzyme, an improved purity of the product is obtained.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a §371 National Stage Application of PCT/EP2013/065347, filed Jul. 19 2013, which claims priority to EP 12177173.7, filed Jul. 19, 2012, EP 12177171.1, filed Jul. 19, 2012, U.S. 61/673,589, filed Jul. 19, 2012 and U.S. 61/673,607, filed Jul 19, 2012.


BACKGROUND
Field of the Invention
The present invention relates to a mutant microbial host cell which has been modified, preferably in its genome, to result in a deficiency in the production of a polypeptide having amylase activity, to a method to produce the mutant microbial host cell and to a method to produce a compound of interest using said mutant microbial host cell.
Description of Related Art
An ever increasing number of products is produced by microbial fermentation at industrial scale. Such products range from primary and secondary metabolites, such as e.g. citric acid and antibiotics, respectively, to proteins, enzymes and even complete microorganisms, e.g. in the form of bakers yeast or biomass.
Different host cell types may be used for different production processes. For example: mammalian cell lines are used for antibody production; fungal cells are preferred organisms for production of polypeptides and secondary metabolites; bacterial cells are preferred for small metabolite and antibiotic production; and plant cells are preferred for taste and flavor compounds. An important aspect in the production of valuable products by microbial fermentation is optimization of the productivity of the microbial cells.
Recombinant techniques are widely employed for optimization of the productivity of such cells and/or the processes in which they are used. This can involve a multitude of options.
Some techniques will aim at the over expression of a gene of interest coding for the product or coding for a compound related to the production of the product by the host cell. Gene expression can be modulated in several ways. For example the gene of interest can be placed in the host cell under the expression control of a strong promoter, or it can be placed under the control of a promoter activated by a transcriptional activator which activator can be up-regulated or down-regulated. In yet an alternative approach gene expression can be improved by increasing the copy number of the gene of interest in the host cell used to express the gene. Yet other approaches aiming at improving the productivity of a compound of interest by a host cell can involve deletion or inactivation of competing pathways, changing compartmentalization of enzymes, increasing protein or metabolite secretion, increasing organelle content and the like.
Another important aspect in the production of valuable products by microbial fermentation is the quality and purity of said products after the fermentation and in the final product formulation. These will depend on the specific product quality demands and the final application of the product. Most commercial products produced by microbial fermentation have requirements in their physical appearance (color, odor). Also impurities related to the process (e.g., the presence of unfermented sugar from the media) and the microorganism used (e.g., metabolites produced during the fermentation) can influence the quality of the product. Dealing with these issues requires incorporating purification steps after recovery of the product from the fermentation broth.
If the valuable product is secreted into the nutrient medium, the product can be isolated directly from the medium. If the product is not secreted, it can be isolated from cell lysates. The product may be isolated from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. A variety of procedures known in the art including, but not limited to, chromatography (e. g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e. g., ammonium sulfate precipitation), or extraction (see, e.g., Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989) can be used at this purpose. The use of several purification steps however will increase production costs. In case the product is an enzyme the presence of other enzyme activities, derived from the production microorganism, should be avoided as it can interfere with the application or, more commonly, negatively affect the product stability.
There is therefore a need to develop microorganisms used to express useful products that combine the capacity for expression of commercial quantities thereof while being deficient in the production of enzymes that can complicate recovery, downstream processing or compromise final purity of the products and/or be detrimental in product applications.
SUMMARY
The present invention relates to a mutant microbial host cell which has been modified, preferably in its genome, to result in a deficiency in the production of a polypeptide having amylase activity, preferably α-amylase activity, selected from the group consisting of:

    
    
        a. a polypeptide according to SEQ ID NO: 3 or 7 or a polypeptide at least 70% identical thereto;
        b. a mature polypeptide comprised in SEQ ID NO: 3 or 7 or a polypeptide at least 70% identical thereto;
        c. a polypeptide encoded by a polynucleotide according to SEQ ID NO: 1, 2, 5 or 6 or encoded by a polynucleotide at least 70% identical to SEQ ID NO: 1, 2, 5 or 6;
        d. a polypeptide encoded by a polynucleotide capable of hybridising to a polynucleotide according to SEQ ID NO: 1, 2, 5 or 6 or capable of hybridising to the complementary strand of SEQ ID NO: 1, 2, 5 or 6;
        if compared with a parent microbial host cell which has not been modified and measured under the same conditions.
    
    


The present invention further relates to a method of producing a mutant microbial host cell according to the invention comprising the steps of:

    
    
        a. providing a parent microbial host cell;
        b. modifying the parent microbial host cell, preferably modifying the genome of the parent microbial host cell to yield a mutant host cell which is deficient in the production of a polypeptide having amylase activity, preferably α-amylase activity, selected from the group consisting of:
        
            i. a polypeptide according to SEQ ID NO: 3 or 7 or a polypeptide at least 70% identical thereto;
            ii. a mature polypeptide comprised in SEQ ID NO: 3 or 7 or a polypeptide at least 70% identical thereto;
            iii. a polypeptide encoded by a polynucleotide according to SEQ ID NO: 1, 2, 5 or 6 or encoded by a polynucleotide at least 70% identical to SEQ ID NO: 1, 2, 5 or 6;
            iv. a polypeptide encoded by a polynucleotide capable of hybridising to a polynucleotide according to SEQ ID NO: 1, 2, 5 or 6 or capable of hybridising to the complementary strand of a polynucleotide according to SEQ ID NO: 1, 2, 5 or 6;

if compared with the parent microbial host cell and measured under the same conditions.

        
        
    
    


The invention relates as well to a method for the production of a compound of interest by microbial fermentation comprising:

    
    
        a. providing a mutant microbial host cell according to the invention or produced according to a method for producing a mutant microbial host cell according to the invention capable of expressing the compound of interest,
        b. culturing said microbial host cell under conditions conducive to the expression of the compound of interest,
        c. optionally isolating the compound of interest from the culture medium.
    
    





BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts pGBTOPGOX-3, the pGBTOP-12 based plasmid used for expression of the Penicillium chrysogenum glucose oxidase enzyme gene with a layout for expression driven by the glucoamylase promoter and targeted integration in the adapted BamHI amplicon.
FIG. 2 depicts pGBDEL-AMY1, the plasmid used for deletion of the amylase encoding agoB gene with a layout representative for other deletion constructs (i.e. pGBDEL-AMY2 and pGBDEL-AMY3).
FIG. 3 depicts relative alpha-amylase activities, as measured in the culture supernatant of the different strains. The activity of the PGOX-2 reference strain at day 4 was set at a level of 100%.
FIG. 4 depicts relative glucose oxidase activities, as measured in the culture supernatant of the different strains. The activity of the PGOX-2 reference strain at day 4 was set at a level of 100%.
FIG. 5 depicts glucose oxidase activities on plate of the different mutant strains. Growth was on 1% maltose and staining with o-anisidine was done after 4 days of growth.



BRIEF DESCRIPTION OF THE SEQUENCE LISTING
SEQ ID NO: 1 sets out the genomic sequence of the amyC amylase gene from Aspergillus niger, including 2 kb upstream and downstream flanking regions. The genomic sequence comprises the cDNA sequence according to SEQ ID NO: 2.
SEQ ID NO: 2 sets out the cDNA sequence of the amyC amylase gene (short sequence) from A. niger. 
SEQ ID NO: 3 sets out the amino acid sequence of the amyC amylase protein (short sequence) from A. niger. 
SEQ ID NO: 4 sets out the amino acid sequence of the AmyC mature amylase protein (short sequence) corresponding to amino acid 17-493 of SEQ ID NO: 3.
SEQ ID NO: 5 sets out the genomic sequence of the amyC amylase gene (long sequence) from Aspergillus niger, including 2 kb upstream and downstream flanking regions. The genomic sequence comprises the cDNA sequence according to SEQ ID NO: 6.
SEQ ID NO: 6 sets out the cDNA sequence of the amyC amylase gene (long sequence) from A. niger. 
SEQ ID NO: 7 sets out the amino acid sequence of the amyC amylase protein (long sequence) from A. niger. 
SEQ ID NO: 8 sets out the amino acid sequence of the AmyC mature amylase protein (long sequence) corresponding to amino acid 17-524 of SEQ ID NO: 7.
SEQ ID NO: 9 sets out the genomic sequence of the agdB gene from Aspergillus niger, including 2 kb upstream and downstream flanking regions. The genomic sequence comprises the cDNA sequence according to SEQ ID NO: 10.
SEQ ID NO: 10 sets out the cDNA sequence of the agdB gene from A. niger. 
SEQ ID NO: 11 sets out the amino acid sequence of the agdB protein from A. niger. 
SEQ ID NO: 12 sets out the genomic sequence of the agdA gene from Aspergillus niger, including 2 kb upstream and downstream flanking regions. The genomic sequence comprises the cDNA sequence according to SEQ ID NO: 13.
SEQ ID NO: 13 sets out the cDNA sequence of the agdA gene from A. niger. 
SEQ ID NO: 14 sets out the amino acid sequence of the agdA protein from A. niger. 
SEQ ID NO: 15 sets out the codon pair optimized cDNA sequence of the glucose oxidase from Penicillium chrysogenum. 
SEQ ID NO: 16 sets out the amino acid sequence of the glucose oxidase from Penicillium chrysogenum. 
SEQ ID NO: 17 sets out the genomic sequence of the agsE gene from Aspergillus niger, including 2 kb upstream and downstream flanking regions. The genomic sequence comprises the cDNA sequence according to SEQ ID NO: 18.
SEQ ID NO: 18 sets out the cDNA sequence of the agsE gene from A. niger. 
SEQ ID NO: 19 sets out the amino acid sequence of the AgsE protein from A. niger. 
SEQ ID NO: 20 sets out the amino acid sequence of the mature AgsE protein corresponding to amino acid 20-2426 of SEQ ID NO: 19.
All nucleotide sequences for A. niger genes and protein sequences and their genomic context can be derived from public databases available for example from the NCBI at http://www.ncbi.nlm.nih.gov/ or EMBL (http://www.ebi.ac.uk/embl/). For example the genome sequence of CBS 513.88 at EMBL has accession numbers no. AM269948-AM270415.
DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT
The present invention relates to a mutant microbial host cell which has a deficiency in the production of the amyC amylase protein. Such a mutant microbial cell is modified, preferably in its genome, to result in a deficiency in the production of a polypeptide having amylase activity, preferably α-amylase activity, selected from the group consisting of:

    
    
        a. a polypeptide according to SEQ ID NO: 3 or 7 or a polypeptide at least 70% identical thereto;
        b. a mature polypeptide comprised in SEQ ID NO: 3 or 7 or a polypeptide at least 70% identical thereto;
        c. a polypeptide encoded by a polynucleotide according to SEQ ID NO: 1, 2, 5 or 6 or encoded by a polynucleotide at least 70% identical to SEQ ID NO: 1, 2, 5 or 6;
        d. a polypeptide encoded by a polynucleotide capable of hybridising to a polynucleotide according to SEQ ID NO: 1, 2, 5 or 6 or capable of hybridising to the complementary strand of a polynucleotide according to SEQ ID NO: 1, 2, 5 or 6;
        if compared with a parent microbial host cell which has not been modified and measured under the same conditions.
    
    


It has been surprisingly found that when the mutant microbial host cell according to the invention and which is capable of expressing a compound of interest is used in a method to produce a compound of interest, for example an enzyme, the amylase side activity in the compound of interest is considerably lowered. This is very advantageous when the compound of interest is an enzyme used in specific (food) applications, e.g. in those applications wherein uncontrolled degradation of starch components is not desirable (e.g. preparation of baked products, mayonnaise light, etcetera).
In particular, it has been found that when a mutant microbial host cell according to the invention (and which is capable of expressing a compound of interest is used in a method to produce a compound of interest, for example an enzyme) also comprises disruptions in the most important and abundant amylases (glucoamylase glaA, alpha-amylase amyBII and ΔamyBI and acid stable alpha-amylase amyA) the effect of the disruption of AmyC is especially dramatic. This is surprising since other amy disruptions showed no pronounced effect on alpha-amylase activity (when used in combination with disruptions to glucoamylase glaA, alpha-amylase amyBII and ΔamyBI and acid stable alpha-amylase amyA).
This means that specifically amyC (An04g06930) disruption results in an important reduction in alpha-amylase activity, especially in the context of a mutant microbial host cell which also has disruptions to glucoamylase glaA, alpha-amylase amyBII and ΔamyBI and acid stable alpha-amylase amyA.
Within the context of the present invention “measured under the same conditions” or “analysed under the same conditions” means that the mutated microbial host cell and the parent microbial host cell are cultivated under the same conditions and that the amount and/or activity of the polypeptide in which the mutant host cell is deficient, if compared to the parent microbial host cell, is measured in the microbial host cell and in the parent host cell, respectively, using the same conditions, preferably by using the same assay and/or methodology, more preferably within the same experiment.
A “mutant microbial host cell” is herewith defined as a microbial host cell derived from a parent host cell and which has been modified, preferably in its genome, if compared to the parent host cell to obtain a different genotype and/or a different phenotype if compared to the parent host cell from which it is derived.
The modification can either be effected by

    
    
        a) subjecting the parent microbial host cell to recombinant genetic manipulation techniques; and/or
        b) subjecting the parent microbial host cell to (classical) mutagenesis; and/or
        c) subjecting the parent microbial host cell to an inhibiting compound or composition.
    
    


A “mutant microbial host cell which has been modified, preferably in its genome, to result in a deficiency in the production of a product”, for example of a product such as a polypeptide having amylase activity according to SEQ ID NO: 3 or 7, is herein defined as a mutant microbial host cell which has been modified, preferably in its genome, to result in a phenotypic feature wherein the cell: a) produces less of the product or produces substantially no product and/or b) produces a product having a decreased activity or decreased specific activity or a product having no activity or no specific activity and combinations of one or more of these possibilities as compared to the parent microbial host cell that has not been modified, when analysed under the same conditions.
In the context of the present invention a polypeptide having amylase activity, preferably α-amylase activity is a polypeptide selected from the group consisting of:

    
    
        a. a polypeptide according to SEQ ID NO: 3 or 7 or a polypeptide at least 70% identical to either thereto;
        b. a mature polypeptide comprised in SEQ ID NO: 3 or 7 or a polypeptide at least 70% identical to either thereto;
        c. a polypeptide encoded by a polynucleotide according to SEQ ID NO: 1, 2, 5 or 6 or encoded by a polynucleotide at least 70% identical to SEQ ID NO: 1, 2, 5 or 6;
        d. a polypeptide encoded by a polynucleotide capable of hybridising to a polynucleotide according to SEQ ID NO: 1, 2, 5 or 6 or to the complementary strand of a polynucleotide according to SEQ ID NO: 1, 2, 5 or 6.
    
    


The polypeptide having amylase activity, preferably having α-amylase activity as defined herein is preferably an acid stable α-amylase. In one embodiment the polypeptide having α-amylase activity has a temperature optimum, as measured according to the Megazyme CERALPHA alpha-amylase assay kit, Megazyme International Ireland Ltd., Co. Wicklow, Ireland, at pH 4, of 45-57° C., more preferably of 48-54° C., even more preferably of about 50° C. Preferably the polypeptide having α-amylase activity has a pH optimum, as measured at 50° C. according to the Megazyme CERALPHA alpha-amylase assay kit, Megazyme International Ireland Ltd., Co. Wicklow, Ireland, of 3.8-5, more preferably of 4-4.5.
A polypeptide according to SEQ ID NO: 3 or 7 corresponds to the amylase AmyC from Aspergillus niger (Yuan X.-L., van der Kaaij R. M., van den Hondel C. A. M. J. J., Punt P. J., van der Marel M. J. E. C., Dijkhuizen L., Ram A. F. J. Mol. Genet. Genomics (2008) 279: 545-561). The polypeptide according to SEQ ID NO: 3 or 7 is encoded by the amylase gene amyC (genomic DNA as depicted in SEQ ID NO: 1 and 5, cDNA as depicted in SEQ ID NO: 2 or SEQ ID NO: 6). The two genomic sequences, SEQ ID NO: 1 and 5 are identical. However, two different cDNAs and consequently two different polypeptides have been identified: a short form (cDNA=SEQ ID NO: 2 and polypeptide=SEQ ID NO: 3); and a long form (cDNA=SEQ ID NO: 6 and polypeptide=SEQ ID NO: 7).
In the context of the present invention a polypeptide having amylase activity, preferably having α-amylase activity, which is at least 70% identical to either of SEQ ID NO: 3 or SEQ ID NO: 7 is a polypeptide characterised by an amino acid sequence comprising one or more substitutions, deletions, and/or insertions of one or more amino acids if compared to the polypeptide of SEQ ID NO: 3 or 7, wherein the polypeptide which is at least 70% identical to SEQ ID NO: 3 or 7 has more or less (α-)amylase activity than the polypeptide according to SEQ ID NO:3 or 7. The polypeptide which is at least 70% identical to SEQ ID NO: 3 or 7 may e.g. be a natural variant, an orthologue or an in vitro generated variant of SEQ ID NO: 3 or 7 obtained using methods well known in the art such as e.g. classical mutagenesis, site-directed mutagenesis, DNA shuffling and in silico design. In the context of the present invention the polypeptide which is at least 70% identical to SEQ ID NO: 3 or 7 has preferably between 20% and 400% amylase activity if compared to SEQ ID NO:3 or 7 and measured under the same conditions, more preferably between 40 and 350% amylase activity, even more preferably between 50 and 300% amylase activity, between 70 and 250% amylase activity, between 80 and 200% amylase activity, most preferably approximately 100% amylase activity of the polypeptide according to SEQ ID NO: 3 or 7. With amylase activity it is herewith intended preferably α-amylase activity. For the measurement of amylase activity in the polypeptide according to SEQ ID NO: 3 or 7 and in the polypeptide at least 70% identical thereto any method known in the art for the measurement of said activity can be used. The only requirement is that the measurement of amylase activity in the polypeptide according to SEQ ID NO: 3 or 7 and in the polypeptide at least 70% identical thereto is preferably performed using the same method and/or assay and under the same conditions, preferably within the same experiment. Alpha-amylase activity may be measured according to methods known to those skilled in the art; e.g. it may be measured according to the well-established Ceralpha method (McCleary, B. V., McNally, M., Monaghan, D. & Mugford, D. C. (2002). “Measurement of α-amylase activity in white wheat flour, milled malt, and microbial enzyme preparations using the Ceralpha Assay”. Collaborative study. J. AOAC International, 85, 1096-1102) using a Megazyme CERALPHA alpha-amylase assay kit, Megazyme International Ireland Ltd., Co. Wicklow, Ireland). In the experimental section a setup using the Megazyme CERALPHA α-amylases assay kit was used allowing a more sensitive determination of alpha-amylase activity. Other suitable methods for the determination of α-amylase are the well-known AACC method 22-01 (AACC. 1983 Approved Methods of the American Association of Cereal Chemists, 8th Edition. St. Paul, Minn.: American association of Cereal Chemists), the ASBC (International method) and the Farrand method.
Preferably the polypeptide having (α)-amylase activity is at least 80% identical to SEQ ID NO: 3 or 7, more preferably at least 85% identical to SEQ ID NO: 3 or 7, even more preferably at least 90% identical to SEQ ID NO: 3 or 7, most preferably at least 91%, for example at least 92%, 93%, 94%, at least 95% identical, at least 96%, 97%, 98%, at least 99% identical to SEQ ID NO: 3 or 7. Preferably the polypeptide having (α)-amylase activity is a polypeptide according to SEQ ID NO: 3 or 7. Preferably sequence identity is measured over the whole polypeptide sequence length.
The polypeptide which production the mutant microbial host cell according to the invention is deficient in, may be a mature polypeptide comprised in SEQ ID NO: 3 or 7. A mature polypeptide is defined herein as a polypeptide in its final form after translation, post-translational modifications, such as N-terminal processing, C-terminal processing, glycosylation, phosphorylation, secretion and optional removal of leader sequences by (proteolytic) cleavage. Signal peptides, propeptides and prepropeptides are in the art sometimes referred to as “leader sequences”. The term “propeptide” is defined herein as a peptide fused in frame to the N-terminus of a polypeptide having biological activity. The resulting polypeptide is known as a propolypeptide which is lacking the polypeptide biological activity and can be converted into a mature, biologically active, polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. A signal peptide and propeptide together are herein referred to as a “prepropeptide”. The “signal sequence” is defined herein as a peptide being fused in frame to the N-terminus of a propeptide and the propeptide being fused in frame to the N-terminus of a polypeptide having biological activity. In some cases the propeptide is lacking and the signal sequence is fused in frame to the N-terminus of the polypeptide. The function of the signal sequence is to direct the polypeptide into the cell secretory pathway.
Therefore SEQ ID NO: 3 or 7 may be the sequence translated from the mRNA and prior to post translational modifications. SEQ ID NO: 3 or 7 may comprise additional amino acids at either the C-terminus and/or the N-terminus if compared to the mature polypeptide comprised therein. SEQ ID NO: 3 or 7 may e.g. comprise the mature polypeptide linked in frame to its signal peptide, propeptide and/or prepropeptide. In a preferred embodiment the mature polypeptide comprised in SEQ ID NO: 3 corresponds to amino acids 17-493 of SEQ ID NO: 3 and is set out in SEQ ID NO: 4. Therefore in one embodiment the mutant microbial host cell according to the invention is deficient in a polypeptide which is the mature polypeptide according to SEQ ID NO: 4. In another preferred embodiment the mature polypeptide comprised in SEQ ID NO: 7 corresponds to amino acids 17-524 of SEQ ID NO: 7 and is set out in SEQ ID NO: 8. Therefore in one embodiment the mutant microbial host cell according to the invention is deficient in a polypeptide which is the mature polypeptide according to SEQ ID NO: 8.
In the context of the present invention the polypeptide which production the mutant microbial cell is deficient in may be a polypeptide at least 70% identical to the mature polypeptide comprised in SEQ ID NO: 3 or 7 and having amylase activity, preferably α-amylase activity as defined herein. Preferably the polypeptide is at least 80% identical to the mature polypeptide as defined herein, more preferably at least 85% identical to the mature polypeptide as defined herein, even more preferably at least 90% identical to the mature polypeptide as defined herein, most preferably at least 91%, for example at least 92%, 93%, 94%, at least 95% identical, at least 96%, 97%, 98%, at least 99% identical to the mature polypeptide as defined herein. Preferably the polypeptide is the mature polypeptide according to SEQ ID NO: 4 or 8. Preferably sequence identity is measured over the whole polypeptide sequence length.
In the context of the present invention a polynucleotide according to SEQ ID NO: 1, 2 5 or 6 or a polynucleotide at least 70% identical to SEQ ID NO: 1, 2, 5 or 6 is a polynucleotide coding for a polypeptide having amylase activity, preferably α-amylase activity as defined herein, according to SEQ ID NO: 3 or 7, for a mature polypeptide comprised in SEQ ID NO: 3 or 7, for a polypeptide according to SEQ ID NO: 4 or 8 or for a polypeptide having amylase activity, preferably α-amylase activity, and having at least 70% identity to SEQ ID NO: 3 or 7, for a polypeptide having at least 70% identity to a mature polypeptide comprised in SEQ ID NO: 3 or 7, for a polypeptide having at least 70% identity to a mature polypeptide according to SEQ ID NO: 4 or 8 as defined above. In the context of the present invention a polynucleotide at least 70% identical to SEQ ID NO: 1, 2, 5 or 6 is a polynucleotide characterised by an nucleotide sequence comprising one or more substitutions, deletions, and/or insertions of one or more nucleotides if compared to the polynucleotide of SEQ ID NO: 1, 2, 5 or 6. Preferably the polynucleotide is at least 80% identical to SEQ ID NO: 1, 2, 5 or 6, more preferably at least 85% identical to SEQ ID NO: 1, 2, 5 or 6, even more preferably at least 90% identical to SEQ ID NO: 1, 2, 5 or 6, most preferably at least 91%, 92%, 93%, 94%, at least 95% identical, at least 96%, 97%, 98%, at least 99% identical to SEQ ID NO: 1, 2, 5 or 6. Preferably the polynucleotide is a polynucleotide according to SEQ ID NO:1, 2, 5 or 6.
For the purpose of this invention, it is defined here that in order to determine the percentage of sequence identity of two amino acid sequences or of two nucleic acid sequences, the 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 can be carried out over the full length of the sequences being compared. Alternatively, the alignment may be carried out over a shorter length, for example over about 20, about 50, about 100 or more nucleic acids/based or amino acids. The sequence identity is the percentage of identical matches between the two sequences over the reported aligned region.
A comparison of sequences and determination of percentage of sequence identity between two sequences can be accomplished using a mathematical algorithm. The skilled person will be aware of the fact that several different computer programs are available to align two sequences and determine the identity 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 percentage of sequence identity between two amino acid sequences or between two nucleotide sequences may 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). Both amino acid sequences and nucleotide sequences can be aligned by the algorithm. 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 sequence, EDNAFULL is used. The optional parameters used 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.
After alignment by the program NEEDLE as described above the percentage of sequence identity between a query sequence and a sequence of the invention is calculated as follows: Number of corresponding positions in the alignment showing an identical amino acid or identical nucleotide in both sequences divided by the total length of the alignment after subtraction of the total number of gaps in the alignment. The identity defined as herein can be obtained from NEEDLE by using the NOBRIEF option and is labeled in the output of the program as “longest-identity”.
The nucleic acid and protein sequences of the present invention 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, wordlength=12 to obtain nucleotide sequences homologous to nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17): 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See the homepage of the National Center for Biotechnology Information at http://www.ncbi.nlm.nih.gov/.
In the context of the present invention a polypeptide having amylase activity preferably α-amylase activity may be a polypeptide encoded by a polynucleotide capable of hybridising to a polynucleotide according to SEQ ID NO: 1, 2, 5 or 6 or capable of hybridising to the complementary strand of a polynucleotide according to SEQ ID NO: 1, 2, 5 or 6, preferably it is capable of hybridising under low stringency conditions, more preferably it is capable of hybridising under medium stringency conditions, even more preferably it is capable of hybridising under high stringency conditions to the complementary strand of a polynucleotide according to SEQ ID NO: 1, 2, 5 or 6.
As used herein, the term “hybridizing” is intended to describe conditions for hybridization and washing under which polynucleotide sequences at least about 60%, 65%, 80%, 85%, 90%, preferably at least 93%, more preferably at least 95% and most preferably at least 98% identical to each other typically remain hybridized to the complement of each other. As used herein, the term “hybridization” means the pairing of substantially complementary strands of oligomeric compounds. One mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleotide bases (nucleotides) of the strands of oligomeric compounds. For example, adenine and thymine are complementary nucleic acids which pair through the formation of hydrogen bonds. Hybridization can occur under varying circumstances. “Stringency hybridization” or “hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions” is used herein to describe conditions for hybridization and washing, more specifically conditions under which an oligomeric compound will hybridize to its target sequence, but to a minimal number of other sequences. So, the oligomeric compound will hybridize to the target sequence to a detectably greater degree than to other sequences. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6:3.6.
The skilled artisan will know which conditions to apply for low, medium and high stringency hybridisation conditions. Additional guidance regarding such conditions is readily available in the art, for example, in Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, N.Y.; and Ausubel et al. (eds.), 1995, Current Protocols in Molecular Biology, (John Wiley & Sons, N.Y.).
Stringency conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Generally, stringency conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the oligomeric compound at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of an oligomeric compound hybridizes to a perfectly matched probe. Stringency conditions may also be achieved with the addition of destabilizing agents such as formamide.
Examples of specific hybridization conditions are as follows: 1) low stringency hybridization conditions in 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed by two washes in 0.2×SSC, 0.1% SDS at least at 50° C. (the temperature of the washes can be increased to 55° C. for low stringency conditions); 2) medium stringency hybridization conditions in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 60° C.; 3) high stringency hybridization conditions in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C.; and 4) very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at 65° C., followed by one or more washes at 0.2×SSC, 1% SDS at 65° C.
Within the context of the present invention the mutant microbial host cell is deficient in the production of a polypeptide having amylase activity, preferably α-amylase activity as defined herein when the host cell comprises a modification, preferably in its genome, which results in a reduced or no production of the polypeptide having amylase activity, preferably α-amylase activity as defined herein if compared to the parent microbial host cell that has not been modified, when analysed under the same conditions and/or comprises a modification which results in a polypeptide derived from the polypeptide having amylase activity, preferably α-amylase activity as described herein with decreased or no (α)-amylase activity if compared to the parent microbial host cell that has not been modified, when analysed under the same conditions. Therefore a mutant microbial host cell as defined herein is deficient in the production of a polypeptide having amylase activity, preferably α-amylase activity as described herein when

    
    
        a) it produces less polypeptide having amylase activity, preferably α-amylase activity as defined herein or it produces no polypeptide having amylase activity, preferably α-amylase activity as defined herein if compared with the parent microbial host cell which has not been modified and measured under the same conditions; and/or
        b) it produces a polypeptide derived from the polypeptide having amylase activity, preferably α-amylase activity as defined herein with decreased or no (α-)amylase activity if compared to the parent microbial host cell that has not been modified, when analysed under the same conditions.
    
    


In one embodiment the mutant microbial host cell produces 1% less polypeptide having amylase activity, preferably α-amylase activity as defined herein if compared with the parent microbial host cell which has not been modified and measured under the same conditions, at least 5% less, at least 10% less, at least 20% less, at least 30% less, at least 40% less, at least 50% less, at least 60% less, at least 70% less, at least 80% less, at least 90% less, at least 91% less, at least 92% less, at least 93% less, at least 94% less at least 95% less, at least 96% less, at least 97% less, at least 98% less, at least 99% less, or at least 99.9% less. Preferably the mutant microbial host cell produces substantially no polypeptide having amylase activity, preferably α-amylase activity as described herein if compared with the parent microbial host cell which has not been modified and measured under the same conditions.
In one embodiment the mutant microbial host cell produces a polypeptide derived from the polypeptide having amylase activity, preferably α-amylase activity as defined herein with 1% less (α-)amylase activity, if compared with the parent microbial host cell which has not been modified and measured under the same conditions, at least 5% less activity, at least 10% less activity, at least 20% less activity, at least 30% less activity, at least 40% less activity, at least 50% less activity, at least 60% less activity, at least 70% less activity, at least 80% less activity, at least 90% less activity, at least 91% less activity, at least 92% less activity, at least 93% less activity, at least 94% less activity, at least 95% less activity, at least 96% less activity, at least 97% less activity, at least 98% less activity, at least 99% less activity, or at least 99.9% less activity. Preferably the mutant microbial host cell produces a polypeptide derived from a polypeptide having amylase activity, preferably α-amylase activity as described herein with substantially no (α-)amylase activity if compared with the parent microbial host cell which has not been modified and analysed under the same conditions.
Deficiency of a mutant microbial host cell according to the invention in the production of a polypeptide having amylase activity as defined herein may be measured by determining the amount and/or (specific) activity of polypeptide having amylase activity, preferably α-amylase activity as defined above produced by the mutated microbial host cell and/or it may be measured by determining the amount of mRNA transcribed from a polynucleotide encoding the polypeptide as described above and/or it may be measured by gene or genome sequencing if compared to the parent host cell which has not been modified.
A modification in the genome can be determined by comparing the DNA sequence of the mutant microbial host cell to the sequence of the parent (non-modified) microbial host cell. Sequencing of DNA and genome sequencing can be done using standard methods known to the person skilled in the art, for example using Sanger sequencing technology and/or next generation sequencing technologies such as Illumina GA2, Roche 454, etc. as reviewed in Elaine R. Mardis (2008), Next-Generation DNA Sequencing Methods, Annual Review of Genomics and Human Genetics, 9: 387-402. (doi:10.1146/annurev.genom 0.9.081307.164359)
Deficiency in the production of the polypeptide having amylase activity preferably α-amylase activity as described herein can be measured using any assay available to the skilled person, such as α-amylase activity measurement as described herein, transcriptional profiling, Northern blotting RT-PCR, Q-PCR and Western blotting. In particular quantifying the amount of mRNA present in a cell may for example be achieved by northern blotting (in Molecular Cloning: A Laboratory Manual, Sambrook et al., New York: Cold Spring Harbour Press, 1989). Quantifying the amount of polypeptide having amylase activity, preferably α-amylase activity described herein present in a cell may for example be achieved by western blotting. The difference in mRNA amount may also be quantified by DNA array analysis (Eisen, M. B. and Brown, P. O. DNA arrays for analysis of gene expression. Methods Enzymol. 1999:303:179-205).
A modification, preferably in the genome, is construed as one or more modifications.
The modification, preferably in the genome, can either be effected by

    
    
        a) subjecting the parent microbial host cell to recombinant genetic manipulation techniques; and/or
        b) subjecting the parent microbial host cell to (classical) mutagenesis; and/or
        c) subjecting the parent microbial host cell to an inhibiting compound or composition.
    
    


Modification of a genome of a (mutant) microbial host cell is herein defined as any event resulting in a change in a polynucleotide sequence in the genome of the cell.
In a preferred embodiment the mutant microbial host cell according to the invention has a modification, preferably in its genome comprising:
a) a modification which results in a reduced or no production of a polypeptide as defined herein if compared to the parent microbial host cell that has not been modified, when analysed under the same conditions and/or
b) a modification which results in a polypeptide derived from a polypeptide as defined herein with decreased or no (enzymatic) activity as defined herein if compared to the parent microbial host cell that has not been modified, when analysed under the same conditions.
In a preferred embodiment the mutant microbial host cell according to the invention has a modification, preferably in its genome comprising:
a) a modification which results in a reduced or no production of a polypeptide having amylase activity, preferably α-amylase activity as defined herein if compared to the parent microbial host cell that has not been modified, when analysed under the same conditions and/or
b) a modification which results in a polypeptide derived from a polypeptide having amylase activity, preferably α-amylase activity as defined herein with decreased or no (α-)amylase activity as defined herein if compared to the parent microbial host cell that has not been modified, when analysed under the same conditions.
Modification can be introduced by classical strain improvement, random mutagenesis followed by selection. Modification can also be introduced by site-directed mutagenesis.
Modification may be accomplished by the introduction (insertion), substitution (replacement) or removal (deletion) of one or more nucleotides in a polynucleotide sequence. A full or partial deletion of a polynucleotide coding for the polypeptide having amylase activity preferably α-amylase activity as defined herein may be achieved. In alternative a polynucleotide coding for the polypeptide as defined herein may be partially or fully replaced with a polynucleotide sequence which does not code for a polypeptide having amylase activity preferably α-amylase activity as defined herein or which code for a partially or fully inactive form of a polypeptide as defined herein. In yet another alternative one or more nucleotides can be inserted into the polynucleotide encoding a polypeptide having amylase activity, preferably α-amylase activity as defined herein resulting in the disruption of said polynucleotide and consequent partial or full inactivation of the polypeptide as defined herein coded by the disrupted polynucleotide.
In one embodiment the mutant microbial host cell according to the invention comprises a modification in its genome selected from
a) a full or partial deletion of a polynucleotide as defined herein,
b) a full or partial replacement of a polynucleotide as defined herein with a polynucleotide sequence which does not code for a polypeptide as defined herein or which code for a partially or fully inactive form of a polypeptide as defined herein
c) a disruption of a polynucleotide as defined herein by the insertion of one or more nucleotides in the polynucleotide sequence and consequent partial or full inactivation of the polypeptide as defined herein coded by the disrupted polynucleotide.
This modification may for example be in a coding sequence or a regulatory element required for the transcription or translation of the polynucleotide as described above. For example, nucleotides may be inserted or removed so as to result in the introduction of a stop codon, the removal of a start codon or a change or a frame-shift of the open reading frame of a coding sequence. The modification of a coding sequence or a regulatory element thereof may be accomplished by site-directed or random mutagenesis, DNA shuffling methods, DNA reassembly methods, gene synthesis (see for example Young and Dong, (2004), Nucleic Acids Research 32, (7) electronic access http://nar.oupjournals.org/cgi/reprint/32/7/e59 or Gupta et al. (1968), Proc. Natl. Acad. Sci USA, 60: 1338-1344; Scarpulla et al. (1982), Anal. Biochem. 121: 356-365; Stemmer et al. (1995), Gene 164: 49-53), or PCR generated mutagenesis in accordance with methods known in the art. Examples of random mutagenesis procedures are well known in the art, such as for example chemical (NTG for example) mutagenesis or physical (UV for example) mutagenesis. Examples of site-directed mutagenesis procedures are the QuickChangeTM site-directed mutagenesis kit (Stratagene Cloning Systems, La Jolla, Calif.), the ‘The Altered Sites® II in vitro Mutagenesis Systems’ (Promega Corporation) or by overlap extension using PCR as described in Gene. 1989 Apr. 15; 77(1):51-9. (Ho S N, Hunt H D, Horton R M, Pullen J K, Pease L R “Site-directed mutagenesis by overlap extension using the polymerase chain reaction”) or using PCR as described in Molecular Biology: Current Innovations and Future Trends. (Eds. A. M. Griffin and H. G. Griffin. ISBN 1-898486-01-8; 1995 Horizon Scientific Press, PO Box 1, Wymondham, Norfolk, U.K.).
Preferred methods of modification are based on recombinant genetic manipulation techniques such as partial or complete gene replacement or partial or complete gene deletion.
For example, in case of replacement of a polynucleotide, nucleic acid construct or expression cassette, an appropriate DNA sequence may be introduced at the target locus to be replaced. The appropriate DNA sequence is preferably present on a cloning vector. Preferred integrative cloning vectors comprise a DNA fragment, which is homologous to the polynucleotide and/or has homology to the polynucleotides flanking the locus to be replaced for targeting the integration of the cloning vector to this pre-determined locus. In order to promote targeted integration, the cloning vector is preferably linearized prior to transformation of the cell. Preferably, linearization is performed such that at least one but preferably either end of the cloning vector is flanked by sequences homologous to the DNA sequence (or flanking sequences) to be replaced. This process is called homologous recombination and this technique may also be used in order to achieve (partial) gene deletion.
For example a polynucleotide corresponding to the endogenous polynucleotide may be replaced by a defective polynucleotide, that is a polynucleotide that fails to produce a (fully functional) polypeptide. By homologous recombination, the defective polynucleotide replaces the endogenous polynucleotide. It may be desirable that the defective polynucleotide also encodes a marker, which may be used for selection of transformants in which the nucleic acid sequence has been modified.
Alternatively or in combination with other mentioned techniques, a technique based on in vivo recombination of cosmids in E. coli can be used, as described in: A rapid method for efficient gene replacement in the filamentous fungus Aspergillus nidulans (2000) Chaveroche, M-K., Ghico, J-M. and d'Enfert C; Nucleic acids Research, vol 28, no 22.
Alternatively, modification, wherein said host cell produces less of or no protein such as the polypeptide having amylase activity, preferably α-amylase activity as described herein and encoded by a polynucleotide as described herein, may be performed by established anti-sense techniques using a nucleotide sequence complementary to the nucleic acid sequence of the polynucleotide. More specifically, expression of the polynucleotide by a host cell may be reduced or eliminated by introducing a nucleotide sequence complementary to the nucleic acid sequence of the polynucleotide, which may be transcribed in the cell and is capable of hybridizing to the mRNA produced in the cell. Under conditions allowing the complementary anti-sense nucleotide sequence to hybridize to the mRNA, the amount of protein translated is thus reduced or eliminated. An example of expressing an antisense-RNA is shown in Appl. Environ. Microbiol. 2000 February; 66(2):775-82. (Characterization of a foldase, protein disulfide isomerase A, in the protein secretory pathway of Aspergillus niger. Ngiam C, Jeenes D J, Punt P J, Van Den Hondel C A, Archer D B) or (Zrenner R, Willmitzer L, Sonnewald U. Analysis of the expression of potato uridinediphosphate-glucose pyrophosphorylase and its inhibition by antisense RNA. Planta. (1993); 190(2):247-52).
In one embodiment the mutant microbial host cell according to the invention is a mutant microbial host cell wherein the modification which results in a reduced or no production of a polypeptide as defined herein is due to a reduced production of the mRNA encoding said polypeptide if compared with a parent microbial host cell which has not been modified and measured under the same conditions.
A modification which results in a reduced amount of the mRNA transcribed from the polynucleotide encoding for the polypeptide having amylase activity, preferably α-amylase activity as described above may be obtained via the RNA interference (RNAi) technique (FEMS Microb. Lett. 237 (2004): 317-324). In this method identical sense and antisense parts of the nucleotide sequence, which expression is to be affected, are cloned behind each other with a nucleotide spacer in between, and inserted into an expression vector. After such a molecule is transcribed, formation of small nucleotide fragments will lead to a targeted degradation of the mRNA, which is to be affected. The elimination of the specific mRNA can be to various extents. The RNA interference techniques described in WO2008/053019, WO2005/05672A1, WO2005/026356A1, Oliveira et al., “Efficient cloning system for construction of gene silencing vectors in Aspergillus niger” (2008) Appl. Microbiol. and Biotechnol. 80 (5): 917-924 and/or Barnes et al., “siRNA as a molecular tool for use in Aspergillus niger” (2008) Biotechnology Letters 30 (5): 885-890 may be used at this purpose.
A modification which results in a polypeptide with decreased or no amylase activity preferably with decreased or no α-amylase activity as defined herein can be obtained by different methods, for example by an antibody directed against such a polypeptide or a chemical inhibitor or a protein inhibitor or a physical inhibitor (Tour O. et al, (2003) Nat. Biotech: Genetically targeted chromophore-assisted light inactivation. Vol. 21. no. 12:1505-1508) or peptide inhibitor or an anti-sense molecule or RNAi molecule (R. S. Kamath_et al, (2003) Nature: Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. vol. 421, 231-237).
In addition of the above-mentioned techniques or as an alternative, it is also possible to inhibiting the activity of a polypeptide as defined herein, or to re-localize the polypeptide as defined herein by means of alternative signal sequences (Ramon de Lucas, J., Martinez O, Perez P., Isabel Lopez, M., Valenciano, S. and Laborda, F. The Aspergillus nidulans carnitine carrier encoded by the acuH gene is exclusively located in the mitochondria. FEMS Microbiol Lett. 2001 Jul. 24; 201(2):193-8) or retention signals (Derkx, P. M. and Madrid, S. M. The foldase CYPB is a component of the secretory pathway of Aspergillus niger and contains the endoplasmic reticulum retention signal HEEL. Mol. Genet. Genomics. 2001 December; 266(4):537-545), or by targeting the polypeptide to a peroxisome which is capable of fusing with a membrane-structure of the cell involved in the secretory pathway of the cell, leading to secretion outside the cell of the polypeptide (e.g. as described in WO2006/040340).
Alternatively or in combination with above-mentioned techniques, inhibition of polypeptide having amylase activity preferably α-amylase activity as defined herein can also be obtained, e.g. by UV or chemical mutagenesis (Mattern, I. E., van Noort J. M., van den Berg, P., Archer, D. B., Roberts, I. N. and van den Hondel, C. A., Isolation and characterization of mutants of Aspergillus niger deficient in extracellular proteases. Mol Gen Genet. 1992 August; 234(2):332-6) or by the use of inhibitors inhibiting enzymatic activity of a polypeptide as described herein (e.g. nojirimycin, which function as inhibitor for β-glucosidases (Carrel F. L. Y. and Canevascini G. Canadian Journal of Microbiology (1991) 37(6): 459-464; Reese E. T., Parrish F. W. and Ettlinger M. Carbohydrate Research (1971) 381-388)).
In an embodiment according to the invention the modification in the genome of the mutant microbial host cell according to the invention is a modification in at least one position of a polynucleotide as defined above encoding for the polypeptide having amylase activity, preferably α-amylase activity as defined above.
In the context of the present invention the “parent microbial host cell” and the “mutant microbial host cell” may be any type of host cell. The specific embodiments of the mutant microbial host cell are hereafter described. It will be clear to those skilled in the art that embodiments applicable to the mutant microbial host cell are as well applicable to the parent microbial host cell unless otherwise indicated.
The mutant microbial host cell according to the present invention may be a prokaryotic cell. Preferably, the prokaryotic host cell is 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. 
According to an embodiment, the mutant microbial host cell according to the invention is a eukaryotic host cell. Preferably, the eukaryotic cell is a mammalian, insect, plant, fungal, or algal cell. Preferred mammalian cells include e.g. Chinese hamster ovary (CHO) cells, COS cells, 293 cells, 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's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK). The filamentous fungi are characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. Filamentous fungal strains include, but are not limited to, strains of Acremonium, Agaricus, Aspergillus, Aureobasidium, Chrysosporium, Coprinus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Panerochaete, Pleurotus, Schizophyllum, Talaromyces, Rasamsonia, Thermoascus, Thielavia, Tolypocladium, and Trichoderma. 
Preferred filamentous fungal cells belong to a species of an Acremonium, Aspergillus, Chrysosporium, Myceliophthora, Penicillium, Talaromyces, Rasamsonia, 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, Rasamsonia emersonii, 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.
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 Wisconsin54-1255(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 C1, Garg 27K, VKM-F 3500 D, Chrysosporium lucknowense C1, Garg 27K, VKM-F 3500 D, ATCC44006 and derivatives thereof.
According to one embodiment of the invention, when the mutant microbial host cell according to the invention is a filamentous fungal host cell the mutant microbial host cell may further comprise one or more modifications in its genome such that the mutant microbial host cell is deficient in the production of at least one product selected from glucoamylase (glaA), acid stable alpha-amylase (amyA), neutral alpha-amylase (amyBI and amyBII), oxalic acid hydrolase (oahA), a toxin, preferably ochratoxin and/or fumonisin, a protease transcriptional regulator prtT, PepA, a product encoded by the gene hdfA and/or hdfB, a non-ribosomal peptide synthase npsE if compared to a parent host cell and measured under the same conditions.
Oxalic acid hydrolase (oahA) is a component of the synthesis pathway of oxalic acid in many host cells. A host cell deficient in oahA will be deficient in oxalic acid. Oxalic acid is an unwanted by-product in many applications such as food-applications. Furthermore, oxalic acid lowers the pH of the medium cultivations of host cell producing this component, resulting in lowered yields; i.e. yield is increased in oxalic acid deficient host cells. It is therefore advantageous if the microbial host cell according to the invention is deficient in oahA. OahA deficient host cells and preferred methods of producing said host cells are extensively described in WO 2000/50576 and WO2004/070022. A preferred method to produce an oahA deficient host cell is the recombinant method of disruption described in WO 2000/50576. Preferably, the mutant microbial host cell according to the invention is deficient in oahA. Preferably, the oahA is a fungal oahA. More preferably, the oahA is the oahA from Aspergillus. Even more preferably the oahA is the oahA from Aspergillus niger. Even more preferably the oahA is the oahA from Aspergillus niger CBS 513.88. Most preferably, the oahA comprises the sequence of An10g00820.
prtT is a transcriptional activator of proteases in eukaryotic cells. Several fungal transcriptional activators of proteases have been recently described in WO 00/20596, WO 01/68864, WO 2006/040312 and WO 2007/062936. These transcriptional activators were isolated from Aspergillus niger (A. niger), Aspergillus fumigatus (A. fumigatus), Penicillium chrysogenum (P. chrysogenum) and Aspergillus oryzae (A. oryzae). These transcriptional activators of protease genes can be used to improve a method for producing a polypeptide in a fungal cell, wherein the polypeptide is sensitive for protease degradation. When the microbial host cell according to the invention is deficient in prtT, the host cell will produce less proteases that are under transcriptional control of prtT. It is therefore advantageous when the host cell according to the invention is deficient in prtT. prtT deficient hosts and preferred methods to produce these hosts are extensively described in WO 01/68864, WO 2006/040312. WO 01/68864 and WO 2006/040312 describe recombinant and classic methods to disrupt the prtT coding sequence. WO 2007/062936 describes disruption of the prtT binding site in a protease promoter. Disruption of the binding site impedes binding of prtT to the binding site. Consequently, the transcription of the protease is not activated by prtT and less protease is produced.
Preferably, the mutant microbial host cell according to the invention comprises a polynucleotide encoding prtT, said polynucleotide comprising a modification, wherein the host cell is deficient in the production of prtT compared to a parent cell it originates from when cultivated under comparable conditions. Preferably, the prtT is a fungal prtT. More preferably, the prtT is the prtT from Aspergillus. Even more preferably the prtT is the prtT from Aspergillus niger. Even more preferably the prtT is the prtT from Aspergillus niger CBS 513.88. Most preferably, the prtT comprises the sequence of An04g06940.
The term “glucoamylase” (glaA) is identical to the term “amyloglucosidase” and is defined herein as an enzyme having dextrin 6-alpha-D-glucanohydrolase activity which catalyses the endo hydrolysis of 1,6-alpha-D-glucoside linkages at points of branching in chains of 1,4-linked alpha-D-glucose residues and terminal 1,4-linked alpha-D-glucose residues. Glucoamylase activity can be measured as AGIU/ml by determining the liberation of paranitrofenol from the substrate p-nitrophenyl-a-D-glucopyranoside (Sigma). This results in a yellow colour, whose absorbance can be measured at 405 nm using a spectrophotometer. 1 AGIU is the quantity of enzyme, which produces 1 μmole of glucose per minute at pH 4.3 and 60° C. from a soluble starch substrate. In WO98/46772 additional details of the assay can be found.
Preferably, the mutant microbial host cell according to the invention comprises a polynucleotide encoding glaA, said polynucleotide comprising a modification, wherein the host cell is deficient in the production of glaA compared to a parent cell it originates from when cultivated under comparable conditions. Preferably, the glaA is a fungal glaA. More preferably, the glaA is the glaA from Aspergillus. Even more preferably the glaA is the glaA from Aspergillus niger. Even more preferably the glaA is the glaA from Aspergillus niger CBS 513.88. Most preferably, the glaA comprises the sequence of An03g06550.
The term “alpha-amylase” is defined herein as 1, 4-alpha-D-glucan glucanohydrolase activity which catalyzes the endohydrolysis of polysaccharides with three or more alpha-1, 4-linked glucose units in the presence of water to malto-oligosaccharides. To determine the (neutral) alpha-amylase activity, the Megazyme cereal alpha-amylase kit is used (Megazyme, CERALPHA alpha amylase assay kit, catalogus. ref. K-CERA, year 2000-2001), according a protocol of the supplier. The measured activity is based on hydrolysis of non-reducing-endblocked ρ-nitrophenyl maltoheptaoside in the presence of excess glucoamylase and α-glucosidase at a pH of 7.0. The amount of formed ρ-nitrophenol is a measure for alpha-amylase activity present in a sample.
The term “acid stable alpha-amylase” (amyA) is defined herein as an enzyme having alpha-amylase activity with optimal activity in the acid pH range. To determine the acid stable alpha-amylase activity, also the Megazyme cereal alpha-amylase kit is used (Megazyme, CERALPHA alpha amylase assay kit, catalogus. ref. K-CERA, year 2000-2001), according a protocol of the supplier but at an acid pH. The measured activity is based on hydrolysis of non-reducing-endblocked ρ-nitrophenyl maltoheptaoside in the presence of excess glucoamylase and α-glucosidase at a pH of 4.5. The amount of formed ρ-nitrophenol is a measure for acid stable alpha-amylase activity present in a sample.
Preferably, the host cell according to the invention comprises a polynucleotide encoding AmyA, said polynucleotide comprising a modification, wherein the host cell is deficient in amyA compared to the parent cell it originates from when cultivated under comparable conditions. Preferably, the amyA is a fungal amyA. More preferably, the amyA is the amyA from Aspergillus. Even more preferably the amyA is the amyA from Aspergillus niger. Even more preferably the amyA is the amyA from Aspergillus niger CBS 513.88. Most preferably, the amyA comprises the sequence of An11g03340.
The term “neutral alpha-amylase activity” (amy) is defined herein as an enzyme having alpha-amylase activity with optimal activity in the neutral pH range.
Preferably, the host cell according to the invention comprises a polynucleotide encoding AmyB, said polynucleotide comprising a modification, wherein the host cell is deficient in amyBI and/or amyBII compared to the parent cell it originates from when cultivated under comparable conditions. More preferably, the microbiaol host cell according to the invention is deficient in amyBI and amy BII. Preferably, the amyB a is a fungal amyB. More preferably, the amyB is the amyB from Aspergillus. Even more preferably the amyB is the amyBI from Aspergillus niger. Even more preferably the amyB is the amyBI from Aspergillus niger CBS 513.88. Most preferably, the amyBI comprises the sequence of An12g06930. Even more preferably the amyB is the amyBII from Aspergillus niger. Even more preferably the amyB is the amyBII from Aspergillus niger CBS 513.88. Most preferably, the amyBII comprises the sequence of An05g02100.
The term toxin associated polynucleotide is defined herein as a gene cluster, a multitude of genes, a gene or part thereof encoding a compound, or biochemical pathway responsible for the biosynthesis or secretion of at least one toxin or toxin intermediate compound. Said compound may e.g. be a polypeptide, which may be an enzyme.
A number of host cells, especially fungi, which are used as host cells in the production of polypeptides of interest possesses genes encoding enzymes involved in the biosynthesis of various toxins. For example, cyclopiazonic acid, kojic acid, 3-nitropropionic acid and aflatoxins are known toxins, which are formed in, e.g., Aspergillus flavus. Similarly, trichothecenes are formed in a number of fungi, e.g., in Fusarium sp. such as Fusarium venenatum and in Trichoderma and ochratoxin may be produced by Aspergillus. Recently, sequencing of the genome of an industrial Aspergillus niger host strain revealed a fumonisin gene cluster (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 formation of such toxins during the fermentation of compounds of interest is highly undesirable as these toxins may present a health hazard to operators, customers and the environment. Consequently, a toxin deficient host cell enables toxin-free production of a compound of interest. The toxin-free compound is easier to produce since no toxin has to be removed from the product. Furthermore, the regulatory approval procedure for the compound is easier.
Preferably, the mutant microbial host cell according to the invention comprises a toxin associated polynucleotide encoding a compound (which may e.g. be a polypeptide which may be an enzyme) or biochemical pathway, said toxin associated polynucleotide comprising a modification, wherein the host cell is deficient in the production of said toxin or a toxin intermediate compound compared to the parent cell it originates from when cultivated under comparable conditions. Preferably, the toxin or toxin intermediate compound is a fungal toxin or toxin intermediate compound. More preferably, the toxin or toxin intermediate compound is a toxin or toxin intermediate compound from Aspergillus. Even more preferably the toxin or the toxin intermediate compound is a toxin or toxin intermediate compound from Aspergillus niger. Even more preferably the toxin or toxin intermediate compound is a toxin or toxin intermediate compound from Aspergillus niger CBS 513.88. Even more preferably, the toxin or the toxin intermediate compound is fumonisin or a fumonisin intermediate compound. Even more preferably, the toxin or the toxin intermediate compound is ochratoxin or an ochratoxin intermediate compound. Most preferably, the toxin or the toxin intermediate compound is ochratoxin or fumonisin or an ochratoxin or a fumonisin intermediate compound.
Preferably, the toxin associated polynucleotide encodes a compound (which may e.g. be a polypeptide which may be an enzyme) or a biochemical pathway which is involved in the production of a fungal toxin or toxin intermediate compound. More preferably, a toxin or toxin intermediate compound from Aspergillus. Even more preferably, a toxin or toxin intermediate compound from Aspergillus niger. Even more preferably, a toxin or toxin intermediate compound from Aspergillus niger CBS 513.88. Even more preferably, a fumonisin or a fumonisin intermediate compound. Even more preferably, a fumonisin-B or a fumonisin-B intermediate compound. Even more preferably, a fumonisin-B2 or a fumonisin-B2 intermediate compound. Even more preferably, the toxin associated polynucleotide comprises the sequence of the fumonisin cluster from An01g06820 until An01g06930. Most preferably, the toxin associated polynucleotide comprises the sequence of An01g06930.
In another preferred embodiment, the toxin associated polynucleotide encodes a compound (which may e.g. be a polypeptide which may be an enzyme) or a biochemical pathway which is involved in ochratoxin or an ochratoxin intermediate compound. More preferably, an ochratoxin A or an ochratoxin A intermediate compound. More preferably, the toxin associated polynucleotide comprises the sequence of the cluster from An15g07880 until An15g07930. Most preferably, the toxin associated polynucleotide comprises the sequence of An15g07910 and/or the sequence of An15g07920.
Preferably, the mutant microbial host cell according to the invention comprises at least one toxin associated polynucleotide encoding a compound (which may e.g. be a polypeptide which may be an enzyme) or biochemical pathway, said toxin associated polynucleotide comprising at least one modification, wherein the host cell is deficient in the production of a toxin or, toxin intermediate compound compared to the parent cell it originates from when cultivated under comparable conditions.
More preferably, the host cell according to the invention comprises two toxin associated polynucleotides, said two toxin associated polynucleotides each comprising at least one modification, wherein the host cell is preferably deficient in the production of fumonisin and ochratoxin compared to the parent cell it originates from when cultivated under comparable conditions.
Even more preferably, the mutant microbial host cell according to the invention comprises three or more toxin associated polynucleotides said three or more toxin associated polynucleotides each comprising at least one modification, wherein the host cell is preferably deficient in the production of fumonisin, ochratoxin and at least one additional toxin or toxin intermediate compound compared to the parent cell it originates from when cultivated under comparable conditions.
Therefore, when the mutant microbial host cell according to the invention is a filamentous fungal host cell the host cell may comprise one or more modifications in its genome to result in a deficiency in the production of the major extracellular aspartic protease PepA. For example the host cell according to the invention may further comprise a disruption of the pepA gene encoding the major extracellular aspartic protease PepA. More preferably, the pepA is the pepA from Aspergillus. Even more preferably the pepA is the pepA from Aspergillus niger. Even more preferably the pepA is the pepA from Aspergillus niger CBS 513.88. Most preferably, the pepA comprises the sequence of An14g04710. Preferably, the efficiency of targeted integration of a polynucleotide to a pre-determined site into the genome of the mutant microbial host cell according to the invention is increased by making the cell deficient in a component in NHR (non-homologous recombination). Preferably, the mutant microbial host cell according to the invention comprises a polynucleotide encoding an NHR component comprising a modification, wherein said host cell is deficient in the production of said NHR component compared to a parent cell it originates from when cultivated under the same conditions.
The NHR component to be modified can be any NHR component known to the person skilled in the art. Preferred NHR components to be modified are selected from the group of filamentous fungal homologues of yeast KU70, KU80, MRE11, RAD50, RAD51, RAD52, XRS2, SIR4, LIG4. More preferred NHR components to be modified are filamentous fungal homologues of yeast KU70 and KU80, preferably hdfA (homologue of yeast KU70) or homologues thereof and hdfB (homologue of yeast KU80) or homologues thereof. The most preferred NHR component to be modified is KU70 or hdfA, or a homologue thereof. Another preferred NHR component to be modified is KU80 or hdfB, or a homologue thereof. Methods to obtain such host cell deficient in a component involved in NHR are known to the skilled person and are extensively described in WO2005/095624. Preferably the hdfA gene is the hdfA gene from A. niger, more preferably the hdfA from A. niger according to SEQ ID NO: 1 of WO2005/095624. In another preferred embodiment the hdfB gene is the hdfB gene from A. niger, more preferably the hdfB from A. niger according to SEQ ID NO: 4 of WO2005/095624.
Therefore when the mutant microbial host cell according to the invention is a filamentous fungal host cell the host cell according to the invention may additionally comprises one or more modifications in its genome to result in a deficiency in the production of the product encoded by the hdf A gene (as depicted in SEQ ID NO: 3 of WO 2005/095624) and/or hdfB gene (as depicted in SEQ ID NO: 6 of WO 2005/095624). For example the host cell according to the invention may further comprise a disruption of the hdfA and/or hdfB gene. Filamentous fungal host cells which are deficient in a product encoded by the hdfA and/or hdfB gene have been described in WO 2005/095624.
When the mutant microbial host cell according to the invention is a filamentous fungal host cell the host cell according to the invention may additionally comprise a modification in its genome which results in the deficiency in the production of the non-ribosomal peptide synthase npsE. Such host cells deficient in the production of non-ribosomal peptide synthase npsE have been described in WO2012/001169 (npsE has a genomic sequence as depicted in SEQ ID NO: 35, a coding sequence depicted in SEQ ID NO: 36, the mRNA depicted in SEQ ID NO: 37 and the nrps protein depicted in SEQ ID NO: 38 of WO2012/001169).
The mutant microbial host cell according to the invention may additionally comprises a modification in its genome which results in the deficiency in the production of the protein product of the agsE gene. Such host cells deficient in this protein product have been described in a co-pending International patent application filed on 19 Jul. 2013 entitled “AgsE-deficient strain” and which claims priority from EP12177172.9 filed on 19 Jul. 2012 and U.S. 61/673596 filed on 19 Jul. 2012. agsE has a genomic sequence as depicted in SEQ ID NO: 1 and a coding sequence depicted in SEQ ID NO: 2 and the AgsE protein as depicted in SEQ ID NO: 3 with the mature AgsE protein shown in SEQ ID NO: 4 of this co-pending International patent application. SEQ ID NOs: 1, 2, 3 and 4 correspond to SEQ ID NOs: 17, 18, 19 and 20 herein respectively.
The deficiency in the production of at least one product selected from glucoamylase (glaA), acid stable alpha-amylase (amyA), neutral alpha-amylase (amyBI and amyBII), oxalic acid hydrolase (oahA), a toxin, preferably ochratoxin and/or fumonisin, a protease transcriptional regulator prtT, PepA, a product encoded by the gene hdfA and/or hdfB, a non-ribosomal peptide synthase npsE, a protein product encoded by AgsE if compared to a parent host cell and measured under the same conditions may already be present in the parent host cell from which the mutant microbial host cell according to the invention is derived.
In one embodiment the mutant microbial cell according to the invention further comprises a deficiency in the production of glaA and optionally at least another product selected from the group consisting of acid stable alpha-amylase (amyA), neutral alpha-amylase (amyBI and amyBII), oxalic acid hydrolase (oahA), a toxin, preferably ochratoxin and/or fumonisin, a protease transcriptional regulator prtT, PepA, a product encoded by the gene hdfA and/or hdfB, a non-ribosomal peptide synthase npsE, a protein product encoded by AgsE if compared to a parent host cell and measured under the same conditions.
In one embodiment the mutant microbial cell according to the invention further comprises a deficiency in the production of glaA, PepA and optionally at least another product selected from the group consisting of acid stable alpha-amylase (amyA), neutral alpha-amylase (amyBI and amyBII), oxalic acid hydrolase (oahA), a toxin, preferably ochratoxin and/or fumonisin, a protease transcriptional regulator prtT, a product encoded by the gene hdfA and/or hdfB, a non-ribosomal peptide synthase npsE, a protein product encoded by AgsE if compared to a parent host cell and measured under the same conditions.
In one embodiment the mutant microbial cell according to the invention further comprises a deficiency in the production of glaA, PepA, acid stable alpha-amylase (amyA) and optionally at least another product selected from the group consisting of neutral alpha-amylase (amyBI and amyBII), oxalic acid hydrolase (oahA), a toxin, preferably ochratoxin and/or fumonisin, a protease transcriptional regulator prtT, a product encoded by the gene hdfA and/or hdfB, a non-ribosomal peptide synthase npsE, a protein product encoded by AgsE if compared to a parent host cell and measured under the same conditions.
In one embodiment the mutant microbial cell according to the invention further comprises a deficiency in the production of glaA, PepA, acid stable alpha-amylase (amyA), neutral alpha-amylase amyBI and optionally at least another product selected from the group consisting of neutral alpha-amylase amyBII, oxalic acid hydrolase (oahA), a toxin, preferably ochratoxin and/or fumonisin, a protease transcriptional regulator prtT, a product encoded by the gene hdfA and/or hdfB, a non-ribosomal peptide synthase npsE, a protein product encoded by AgsE if compared to a parent host cell and measured under the same conditions.
In one embodiment the mutant microbial cell according to the invention further comprises a deficiency in the production of glaA, PepA, acid stable alpha-amylase (amyA), neutral alpha-amylase amyBI and amyBII, and optionally at least another product selected from the group consisting of oxalic acid hydrolase (oahA), a toxin, preferably ochratoxin and/or fumonisin, a protease transcriptional regulator prtT, a product encoded by the gene hdfA and/or hdfB, a non-ribosomal peptide synthase npsE, a protein product encoded by AgsE if compared to a parent host cell and measured under the same conditions.
In one embodiment the mutant microbial cell according to the invention further comprises a deficiency in the production of glaA, PepA, acid stable alpha-amylase (amyA), neutral alpha-amylase amyBI and amyBII, a product encoded by the gene hdfA and optionally at least another product selected from the group consisting of oxalic acid hydrolase (oahA), a toxin, preferably ochratoxin and/or fumonisin, a protease transcriptional regulator prtT, a product encoded by the gene hdfB, a non-ribosomal peptide synthase npsE, a protein product encoded by AgsE if compared to a parent host cell and measured under the same conditions.
In one embodiment the mutant microbial cell according to the invention further comprises a deficiency in the production of glaA, PepA, acid stable alpha-amylase (amyA), neutral alpha-amylase amyBI and amyBII, a product encoded by the gene hdfA, oxalic acid hydrolase (oahA) and optionally at least another product selected from the group consisting of, a toxin, preferably ochratoxin and/or fumonisin, a protease transcriptional regulator prtT, a product encoded by the gene hdfB, a non-ribosomal peptide synthase npsE, a protein product encoded by AgsE if compared to a parent host cell and measured under the same conditions.
In one embodiment the mutant microbial cell according to the invention further comprises a deficiency in the production of glaA, PepA, acid stable alpha-amylase (amyA), neutral alpha-amylase amyBI and amyBII, a product encoded by the gene hdfA, oxalic acid hydrolase (oahA), ochratoxin, fumonisin, and optionally at least another product selected from the group consisting of a protease transcriptional regulator prtT, a product encoded by the gene hdfB, a non-ribosomal peptide synthase npsE, a protein product encoded by AgsE if compared to a parent host cell and measured under the same conditions.
In one embodiment the mutant microbial cell according to the invention further comprises a deficiency in the production of glaA, PepA, acid stable alpha-amylase (amyA), neutral alpha-amylase amyBI and amyBII, a product encoded by the gene hdfA, oxalic acid hydrolase (oahA), ochratoxin, fumonisin, a protease transcriptional regulator prtT and optionally at least another product selected from the group consisting of a product encoded by the gene hdfB, a non-ribosomal peptide synthase npsE, a protein product encoded by AgsE if compared to a parent host cell and measured under the same conditions.
In one embodiment the mutant microbial cell according to the invention further comprises a deficiency in the production of glaA, PepA, acid stable alpha-amylase (amyA), neutral alpha-amylase amyBI and amyBII, a product encoded by the gene hdfA, oxalic acid hydrolase (oahA), ochratoxin, fumonisin, a protease transcriptional regulator prtT, a non-ribosomal peptide synthase npsE and optionally at least another product selected from the group consisting of a product encoded by the gene hdfB, a protein product encoded by AgsE if compared to a parent host cell and measured under the same conditions.
In one embodiment the mutant microbial cell according to the invention further comprises a deficiency in the production of glaA, PepA, acid stable alpha-amylase (amyA), neutral alpha-amylase amyBI and amyBII, a product encoded by the gene hdfA, oxalic acid hydrolase (oahA), ochratoxin, fumonisin, a protease transcriptional regulator prtT, a protein product encoded by AgsE and optionally at least another product selected from the group consisting of a product encoded by the gene hdfB, a non-ribosomal peptide synthase npsE, if compared to a parent host cell and measured under the same conditions.
In a more preferred embodiment the mutant microbial cell according to the invention further has a reduced amylase background and comprises a deficiency in the production of glaA, acid stable alpha-amylase (amyA), neutral alpha-amylase amyBI and amyBII, if compared to a parent host cell and measured under the same conditions. Such a microbial mutant cell may also comprise a deficiency in the production of a filamentous fungal homolog of KU70 or KU80. Such a microbial mutant cell may also comprise a deficiency in the production of a toxin. Such a microbial mutant cell may also comprise a deficiency in the production of a filamentous fungal homolog of KU70 or KU80 and a deficiency in the production of a toxin.
In an even more preferred embodiment the mutant microbial cell according to the invention has a reduced amylase background and further comprises a deficiency in the production of glaA, acid stable alpha-amylase (amyA), neutral alpha-amylase amyBI, amyBII and a protein product encoded by AgsE if compared to a parent host cell and measured under the same conditions. Such a microbial mutant cell may also comprise a filamentous fungal homolog of KU70 or KU80. Such a microbial mutant cell may also comprise a deficiency in the production of a toxin. Such a microbial mutant cell may also comprise a deficiency in the production of a filamentous fungal homolog of KU70 or KU80 and a deficiency in the production of a toxin.
In a most preferred embodiment the mutant microbial cell according to the invention further has a reduced alpha-amylase background and comprises a deficiency in the production acid stable alpha-amylase (amyA), neutral alpha-amylase amyBI and amyBII, if compared to a parent host cell and measured under the same conditions. Such a microbial mutant cell may also comprise a filamentous fungal homolog of KU70 or KU80. Such a microbial mutant cell may also comprise a deficiency in the production of a toxin. Such a microbial mutant cell may also comprise a deficiency in the production of a filamentous fungal homolog of KU70 or KU80 and a deficiency in the production of a toxin.
When the mutant microbial host cell according to the invention is a filamentous fungal host cell the host cell may additionally comprise at least two substantially homologous DNA domains suitable for integration of one or more copies of a polynucleotide encoding a compound of interest wherein at least one of the at least two substantially homologous DNA domains is adapted to have enhanced integration preference for the polynucleotide encoding a compound of interest compared to the substantially homologous DNA domain it originates from, and wherein the substantially homologous DNA domain where the adapted substantially homologous DNA domain originates from has a gene conversion frequency that is at least 10% higher than one of the other of the at least two substantially homologous DNA domains. These cells have been described in WO2011/009700. Strains containing two or more copies of these substantially homologous DNA domains are also referred hereafter as strain containing two or more amplicons. Examples of host cells comprising such amplicons are e.g. described in van Dijck et al, 2003, Regulatory Toxicology and Pharmacology 28; 27-35: On the safety of a new generation of DSM Aspergillus niger enzyme production strains. In van Dijck et al, an Aspergillus niger strain is described that comprises 7 amplified glucoamylase gene loci, i.e. 7 amplicons. Preferred host cells within this context are filamentous fungus host cells, preferably A. niger host cells, comprising two or more amplicons, preferably two or more ΔglaA amplicons (preferably comprising 3, 4, 5, 6, 7 ΔglaA amplicons) wherein the amplicon which has the highest frequency of gene conversion, has been adapted to have enhanced integration preference for the polynucleotide encoding a compound of interest compared to the amplicon it originates from. Adaptation of the amplicon can be performed according to any one of the methods described in WO2011/009700 (which is here fully incorporated by reference). An example of these host cells, described in WO2011/009700, are host cells comprising three ΔglaA amplicons being a BamHI truncated amplicon, a SalI truncated amplicon and a Bg/II truncated amplicon and wherein the BamHI amplicon has been adapted to have enhanced integration preference for a polynucleotide encoding a compound of interest compared to the BamHI amplicon it originates from. Host cells comprising two or more amplicons wherein one amplicon has been adapted to have enhanced integration preference for a polynucleotide encoding a compound of interest compared to the amplicon it originates from are hereafter referred as host cells comprising an adapted amplicon.
When the mutant microbial host cell according to the invention is a filamentous fungal host cell, the host cell according to the invention may additionally comprises a modification of Sec61. A preferred SEC61 modification is a modification which results in a one-way mutant of SEC61; i.e. a mutant wherein the de novo synthesized protein can enter the ER via SEC61, but the protein cannot leave the ER via SEC61. Such modifications are extensively described in WO2005/123763. In a preferred embodiment the mutant microbial host cell comprises a modification in a Sec61 as depicted in SEQ ID NO: 3 of WO2005/123763. Most preferably, the SEC 61 modification is the S376W mutation in which Serine 376 is replaced by Tryptophan in SEQ ID NO: 3 of WO2005/123763.
In a preferred embodiment, the mutant microbial host cell according to the invention comprises at least one polynucleotide coding for a compound of interest or at least one polynucleotide coding for a compound involved in the production of a compound of interest by the cell.
The compound of interest can be any biological compound. The biological compound may be biomass or a biopolymer or metabolite. The biological compound may be encoded by a single polynucleotide or a series of polynucleotides composing a biosynthetic or metabolic pathway or may be the direct result of the product of a single polynucleotide or products of a series of polynucleotides. The biological compound may be native to the host cell or heterologous.
The term “heterologous biological compound” is defined herein as a biological compound which is not native to the cell; or a native biological compound in which structural modifications have been made to alter the native biological compound.
The term “biopolymer” is defined herein as a chain (or polymer) of identical, similar, or dissimilar subunits (monomers). The biopolymer may be any biopolymer. The biopolymer may for example be, but is not limited to, a nucleic acid, polyamine, polyol, polypeptide (or polyamide), or polysaccharide.
The biopolymer may be a polypeptide. The polypeptide may be any polypeptide having a biological activity of interest. The term “polypeptide” is not meant herein to refer to a specific length of the encoded product and, therefore, encompasses peptides, oligopeptides, and proteins. Polypeptides further include naturally occurring allelic and engineered variations of the above-mentioned polypeptides and hybrid polypeptides. The polypeptide may be native or may be heterologous to the host cell. 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, protein involved in secretion process, protein involved in folding process, chaperone, peptide amino acid transporter, glycosylation factor, transcription factor, synthetic peptide or oligopeptide, intracellular protein. The intracellular protein may be an enzyme such as, a protease, ceramidases, epoxide hydrolase, aminopeptidase, acylases, aldolase, hydroxylase, aminopeptidase, lipase. The polypeptide may also be an enzyme secreted extracellularly. Such enzymes may belong to the groups of oxidoreductase, transferase, hydrolase, lyase, isomerase, ligase, catalase, cellulase, chitinase, cutinase, deoxyribonuclease, dextranase, esterase. The enzyme may be a carbohydrase, 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, a maltogenic amylase, carbohydrase, carboxypeptidase, endo-protease, metallo-protease, serine-protease catalase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, haloperoxidase, protein deaminase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phospholipase, galactolipase, chlorophyllase, polyphenoloxidase, ribonuclease, transglutaminase, or glucose oxidase, hexose oxidase, monooxygenase.
Preferably the compound of interest is a glucose oxidase.
According to the present invention, a polypeptide or enzyme also can be a product as described in WO2010/102982. According to the present invention, a polypeptide can also be a fused or hybrid polypeptide to which another polypeptide is fused at the N-terminus or the C-terminus of the polypeptide or fragment thereof. A fused polypeptide is produced by fusing a nucleic acid sequence (or a portion thereof) encoding one polypeptide to a nucleic acid sequence (or a portion thereof) encoding another polypeptide.
Techniques for producing fusion polypeptides are known in the art, and include, ligating the coding sequences encoding the polypeptides so that they are in frame and expression of the fused polypeptide is under control of the same promoter (s) and terminator. The hybrid polypeptides may comprise a combination of partial or complete polypeptide sequences obtained from at least two different polypeptides wherein one or more may be heterologous to the host cell. Example of fusion polypeptides and signal sequence fusions are for example as described in WO2010/121933.
The biopolymer may be a polysaccharide. The polysaccharide may be any polysaccharide, including, but not limited to, a mucopolysaccharide (e. g., heparin and hyaluronic acid) and nitrogen-containing polysaccharide (eg., chitin). In a more preferred option, the polysaccharide is hyaluronic acid.
The polynucleotide coding for the compound of interest or coding for a compound involved in the production of the compound of interest according to the invention may encode an enzyme involved in the synthesis of a primary or secondary metabolite, such as organic acids, carotenoids, (beta-lactam) antibiotics, and vitamins. Such metabolite may be considered as a biological compound according to the present invention.
The term “metabolite” encompasses both primary and secondary metabolites; the metabolite may be any metabolite. Preferred metabolites are citric acid, gluconic acid, adipic acid, fumaric acid, itaconic acid and succinic acid.
The metabolite may be encoded by one or more genes, such as in a biosynthetic or metabolic pathway. Primary metabolites are products of primary or general metabolism of a cell, which are concerned with energy metabolism, growth, and structure. Secondary metabolites are products of secondary metabolism (see, for example, R. B. Herbert, The Biosynthesis of Secondary Metabolites, Chapman and Hall, New York, 1981).
The primary metabolite may be, but is not limited to, an amino acid, fatty acid, nucleoside, nucleotide, sugar, triglyceride, or vitamin.
The secondary metabolite may be, but is not limited to, an alkaloid, coumarin, flavonoid, polyketide, quinine, steroid, peptide, or terpene. The secondary metabolite may be an antibiotic, antifeedant, attractant, bacteriocide, fungicide, hormone, insecticide, or rodenticide. Preferred antibiotics are cephalosporins and beta-lactams. Other preferred metabolites are exo-metabolites. Examples of exo-metabolites are Aurasperone B, Funalenone, Kotanin, Nigragillin, Orlandin, Other naphtho-γ-pyrones, Pyranonigrin A, Tensidol B, Fumonisin B2 and Ochratoxin A.
The biological compound may also be the product of a selectable marker. A selectable marker is a product of a polynucleotide of interest which product provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. Selectable markers include, but are not limited to, amdS (acetamidase), argB (ornithinecarbamoyltransferase), bar (phosphinothricinacetyltransf erase), hygB (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase), trpC (anthranilate synthase), ble (phleomycin resistance protein), hyg (hygromycin), NAT or NTC (Nourseothricin) as well as equivalents thereof.
According to the invention, the compound of interest is preferably a polypeptide as described in the list of compounds of interest.
Preferably, the polypeptide is an enzyme as described in the list of compounds of interest. Preferably a glucose oxidase.
According to another embodiment of the invention, the compound of interest is preferably a metabolite.
The mutant microbial cell may already be capable of producing the compound of interest. The mutant microbial host cell may also be provided with a homologous or heterologous nucleic acid construct that encodes a polypeptide wherein the polypeptide may be the compound of interest or a polypeptide involved in the production of the compound of interest. The person skilled in the art knows how to modify a microbial host cell such that it is capable of producing the compound of interest.
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 an RNA or an mRNA and optionally of a polypeptide translated from said (m)RNA.
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.
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. The man skilled in the art knows which types of terminators can be used in the microbial host cell as described herein.
Preferred terminator sequences for filamentous fungal cells are obtained from any terminator sequence of a filamentous fungal gene, more preferably from Aspergillus genes, even more preferably from the gene A. oryzae TAKA amylase, the genes encoding A. niger glucoamylase (glaA), A. nidulans anthranilate synthase, A. niger alpha-glucosidase, trpC and/or Fusarium oxysporum trypsin-like protease.
The control sequence may also be an optimal translation initiation sequences (as described in Kozak, 1991, J. Biol. Chem. 266:19867-19870), or a 5′-untranslated sequence, a non-translated region of a mRNA which is important for translation by the mutated microbial host cell. The translation initiation sequence or 5′-untranslated sequence is operably linked to the 5′-terminus of the coding sequence encoding the polypeptide. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Control sequences may be optimized to their specific purpose.
Suitable 5′-untranslated sequences may be those polynucleotides preceeding the fungal amyloglucosidase (AG) gene, A. oryzae TAKA amylase and Aspergillus triose phosphate isomerase genes and A. niger glucoamylase glaA, alpha-amylase, xylanase and phytase encoding genes.
The control sequence may also be a non-translated region of a mRNA which is important for translation by the mutated microbial host 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.
Leader sequences may be those originating from the fungal amyloglucosidase (AG) gene (glaA-both 18 and 24 amino acid versions e. g. from Aspergillus), the α-factor gene (yeasts e. g. Saccharomyces and Kluyveromyces) or the α-amylase (amyE, amyQ and amyL) and alkaline protease aprE and natural protease genes (Bacillus), or signal sequences ad described in WO2010/121933.
Preferred leaders for filamentous fungal cells are obtained from the polynucleotides preceding 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 microbial host cell (mutated or parent) 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.
In the mutant microbial host cell according to the invention the at least one polynucleotide coding for the compound of interest or the at least one polynucleotide coding for a compound involved in the production of a compound of interest may be operably linked to a promoter.
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.
The promoter may be an inducible promoter. The promoter may be a carbohydrate inducible promoter. Carbohydrate inducible promoters that can be used are a starch-, cellulose-, hemicellulose (such as xylan- and/or xylose-inducible) promoters. Other inducible promoters are copper-, oleic acid-inducible promoters. Promoters suitable in filamentous fungi are promoters which may be selected from the group, which includes but is not limited to promoters obtained from the polynucleotides encoding A. oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A. niger neutral alpha-amylase, A. niger acid stable alpha-amylase, A. niger or A. awamori glucoamylase (glaA), A. niger or A. awamori endoxylanase (xlnA) or beta-xylosidase (xlnD), T. reesei cellobiohydrolase I (CBHI), R. miehei lipase, A. oryzae alkaline protease, A. oryzae triose phosphate isomerase, A. nidulans acetamidase, Fusarium venenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Dania (WO 00/56900), Fusarium venenatum Quinn (WO 00/56900), Fusarium oxysporum trypsin-like protease (WO 96/00787), Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase IV, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpi promoter (a hybrid of the promoters from the polynucleotides encoding A. niger neutral alpha-amylase and A. oryzae triose phosphate isomerase), and mutant, truncated, and hybrid promoters thereof. Other examples of promoters are the promoters described in WO2006/092396 and WO2005/100573, which are herein incorporated by reference. An even other example of the use of promoters is described in WO2008/098933. Promoters can also be constitutive promoters.
Examples of promoters from Gram-positive microorganisms include, but are not limited to, gnt (gluconate operon promoter); penP from Bacillus licheniformis; glnA (glutamine synthetase); xylAB (xylose operon); araABD (L-arabinose operon) and Pspac promoter, a hybrid SPO1/lac promoter that can be controlled by inducers such as isopropyl-β-D-thiogalactopyranoside [IPTG] ((Yansura D. G., Henner D. J. Proc Natl Acad Sci USA. 1984 81(2):439-443). Activators are also sequence-specific DNA binding proteins that induce promoter activity. Examples of such promoters from Gram-positive microorganisms include, but are not limited to, two-component systems (PhoP-PhoR, DegU-DegS, SpoOA-Phosphorelay), LevR, Mry and GltC. (ii) Production of secondary sigma factors can be primarily responsible for the transcription from specific promoters. Examples from Gram-positive microorganisms include, but are not limited to, the promoters activated by sporulation specific sigma factors: σF, σE, σG and σK and general stress sigma factor, σB. The σB-mediated response is induced by energy limitation and environmental stresses (Hecker M, Völker U. Mol Microbiol. 1998; 29(5):1129-1136). (iii) Attenuation and antitermination also regulates transcription. Examples from Gram-positive microorganisms include, but are not limited to, trp operon and sacB gene. (iv) Other regulated promoters in expression vectors are based the sacR regulatory system conferring sucrose inducibility (Klier A F, Rapoport G. Annu Rev Microbiol. 1988; 42:65-95).
Suitable inducible promoters useful in bacteria, such as Bacilli, include: promoters from Gram-positive microorganisms such as, but are not limited to, SP01-26, SP01-15, veg, pyc (pyruvate carboxylase promoter), and amyE. Examples of promoters from Gram-negative microorganisms include, but are not limited to, tac, tet, trp-tet, lpp, lac, lpp-lac, laclq, T7, T5, T3, gal, trc, ara, SP6, λ-PR, and λ-PL.
Additional examples of promoters useful in bacterial cells, such as Bacilli, include the α-amylase and SPo2 promoters as well as promoters from extracellular protease genes.
Other example of a suitable promoter are the promoter obtained from the E. coli lac operon. Another example is the promoter of the Streptomyces coelicolor agarase gene (dagA). Another example is the promoter of the Bacillus lentus alkaline protease gene (aprH). Another example is the promoter of the Bacillus licheniformis alkaline protease gene (subtilisin Carlsberg gene). Another example is the promoter of the Bacillus subtilis levansucrase gene (sacB). Another example is the promoter of the Bacillus subtilis alphaamylase gene (amyF). Another example is the promoter of the Bacillus licheniformis alphaamylase gene (amyL). Another example is the promoter of the Bacillus stearothermophilus maltogenic amylase gene (amyM). Another example is the promoter of the Bacillus amyloliquefaciens alpha-amylase gene (amyQ). Another example is a “consensus” promoter having the sequence TTGACA for the “-35” region and TATAAT for the “-10” region. Another example is the promoter of the Bacillus licheniformis penicillinase gene (penP). Another example are the promoters of the Bacillus subtilis xylA and xylB genes.
Preferably the promoter sequence is from a highly expressed gene. Examples of preferred highly expressed genes from which promoters may be selected and/or which are comprised in preferred predetermined target loci for integration of expression constructs, include but are not limited to genes encoding glycolytic enzymes such as triose-phosphate isomerases (TPI), glyceraldehyde-phosphate dehydrogenases (GAPDH), phosphoglycerate kinases (PGK), pyruvate kinases (PYK or PKI), alcohol dehydrogenases (ADH), as well as genes encoding amylases, glucoamylases, proteases, xylanases, cellobiohydrolases, β-galactosidases, alcohol (methanol) oxidases, elongation factors and ribosomal proteins. Specific examples of suitable highly expressed genes include e. g. the LAC4 gene from Kluyveromyces sp., the methanol oxidase genes (AOX and MOX) from Hansenula and Pichia, respectively, the glucoamylase (glaA) genes from A. niger and A. awamori, the A. oryzae TAKA-amylase gene, the A. nidulans gpdA gene and the T. reesei cellobiohydrolase genes.
Promoters which can be used in yeast include e.g. promoters from glycolytic genes, such as the phosphofructokinase (PFK), triose phosphate isomerase (TPI), glyceraldehyde-3-phosphate dehydrogenase (GPD, TDH3 or GAPDH), pyruvate kinase (PYK), phosphoglycerate kinase (PGK) promoters from yeasts or filamentous fungi; more details about such promoters from yeast may be found in (WO 93/03159). Other useful promoters are ribosomal protein encoding gene promoters, the lactase gene promoter (LAC4), alcohol dehydrogenase promoters (ADHI, ADH4, and the like), and the enolase promoter (ENO). Other promoters, both constitutive and inducible, and enhancers or upstream activating sequences will be known to those of skill in the art. The promoters used in the host cells of the invention may be modified, if desired, to affect their control characteristics. Suitable promoters in this context include both constitutive and inducible natural promoters as well as engineered promoters, which are well known to the person skilled in the art. Suitable promoters in eukaryotic host cells may be GAL7, GAL10, or GAL1, CYC1, HIS3, ADH1, PGL, PH05, GAPDH, ADC1, TRP1, URA3, LEU2, ENO1, TPI1, and AOX1. Other suitable promoters include PDC1, GPD1, PGK1, TEF1, and TDH3
In order to facilitate expression, the polynucleotide encoding the polypeptide being the compound of interest or the polypeptide involved in the production of the compound of interest may be a synthetic polynucleotide. The synthetic polynucleotides may be optimized in codon use, preferably according to the methods described in WO2006/077258 and/or PCT/EP2007/055943 (published as WO2008/000632), which are herein incorporated by reference. PCT/EP2007/055943 addresses codon-pair optimization. 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.
In order to facilitate expression and/or translation, the polynucleotide encoding the polypeptide being the compound of interest or encoding the polypeptide involved in the production of the compound of interest may be comprised in an expression vector such that the gene encoding the polypeptide product is operably linked to the appropriate control sequences for expression and/or translation in vitro, or in the mutant microbial host cell.
The expression vector may be any vector (e.g., a plasmid or virus), which can be conveniently subjected to recombinant DNA procedures and can bring about the expression of the polynucleotide encoding the polypeptide. The choice of the vector will typically depend on the compatibility of the vector with the cell into which the vector is to be introduced. The vectors may be linear or closed circular plasmids. The vector may be an autonomously replicating vector, i. e., a vector, which exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extra-chromosomal element, a mini-chromosome, or an artificial chromosome. An autonomously maintained cloning vector may comprise the AMA1-sequence (see e.g. Aleksenko and Clutterbuck (1997), Fungal Genet. Biol. 21: 373-397).
Alternatively, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. The integrative cloning vector may integrate at random or at a predetermined target locus in the chromosomes of the host cell. In a preferred embodiment of the invention, the integrative cloning vector comprises a DNA fragment, which is homologous to a DNA sequence in a predetermined target locus in the genome of host cell for targeting the integration of the cloning vector to this predetermined locus. In order to promote targeted integration, the cloning vector is preferably linearized prior to transformation of the cell. Linearization is preferably performed such that at least one but preferably either end of the cloning vector is flanked by sequences homologous to the target locus. The length of the homologous sequences flanking the target locus is preferably at least 30 bp, preferably at least 50 bp, preferably at least 0.1 kb, even preferably at least 0.2 kb, more preferably at least 0.5 kb, even more preferably at least 1 kb, most preferably at least 2 kb. Preferably, the efficiency of targeted integration into the genome of the host cell, i.e. integration in a predetermined target locus, is increased by augmented homologous recombination abilities of the host cell.
Preferably, the homologous flanking DNA sequences in the cloning vector, which are homologous to the target locus, are derived from a highly expressed locus meaning that they are derived from a gene, which is capable of high expression level in the host cell. A gene capable of high expression level, i.e. a highly expressed gene, is herein defined as a gene whose mRNA can make up at least 0.5% (w/w) of the total cellular mRNA, e.g. under induced conditions, or alternatively, a gene whose gene product can make up at least 1% (w/w) of the total cellular protein, or, in case of a secreted gene product, can be secreted to a level of at least 0.1 g/l (as described in EP 357 127 B1).
A number of preferred highly expressed fungal genes are given by way of example: the amylase, glucoamylase, alcohol dehydrogenase, xylanase, glyceraldehyde-phosphate dehydrogenase or cellobiohydrolase (cbh) genes from Aspergilli, Chrysosporium or Trichoderma. Most preferred highly expressed genes for these purposes are a glucoamylase gene, preferably an A. niger glucoamylase gene, an A. oryzae TAKA-amylase gene, an A. nidulans gpdA gene, a Trichoderma reesei cbh gene, preferably cbh1, a Chrysosporium lucknowense cbh gene or a cbh gene from P. chrysogenum. 
More than one copy of a nucleic acid sequence may be inserted into the mutated microbial host cell to increase production of the product (over-expression) encoded by said sequence. This can be done, preferably by integrating into its genome copies of the DNA sequence, more preferably by targeting the integration of the DNA sequence at one of the highly expressed loci defined in the former paragraph. Alternatively, this can be done by including an amplifiable selectable marker gene with the nucleic acid sequence where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the nucleic acid sequence, can be selected for by cultivating the cells in the presence of the appropriate selectable agent. To increase even more the number of copies of the DNA sequence to be over expressed the technique of gene conversion as described in WO98/46772 may be used.
The vector system may be a single vector or plasmid or two or more vectors or plasmids, which together contain the total DNA to be introduced into the genome of the host cell, or a transposon.
The vectors preferably contain one or more selectable markers, which permit easy selection of transformed cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. The selectable marker may be introduced into the cell on the expression vector as the expression cassette or may be introduced on a separate expression vector.
A selectable marker for use in a filamentous fungal cell may be selected from the group including, but not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), bleA (phleomycin binding), hygB (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase), NAT or NTC (Nourseothricin) and trpC (anthranilate synthase), as well as equivalents from other species. Preferred for use in an Aspergillus and Penicillium cell are the amdS (see for example EP 635574 B1, EP0758020A2, EP1799821A2, WO 97/06261A2) and pyrG genes of A. nidulans or A. oryzae and the bar gene of Streptomyces hygroscopicus. More preferably an amdS gene is used, even more preferably an amdS gene from A. nidulans or A. niger. A most preferred selectable marker gene is the A. nidulans amdS coding sequence fused to the A. nidulans gpdA promoter (see EP 635574 B1). Other preferred AmdS markers are those described in WO2006/040358. AmdS genes from other filamentous fungi may also be used (WO 97/06261).
Markers which can be used in bacteria include ATP synthetase, subunit 9 (oliC), orotidine-5′-phosphatedecarboxylase (pvrA), the bacterial G418 resistance gene (this may also be used in yeast, but not in filamentous fungi), the ampicillin resistance gene (E. coli), resistance genes for, neomycin, kanamycin, tetracycline, spectinomycin, erythromycin, chloramphenicol, phleomycin (Bacillus) and the E. coli uidA gene, coding for β-glucuronidase (GUS). Vectors may be used in vitro, for example for the production of RNA or used to transfect or transform a host cell.
Versatile marker genes that can be used for transformation of most filamentous fungi and yeasts such as acetamidase genes or cDNAs (the amdS, niaD, facA genes or cDNAs from A. nidulans, A. oryzae or A. niger), or genes providing resistance to antibiotics like G418, hygromycin, bleomycin, kanamycin, methotrexate, phleomycin orbenomyl resistance (benA). Alternatively, specific selection markers can be used such as auxotrophic markers which require corresponding mutant host strains: e. g. D-alanine racemase (from Bacillus), URA3 (from S. cerevisiae or analogous genes from other yeasts), pyrG or pyrA (from A. nidulans or A. niger), argB (from A. nidulans or A. niger) or trpC. In a preferred embodiment the selection marker is deleted from the transformed host cell after introduction of the expression construct so as to obtain transformed host cells capable of producing the polypeptide which are free of selection marker genes.
The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to one skilled in the art (see, e.g. Sambrook & Russell, Molecular Cloning: A Laboratory Manual, 3rd Ed., CSHL Press, Cold Spring Harbor, N.Y., 2001; and Ausubel et al., Current Protocols in Molecular Biology, Wiley InterScience, NY, 1995).
Furthermore, standard molecular cloning techniques such as DNA isolation, gel electrophoresis, enzymatic restriction modifications of nucleic acids, Southern analyses, transformation of cells, etc., are known to the skilled person and are for example described by Sambrook et al. (1989) “Molecular Cloning: a laboratory manual”, Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y. and Innis et al. (1990) “PCR protocols, a guide to methods and applications” Academic Press, San Diego.
A nucleic acid may be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
Preferably, the mutant microbial host cell is modified to improve the expression of the polynucleotides to enhance production of the polypeptides being the compound of interest or a polypeptide involved in the production of a compound of interest.
Preferably, the efficiency of targeted integration into the genome of the host cell, i.e. integration in a predetermined target locus, is increased by augmented homologous recombination abilities of the host cell. Such phenotype of the cell preferably involves a deficient hdfA or hdfB as described in WO2005/095624. WO2005/095624 discloses a preferred method to obtain a filamentous fungal cell comprising increased efficiency of targeted integration.
Optionally, the host cell has been modified to comprise an elevated unfolded protein response (UPR) to enhance production abilities of a polypeptide of interest. UPR may be increased by techniques described in US2004/0186070A1 and/or US2001/0034045A1 and/or WO01/72783A2 and/or WO2005/123763. More specifically, the protein level of HAC1 and/or IRE1 and/or PTC2 may be modulated, and/or the SEC61 protein may be engineered in order to obtain a host cell having an elevated UPR.
The person skilled in the art knows how to transform cells with the one or more expression cassettes and the selectable marker. For example, the skilled person may use one or more expression vectors, wherein the one or more cloning vectors comprise the expression cassettes and the selectable marker.
Transformation of the mutant microbial host cell may be conducted by any suitable known methods, including e.g. electroporation methods, particle bombardment or microprojectile bombardment, protoplast methods and Agrobacterium mediated transformation (AMT). Preferably the protoplast method is used. Procedures for transformation are described by J. R. S. Fincham, Transformation in fungi. 1989, Microbiological reviews. 53, 148-170.
Transformation of the mutant microbial host cell by introduction of a polynucleotide an expression vector or a nucleic acid construct into the cell is preferably performed by techniques well known in the art (see Sambrook & Russell; Ausubel, supra).
Transformation may involve a process consisting of protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus cells are described in EP 238 023 and Yelton et al., 1984, Proceedings of the National Academy of Sciences USA 81:1470-1474. Suitable procedures for transformation of Aspergillus and other filamentous fungal host cells using Agrobacterium tumefaciens are described in e.g. De Groot et al., Agrobacterium tumefaciens-mediated transformation of filamentous fungi. Nat Biotechnol. 1998, 16:839-842. Erratum in: Nat Biotechnol 1998 16:1074. A suitable method of transforming Fusarium species is described by Malardier et al., 1989, Gene 78:147156 or in WO 96/00787. Other methods can be applied such as a method using biolistic transformation as described in: Christiansen et al., Biolistic transformation of the obligate plant pathogenic fungus, Erysiphe graminis fsp. hordei. 1995, Curr Genet. 29:100-102. Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito et al., 1983, Journal of Bacteriology 153: 163; and Hinnen et al., 1978, Proceedings of the National Academy of Sciences USA 75: 1920.
In order to enhance the amount of copies of the polynucleotide coding for the compound of interest or coding for a compound involved in the production by the cell of the compound of interest (the gene) in the mutated microbial host cell, multiple transformations of the host cell may be required. In this way, the ratios of the different enzymes produced by the host cell may be influenced. Also, an expression vector may comprise multiple expression cassettes to increase the amount of copies of the polynucleotide(s) to be transformed.
Another way could be to choose different control sequences for the different polynucleotides, which—depending on the choice—may cause a higher or a lower production of the desired polypeptide(s).
The cells transformed with the selectable marker can be selected based on the presence of the selectable marker. In case of transformation of (Aspergillus) cells, usually when the cell is transformed with all nucleic acid material at the same time, when the selectable marker is present also the polynucleotide(s) encoding the desired polypeptide(s) are present.
The invention also provides a method of producing a mutant microbial host cell according to the invention comprising the steps of:

    
    
        a. providing a parent microbial host cell as described herein;
        b. modifying the parent microbial host cell, preferably modifying the genome of the parent microbial host cell, to yield a mutant microbial host cell as described herein which is deficient in the production of a polypeptide as described herein having amylase activity, preferably α-amylase activity selected from the group consisting of:
        
            i. a polypeptide according to SEQ ID NO: 3 or 7 or a polypeptide at least 70% identical thereto;
            ii. a mature polypeptide comprised in SEQ ID NO: 3 or 7 or a polypeptide at least 70% identical thereto
            iii. a polypeptide encoded by a polynucleotide according to SEQ ID NO: 1, 2, 5 or 6 or encoded by a polynucleotide at least 70% identical to SEQ ID NO: 1, 2, 5 or 6;
            iv. a polypeptide encoded by a polynucleotide capable of hybridising to a polynucleotide according to SEQ ID NO: 1, 2, 5 or 6 or capable of hybridising to the complementary strand of a polynucleotide according to SEQ ID NO: 1, 2, 5 or 6;
            if compared with the parent microbial host cell and measured under the same conditions.
        
        
    
    


Within this context it will be clear to those skilled in the art that the specific embodiments that may be applicable to the mutant microbial host cell according to the invention may also be applicable to the other aspects of the invention.
The invention further provides a method for the production of a compound of interest by microbial fermentation comprising:

    
    
        a. providing a mutant microbial host cell according to the invention capable of expressing the compound of interest,
        b. culturing said microbial host cell under conditions conducive to the expression of the compound of interest,
        c. optionally isolating the compound of interest from the culture medium.
    
    


In step a. a mutant microbial host cell may be a mutant host cell as described herein.
In step b. the mutant microbial host cell of step a. is cultured under conditions conducive to the expression of the compound of interest as described herein. The mutant microbial cells are cultivated in a nutrient medium suitable for production of the compound of interest using methods known in the art. For example, the cells may be cultivated by shake flask cultivation, small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the compound of interest to be produced and/or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art (see, e. g., Bennett, J. W. and LaSure, L., eds., More Gene Manipulations in Fungi, Academic Press, CA, 1991). Suitable media are available from commercial suppliers or may be prepared using published compositions (e. g., in catalogues of the American Type Culture Collection). If the compound of interest is secreted into the nutrient medium, the compound can be isolated directly from the medium. If the compound of interest is not secreted, it can be isolated from cell lysates.
In step c. the compound of interest may be optionally isolated. The compound of interest as described herein may be isolated by methods known in the art. For example, the compound of interest may be isolated from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. The isolated compound of interest may then be further purified by a variety of procedures known in the art including, but not limited to, chromatography (e. g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e. g., ammonium sulfate precipitation), or extraction (see, e.g., Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989). In some applications the compound of interest may be used without substantial isolation from the culture broth; separation of the culture medium from the biomass may be adequate. The method for producing a compound of interest according to the invention offers the great advantage that less purification steps to eliminate amylase enzymatic side activities may be necessary, making such a method economically attractive.
A mutant microbial host cell as defined herein may be used in the method for the production of a compound of interest of the invention.
The compound of interest produced in the method for the production of a compound of interest by microbial fermentation may be any compound of interest as described herein.
Preferred Embodiments of the Invention

    1. A mutant microbial host cell which has been modified, preferably in its genome, to result in a deficiency in the production of a polypeptide having amylase activity, preferably α-amylase activity selected from the group consisting of:
    
        a. a polypeptide according to SEQ ID NO: 3 or 7 or a polypeptide at least 70% identical thereto;
        b. a mature polypeptide comprised in SEQ ID NO: 3 or 7 or a polypeptide at least 70% identical thereto;
        c. a polypeptide encoded by a polynucleotide according to SEQ ID NO: 1, 2, 5 or 6 or encoded by a polynucleotide at least 70% identical to SEQ ID NO: 1, 2, 5 or 6;
        d. a polypeptide encoded by a polynucleotide capable of hybridising to a polynucleotide according to SEQ ID NO: 1, 2, 5 or 6 or capable of hybridising to the complementary strand of a polynucleotide according to SEQ ID NO: 1, 2, 5 or 6;
        if compared with a parent microbial host cell which has not been modified and measured under the same conditions.
    
    
    2. A mutant microbial host cell according to embodiment 1 wherein the mature polypeptide comprised in SEQ ID NO: 3 or 7 is a mature polypeptide according to SEQ ID NO: 4 or 8 respectively.
    3. The mutant microbial host cell according to embodiment 1 or 2 wherein the modification comprises:
    
        a) a modification which results in a reduced or no production of a polypeptide having amylase activity, preferably α-amylase activity as defined in embodiment 1 a. to 1.d. if compared to the parent microbial host cell that has not been modified, when analysed under the same conditions; and/or
        b) a modification which results in a polypeptide, derived from a polypeptide as defined in embodiment 1 a. to 1 d., but with decreased or no amylase activity, preferably with decreased or no α-amylase activity as compared to the parent microbial host cell that has not been modified, when analysed under the same conditions.
    
    
    4. The mutant microbial host cell according to any one of embodiments 1 to 3 wherein the mutant microbial host cell
    
        a. produces less polypeptide as defined in embodiment 1 a. to 1 d. or it produces no polypeptide as defined in embodiment 1 a. to 1 d if compared with the parent microbial host cell which has not been modified and measured under the same conditions; and/or
        b. produces a polypeptide derived from the polypeptide as defined in embodiment 1 a. to 1 d, but with decreased or no amylase activity, preferably with decreased or no α-amylase activity if compared to the parent microbial host cell that has not been modified, when analysed under the same conditions.
    
    
    5. The mutant microbial host cell according to any one of embodiments 1 to 4 wherein the mutant microbial host cell produces 1% less polypeptide having amylase activity, preferably α-amylase activity as defined in embodiment 1 a. to 1 d. if compared with the parent microbial host cell which has not been modified and measured under the same conditions, at least 5% less, at least 10% less, at least 20% less, at least 30% less, at least 40% less, at least 50% less, at least 60% less, at least 70% less, at least 80% less, at least 90% less, at least 91% less, at least 92% less, at least 93% less, at least 94% less at least 95% less, at least 96% less, at least 97% less, at least 98% less, at least 99% less, or at least 99.9% less, preferably the mutant microbial host cell produces substantially no polypeptide as defined in embodiment 1 a. to 1 d. if compared with the parent microbial host cell which has not been modified and measured under the same conditions.
    6. The mutant microbial host cell according to any one of embodiments 1 to 5 wherein the mutant microbial host cell produces a polypeptide derived from the polypeptide having amylase activity, preferably α-amylase activity as defined in embodiment 1 a. to 1 d. with 1% less (α-)amylase activity, if compared with the parent microbial host cell which has not been modified and measured under the same conditions, at least 5% less activity, at least 10% less activity, at least 20% less activity, at least 30% less activity, at least 40% less activity, at least 50% less activity, at least 60% less activity, at least 70% less activity, at least 80% less activity, at least 90% less activity, at least 91% less activity, at least 92% less activity, at least 93% less activity, at least 94% less activity, at least 95% less activity, at least 96% less activity, at least 97% less activity, at least 98% less activity, at least 99% less activity, or at least 99.9% less activity, preferably the mutant microbial host cell produces a polypeptide derived from a polypeptide as defined in embodiment 1 a. to 1 d. with substantially no (α-)amylase activity if compared with the parent microbial host cell which has not been modified and analysed under the same conditions.
    7. The mutant microbial host cell according to any one of embodiments 1 to 6 wherein the modification in its genome is selected from:
    
        a) a full or partial deletion of a polynucleotide as defined in embodiment 1 c. or 1 d.,
        b) a full or partial replacement of a polynucleotide as defined in embodiment 1 c. or 1 d. with a polynucleotide sequence which does not code for a polypeptide as defined in claims 1 a. to 1 d. or which code for a partially or fully inactive form of a polypeptide as defined in embodiment 1 a. to 1 d.;
        c) a disruption of a polynucleotide as defined in embodiment 1 c. or 1 d. by the insertion of one or more nucleotides in the polynucleotide sequence and consequent partial or full inactivation of the polypeptide as defined in embodiment 1 a. to 1 d.
    
    
    8. The mutant microbial host cell according to any one of embodiments 1 to 7 wherein the modification which results in a reduced or no production of a polypeptide as defined in embodiment 1 a. to 1 d. is due to a reduced production of the mRNA encoding said polypeptide.
    9. The mutant microbial host cell according to any one of embodiments 1 to 8 comprising at least one polynucleotide coding for a compound of interest or at least one polynucleotide coding for a compound involved in the production of a compound of interest.
    10. The mutant microbial host cell according to embodiment 9 wherein the at least one polynucleotide coding for the compound of interest or the at least one polynucleotide coding for a compound involved in the production of a compound of interest is operably linked to a promoter.
    11. The mutant microbial host cell according to any one of embodiments 1 to 10 which is a eukaryotic cell, more preferably a fungal cell, even more preferably the mutant microbial host cell is a filamentous fungus.
    12. The mutant microbial host cell according to embodiment 11 which is a filamentous fungus selected from Aspergillus, Acremonium, Myceliophthora, Thielavia Chrysosporium, Penicillium, Talaromyces, Rasamsonia, Fusarium or Trichoderma, preferably a species of Aspergillus niger, Aspergillus awamori, Aspergillus foetidus, Aspergillus sojae, Aspergillus fumigatus, Aspergillus oryzae, Acremonium alabamense, Myceliophthora thermophila, Thielavia terrestris, Chrysosporium lucknowense, Fusarium oxysporum, Rasamsonia emersonii, Talaromyces emersonii, Trichoderma reesei or Penicillium chrysogenum. 
    13. The mutant microbial host cell according to any one of the preceding embodiments which has been modified, preferably in its genome, to result in a deficiency in the production of one or more of a polypeptide having glucoamylase glaA activity, a polypeptide having alpha-amylase amyBII activity, a polypeptide having ΔamyBI activity and a polypeptide having acid stable alpha-amylase amyA activity, preferably a cell which has been modified, preferably in its genome, to result in a deficiency in the production of glucoamylase glaA activity, a polypeptide having alpha-amylase amyBII activity, a polypeptide having ΔamyBI activity and a polypeptide having acid stable alpha-amylase amyA activity.
    14. A method of producing a mutant microbial host cell comprising the steps of:
    
        a. providing a parent microbial host cell;
        b. modifying the parent microbial host cell, preferably modifying the genome of the parent microbial host cell, to yield a mutant microbial host cell which is deficient in the production of a polypeptide having amylase activity, preferably α-amylase activity selected from the group consisting of:
        
            i. a polypeptide according to SEQ ID NO: 3 or 7 or a polypeptide at least 70% identical thereto;
            ii. a mature polypeptide comprised in SEQ ID NO: 3 or 7 or a polypeptide at least 70% identical thereto;
            iii. a polypeptide encoded by a polynucleotide according to SEQ ID NO: 1, 2, 5 or 6 or encoded by a polynucleotide at least 70% identical to SEQ ID NO: 1, 2, 5 or 6;
            iv. a polypeptide encoded by a polynucleotide capable of hybridising to the polynucleotide according to SEQ ID NO: 1, 2, 5 or 6 or capable of hybridising to the complementary strand of a polynucleotide according to SEQ ID NO: 1, 2, 5 or 6;
            if compared with the parent microbial host cell and measured under the same conditions.
        
        
    
    
    15. The method according to embodiment 14 wherein the mutant microbial host cell is a mutant microbial host cell according to any one of embodiments 1 to 13.
    16. A method for the production of a compound of interest by microbial fermentation comprising:
    
        a. providing a mutant microbial host cell according to any one of embodiments 1 to 13 or produced by a method according to embodiments 14 or 15 capable of expressing the compound of interest,
        b. culturing said mutant microbial host cell under conditions conducive to the expression of the compound of interest,
        c. optionally isolating the compound of interest from the culture medium.
    
    
    17. The method according to embodiment 16 wherein the compound of interest is a biological compound selected from the group consisting of biomass, a biopolymer, a metabolite, preferably the compound of interest is selected from a biopolymer or a metabolite.
    18. The method according to embodiment 17 wherein the biopolymer is selected from a nucleic acid, a polyamine, a polyol, a polypeptide (such as a protein, preferably an enzyme) or a polyamide, or a polysaccharide or a metabolite is selected from a primary or secondary metabolite.
    19. The method according to embodiment 18 wherein the compound of interest is an enzyme, preferably glucose oxidase.


Hereafter the invention will be illustrated by examples which however should not be interpreted as limiting the scope of the invention.
EXAMPLES
Strains
WT 1: This Aspergillus niger strain is used as a wild-type strain. This strain is deposited at the CBS Institute under the deposit number CBS 513.88.
GBA 306: The construction of GBA 306 using WT1 as starting strain has been described in detail in WO2011/009700. This GBA 306 strain has the following genotype: ΔglaA, ΔpepA, ΔhdfA, an adapted BamHI amplicon, ΔamyBII, ΔamyBI, and ΔamyA.
PGOX-2: This A. niger strain is a GBA306 strain expressing the Penicillium chrysogenum glucose oxidase enzyme. The PGOX-2 strain was constructed using the pGBTOPGOX-3 expression vector (see FIG. 1—pGBTOP12 expression vector (WO2011/009700) with a codon pair optimized Penicillium chrysogenum glucose oxidase (as depicted in SEQ ID NO: 29 of WO2012/001169) coding sequence cloned in), which was introduced by co-transformation with the amdS selectable marker-gene containing vector pGBAAS-3 using the method as described in WO2011/009700 and WO2012/001169. After transformation and counter-selection (as also described in WO98/46772 and WO99/32617), followed by selection of strains with multiple copies, 1 multi-copy enzyme-producing strain was selected and named PGOX-2. This strain is used as the glucose oxidase enzyme producing strain in subsequent experiments.
Molecular Biology Techniques
In these strains, using molecular biology techniques known to the skilled person (see: Sambrook & 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. Examples of the general design of expression vectors for gene over expression and disruption vectors for down-regulation, transformation, use of markers and selective media can be found in WO199846772, WO199932617, WO2001121779, WO2005095624, WO2006040312, EP 635574B, WO2005100573, WO2011009700 and WO2012001169. All gene replacement 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.
A. niger Shake Flask Fermentations
A. niger strains were pre-cultured and cultured at 34° C. and 170 rpm as described in WO2010/102982. Pre-culture was in 20 ml CSL pre-culture medium and after overnight growth 10 ml of this culture was transferred to 100 ml fermentation medium (FM) as described in more detail in WO2010/102982 with a cultivation time as indicated in the examples.
Enzyme Activity Measurements
Glucose oxidase (GOX) activity and the GOX activity plate assay (using o-anisidine) were measured as described in Witteveen et al. 1990, “Glucose oxidase overproducing and negative mutants of Aspergillus niger”, Appl Microbiol Biotechnol 33:683-686.
Fungal alpha-amylase measurement was determined in A. niger culture broth, using the Megazyme cereal alpha-amylase kit (Megazyme, CERALPHA alpha amylase assay kit, catalogus. ref. K-CERA, year 2000-2001), according to basic instructions of the supplier with some modifications. The measured activity is based on hydrolysis of non-reducing-end-blocked ρ-nitrophenyl maltoheptaoside in the presence of excess glucoamylase and α-glucosidase. The amount of formed ρ-nitrophenol is a measure for alpha-amylase activity present in a sample. To make this assay sensitive for the amount of enzyme present in the culture samples, the following setup for measurement was used in the assay: 20 μl culture sample was mixed with 90 μl substrate solution (Ceralpha reagent), dissolved in 10 ml water with 8 ml of buffer added). Incubation was done for one hour at 37° C., after which the reaction was stopped with 20 μl 0.635 M Tris and measurement of the absorption at 405 nm. Activity was calculated using an enzymatic Fungal Amylase standard in the range of 0.00005-0.0005 FAUs/ml (Amylase enzyme was diluted in 50 mM malic acid buffer pH 5.2 (50 mM NaCl, 2 mM CaCl2, 0.05% BSA).
Example 1
Construction Approach of Aspergillus Niger PGOX-2 Strains, Containing Glycoside Hydrolase Gene Deletions
To be able to disrupt the glycoside hydrolase (GH)-related genes (also known under the gene codes: An01g10930, An04g06920, and An04g06930 encoding amylase and/or (putative) alpha-glucosidase enzymes), a gene replacement vector was designed for each of the three genes as described above. Details of the amylase encoding genes can be found in Table 1.







TABLE 1







Gene and strain details for respective


GH disruption strain constructed









PCR results











Strain code for
Amy gene
Disruption

Amy


disruption strain
disrupted
vector
amdS
gene





PGOX-2
—
—
−
−


PGOX-2_AMY1
AgdB -
pGBDEL-AMY1
+
−



An01g10930


PGOX-2_AMY2
AgdA -
pGBDEL-AMY2
+
−



An04g06920


PGOX-2_AMY3
AmyC -
pGBDEL-AMY3
+
−



An04g06930









Vector pGBDEL-AMY1 (FIG. 2) and the other pGBDEL variants, which comprise approximately 1 kb flanking regions of the respective amylase encoding ORF's for homologous recombination, were used to transform Aspergillus niger PGOX-2. After verification of the truthful recombination events and correctness of the strains, the resulting correct strains PGOX-2, PGOX-2_AMY1, PGOX-2_AMY2, PGOX-2_AMY3-1, and PGOX-2_AMY3-2, were selected as representative strains with the respective amy genes (Table 1) inactivated in the PGOX-2 strain background.
Example 2
Analysis of the A. niger PGOX-2 Derived Strains for the Amount of Glucose Oxidase Enzyme Product Produced
To be able to assess the effect of the GH gene disruptions, shake-flask analysis in FM1 medium of these transformants was analysed. At day 4 and 6 after inoculation, medium samples were taken. Both the alpha-amylase activities and the glucose oxidase levels were analysed in the culture supernatant. In the PGOX-2 based strain background, the most important and abundant amylases, being glucoamylase glaA, alpha-amylase amyBII and ΔamyBI and acid stable alpha-amylase amyA were already disrupted. Surprisingly, upon disruption of AmyC-An04g06930, the alpha-amylase activity was reduced dramatically in PGOX-2_AMY3-1 and PGOX-2_AMY3-2 strains, whereas the other amy disruptions showed no pronounced effect in alpha-amylase activity (FIG. 3). This means that specifically amyC-An04g06930 disruption results in an important reduction in alpha-amylase activity.
For glucose oxidase production, (FIG. 4) it can be seen that both the PGOX-2_AMY3-1 and PGOX-2_AMY3-2 strain, as identified from glucose oxidase activities in the culture supernatant, had an activity on both sampling days (day 4 and 6) comparable with the original strain. This was confirmed by analyzing GOX expression on plate (FIG. 5) for random transformants, which were isolated as described in Example 1.

## Claims

1. A mutant microbial host cell which has been modified to result in a deficiency in production of a polypeptide having α-amylase activity selected from the group consisting of:
a. a polypeptide according to SEQ ID NO: 3 or 7 or a polypeptide at least 90% identical thereto;
b. a mature polypeptide comprised in SEQ ID NO: 3 or 7 or a polypeptide at least 90% identical thereto;
c. a polypeptide encoded by a polynucleotide according to SEQ ID NO: 1, 2, 5 or 6 or encoded by a polynucleotide at least 90% identical to SEQ ID NO: 1, 2, 5 or 6;
d. a polypeptide encoded by a polynucleotide capable of hybridising to a polynucleotide according to SEQ ID NO: 1, 2, 5 or 6 or capable of hybridising to the complementary strand of a polynucleotide according to SEQ ID NO: 1, 2, 5 or 6;
wherein the modification comprises a modification which results in a reduced or no production of a polypeptide having α-amylase activity as defined in a)-d) compared to the parent microbial host cell that has not been modified;
wherein the modification is made in the genome of the microbial host cell;
wherein the modification is selected from the group consisting of:
1) a full or partial deletion of a polynucleotide as defined in c) or d);
2) a full or partial replacement of a polynucleotide as defined in c) or d) with a polynucleotide sequence which does not code for a polypeptide as defined in a)-d) or which code for a partially or fully inactive form of a polypeptide as defined in a)-d);
3) a disruption of a polynucleotide as defined in c) or d) by the insertion of one or more nucleotides in the polynucleotide sequence and consequent partial or full inactivation of the polypeptide as defined in a)-d);
wherein the mutant microbial host cell is an Aspergillus niger cell; wherein the mutant microbial host cell has been further modified to result in a deficiency in the production of at least one of a polypeptide selected from the group consisting of: a polypeptide having glucoamylase glaA activity, a polypeptide having alpha-amylase amyBII activity, a polypeptide having amyBI activity, and a polypeptide having acid stable alpha-amylase amyA activity, if compared with a parent microbial host cell which has not been modified and measured under the same conditions.

2. A mutant microbial host cell according to claim 1, wherein the mature polypeptide comprised in SEQ ID NO: 3 or 7 is a mature polypeptide according to SEQ ID NO: 4 or 8.

3. The mutant microbial host cell according to claim 1, wherein the mutant microbial host cell
1) produces less of the polypeptide having α-amylase activity or produces none of the polypeptide having a-amylase activity compared with the parent microbial host cell which has not been modified and measured under the same conditions; and/or
2) produces a polypeptide derived from the polypeptide as defined in a) to d), but with decreased or no α-amylase activity if compared to the parent microbial host cell that has not been modified, when analysed under the same conditions.

4. The mutant microbial host cell according to claim 1, wherein the mutant microbial host cell produces 1% less polypeptide having α-amylase activity as defined in a) to d) compared with the parent microbial host cell which has not been modified and measured under the same conditions.

5. The mutant microbial host cell according to claim 1, wherein the mutant microbial host cell produces a polypeptide derived from the polypeptide having α-amylase activity with at least 1% less α-amylase activity, if compared with the parent microbial host cell which has not been modified and measured under the same condition.

6. The mutant microbial host cell according to claim 1, wherein a modification which results in a reduced or no production of the polypeptide having α-amylase activity is due to a reduced production of mRNA encoding said polypeptide.

7. The mutant microbial host cell according to claim 1, further comprising at least one polynucleotide coding for a compound of interest or at least one polynucleotide coding for a compound involved in the production of a compound of interest.

8. The mutant microbial host cell according to claim 7, wherein the at least one polynucleotide coding for the compound of interest or the at least one polynucleotide coding for a compound involved in the production of a compound of interest is operably linked to a promoter.

9. The mutant microbial host cell according to claim 1 which has been modified to result in a deficiency in production of glucoamylase glaA activity, a polypeptide having alpha-amylase amylase amyBII activity, a polypeptide having ΔamyBI activity and a polypeptide having acid stable alpha-amylase amyA activity.

10. A method of producing a mutant microbial host cell comprising:
a. providing a parent microbial host cell;
b. modifying the parent microbial host cell, optionally modifying the genome of the parent host cell, to yield a mutant host cell which is deficient in the production of a polypeptide having α-amylase activity selected from the group consisting of:
i. a polypeptide according to SEQ ID NO: 3 or 7 or a polypeptide at least 90% identical thereto;
ii. a mature polypeptide comprised in SEQ ID NO: 3 or 7 or a polypeptide at least 90% identical thereto;
iii. a polypeptide encoded by a polynucleotide according to SEQ ID NO: 1, 2, 5 or 6 or encoded by a polynucleotide at least 90% identical to SEQ ID NO: 1, 2, 5 or 6;
iv. a polypeptide encoded by a polynucleotide capable of hybridising to the polynucleotide according to SEQ ID NO: 1, 2, 5 or 6 or capable of hybridising to the complementary strand of a polynucleotide according to SEQ ID NO: 1, 2, 5 or 6;

wherein the modification comprises a modification which results in a reduced or no production of a polypeptide having α-amylase activity as defined in a)-d) compared to the parent microbial host cell that has not been modified;
wherein the modification is made in the genome of the microbial host cell;
wherein the modification is selected from the group consisting of:
1) a full or partial deletion of a polynucleotide as defined in c) or d);
2) a full or partial replacement of a polynucleotide as defined in c) or d) with a polynucleotide sequence which does not code for a polypeptide as defined in a)-d) or which code for a partially or fully inactive form of a polypeptide as defined in a)-d);
3) a disruption of a polynucleotide as defined in c) or d) by the insertion of one or more nucleotides in the polynucleotide sequence and consequent partial or full inactivation of the polypeptide as defined in a)-d);
wherein the mutant microbial host cell is an Aspergillus niger cell; wherein the mutant microbial host cell has been modified to result in a deficiency in the production of at least one of a polypeptide selected from the group consisting of: a polypeptide having glucoamylase glaA activity, a polypeptide having alpha-amylase amyBII activity, a polypeptide having amyBI activity, and a polypeptide having acid stable alpha-amylase amyA activity, if compared with the parent microbial host cell and measured under the same conditions.

11. A method for production of a compound of interest by microbial fermentation comprising:
a. providing a mutant microbial host cell according to claim 1 capable of expressing the compound of interest,
b. culturing said mutant microbial host cell under conditions conducive to expression of the compound of interest,
c. optionally isolating the compound of interest from a culture medium.

12. The method according to claim 11 wherein the compound of interest is a biological compound selected from the group consisting of biomass, a biopolymer, a metabolite, preferably the compound of interest is selected from a biopolymer or a metabolite.

13. The method according to claim 12, wherein the biopolymer is selected from a nucleic acid, a polyamine, a polyol, a polypeptide, a protein, optionally an enzyme or a polyamide, or a polysaccharide or a metabolite is selected from a primary or secondary metabolite.

14. The method according to claim 13, wherein the compound of interest is an enzyme.

15. The mutant microbial host cell according to claim 4, wherein the mutant microbial host cell produces at least 95% less polypeptide having α-amylase activity as defined in a) to d) compared with the parent microbial host cell which has not been modified and measured under the same conditions.

16. The mutant microbial host cell according to claim 5, wherein the mutant microbial host cell produces a polypeptide derived from the polypeptide having α-amylase activity with at least 95% less α-amylase activity, if compared with the parent microbial host cell which has not been modified and measured under the same conditions.

17. The method according to claim 14, wherein the enzyme is a glucose oxidase.