WO2014202624A2

WO2014202624A2 - Rasamsonia gene and use thereof

Info

Publication number: WO2014202624A2
Application number: PCT/EP2014/062746
Authority: WO
Inventors: Alrik Pieter Los; Sacha DALHUIJSEN; Stefanus Cornelis Hendrikus Jozef Turk
Original assignee: Dsm Ip Assets B.V.
Priority date: 2013-06-19
Filing date: 2014-06-17
Publication date: 2014-12-24
Also published as: WO2014202624A9

Abstract

The invention relates to a polypeptide which comprises the amino acid sequence set out in SEQ ID NO: 4, 8, 9, 14 or 18or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, 2, 3, 5, 6, 7, 11, 12, 13, 15, 16 or 17or a variant polypeptide, wherein the variant polypeptide (i) has at least 70% sequence identity with the sequence set out in SEQ ID NO: 4, 8, 9, 14 or 18or (ii) has an amino acid sequence that differs in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acids from the amino acid sequence of SEQ ID NO: 4, 8, 9, 14 or 18. The invention features the full length coding sequence of the novel gene as well as the amino acid sequence of the full-length functional polypeptide and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes.

Description

RASAMSONIA GENE AND USE THEREOF

Field of the invention

The invention relates to sequences comprising genes that encode polypeptides influencing Iignocellulosic material degrading activity. The invention features the full- length coding sequence of the novel gene as well as the amino acid sequence of the full- length functional protein, and variants and fragments of the gene or the amino acid sequence. The invention also relates to methods for using these proteins in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins. Also the invention relates to the successful expression of the genes that encode polypeptides influencing Iignocellulosic material degrading activity in a host organism such as Aspergillus niger and/or Rasamsonia emersonii.

Background of the invention

Carbohydrates constitute the most abundant organic compounds on earth. However, much of this carbohydrate is sequestered in complex polymers including starch (the principle storage carbohydrate in seeds and grain), and a collection of carbohydrates and lignin known as lignocellulose. The main carbohydrate components of lignocellulose are cellulose, hemicellulose, and pectins. These complex polymers are often referred to collectively as lignocellulose.

Bioconversion of renewable Iignocellulosic biomass to a fermentable sugar that is subsequently fermented to produce alcohol (e.g., ethanol) as an alternative to liquid fuels has attracted an intensive attention of researchers since 1970s, when the oil crisis broke out because of decreasing the output of petroleum by OPEC. Ethanol has been widely used as a 10% blend to gasoline in the USA or as a neat fuel for vehicles in Brazil in the last two decades. More recently, the use of E85, an 85% ethanol blend has been implemented especially for clean city applications. The importance of fuel bioethanol will increase in parallel with increases in prices for oil and the gradual depletion of its sources. Additionally, fermentable sugars are being used to produce plastics, polymers and other bio-based products and this industry is expected to grow substantially therefore increasing the demand for abundant low cost fermentable sugars which can be used as a feed stock in lieu of petroleum based feedstocks.

The sequestration of such large amounts of carbohydrates in plant biomass provides a plentiful source of potential energy in the form of sugars, both five carbon and six carbon sugars that could be utilized for numerous industrial and agricultural processes. However, the enormous energy potential of these carbohydrates is currently under-utilized because the sugars are locked in complex polymers, and hence are not readily accessible for fermentation. Methods that generate sugars from plant biomass would provide plentiful, economically-competitive feedstocks for fermentation into chemicals, plastics, such as for instance succinic acid and (bio) fuels, including ethanol, methanol, butanol synthetic liquid fuels and biogas.

Regardless of the type of cellulosic feedstock, the cost and hydrolytic efficiency of enzymes are major factors that restrict the commercialization of the biomass bioconversion processes. The production costs of microbially produced enzymes are tightly connected with a productivity of the enzyme-producing strain, the specific activity of the enzymes, the mode of action of the enzyme and the final activity yield in the fermentation broth.

In spite of the continued research of the last few decades to understand enzymatic lignocellulosic biomass degradation and cellulase production, it remains desirable to discover or to engineer new highly active cellulases and hemicellulases. It would also be highly desirable to construct highly efficient enzyme compositions capable of performing rapid and efficient biodegradation of lignocellulosic materials, in particular such cellulases and hemicellulases that have increased thermostability.

Such enzymes may be used to produce sugars for fermentation into chemicals, plastics, such as for instance succinic acid and (bio) fuels, including ethanol, methanol, butanol, synthetic liquid fuels and biogas, for ensiling, and also as enzyme in other industrial processes, for example in the food or feed, textile, pulp or paper or detergent industries and other industries.

Summary of the invention

The present invention provides a polypeptide which comprises the amino acid sequence set out in SEQ ID NO: 4, 8, 9, 14 or 18 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1 , 2, 3, 5, 6, 7, 1 1 , 12, 13, 15, 16 or 17 or a variant polypeptide , wherein the variant polypeptide (i) has at least 70% sequence identity with the sequence set out in SEQ ID NO: 4, 8, 9, 14 or 18 or (ii) has an amino acid sequence that differs in 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 or 12 amino acids from the amino acid sequence of SEQ ID NO: 4, 8, 9, 14 or 18. Preferably the polypeptide of the invention is a polypeptide having saccharide transporter or sugar transporter activity, such as a saccharide transporter or sugar transporter.

The invention also provides a polynucleotide having a nucleic acid sequence coding for a polypeptide, whereby the nucleic acid sequence is selected from the group consisting of:

(a) a nucleic acid sequence having at least 70% identity with the nucleic acid sequence of SEQ ID NO: 1 , 2, 3, 5, 6, 7, 1 1 , 12, 13, 15, 16 or 17;

(b) a nucleic acid sequence hybridizing with the complement of the nucleic acid sequence of SEQ ID NO: 1 , 2, 3, 5, 6, 7, 1 1 , 12, 13, 15, 16 or 17;

(c) a nucleic acid sequence encoding (i) the amino acid sequence of SEQ ID NO: 4, 8, 9, 14 or 18, (ii) an amino acid sequence having at least 70% identity with the amino acid sequence of SEQ ID NO: 4, 8, 9, 14 or 18, or (iii) an amino acid sequence that differs in 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 or 12 amino acids from the amino acid sequence of SEQ ID NO: 4, 8, 9, 14 or 18; or

(d) a nucleotide sequence which is the reverse complement of a nucleotide sequence as defined in (a), (b) or (c).

Preferably the polynucleoide of the invention has a nucleic acid sequence coding for a polypeptide which is a polypeptide having saccharide transporter or sugar transporter activity, such as a saccharide transporter or sugar transporter The present invention enables the protein production characteristics of a microorganism.

The invention also provides a nucleic acid construct or vector comprising the polynucleotide of the invention and a recombinant cell comprising the polynucleotide of the invention or a nucleic acid construct or vector of the invention.

The invention further provides a cell wherein the polynucleotide according to the invention is mutated or deleted from the genome to obtain lower or no activity of the polypeptide compared to the cell wherein the polynucleotide of the invention is not mutated or deleted from the genome. The invention further provides a mutant cell wherein the polynucleotide according to the invention is overexpressed compared to the native or starting cell. Overexpression can be obtained for example by altering the promoter or by introducing one or more copies of polynucleotide according to the invention in the cell. According to a preferred embodiment the polypeptide having saccharide transporter or sugar transporter activity, preferably TEMER01209, TEMER09077, TEMER061 18 or TEMER03835 is overexpressed compared to the native or starting cell.

According to another aspect of the invention the cell of the invention is a fungal cell, preferably a fungal cell selected from the group consisting of the genera Acremonium, Agaricus, Aspergillus, Aureobasidium, Chrysosporium, Coprinus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Panerochaete, Pleurotus, Schizophyllum, Talaromyces, Rasamsonia, Saccharomyces, Thermoascus, Thielavia, Tolypocladium, and Trichoderma. Preferably the cell of the invention is a recombinant cell.

In the cell of the invention one or more gene can be deleted, knocked-out or disrupted in full or in part, wherein optionally the gene encodes for a protease.

The invention further discloses a recombinant cell wherein the polynucleotide according to the invention is overexpressed compared to the native or starting cell, preferably by altering the promoter or by introducing one or more copies of polynucleotide according to the invention in the cell, more preferably wherein the polypeptide having saccharide transporter or sugar transporter activity, preferably TEMER01209, TEMER09077, TEMER061 18 or TEMER03835 is overexpressed compared to the native or starting cell.

The invention also provides a method of producing an enzyme or enzyme composition comprising the fermentation of the cell according of the invention and optionally recovery of the enzyme or enzyme composition.

Brief description of the drawings

Fig 1 : shows the strategy used to overexpress the gene of interest (GOI) encoding the protein of interest in R. emersonii. The deletion vectors comprise the overlapping nonfunctional ble selection marker fragments (split marker) flanked by loxP sites and 5' and 3' homologous regions of the pepA gene for targeting. The constructs integrate through triple homologous recombination (X) at the pepA locus and at the overlapping homologous nonfunctional ble selection marker fragment and replaces the genomic pepA gene copy.

Fig. 2 shows a schematic diagram of plasmid pEBA1006 that was used in bipartite gene-targeting method in combination with the pEBA expression vector, containing either TEMER01209, TEMER09077, TEMER061 18 or TEMER03835, with the goal to replace the RePepA ORF and approximately 1500 nucleotides upstream of the start ATG codon by the expression cassette of TEMER01209, TEMER09077, TEMER061 18 or TEMER03835 in Rasamsonia emersonii. The vector comprises the 3' part of the ble coding region, the A.nidulans trpC terminator, a lox71 site, a 2500 bp 3' flanking region of the RePepA ORF, and the backbone of pUC19 (Invitrogen, Breda, The Netherlands). The E. coli DNA was removed by digestion with restriction enzyme A/oil, prior to transformation of the R. emersonii strains.

Fig. 3 shows a schematic diagram of pEBA expression plasmid, containing TEMER01209, TEMER09077, TEMER061 18 or TEMER03835, that was used in bipartite gene-targeting method in combination with the pEBA1006 vector with the goal to replace the RePepA ORF and approximately 1500 nucleotides upstream of the start ATG codon by the expression cassette of TEMER01209, TEMER09077, TEMER061 18 or TEMER03835 in Rasamsonia emersonii. The vector comprises a 1500 bp 5' flanking region 1 .5 kb upstream of the RePepA ORF for targeting in the RePepA locus, TEMER01209, TEMER09077, TEMER061 18 or TEMER03835 expression cassette consisting of R. emersonii promoter 2, TEMER01209, TEMER09077, TEMER061 18 or TEMER03835 coding region and the A.nidulans amdS terminator (TamdS), a lox66 site, the nonfunctional 5' part of the ble coding region (5' ble) driven by the A.nidulans gpdA promoter. The E. coli DNA was removed by digestion with restriction enzyme A/oil, prior to transformation of the R. emersonii strains.

Fig.4 shows a protein gel on which equal amounts of extracellular proteins of the wild-type strain TEC-221 and the strain overexpressing TEMER01209 (Tec-221 - EBA01209) were loaded.

Fig.5 shows a protein gel on which equal amouns of extracellular proteins of the wild-type strain TEC-221 and the strains overexpressing TEMER03835 (Tec-221 - EBA03835) were loaded.

Fig.6 shows a protein gel on which equal amounts of extracellular proteins of the wild-type strain TEC-221 and the strains overexpressing either TEMER061 18 (Tec-221 - EBA061 18) or TEMER09077 (Tec-221 -EBA09077) were loaded.

Brief description of the sequence listing

SEQ ID NO: 1 Genomic DNA sequence of TEMER01290

SEQ ID NO: 2 cDNA sequence of TEMER01290

SEQ ID NO: 3 Codon-pair optimised (CPO) coding sequence of TEMER01290 SEQ ID NO 4 Amino acid sequence of TEMER01290

SEQ ID NO 5 Genomic DNA sequence of TEMER09077

SEQ ID NO 6 cDNA sequence of TEMER09077

SEQ ID NO 7 Codon-pair optimised (CPO) coding sequence of TEMER09077

SEQ ID NO 8 Amino acid sequence of TEMER09077

SEQ ID NO 9 Mature amin acid sequence of TEMER09077

SEQ ID NO 10 Signal sequence of TEMER09077

SEQ ID NO 1 1 Genomic DNA sequence of TEMER061 18

SEQ ID NO 12 cDNA sequence of TEMER061 18

SEQ ID NO 13 Codon-pair optimised (CPO) coding sequence of TEMER061 18

SEQ ID NO 14 Amino acid sequence of TEMER061 18

SEQ ID NO 15 Genomic DNA sequence of TEMER03835

SEQ ID NO 16 cDNA sequence of TEMER03835

SEQ ID NO 17 Codon-pair optimised (CPO) coding sequence of TEMER03835

SEQ ID NO 18 Amino acid sequence of TEMER03835

SEQ ID NO 19 Rasamsonia. emersonii promoter 2

SEQ ID NO 20 Aspergillus nidulans AmdS terminator

SEQ ID NO 21 Forward (FW) PCR primer for Rasamsonia. emersonii pepA locus

SEQ ID NO 22 Reverse (Rev) PCR primer for Rasamsonia. emersonii pepA locus

SEQ ID NO 23 Forward (FW) PCR primer for phleomycin resistance gene

SEQ ID NO 24 Reverse (Rev) PCR primer for phleomycin resistance gene

Table 1

Detailed description of the invention Throughout the present specification and the accompanying claims, the words "comprise" and "include" and variations such as "comprises", "comprising", "includes" and "including" are to be interpreted inclusively. That is, these words are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context allows.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e. to one or at least one) of the grammatical object of the article. By way of example, "an element" may mean one element or more than one element.

The term "derived from" also includes the terms "originated from," "obtained from," "obtainable from," "isolated from," and "created from," and generally indicates that one specified material find its origin in another specified material or has features that can be described with reference to the another specified material. As used herein, a substance (e.g., a nucleic acid molecule or polypeptide) "derived from" a microorganism preferably means that the substance is native to that microorganism. The present invention provides polynucleotides encoding polypeptides, e.g. sugar transporters which play a role in the production of enzymes which have the ability to modify, for example degrade, a carbohydrate material. Overexpression of the present transporters leads to an improved production of desired enzymes such as cellulases. A carbohydrate material is a material which comprises, consists of or substantially consists of one or more carbohydrates. Enzymes are herein a subclass of polypeptides.

Substrate (also called feedstock, lignocellulosic material, biomass) herein is used to refer to a substance that comprises carbohydrate material, which may be treated with enzymes produced using the invention, so that the carbohydrate material therein is modified. The substrate may be pretreated or non-pretreated substrate. In addition to the carbohydrate material the substrate may contain any other component, including but not limited to non-carbohydrate material and starch.

A carbohydrate material is a material which comprises, consists of or substantially consists of one or more carbohydrates.

Typically, a polynucleotide of the invention encodes a polypeptide which is preferably a polypeptide having saccharide transporter or sugar transporter activity, such as a saccharide transporter or sugar transporter that is preferably capable to alter or influence the production of a protein such as oxidoreductase, transferase, hydrolase, lyase, isomerase or ligase, still more preferably is capable to alter or influence the production of a carbohydrate degrading enzyme and/or carbohydrate hydrolysing enzyme and most preferably having an amino acid sequence according to SEQ ID NO: 4, 8, 9, 14 or 18, or a sequence which is a variant thereof, typically functionally equivalent to the polypeptide having the sequence of SEQ ID NO: 4, 8, 9, 14 or 18, or a sequence which is a fragment of either thereof.

A polypeptide of the invention is preferably a polypeptide having saccharide transporter or sugar transporter activity, such as a saccharide transporter or sugar transporter and that is preferably capable to alter or influence the production of a protein such as an enzyme, more preferably is an oxidoreductase, transferase, hydrolase, lyase, isomerase or ligase or is capable to alter or influence the expression of an oxidoreductase, transferase, hydrolase, lyase, isomerase or ligase, still more preferably is a carbohydrate degrading enzyme and/or carbohydrate hydrolysing enzyme. A polypeptide of the invention may have one or more alternative and/or additional activities.

Carbohydrate in this context includes all saccharides, for example polysaccharides, oligosaccharides, disaccharides or monosaccharides.

The invention provides the use of the polypeptide according to the invention and compositions useful in industrial processes.

Despite the long term experience obtained with these processes, the polypeptide of the invention may feature a number of significant advantages over polypeptides currently used. Depending on the specific application, these advantages may include aspects such as lower production costs, higher protein production of the microorganism expressing the polypeptide of the invention, higher specificity towards the substrate, reduced antigenicity, fewer undesirable side activities, higher yields when produced in a suitable microorganism, more suitable pH and temperature ranges, non- inhibition by hydrophobic, lignin-derived products or less product inhibition or, in the case of the food industry a better taste or texture of a final product as well as food grade and kosher aspects.

Advantageously the amended deletion or overexpression of the polypeptide of the invention may result in a (productivity) yield (of enzymes) increasing effect when expressed for example in TEC-147 or TEC-210. This (productivity) yield or enzyme production can be shown for example as an increase, compared to the native strain, of the amount of extracellular proteinproduced per ml when grown with a fixed amount of sugar or as the amount of protein produced per gram of sugar added compared to the native strain. Advantageously the amended overexpression of the polypeptide of the invention may result in a (productivity) yield (of enzymes) increasing effect when expressed for example in TEC-147 or TEC-210. This (productivity) yield or enzyme production can be shown for example as an increase of the amount of protein produced or as the amount of protein produced per gram of sugar added compared to the native strain..

Advantageously the protein composition produced with the microorganism expressing the polypeptide of the invention may have a yield increasing effect in an enzyme composition designed for (feedstock) hydrolysis such as the compositions produced by TEC-147, TEC-210 or 4E mix (see Examples) or when the composition of the invention is added to other suitable compositions including commercial compositions such as Celluclast ® combined with Novozyme 188 (obtainable from Novozymes, Denmark or Sigma-Aldrich®, USA), Accellerase ® 1000 (obtainable from Genencor, USA or Sigma-Aldrich®, USA), and Methaplus ® (obtainable from DSM, Netherlands). This yield increasing effect may be shown by replacing part of the enzyme composition by an equal amount (on protein) of a composition produced by a microorganism expressing the polypeptides of the invention. This yield increase is even possible in case of feedstock such as corn stover. This yield increase can be shown for example as an increase of the amount of glucose released during a fixed reaction (hydrolysis) period of time compared to the situation without the addition of the present composition, or this yield increase can be shown as an similar amount of glucose production with a lower dosage of the 4E or TEC-210 compared to the situation without the addition of the present composition to the regular dosage of the 4E or TEC-210.

Suitable carbohydrate materials

Lignocellulolytic or lignocellulosic materials or biomass are abundant in nature and have great value as alternative energy source. Second generation biofuels, also known as advanced biofuels, are fuels that can be manufactured from various types of biomass. Biomass is a wide-ranging term meaning any source of organic carbon that is renewed rapidly as part of the carbon cycle. Biomass is derived from plant materials but can also include animal materials. The composition of lignocellulosic biomass varies, the major component is cellulose (in general 35-50%), followed by xylan (in general 20-35%, a type of hemicellulose) and lignin (in general 10-25%), in addition to minor components such as proteins, oils and ash that make up the remaining fraction of lignocellulosic biomass. Lignocellulosic biomass contains a variety of carbohydrates. The term carbohydrate is most common in biochemistry, where it is a synonym of saccharide. Carbohydrates (saccharides) are divided into four chemical groupings: monosaccharides, disaccharides, oligosaccharides, and polysaccharides. In general, monosaccharides and disaccharides, which are smaller (lower molecular weight) carbohydrates, are commonly referred to as sugars.

A non-starch carbohydrate suitable for modification by an enzyme composition produced using the polypeptide of the invention is lignocellulose. The major polysaccharides comprising different lignocellulosic residues, which may be considered as a potential renewable feedstock, are cellulose (glucans), hemicelluloses (xylans, heteroxylans and xyloglucans). In addition, some hemicellulose may be present as glucomannans, for example in wood-derived feedstocks. The enzymatic hydrolysis of these polysaccharides to soluble sugars, for example glucose, xylose, arabinose, galactose, fructose, mannose, rhamnose, ribose, D-galacturonic acid and other hexoses and pentoses occurs under the action of different enzymes acting in concert.

Lignin fills the spaces in the cell wall between cellulose, hemicellulose, and pectin components, especially in xylem tracheids, vessel elements and sclereid cells. It is covalently linked to hemicellulose and, therefore, crosslinks different plant polysaccharides, conferring mechanical strength to the cell wall and by extension the plant as a whole. Lignin is a highly hydrophobic crosslinked aromatic polymeric material that is formed by different monolignol monomers, which can be methoxylated to various degrees. There are three monolignol monomers, methoxylated to various degrees: p- coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol. These lignols are incorporated into lignin in the form of the phenylpropanoids p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S), respectively. Biodegradation of lignin is a prerequisite for processing biofuel from plant raw materials. Lignin can be degraded by applying different pretreatment methods, or by using ligninases or lignin-modifying enzymes (LME's). The improving of lignin degradation would drive the output from biofuel processing to better gain or better efficiency factor, for example by improving the accessibility to the (hemi)cellulosic components or by removing lignin-(hemi)cellulose linkages in oligosaccharides released by the action of (hemi)cellulases.

In addition, pectins and other pectic substances such as arabinans may make up considerably proportion of the dry mass of typically cell walls from non-woody plant tissues (about a quarter to half of dry mass may be pectins). Cellulose is a linear polysaccharide composed of glucose residues linked by β- 1 ,4 bonds. The linear nature of the cellulose fibers, as well as the stoichiometry of the β- linked glucose (relative to a) generates structures more prone to interstrand hydrogen bonding than the highly branched olinked structures of starch. Thus, cellulose polymers are generally less soluble, and form more tightly bound fibers than the fibers found in starch.

Hemicellulose is a complex polymer, and its composition often varies widely from organism to organism and from one tissue type to another. In general, a main component of hemicellulose is β-1 ,4-linked xylose, a five carbon sugar. However, this xylose is often branched at 0-3 and/or 0-2 and can be substituted with linkages to arabinose, galactose, mannose, glucuronic acid, galacturonic acid or by esterification to acetic acid (and esterification of ferulic acid to arabinose). Hemicellulose can also contain glucan, which is a general term for β-linked six carbon sugars (such as the β- (1 ,3)(1 ,4) glucans and heteroglucans mentioned previously) and additionally glucomannans (in which both glucose and mannose are present in the linear backbone, linked to each other by β-linkages).

The composition, nature of substitution, and degree of branching of hemicellulose is very different in dicotyledonous plants (dicots, i.e., plant whose seeds have two cotyledons or seed leaves such as lima beans, peanuts, almonds, peas, kidney beans) as compared to monocotyledonous plants (monocots; i.e., plants having a single cotyledon or seed leaf such as corn, wheat, rice, grasses, barley). In dicots, hemicellulose is comprised mainly of xyloglucans that are 1 ,4-3-linked glucose chains with 1 ,θ-β-linked xylosyl side chains. In monocots, including most grain crops, the principal components of hemicellulose are heteroxylans. These are primarily comprised of 1 ,4"3-linked xylose backbone polymers with 1 ,3 -a linkages to arabinose, galactose, mannose and glucuronic acid or 4-O-methyl-glucuronic acid as well as xylose modified by ester-linked acetic acids. Also present are β glucans comprised of 1 ,3- and 1 ,4-β- linked glucosyl chains. In monocots, cellulose, heteroxylans and β-glucans may be present in roughly equal amounts, each comprising about 15-25% of the dry matter of cell walls. Also, different plants may comprise different amounts of, and different compositions of, pectic substances. For example, sugar beet contains about 19% pectin and about 21 % arabinan on a dry weight basis.

Accordingly, an enzyme composition produced using the invention may be tailored in view of the particular feedstock (also called substrate) which is to be used. That is to say, the spectrum of activities in a composition produced using the invention may vary depending on the feedstock in question.

Enzyme combinations or physical treatments can be administered concomitantly or sequentially. The enzymes can be produced either exogenously in microorganisms, yeasts, fungi, bacteria or plants, then isolated and added to the lignocellulosic feedstock or lignocellulosic material. Alternatively, the enzymes are produced, but not isolated, and crude cell mass fermentation broth are added to the feedstock. Alternatively, the crude cell mass or enzyme production medium or plant material may be treated to prevent further microbial growth (for example, by heating or addition of antimicrobial agents), then added to the feedstock. These crude enzyme mixtures may include the organism producing the enzyme. Alternatively, the enzyme may be produced in a fermentation that uses feedstock (such as corn stover) to provide nutrition to an organism that produces an enzyme(s). In this manner, plants that produce the enzymes may serve as the lignocellulosic feedstock and be added into lignocellulosic feedstock.

Enzymatic activity

Endo-1 ,4-3-glucanases (EG) and exo-cellobiohydrolases (CBH) catalyze the hydrolysis of insoluble cellulose to cellooligosaccharides (cellobiose as a main product), while β-glucosidases (BGL) convert the oligosaccharides, mainly cellobiose and cellotriose to glucose.

Xylanases together with other accessory enzymes, for example a-L- arabinofuranosidases, feruloyl and acetylxylan esterases, glucuronidases, and β- xylosidases) catalyze the hydrolysis of part of the hemicelluloses.

Pectic substances include pectins, arabinans, galactans and arabinogalactans. Pectins are the most complex polysaccharides in the plant cell wall. They are built up around a core chain of a(1 ,4)-linked D-galacturonic acid units interspersed to some degree with L-rhamnose. In any one cell wall there are a number of structural units that fit this description and it has generally been considered that in a single pectic molecule, the core chains of different structural units are continuous with one another.

Pectinases include, for example an endo-polygalacturonase, a pectin methyl esterase, an endo-galactanase, a β-galactosidase, a pectin acetyl esterase, an endo- pectin lyase, pectate lyase, a-rhamnosidase, an exo-galacturonase, an exo- polygalacturonate lyase, a rhamnogalacturonan hydrolase, a rhamnogalacturonan lyase, a rhamnogalacturonan acetyl esterase, a rhamnogalacturonan galacturonohydrolase, a xylogalacturonase, an oarabinofuranosidase.

The principal types of structural unit are: galacturonan (homogalacturonan), which may be substituted with methanol on the carboxyl group and acetate on 0-2 and 0-3; rhamnogalacturonan I (RGI), in which galacturonic acid units alternate with rhamnose units carrying (1 ,4)-linked galactan and (1 ,5)-linked arabinan side-chains. The arabinan side-chains may be attached directly to rhamnose or indirectly through the galactan chains; xylogalacturonan, with single xylosyl units on 0-3 of galacturonic acid (closely associated with RGI); and rhamnogalacturonan II (RGI I), a particularly complex minor unit containing unusual sugars, for example apiose. An RGII unit may contain two apiosyl residues which, under suitable ionic conditions, can reversibly form esters with borate.

As set out above, a polypeptide of the invention is preferably a polypeptide having saccharide transporter or sugar transporter activity, such as a saccharide transporter or sugar transporter.

. However, a polypeptide of the invention may have one or more of the activities set out above in addition to or alternative to that activity. Also, a composition of the invention as described herein may have one or more of the activities mentioned above in addition to that provided by the polypeptide of the invention.

Polynucleotide sequence

The invention provides genomic polynucleotide sequences comprising the gene encoding the TEMER01209, TEMER09077, TEMER061 18 or TEMER03835 as well as its coding sequence. Accordingly, the invention relates to an isolated polynucleotide comprising the genomic nucleotide sequence according to the coding nucleotide sequence according to SEQ ID NO: 1 , 2, 3, 5, 6, 7, 1 1 , 12, 13, 15, 16 or 17 and to variants, such as functional equivalents, of either thereof.

In particular, the invention relates to an isolated polynucleotide which is capable of hybridizing selectively, for example under stringent conditions, preferably under highly stringent conditions, with the reverse complement of a polynucleotide comprising the sequence set out in SEQ ID NO: 1 , 2, 3, 5, 6, 7, 1 1 , 12, 13, 15, 16 or 17.

More specifically, the invention relates to a polynucleotide comprising or consisting essentially of a nucleotide sequence according to SEQ ID NO: 1 , 2, 3, 5, 6, 7, 1 1 , 12, 13, 15, 16 or 17. The invention also relates to an isolated polynucleotide comprising or consisting essentially of a sequence which encodes at least one functional domain of a polypeptide according to SEQ ID NO: 4, 8, 9, 14 or 18 or a variant thereof, such as a functional equivalent, or a fragment of either thereof.

In one embodiment, a TEMER01209, TEMER09077, TEMER061 18 or TEMER03835 nucleic acid of the invention is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more homologous to a nucleic acid sequence shown in SEQ ID NO: 1 , 2, 3, 5, 6, 7, 1 1 , 12, 13, 15, 16 or 17 or the complement thereof.

The term "gene" as used herein refers to a segment of a nucleic acid molecule coding for a polypeptide chain, that may or may not include gene regulatory sequences preceding and following the coding sequence, e.g. promoters, enhancers, etc., as well as intervening sequences (introns) between individual coding segments (exons). It will further be appreciated that the definition of gene can include nucleic acids that do not encode polypeptide, but rather provide templates for transcription of functional RNA molecules such as tRNAs, rRNAs, etc.

A nucleic acid molecule of the present invention, such as a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1 , 2, 3, 5, 6, 7, 1 1 , 12, 13, 15, 16 or 17 or a variant thereof, such as a functional equivalent, can be isolated using standard molecular biology techniques and the sequence information provided herein. For example, using all or a portion of the nucleic acid sequence of SEQ ID NO: 1 , 2, 5, 6, 1 1 , 12, 15 or 16 as a hybridization probe, nucleic acid molecules according to the invention can be isolated using standard hybridization and cloning techniques (e. g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

Moreover, a nucleic acid molecule encompassing all or a portion of SEQ ID NO: 1 , 2, 3, 5, 6, 7, 1 1 , 12, 13, 15, 16 or 17 may be isolated by the polymerase chain reaction (PCR) using synthetic oligonucleotide primers designed based upon the sequence information contained in SEQ ID NO: 1 , 2, 3, 5, 6, 7, 1 1 , 12, 13, 15, 16 or 17.

A nucleic acid of the invention can 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. Furthermore, oligonucleotides corresponding to or hybridizable to a nucleotide sequence according to the invention can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.

In a preferred embodiment, an isolated nucleic acid molecule of the invention comprises the nucleotide sequence shown in SEQ ID NO: 1 , 2, 3, 5, 6, 7, 1 1 , 12, 13, 15, 16 or 17.

In another preferred embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule which is the reverse complement of the nucleotide sequence shown in SEQ ID NO: 1 , 2, 3, 5, 6, 7, 1 1 , 12, 13, 15, 16 or 17 or a variant, such as a functional equivalent, of either such nucleotide sequence.

A nucleic acid molecule which is complementary to another nucleotide sequence is one which is sufficiently complementary to the other nucleotide sequence such that it can hybridize to the other nucleotide sequence thereby forming a stable duplex. The term "cDNA" (complementary DNA) is defined herein as a DNA molecule which can be prepared by reverse transcription from a mRNA molecule. In prokaryotes the mRNA molecule is obtained from the transcription of the genomic DNA of a gene present in a cell. In eukaryotic cells genes contain both exons, i.e. coding sequences, and introns, i.e. intervening sequences located between the exons. Therefore in eukaryotic cell the initial, primary RNA obtained from transcription of the genomic DNA of a gene is processed through a series of steps before appearing as mRNA. These steps include the removal of intron sequences by a process called splicing. cDNA derived from mRNA only contains coding sequences and can be directly translated into the corresponding polypeptide product. The term "complementary strand" can be used interchangeably with the term "complement". The complement of a nucleic acid strand can be the complement of a coding strand or the complement of a non-coding strand. When referring to double- stranded nucleic acids, the complement of a nucleic acid encoding a polypeptide refers to the complementary strand of the strand encoding the amino acid sequence or to any nucleic acid molecule containing the same.

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. Aqueous and non-aqueous methods are described in that reference and either can be used. Stringency conditions are sequence-dependent and will be different in different circumstances. 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 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by two washes in 0.2X 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 6X SSC at about 45°C, followed by one or more washes in 0.2X SSC, 0.1 % SDS at 60°C; 3) high stringency hybridization conditions in 6X SSC at about 45°C, followed by one or more washes in 0.2X 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 0.2X SSC, 1 % SDS at 65°C.

In general, high stringency conditions, such as high hybridization temperature and optionally low salt concentrations, permit only hybridization between sequences that are highly similar, whereas low stringency conditions, such as low hybridization temperature and optionally high salt concentrations, allow hybridization when the sequences are less similar.

One aspect of the invention pertains to isolated nucleic acid molecules that encode a polypeptide of the invention or a variant, such as a functional equivalent thereof, for example a biologically active fragment or domain, as well as nucleic acid molecules sufficient for use as hybridization probes to identify nucleic acid molecules encoding a polypeptide of the invention and fragments of such nucleic acid molecules suitable for use as PCR primers for the amplification or mutation of nucleic acid molecules.

Moreover, an "isolated nucleic acid fragment" is a nucleic acid fragment that is not naturally occurring as a fragment and would not be found in the natural state.

The term "naturally-occurring" as used herein refers to processes, events, or things that occur in their relevant form in nature. By contrast, "not naturally-occurring" refers to processes, events, or things whose existence or form involves the hand of man. Generally, the term "naturally-occurring" with regard to polypeptides or nucleic acids can be used interchangeable with the term "wild-type" or "native". It refers to polypeptide or nucleic acids encoding a polypeptide, having an amino acid sequence or polynucleotide sequence, respectively, identical to that found in nature. Naturally occurring polypeptides include native polypeptides, such as those polypeptides naturally expressed or found in a particular host. Naturally occurring polynucleotides include native polynucleotides such as those polynucleotides naturally found in the genome of a particular host. Additionally, a sequence that is wild-type or naturally-occurring may refer to a sequence from which a variant or a synthetic sequence is derived.

As used herein, a "synthetic" molecule is produced by in vitro chemical or enzymatic synthesis. It includes, but is not limited to, variant nucleic acids made with optimal codon usage for host organisms of choice.

The term "recombinant" when used in reference to a cell, nucleic acid, protein or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all. The term "recombinant" is synonymous with "genetically modified".

The term "isolated polypeptide" as used herein means a polypeptide or protein that is removed from at least one component, e.g. other polypeptide material, with which it is naturally associated. Thus, an isolated polypeptide may contain at most 10%, at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, even more preferably at most 2%, even more preferably at most 1 % and most preferably at most 0.5% as determined by SDS-PAGE of other polypeptide material with which it is natively associated. The isolated polypeptide may be free of any other impurities. The isolated polypeptide may be at least 50% pure, e.g., at least 60% pure, at least 70% pure, at least 75% pure, at least 80% pure, at least 85% pure, at least 80% pure, at least 90% pure, or at least 95% pure, 96%, 97%, 98%, 99%, 99.5%, 99.9% as determined by SDS-PAGE or any other analytical method suitable for this purpose and known to the person skilled in the art. An "isolated polynucleotide" or "isolated nucleic acid" is a polynucleotide removed from other polynucleotides with which it is naturally associated. Thus, an isolated polynucleotide may contain at most 10%, at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, even more preferably at most 2%, even more preferably at most 1 % and most preferably at most 0,5% by weight of other polynucleotide material with which it is naturally associated. The isolated polynucleotide may be free of any other impurities. The isolated polynucleotide may be at least 50% pure, e.g., at least 60% pure, at least 70% pure, at least 75% pure, at least 80% pure, at least 85% pure, at least 90% pure, or at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% pure by weight.

The term "substantially pure" with regard to polypeptides refers to a polypeptide preparation which contains at the most 50% by weight of other polypeptide material. The polypeptides disclosed herein are preferably in a substantially pure form. In particular, it is preferred that the polypeptides disclosed herein are in "essentially pure form", i.e. that the polypeptide preparation is essentially free of other polypeptide material. Optionally, the polypeptide may also be essentially free of non-polypeptide material such as nucleic acids, lipids, media components, and the like. Herein, the term "substantially pure polypeptide" is synonymous with the terms "isolated polypeptide" and "polypeptide in isolated form". The term "substantially pure" with regard to polynucleotide refers to a polynucleotide preparation which contains at the most 50% by weight of other polynucleotide material. The polynucleotides disclosed herein are preferably in a substantially pure form. In particular, it is preferred that the polynucleotide disclosed herein are in "essentially pure form", i.e. that the polynucleotide preparation is essentially free of other polynucleotide material. Optionally, the polynucleotide may also be essentially free of non-polynucleotide material such as polypeptides, lipids, media components, and the like. Herein, the term "substantially pure polynucleotide" is synonymous with the terms "isolated polynucleotide" and "polynucleotide in isolated form". The term "nucleic acid" as used in the present invention refers to a nucleotide polymer including at least 5 nucleotide units. A nucleic acid refers to a ribonucleotide polymer (RNA), deoxynucleotide polymer (DNA) or a modified form of either type of nucleic acid or synthetic form thereof or mixed polymers of any of the above. Nucleic acids may include either or both naturally-occurring and modified nucleic acids linked together by naturally-occurring and/or non-naturally occurring nucleic acid linkages. The nucleic acid molecules may be modified chemically or biochemically or may contain non- natural or derivatized nucleic acid bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleic acids with an analog, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, etc.) The term nucleic acid is also intended to include any topological conformation, including single-stranded (sense strand and antisense strand), double-stranded, partially duplexed, triplex, hairpinned, circular and padlocked conformations. Also included are synthetic molecules that mimic nucleic acids in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule. A reference to a nucleic acid sequence encompasses its complement unless otherwise specified. Thus, a reference to a nucleic acid molecule having a particular sequence should be understood to encompass its complementary strand, with its complementary sequence. The complementary strand is also useful, e.g., for antisense therapy, hybridization probes and PCR primers. The term "nucleic acid", "nucleic acid molecule" and "polynucleotide" can be used interchangeably herein.

A "substitution", as used herein in relation to polypeptides or nucleic acids, denotes the replacement of one or more amino acids in a polypeptide sequence or of one or more nucleotides in a polynucleotide sequence, respectively, by different amino acids or nucleotides, respectively

Another embodiment of the invention provides an isolated nucleic acid molecule which is antisense to a TEMER01209, TEMER09077, TEMER061 18 or TEMER03835 nucleic acid molecule, e.g., the coding strand of a TEMER01209, TEMER09077, TEMER061 18 or TEMER03835 nucleic acid molecule. Also included within the scope of the invention are the complementary strands of the nucleic acid molecules described herein.

Unless otherwise indicated, all nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer and all amino acid sequences of polypeptides encoded by DNA molecules determined herein were predicted by translation of a DNA sequence determined as above. Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule.

The actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art. As is also known in the art, a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion. The term "deletion", as used herein, denotes a change in either amino acid or nucleic acid sequence in which one or more amino acid or nucleotide, respectively, are absent as compared to the parent, often the naturally occurring, amino acid or nucleic acid sequence. The term "insertion", also known as the term "addition", denotes a change in an amino acid or nucleic acid sequence resulting in the addition of one or more amino acid or nucleotide, respectively, as compared to the parent, often the naturally occurring, amino acid or nucleic acid sequence.

The person skilled in the art is capable of identifying such erroneously identified bases and knows how to correct for such errors for example by sequencing the relevant gene of TEC-101 or the genome of TEC-101 (CBS 127450) as described in WO201 1/000949. A nucleic acid molecule according to the invention may comprise only a portion or a fragment of the nucleic acid sequence shown in SEQ ID NO: 1 , 2, 3, 5, 6, 7, 1 1 , 12, 13, 15, 16 or 17 (or of a variant of either thereof), for example a fragment which can be used as a probe or primer or a fragment encoding a portion of a TEMER01209, TEMER09077, TEMER061 18 or TEMER03835 protein. The nucleotide sequence determined from the cloning of the TEMER01209, TEMER09077, TEMER061 18 or TEMER03835 gene and cDNA allows for the generation of probes and primers designed for use in identifying and/or cloning other TEMER01209, TEMER09077, TEMER061 18 or TEMER03835 family members, as well as TEMER01209, TEMER09077, TEMER061 18 or TEMER03835 homologues from other species.

The probe/primer typically comprises a substantially purified oligonucleotide which typically comprises a region of nucleotide sequence that hybridizes preferably under highly stringent conditions to at least from about 12 to about 15, preferably from about 18 to about 20, preferably from about 22 to about 25, more preferably about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, or about 75 or more consecutive nucleotides of a nucleotide sequence shown in SEQ ID NO: 1 , 2, 3, 5, 6, 7, 1 1 , 12, 13, 15, 16 or 17 or of a variant, such as a functional equivalent, of either thereof.

Probes based on the TEMER01209, TEMER09077, TEMER061 18 or TEMER03835 nucleotide sequences can be used to detect transcripts or genomic TEMER01209, TEMER09077, TEMER061 18 or TEMER03835 sequences encoding the same or homologous proteins for instance in other organisms. In preferred embodiments, the probe further comprises a label group attached thereto, e.g., the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor. Such probes can also be used as part of a diagnostic test kit for identifying cells which express a TEMER01209, TEMER09077, TEMER061 18 or TEMER03835 protein.

The polynucleotides herein may be synthetic polynucleotides. The synthetic polynucleotides may be optimized in codon use, preferably according to the methods described in WO2006/077258 and/or PCT/EP2007/055943, 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. Those skilled in the art will know that the codon usage needs to be adapted depending on the host species, possibly resulting in variants with significant homology deviation from SEQ ID NO: 1 , 2, 3, 5, 6, 7, 1 1 , 12, 13, 15, 16 or 17 but still encoding the polypeptide of the invention.

The term "expression" includes any step involved in the production of the polypeptide including, but not limited to, transcription, post transcriptional modification, translation, post-translational modification, targeting and secretion.

The invention further relates to a nucleic acid construct comprising the polynucleotide as described before. 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 "coding sequence" as defined herein is a sequence, which is transcribed into mRNA and translated into a transcriptional activator of a protease promoter of the invention. The boundaries of the coding sequence are generally determined by the ATG start codon at the 5'end of the mRNA and a translation stop codon sequence terminating the open reading frame at the 3' end of the mRNA. A coding sequence can include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences. Preferably, the nucleic acid has high GC content. The GC content herein indicates the number of G and C nucleotides in the construct, divided by the total number of nucleotides, expressed in %. The GC content is preferably 56% or more, 57% or more, 58% or more, 59% or more, 60% or more, or in the range of 56-70% or the range of 58-65%. Preferably, the DNA construct comprises a promoter DNA sequence, a coding sequence in operative association with said promoter DNA sequence and control sequences such as:- one translational termination sequence orientated in 5' towards 3' direction selected from the following list of sequences: TAAG, TAGA and TAAA, preferably TAAA, and/or- one translational initiator coding sequence orientated in 5' towards 3' direction selected from the following list of sequences: GCTACCCCC; GCTACCTCC; GCTACCCTC; GCTACCTTC; GCTCCCCCC; GCTCCCTCC; GCTCCCCTC; GCTCCCTTC; GCTGCCCCC; GCTGCCTCC; GCTGCCCTC; GCTGCCTTC; GCTTCCCCC; GCTTCCTCC; GCTTCCCTC; and GCTTCCTTC, preferably GCT TCC TTC, and/or one translational initiator sequence selected from the following list of sequences: 5'-mwChkyCAAA-3'; 5'-mwChkyCACA-3' or 5'-mwChkyCAAG-3\ using ambiguity codes for nucleotides: m (A/C); w (A/T); y (C/T); k (G/T); h (A/C/T), preferably 5'-CACCGTCAAA-3' or 5'-CGCAGTCAAG-3'.

In the context of this invention, the term "translational initiator coding sequence" is defined as the nine nucleotides immediately downstream of the initiator or start codon of the open reading frame of a DNA coding sequence. The initiator or start codon encodes for the AA methionine. The initiator codon is typically ATG, but may also be any functional start codon such as GTG.

In the context of this invention, the term "translational termination sequence" is defined as the four nucleotides starting from the translational stop codon at the 3' end of the open reading frame or nucleotide coding sequence and oriented in 5' towards 3' direction.

In the context of this invention, the term "translational initiator sequence" is defined as the ten nucleotides immediately upstream of the initiator or start codon of the open reading frame of a DNA sequence coding for a polypeptide. The initiator or start codon encodes for the AA methionine. The initiator codon is typically ATG, but may also be any functional start codon such as GTG. It is well known in the art that uracil, U, replaces the deoxynucleotide thymine, T, in RNA.

Homology and identity

The terms "sequence homology" or "sequence identity" are used interchangeably herein. For the purpose of this invention, it is defined here that in order to determine the percentage of sequence homology or 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 percent 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. LongdenJ. and BleasbyA Trends in Genetics 16, (6) pp276— 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, word-lengt = 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, word-lengt = 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/.

Vectors

An expression vector comprises a polynucleotide coding for a polypeptide, operably linked to the appropriate control sequences (such as a promoter, and transcriptional and translational stop signals) for expression and/or translation in vitro, or in the host cell of the polynucleotide. Certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.

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. 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. 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.

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.

Another aspect of the invention pertains to vectors, including cloning and expression vectors, comprising a polynucleotide of the invention encoding a TEMER01209, TEMER09077, TEMER061 18 or TEMER03835 protein or a functional equivalent thereof and methods of growing, transforming or transfecting such vectors in a suitable host cell, for example under conditions in which expression of a polypeptide of the invention occurs. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Thus in a further embodiment, the invention provides a method of making polynucleotides of the invention by introducing a polynucleotide of the invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about replication of the vector. The vector may be recovered from the host cell. Suitable host cells are described below.

One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. The terms "plasmid" and "vector" can be used interchangeably herein as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as cosmid, viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses) and phage vectors which serve equivalent functions.

Vectors according to the invention may be used in vitro, for example for the production of RNA or used to transfect or transform a host cell.

A vector of the invention may comprise two or more, for example three, four or five, polynucleotides of the invention, for example for overexpression.

The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vector includes one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operably linked to the nucleic acid sequence to be expressed.

The term "operably linked", "operatively linked" or "in operative association" as used herein refers to two or more nucleic acid sequence elements that are physically linked and are in a functional relationship with each other. For instance, a promoter is operably linked to a coding sequence if the promoter is able to initiate or regulate the transcription or expression of a coding sequence, in which case the coding sequence should be understood as being "under the control of" the promoter. Generally, when two nucleic acid sequences are operably linked, they will be in the same orientation and usually also in the same reading frame. They usually will be essentially contiguous, although this may not be required.

A vector or expression construct for a given host cell may thus comprise the following elements operably linked to each other in a consecutive order from the 5'-end to 3'-end relative to the coding strand of the sequence encoding the polypeptide of the first invention: (1 ) a promoter sequence capable of directing transcription of the nucleotide sequence encoding the polypeptide in the given host cell ; (2) optionally, a signal sequence capable of directing secretion or targetting of the polypeptide from the given host cell into a culture medium or membrane, respectively; (3) a DNA sequence of the invention encoding a mature and preferably active form of a polypeptide which is preferably a polypeptide having saccharide transporter or sugar transporter activity, such as a saccharide transporter or sugar transporter ; and preferably also has (4) a transcription termination region (terminator) capable of terminating transcription downstream of the nucleotide sequence encoding the polypeptide.

The term "mature polypeptide" or "mature form of a polypeptide" is defined herein as a polypeptide in its final form and is obtained after translation of a mRNA into polypeptide and post-translational modifications of said polypeptide. Post-translational modification include N-terminal processing, C-terminal truncation, glycosylation, phosphorylation and removal of leader sequences such as signal peptides, pro-peptides and/or prepro-peptides as defined herein by cleavage.

Downstream of the nucleotide sequence according to the invention there may be a 3' untranslated region containing one or more transcription termination sites (e. g. a terminator). The terminator can, for example, be native to the DNA sequence encoding the polypeptide. However, preferably a yeast terminator is used in yeast host cells and a filamentous fungal terminator is used in filamentous fungal host cells. More preferably, the terminator is endogenous to the host cell (in which the nucleotide sequence encoding the polypeptide is to be expressed). In the transcribed region, a ribosome binding site for translation may be present. The coding portion of the mature transcripts expressed by the constructs will include a translation initiating AUG at the beginning and a termination codon appropriately positioned at the end of the polypeptide to be translated.

Enhanced expression of the polynucleotide of the invention may also be achieved by the selection of heterologous regulatory regions, e. g. promoter, targatting or secretion leader and/or terminator regions, which may serve to increase expression and, if desired, targeting or secretion levels of the protein of interest from the expression host and/or to provide for the inducible control of the expression of a polypeptide of the invention.

It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The vectors, such as expression vectors, of the invention can be introduced into host cells to thereby produce proteins or polypeptides, encoded by nucleic acids as described herein (e.g. TEMER01209, TEMER09077, TEMER061 18 or TEMER03835 proteins, mutant forms of TEMER01209, TEMER09077, TEMER061 18 or TEMER03835 proteins, fragments, variants or functional equivalents thereof. The vectors, such as recombinant expression vectors, of the invention can be designed for expression of TEMER01209, TEMER09077, TEMER061 18 or TEMER03835 proteins in prokaryotic or eukaryotic cells.

Appropriate culture mediums and conditions for the above-described host cells are known in the art.

The recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

For most filamentous fungi and yeast, the vector or expression construct is preferably integrated in the genome of the host cell in order to obtain stable transformants. However, for certain yeasts also suitable episomal vectors are available into which the expression construct can be incorporated for stable and high level expression, examples thereof include vectors derived from the 2μ and pKD1 plasmids of Saccharomyces and Kluyveromyces, respectively, or vectors containing an AMA sequence (e.g. AMA1 from Aspergillus). In case the expression constructs are integrated in the host cells genome, the constructs are either integrated at random loci in the genome, or at predetermined target loci using homologous recombination, in which case the target loci preferably comprise a highly expressed gene.

Accordingly, expression vectors useful in the present invention include chromosomal-, episomal- and virus-derived vectors e.g., vectors derived from bacterial plasmids, bacteriophage, yeast episome, yeast chromosomal elements, viruses such as baculoviruses, papova viruses, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. The term "control sequence" or "regulatory sequence" can be used interchangeably with the term "expression-regulating nucleic acid sequence". The term as used herein refers to nucleic acid sequences necessary for and/or affecting the expression of an operably linked coding sequence in a particular host organism or in vitro. When two nucleic acid sequences are operably linked, they usually will be in the same orientation and also in the same reading frame. They usually will be essentially contiguous, although this may not be required. The expression-regulating nucleic acid sequences, such as inter alia appropriate transcription initiation, termination, promoter, leader, signal peptide, pro-peptide, prepro-peptide, or enhancer sequences; Shine- Delgarno sequence, repressor or activator sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., ribosome binding sites); sequences that enhance protein stability; and when desired, sequences that enhance protein targeting or secretion, can be any nucleic acid sequence showing activity in the host organism of choice and can be derived from genes encoding proteins, which are either homologous or heterologous to the host organism. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. When desired, the control sequence 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. Control sequences may be optimized to their specific purpose.

The control sequence may be an appropriate promoter sequence, a nucleic acid sequence, which is recognized by a host cell for expression of the nucleic acid sequence. The promoter sequence contains transcriptional control sequences, which mediate the expression of the polypeptide. The promoter may be any nucleic acid sequence, which shows transcriptional activity in the cell including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the cell.

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.

Preferably the promoter is an inducible promoter. More preferably the promoter is a carbohydrate inducible promoter. Carbohydrate inducible promoters that are preferably used are selected from a starch-inducible promoter (i.e. a promoter inducible by starch, a monomer, a dimer, a oligomer thereof, such as for example a maltose-inducible promoter, an isomaltose-inducible promoter), a cellulose-inducible promoter (i.e. a promoter inducible by cellulose, a monomer, a dimer and/or oligomer thereof, such as for example a cellobiose-inducible promoter, a sophorose-inducible promoter), a hemicellulose inducible promoter (i.e. a promoter inducible by hemicellulose, a monomer, a dimer, and/or a oligomer thereof, such as e.g. a xylan-inducible promoter, an arabionose-inducible promoter, a xylose-inducible promoter), a pectin-inducible promoter (i.e. a promoter inducible by pectin, a monomer, a dimer and/or an oligomer thereof such as for example a galacturonic acid-inducible promoter, a rhamnose- inducible promoter), an arabinan-inducible promoter (i.e. a promoter inducible by arabinan, a monomer, a dimer, and/or an oligomer thereof such as for example an arabinose-inducible promoter), a glucose-inducible promoter, a lactose-inducible promoter, a galactose-inducible promoter. 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, Aspergillus gpdA promoter, 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 (x/nD), 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. Even other examples of the use of promoters are described in WO2008/098933 and co-pending patent application no. EP12172605. Preferred carbohydrate inducible promoters which can be used in filamentous fungi are Rasamsonia promoters such Rasamsonia emersonii beta-glucosidase, Rasamsonia emersonii cellobiohydrolase I, Rasamsonia emersonii cellobiohydrolase II, Rasamsonia emersonii endoglucanase IV(GH61 ), Rasamsonia emersonii acetyl xylan esterase promoter, A. oryzae TAKA amylase, 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 (x/nD), T, 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) as defined above.

Also promoters disclosed in WO2009150195 can be used, these promoters direct expression in a wide range of industrially relevant species, both prokaryotes and eukaryotes. When the polynucleotide sequences of the invention are applied in combination with selection marker genes it is possible to perform selectable cloning in a laboratory host and use the same construct in the final host.

Examples of such 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 SP01/lac promoter that can be controlled by inducers such as isopropyl^-D-thiogalactopyranoside [IPTG] ((Yansura D.G., Henner D.J. Proc Natl Acad Sci U S A. 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: aF, σΕ, aG and σΚ and general stress sigma factor, σΒ. The σΒ-mediated response is induced by energy limitation and environmental stresses (Hecker M, Volker U. Mol Microbiol. 1998; 29(5):1 129-1 136.). (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 AF, 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, Ipp, lac, Ipp-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 a-amylase and SPo2 promoters as well as promoters from extracellular protease genes.

Another example of a suitable promoter is 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, EN01, TPI1, and AOX1. Other suitable promoters include PDC1, GPD1, PGK1, TEF1, and TDH3. Examples of carbohydrate inducible promoters which can be used are GAL promoters, such as GAL1 or GAL10 promoters. All of the above-mentioned promoters are readily available in the art.

Transcription of the DNA encoding the polypeptides of the present invention by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act to increase transcriptional activity of a promoter in a given host cell-type. Examples of enhancers include the SV40 enhancer, which is located on the late side of the replication origin at bp 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

The control sequence may also be a suitable transcription 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 control sequence may also be a terminator. Preferred terminators for filamentous fungal cells are obtained from the genes encoding A. oryzae TAKA amylase, A. niger glucoamylase (glaA), A. nidulans anthranilate synthase, A. niger alpha- glucosidase, trpC gene and Fusarium oxysporum trypsin-like protease.

The control sequence may also include a suitable leader sequence, a non- translated region of a mRNA which is important for translation by the filamentous fungal cell. The leader sequence is operably linked to the 5' terminus of the nucleic acid sequence encoding the polypeptide. Any leader sequence, which is functional in the cell, may be used in the present invention. Preferred leaders for filamentous fungal cells are obtained from the genes encoding A. oryzae TAKA amylase and A. nidulans triose phosphate isomerase and A. niger glaA. Other preferred sequences are isolated and/or disclosed in WO2006/077258.

Other control sequences may be isolated from the Penicillium IPNS gene, or pcbC gene, the beta tubulin gene. All the control sequences cited in WO 01/21779 are herewith incorporated by reference.

The control sequence may also be a polyadenylation sequence, a sequence which is operably linked to the 3' terminus of the nucleic acid sequence and which, when transcribed, is recognized by the filamentous fungal cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence, which is functional in the cell, may be used in the present invention. Preferred polyadenylation sequences for filamentous fungal cells are obtained from the genes encoding A. oryzae TAKA amylase, A. niger glucoamylase, A. nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease and A. niger alpha-glucosidase.

When the polypeptide according to the invention is to be targeted to the (outer) membrane of the host cell, an appropriate signal sequence can be added to the polypeptide in order to direct the de novo synthesized polypeptide to the secretion route of the host cell. The person skilled in the art knows to select an appropriate signal sequence for a specific host. The signal sequence may be native to the host cell, or may be foreign to the host cell. As an example, a signal sequence from a protein native to the host cell can be used. Preferably, said native protein is a highly targeted or secreted protein, i.e. a protein that is targeted or secreted in amounts higher than 10% of the total amount of protein being targeted or secreted. The signal sequences preferably used according to the invention are for example: pmeA.

As an alternative for a signal sequence, the polypeptide of the invention can be fused to a targeted or secreted carrier protein, or part thereof. Such chimeric construct is directed to the secretion route by means of the signal sequence of the carrier protein, or part thereof. In addition, the carrier protein will provide a stabilizing effect to the polypeptide according to the invention and or may enhance solubility. Such carrier protein may be any protein. Preferably, a highly targeted or secreted protein is used as a carrier protein. The carrier protein may be native or foreign to the polypeptide according to the invention. The carrier protein may be native of may be foreign to the host cell. Examples of such carrier proteins are glucoamylase, prepro sequence of alpha-Mating factor, cellulose binding domain of Clostridium cellulovorans cellulose binding protein A, glutathione S-transferase, chitin binding domain of Bacillus circulans chitinase A1 , maltose binding domain encoded by the malE gene of E. coli K12, beta-galactosidase, and alkaline phosphatase. A preferred carrier protein for expression of such chimeric construct in Aspergillus cells is glucoamylase. The carrier protein and polypeptide according to the invention may contain a specific amino acid motif to facilitate isolation of the polypeptide; the polypeptide according to the invention may be released by a special releasing agent. The releasing agent may be a proteolytic enzyme or a chemical agent. An example of such amino acid motif is the KEX protease cleavage site, which is well- known to the person skilled in the art.

A signal sequence can be used to facilitate targeting to the cell membrane of a protein or polypeptide of the invention. Signal sequences are typically characterized by a core of hydrophobic amino acids, which are generally cleaved from the mature protein during secretion in one or more cleavage events. Such signal peptides contain processing sites that allow cleavage of the signal sequence from the mature proteins as they pass through the secretory pathway. The signal sequence directs targeting or secretion of the protein, such as from a eukaryotic host into which the expression vector is transformed, and the signal sequence is subsequently or concurrently cleaved..

As an alternative for a signal sequence, the polypeptide of the invention can be fused to a targeted or secreted carrier protein, or part thereof. Such chimeric construct is directed to the secretion route by means of the signal sequence of the carrier protein, or part thereof. In addition, the carrier protein will provide a stabilizing effect to the polypeptide according to the invention and or may enhance solubility. Such carrier protein may be any protein. Preferably, a highly targeted or secreted protein is used as a carrier protein. The carrier protein may be native or foreign to the polypeptide according to the invention. The carrier protein may be native of may be foreign to the host cell. Examples of such carrier proteins are glucoamylase, prepro sequence of alpha-Mating factor, cellulose binding domain of Clostridium cellulovorans, cellulose binding protein A, glutathione S-transferase, chitin binding domain of Bacillus circulans chitinase A1 , maltose binding domain encoded by the malE gene of E. coli K12, beta-galactosidase, and alkaline phosphatase. A preferred carrier protein for expression of such chimeric construct in Aspergillus cells is glucoamylase.

(Over)expression

In a preferred embodiment, the polynucleotides of the present invention as described herein may be over-expressed in a microbial strain of the invention compared to the parent microbial strain in which said gene is not over-expressed. Over-expression of a polynucleotide sequence is defined herein as the expression of the said sequence gene which results in an activity of the polypeptide encoded by the said sequence in a microbial strain being at least 1 .1 , at least 1.25 or at least 1 .5-fold the activity of the polypeptide in the parent microbial; preferably the activity of said polypeptide is at least 2-fold, more preferably at least 3-fold, more preferably at least 4-fold, more preferably at least 5-fold, even more preferably at least 10-fold and most preferably at least 20-fold the activity of the polypeptide in the parent microbial.

The vector may further include sequences flanking the polynucleotide giving rise to RNA which comprise sequences homologous to eukaryotic genomic sequences or viral genomic sequences. This will allow the introduction of the polynucleotides of the invention into the genome of a host cell. An integrative cloning vector may integrate at random or at a predetermined target locus in the chromosome(s) of the host cell into which it is to be integrated. In a preferred embodiment of the invention, an integrative cloning vector may comprise 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 may be preferably linearized prior to transformation of the host cell. Linearization may preferably be 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 about 0.1 kb, such as about at least 0.2kb, more preferably at least about 0.5 kb, even more preferably at least about 1 kb, most preferably at least about 2 kb. Preferably, the parent host strains may be modified for improved frequency of targeted DNA integration as described in WO05/095624 and/or WO2007/1 15886.

Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, transduction, infection, lipofection, cationic lipid-mediated transfection or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual, 2^nd, ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), Davis et al., Basic Methods in Molecular Biology (1986) and other laboratory manuals.

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 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 f.sp. 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 polypeptides 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.

For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include, but are not limited to, those which confer resistance to drugs or which complement a defect in the host cell. A selectable marker is a gene which allow for selection of cells transformed with such gene and 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.

Preferred selectable markers include, but are not limited to, those which confer resistance to drugs or which complement a defect in the host cell. They include e. g. 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, nourseothricin, methotrexate, phleomycin orbenomyl resistance (benA).

Alternatively, specific selection markers can be used such as auxotrophic markers which require corresponding mutant host strains: e. g.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 which are free of selection marker genes.

Other markers 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 fungi), the ampicillin resistance gene (E. coli), the neomycin resistance gene (Bacillus) and the E. coli uidA gene, coding for β-glucuronidase (GUS).

The term selectable marker extends to a marker gene used for screening, i.e. marker gene that, once introduced into a host cell confers to the cell a visible phenotype and causes the cell look different. An example of marker for screening is the gene coding for the Green fluorescent protein which causes cells glow green under UV light.

Expression of proteins in prokaryotes is often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, e.g. to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: 1 ) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.

As indicated, the expression vectors will preferably contain selectable markers. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracyline or ampicillin resistance for culturing in E. coli and other bacteria.

Vectors preferred for use in bacteria are for example disclosed in WO-A1 - 2004/074468, which are hereby enclosed by reference. Other suitable vectors will be readily apparent to the skilled artisan.

The TEMER01209, TEMER09077, TEMER061 18 or TEMER03835 polypeptide may be expressed in a modified form, such as a fusion protein, and may include not only targeting or secretion signals but also additional heterologous functional regions. Thus, for instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification or during subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification

The invention provides an recombinant polypeptide having the amino acid sequence according to SEQ ID NO: 4, 8, 9, 14 or 18 and an amino acid sequence obtainable by expressing the polynucleotide of SEQ ID NO: 1 , 2, 3, 5, 6, 7, 1 1 , 12, 13, 15, 16 or 17 in an appropriate host. Also, a peptide or polypeptide comprising a variant of the above polypeptides, such as a functional equivalent, is comprised within the present invention. The above polypeptides are collectively comprised in the term "polypeptides according to the invention"

As used herein, the terms "variant, "derivative", "mutant" or "homologue" can be used interchangeably. They can refer to either polypeptides or nucleic acids. Variants include substitutions, insertions, deletions, truncations, transversions, and/or inversions, at one or more locations relative to a reference sequence. Variants can be made for example by site-saturation mutagenesis, scanning mutagenesis, insertional mutagenesis, random mutagenesis, site-directed mutagenesis, and directed-evolution, as well as various other recombination approaches. Variant polypeptides may differ from a reference polypeptide by a small number of amino acid residues and may be defined by their level of primary amino acid sequence homology/identity with a reference polypeptide. In general related polypeptides may have several essential amino acids in common (which are sometimes referred to as motif). The identity of those essential amino acids can be identified from the alignment of related polypeptides. Mutating of one or more of the essential amino acids may change the properties of the polypeptide such as activity, substrate specificity, thermostability or change of pH optimum. Mutating of one or more of the non-essential amino acids may have smaller effect on the properties of the polypeptide such as substrate specificity, thermostability or change of pH optimum. Preferably, variant polypeptides have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99% amino acid sequence identity with a reference polypeptide. Methods for determining percent identity are known in the art and described herein. Generally, the variants retain the characteristic nature of the reference polypeptide, but have altered properties in some specific aspects. For example, a variant may have a modified pH optimum, a modified substrate binding ability, a modified resistance to enzymatic degradation or other degradation, an increased or decreased activity, a modified temperature or oxidative stability, but retains its characteristic functionality. Variants further include polypeptides with chemical modifications that change the characteristics of a reference polypeptide.

With regard to nucleic acids, the terms refer to a nucleic acid that encodes a variant polypeptide, that has a specified degree of homology/identity with a reference nucleic acid, or that hybridizes under stringent conditions to a reference nucleic acid or the complement thereof. Preferably, a variant nucleic acid has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99% nucleic acid sequence identity with a reference nucleic acid. Methods for determining percent identity are known in the art and described herein.

As used herein, the term "polypeptide" refers to a molecule comprising amino acid residues linked by peptide bonds and containing more than five amino acid residues. The amino acids are identified by either the single-letter or three-letter designations. The term "protein" as used herein is synonymous with the term "polypeptide" and may also refer to two or more polypeptides. Thus, the terms "protein", "peptide" and "polypeptide" can be used interchangeably. Polypeptides may optionally be modified (e.g., glycosylated, phosphorylated, acylated, farnesylated, prenylated, sulfonated, and the like) to add functionality. Polypeptides exhibiting activity may be referred to as enzymes. It will be understood that, as a result of the degeneracy of the genetic code, a multitude of nucleotide sequences encoding a given polypeptide may be produced.

The term "a polypeptide having biological activity" refers to a polypeptide which is encoded by a polynucleotide or a series of polynucleotides (contiguous or noncontiguous) and has an activity or function on other compounds or on organisms. For example an enzyme (for example a lipase) has a catalytic effect (for example on lipids). A signal sequence has a function on its corresponding or fused polypeptide such as a mature protein. Examples of use of the polypeptide or polynucleotide produced using the invention can be found in the production of a desired protein such as an enzyme which is industrially useful or by the use of the polypeptide or polynucleotide of the invention to affect directly and indirectly processes within the cell which may result in an industrial advantage for example by an improved, more efficient, or more pure production of a desired product such as an enzyme by the cell. These proteins or polypeptides having biological activity can be for example categorized according to FunCat (Ruepp, A et al, Nucleic Acids Research, 2004, vol. 32, no. 18, p. 5539) in proteins involved in the metabolism (metabolism, energy, storage protein), information pathways (cell cycle and DNA processing, transcription, protein synthesis, protein fate [folding, modification and destination], protein with binding function or cofactor requirement [structural or catalytic], protein activity regulation), transport (cellular transport, transport facilitation and transport routes), perception and response to stimuli (cellular communication/signal transduction mechanism, cell rescue, defense and virulence, interaction with the cellular environment, interaction with the environment [systemic], transposable elements, viral and plasmid proteins), developmental processes (cell fate, development [systemic], biogenesis of cellular components, cell type differentiation, tissue differentiation, organ differentiation), localization (subcellular localization, cell type localization, tissue localization, organ localization, ubiquitous expression), experimentally uncharacterized or hypothetical (or conserved hypothetical) proteins (classification not yet clear-cut, and unclassified proteins).

An example of a protein or polypeptide having biological activity in an industrial application is an enzyme. Enzymes are used in the chemical industry and other industrial applications when specific catalysts are required. Enzymes in general are limited in the number of reactions they have evolved to catalyze and their deactivation at high temperatures. As a consequence, protein engineering is an active area of research and involves attempts to create new enzymes with novel properties, either through rational design or in vitro evolution. These efforts have begun to be successful, and a few enzymes have now been designed to improve enzymatic reactions. For designing it is essential to have starting sequences from useful microorganisms especially thermophilic microorganisms like fungi.

Enzymes can be categorized using their Enzyme Commission number (EC number) which is a numerical classification scheme for enzymes, based on the chemical reactions they catalyze. As a system of enzyme nomenclature, every EC number is associated with a recommended name for the respective enzyme.

Strictly speaking, EC numbers do not specify enzymes, but enzyme-catalyzed reactions. If different enzymes (for instance from different organisms) catalyze the same reaction, then they receive the same EC number (Moss, G.P. "Recommendations of the Nomenclature Committee". International Union of Biochemistry and Molecular Biology on the Nomenclature and Classification of Enzymes by the Reactions they Catalyse) .By contrast, UniProt identifiers uniquely specify a protein by its amino acid sequence.

Top-level EC numbers:

EC 1 (Oxidoreductases) To catalyze oxidation/reduction reactions; transfer of H and O atoms or electrons from one substance to another.

EC 2 (Transferases) Transfer of a functional group from one substance to another. The group may be methyl-, acyl-, amino- or phosphate group.

EC 3 (Hydrolases) Formation of two products from a substrate by hydrolysis.

EC 4 (Lyases) Non-hydrolytic addition or removal of groups from substrates. C-C, C-N, C-0 or C-S bonds may be cleaved

EC 5 (Isomerases) Intramolecule rearrangement, i.e. isomerization changes within a single molecule.

EC 6 (Ligases) Join together two molecules by synthesis of new C-O, C-S, C-N or C-C bonds with simultaneous breakdown of ATP.

The polypeptide of the invention is preferably is preferably a polypeptide having saccharide transporter or sugar transporter activity, such as a saccharide transporter or sugar transporter.

A polypeptide which is capable to alter or influence the expression of another polypeptide may be, but is not limited to, a polypeptide having transcriptional activation activity or having transcription repressing activity (transcriptor factor or repressor, ) or a polypeptide having saccharide transporter activity.

By a polypeptide having transcriptional activation activity or transcriptional activator is meant a polypeptide which has the capability to activate transcription from a specific promoter or a set of promoters, for example a cellulase promoter, said activator being necessary for the initiation of transcription of the matching protein (such as cellulase(s)) encoding sequence to which the promoter(s) is (are) operably linked to.

By a polypeptide having transcriptional repressing activity or transcriptional repressor is meant a polypeptide which has the capability to repress transcription from a specific promoter or a set of promoters, for example a cellulase promoter, said repressor being necessary for the regulation of transcription of the matching protein (such as cellulase(s)) encoding sequence to which the promoter(s) is (are) operably linked to.

By a polypeptide having saccharide transporter activity or sugar transporter activity or saccharide transporter or sugar transporter is meant a polypeptide which has the capability to transport saccharides between the extracellular environment across the plasma membrane and the host cell. Transporters may influence processes like expression in multiple ways, for example the saccharide transport via saccharide transporters may influence induction of cellulases. By saccharides is meant all saccharides including mono-, di-, oligo- and polysaccharides.

Direct or indirect measurement of the activity of the polypeptide of the invention is a useful way to determine the activity of the polypeptide of the invention.

The biological activity of a polypeptide which is capable to alter or influence the expression of a polypeptide can be determined by measuring the expression, or is preferably determined indirectly through measurement of the activity of the expressed polypeptide such as a cellulase as for example described in the example section herein for determination of the cellulase activity using corn stover as substrate.

The polypeptide produced using the invention may be comprised in a composition. Preferably, the composition is enriched in such a polypeptide. By "enriched" is meant that the polypeptide in the composition is increased, for example with at least a factor of 1.1 , preferably 1 .5, more preferably 2 on protein compared to the composition without the overexpressed polypeptide of the invention. The composition may comprise a polypeptide produced using the present invention as the major enzymatic component, e.g., a mono-component composition. Alternatively, the composition may comprise multiple enzymatic activities. The polypeptide compositions may be prepared in accordance with methods known in the art and may be in the form of a liquid or a dry composition. For instance, the polypeptide composition may be in the form of a granulate or a microgranulate. The polypeptide to be included in the composition may be stabilized in accordance with methods known in the art. The dosage of the polypeptide composition produced using the invention and other conditions under which the composition is used depend on the ultimate use of the composition.

The term "polypeptide fragment" is defined herein as a polypeptide having one or more amino acids deleted from the amino and/or carboxyl terminus of the parent polypeptide or a homologous sequence thereof.

The term "prepro-peptide" is defined herein as a signal peptide and propeptide present at the amino terminus of a polypeptide, where the propeptide is linked (or fused) in frame to the amino terminus of a polypeptide and the signal peptide is linked in frame (or fused) to the amino terminus of the propeptide region. The term "signal peptide" is defined herein as a peptide linked (fused) in frame to the amino terminus of a polypeptide and directs the polypeptide into the cell" secretory pathway. A pro-peptide may be present between the signal peptide and the amino terminus of the polypeptide. The term "pro-peptide" is an amino acid sequence linked (fused) in frame to the amino terminus of a polypeptide having biological activity, wherein the resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases), A propolypeptide is generally biologically inactive and can be converted to a mature active polypeptide by catalytic or autocatalitic cleavage of the propeptide from the propolypeptide.

The enzyme or polypeptide produced using the invention can be recovered and purified from recombinant cell cultures by methods known in the art. Most preferably, high performance liquid chromatography ("HPLC") is employed for purification.

Polypeptides produced using the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides produced using the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides produced using the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes. The invention also features biologically active fragments of the polypeptides according to the invention.

Biologically active fragments of a polypeptide of the invention include polypeptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of the TEMER01209, TEMER09077, TEMER061 18 or TEMER03835 protein (e.g., the amino acid sequence of SEQ ID NO: 4, 8, 9, 14 or 18), which include fewer amino acids than the full length protein but which exhibit at least one biological activity of the corresponding full-length protein. Typically, biologically active fragments comprise a domain or motif with at least one activity of the TEMER01209, TEMER09077, TEMER061 18 or TEMER03835 protein.

A biologically active fragment of a protein of the invention can be a polypeptide which is, for example, about 10, about 25, about 50, about 100 or more amino acids in length or at least about 100 amino acids, at least 150, 200, 250, 300, 350, 400 amino acids in length, or of a length up the total number of amino acids of polypeptide of the invention.

Moreover, other biologically active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the biological activities of the native form of a polypeptide of the invention. The invention also features nucleic acid fragments which encode the above biologically active fragments of the TEMER01209, TEMER09077, TEMER061 18 or TEMER03835 protein.

In another aspect of the invention, improved polypeptides of the invention are provided. Improved polypeptides of the invention are polypeptides wherein at least one biological activity is improved. Such polypeptides may be obtained by randomly introducing mutations along all or part of the coding sequence of the polypeptide of the invention, such as by saturation mutagenesis, and the resulting mutants can be expressed recombinantly and screened for biological activity. For instance, the art provides for standard assays for measuring the activity of the polypeptide according to the invention and thus improved proteins may easily be selected. Improved variants of the amino acid sequences of the present invention leading to an improved activity of the polypeptide of the invention may be obtained by the corresponding genes of the present invention. Among such modifications are included: 1 . Error prone PCR to introduce random mutations, followed by a screening of obtained variants and isolating of variants with improved kinetic properties

2. Family shuffling of related variants of the genes encoding the polypeptide according to the invention, followed by a screening of obtained variants and isolating of variants with improved kinetic properties

Variants of the genes of the present invention leading to an increased level of mRNA and/or protein, resulting in more activity may be obtained by the polynucleotide sequences of said genes. Among such modifications are included:

1 . Improving the codon usage in such a way that the codons are (optimally) adapted to the parent microbial host.

2. Improving the codon pair usage in such a way that the codons are (optimally)adapted to the parent microbial host

3. Addition of stabilizing sequences to the genomic information encoding the polypeptide according to the invention resulting in mRNA molecules with an increased half life

Preferred methods to isolate variants with improved catalytic properties or increased levels of mRNA or protein are described in WO03/010183 and WO03/0131 1. Preferred methods to optimize the codon usage in parent microbial strains are described in PCT/EP2007/05594. Preferred methods for the addition of stabilizing elements to the genes encoding the polypeptide of the invention are described in WO2005/059149.

In a preferred embodiment the protein of the invention has an amino acid sequence according to SEQ ID NO: 4, 8, 9, 14 or 18. In another embodiment, the polypeptide of the invention is substantially homologous to the amino acid sequence according to SEQ ID NO: 4, 8, 9, 14 or 18 and retains at least one biological activity of a polypeptide according to SEQ ID NO: 4, 8, 9, 14 or 18, yet differs in amino acid sequence due to natural variation or mutagenesis as described.

In a further preferred embodiment, the protein of the invention has an amino acid sequence encoded by an isolated nucleic acid fragment capable of hybridizing to a nucleic acid according to SEQ ID NO: 1 , 2, 3, 5, 6, 7, 1 1 , 12, 13, 15, 16 or 17, preferably under highly stringent hybridization conditions.

Accordingly, the protein of the invention is preferably a protein which comprises an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, 92%, 93%, 94%, 95%, 9695%, 96%, 97%, 98%, 97%, 98%, 99% or more homologous to the amino acid sequence shown in SEQ ID NO: 4, 8, 9, 14 or 18 and, typically, retains at least one functional activity of the polypeptide according to SEQ ID NO: 4, 8, 9, 14 or 18.

According to one aspect of the invention the polypeptide of the invention may comprise the amino acid sequence set out in SEQ ID NO: 4, 8, 9, 14 or 18 or an amino acid sequence that differs in 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 or 12 amino acids from the amino acid sequence set out in SEQ ID NO: 4, 8, 9, 14 or 18 and whereby the polypeptide still has the activity or function of the polypeptide of the invention. The skilled person will appreciate that these minor amino acid changes in the polypeptide of the invention may be present (for example naturally occurring mutations) or made (for example using r-DNA technology) without loss of the protein function or activity. In case these mutations are present in a binding domain, active site, or other functional domain of the polypeptide a property of the polypeptide may change (for example its thermostability) but the polypeptide may keep its activity. In case a mutation is present which is not close to the active site, binding domain, or other functional domain, less effect may be expected.

Functional equivalents of a polypeptide according to the invention can also be identified e.g. by screening combinatorial libraries of mutants, e.g. truncation mutants, of the polypeptide of the invention for the biological activity of the polypeptide of the invention. In one embodiment, a variegated library of variants is generated by combinatorial mutagenesis at the nucleic acid level. A variegated library of variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential protein sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g. for phage display). There are a variety of methods that can be used to produce libraries of potential variants of the polypeptides of the invention from a degenerate oligonucleotide sequence. Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 1 1 :477). The term "degenerate nucleic acid sequence" or "degenerate (oligo)nucleotide sequence" denotes a sequence of nucleic acids that includes one or more degenerate codons (as compared to a reference nucleic acid molecule that encodes a polypeptide). Degenerate codons contain different triplets of nucleic acids, but encode the same amino acid residue (i.e., GAU and GAC triplets each encode Asp). The codon degeneracy refers to the nature of the genetic code permitting variation of the nucleic acid sequence without affecting the aminoacid sequence of an encoded polypeptide. The skilled artisan is well aware of the "codon-bias" exhibited by a specific host cell in usage of nucleic acid codons to specify a given amino acid.

In addition, libraries of fragments of the coding sequence of a polypeptide of the invention can be used to generate a variegated population of polypeptides for screening a subsequent selection of variants. For example, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of the coding sequence of interest with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S1 nuclease, and ligating the resulting fragment library into an expression vector. By this method, an expression library can be derived which encodes N-terminal and internal fragments of various sizes of the protein of interest.

Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations of truncation, and for screening cDNA libraries for gene products having a selected property. The most widely used techniques, which are amenable to high through-put analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify variants of a protein of the invention (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA 89:781 1 - 7815; Delgrave et al. (1993) Protein Engineering 6(3): 327-331 ).

In addition to the TEMER01209, TEMER09077, TEMER061 18 or TEMER03835 gene sequence shown in SEQ ID NO: 1 , 2, 3, 5, 6, 7, 1 1 , 12, 13, 15, 16 or 17, it will be apparent for the person skilled in the art that DNA sequence polymorphisms may exist within a given population, which may lead to changes in the amino acid sequence of the TEMER01209, TEMER09077, TEMER061 18 or TEMER03835 protein. Such genetic polymorphisms may exist in cells from different populations or within a population due to natural allelic variation. Allelic variants may also include functional equivalents. Fragments of a polynucleotide according to the invention may also comprise polynucleotides not encoding functional polypeptides. Such polynucleotides may function as probes or primers for a PCR reaction.

Methods of inactivation

As used herein the expression "inactivated mutant" or "inactivated cell" means a genetically engineered or a classical mutated cell having a gene which inactivated by a non-reversible inactivation, the inactivation includes inactivation in the protein-coding region. Inactivation of a microbial host cell in the production of the protein of the invention is herein defined as a phenotypic feature wherein the cell, due to modification in the genome: a) produces less of the protein of the invention as compared to the parent microbial host cell that has not been modified in its genome according to the invention, when analyzed under substantially identical conditions.

Inactivation of a gene of a microbial host cell may be a result of a change or modification in a polynucleotide sequence in the genome of the cell. Inactivation includes any method that prevents the functional expression of a selected protein, wherein the gene or the gene product is unable to carry out its function. Modification can be introduced by classical strain improvement, random mutagenesis followed by selection. Modification may be accomplished by the introduction (insertion), substitution or removal (deletion) of one or more nucleotides in a nucleotide sequence. This modification may for example be in a coding sequence or a regulatory element required for the transcription or translation of the polynucleotide. 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.oupjournais.Org/cai/reDrint/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 directed mutagenesis procedures are the QuickChangea site-directed mutagenesis kit (Stratagene Cloning Systems, La Jolla, CA), 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 SN, Hunt HD, Horton RM, Pullen JK, Pease LR "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 or inactivation are based on techniques of gene replacement, gene deletion, or gene disruption.

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 or has homology to the polynucleotides flanking the locus to be replaced 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. 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 or gene disruption.

For example, for gene disruption, 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) protein. 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 or inactivation, wherein said host cell produces less of or is deficient in the production of a protein such as the protein of the invention encoded by a polynucleotide 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 Feb; 66(2)775-82. (Characterization of a foldase, protein disulfide isomerase A, in the protein secretory pathway of Aspergillus niger. Ngiam C, Jeenes DJ, Punt PJ, Van Den Hondel CA, Archer DB) 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.).

Furthermore, modification, downregulation or inactivation of a polynucleotide 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 for downregulation, modification or inactivation of a polynucleotide.

The invention further provides a cell wherein the polynucleotide according to the invention is mutated or deleted from the genome to obtain lower or no expression of the polypeptide encoded by said polynucleotide compared to the cell wherein the polynucleotide of the invention is not mutated or deleted from the genome. Preferably, the downregulation, modification or inactivation in the genome of the microbial host cell used according to the invention is a modification in the genome on at least one position of at least one nucleic acid sequence encoding a protein of interest having at least 70% identity, even more preferably at least 75% identity, even more preferably at least 80% identity, even more preferably at least 85% identity, even more preferably at least 90% identity, for example at least 91 % identity, for example at least 92% identity, for example at least 93% identity, for example at least 94% identity, for example at least 95% identity, for example at least 96% identity, for example at least 97% identity, for example at least 98% identity, for example at least 99% identity, for example 100% identity with a polypeptide according to SEQ ID NO: 4, 8, 9, 14 or 18 and/or the modification in the genome of the microbial host cell in the method according to the invention is a modification resulting in the reduction of the amount of at least one mRNA having at least 60% identity, even more preferably at least 65% identity, even more preferably at least 70% identity, even more preferably at least 75% identity, even more preferably at least 80% identity, even more preferably at least 85% identity, even more preferably at least 90% identity, for example at least 91 % identity, for example at least 92% identity, for example at least 93% identity, for example at least 94% identity, for example at least 95% identity, for example at least 96% identity, for example at least 97% identity, for example at least 98% identity, for example at least 99% identity, for example 100% identity with an mRNA according to SEQ ID NO: 1 , 2, 3, 5, 6, 7, 1 1 , 12, 13, 15, 16 or 17.

Therefore inactivation of a microbial host cell may be measured by determining the amount and/or (specific) activity of the protein produced using the invention produced by the microbial host cell modified in its genome and/or it may be measured by determining the amount of mRNA transcribed from a gene encoding the protein of the invention and/or it may be measured by determining the amount of a product produced by the protein of the invention in a microbial host cell modified in its genome as defined above and/or it may be measured by gene or genome sequencing if compared to the parent host cell which has not been modified in its genome. Inactivation in the production of the protein of the invention can be measured using any assay available to the skilled person, such as transcriptional profiling, Southern blotting, Northern blotting, RT-PCR, Q-PCR, MALDI-TOF analysis, LC-MS, LC/MS-MS and Western blotting. The modification in the DNA sequence can also be determined by comparing the DNA sequence of the modified cell to the sequence of the non-modified 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 lllumina GA2, Roche 454, and the like, as reviewed in Elaine R. Mardis (2008), Next-Generation DNA Sequencing Methods, Annual Review of Genomics and Human Genetics 9: 387-402. The modification in the RNA sequence can also be determined by comparing the RNA sequence of the modified cell to the sequence of the non-modified cell. Sequencing of RNA can be done using standard methods known to the person skilled in the art, for example using next generation sequencing technologies such as lllumina GA2, Roche 454, and the like, as reviewed in Pareek et al., 201 1 Sequencing technologies and genome sequencing, J Appl Genetics 52:413-435.

Host cells

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.

Provided also are host cells comprising a polynucleotide or vector of the invention. The polynucleotide may be heterologous to the genome of the host cell.

The term "heterologous" as used herein refers to nucleic acid or amino acid sequences not naturally occurring in a host cell. In other words, the nucleic acid or amino acid sequence is not identical to that naturally found in the host cell. As used herein, the term "endogenous" or "homologous" refers to a nucleic acid or amino acid sequence naturally occurring in a host.

In another embodiment, the invention features cells, e.g., transformed host cells or recombinant host cells that contain a nucleic acid encompassed by the invention. A "transformed cell" or "recombinant cell" is a cell into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a nucleic acid according to the invention. Both prokaryotic and eukaryotic cells are included, e.g., bacteria, fungi, yeast, and the like, especially preferred are cells from filamentous fungi, such as Aspergillus niger.

As used herein, the terms "transformed" or "transgenic" with reference to a cell mean that the cell has a non-native (heterologous) nucleic acid sequence integrated into its genome or has an episomal plasmid that is maintained through multiple generations. The term is synonymous with the term "recombinant" or "genetically modified".

A host cell can be chosen that modulates the expression of the inserted sequences, or modifies and processes the gene product in a specific, desired fashion. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may facilitate optimal functioning of the protein.

Various host cells have characteristic and specific mechanisms for post- translational processing and modification of proteins and gene products. Appropriate cell lines or host systems familiar to those of skill in the art of molecular biology and/or microbiology can be chosen to ensure the desired and correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such host cells are well known in the art.

If desired, a cell as described above may be used to in the preparation of a polypeptide produced using the invention. Such a method typically comprises cultivating a host cell (e. g. transformed or transfected with an expression vector as described above) under conditions to provide for expression (by the vector) of a coding sequence encoding the polypeptide, and optionally recovering the expressed polypeptide. Polynucleotides of the invention can be incorporated into a recombinant replicable vector, e. g. an expression vector. The vector may be used to replicate the nucleic acid in a compatible host cell. Thus in a further embodiment, the invention provides a method of making a polynucleotide of the invention by introducing a polynucleotide of the invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about the replication of the vector. The vector may be recovered from the host cell.

Optionally the polypeptide is produced as a targetted protein in which case the nucleotide sequence encoding a mature form of the polypeptide in the expression construct is operably linked to a nucleotide sequence encoding a signal sequence. Preferably this signal sequence is native (homologous) to the nucleotide sequence encoding the polypeptide. Alternatively the signal sequence is foreign (heterologous) to the nucleotide sequence encoding the polypeptide, in which case the signal sequence is preferably endogenous to the host cell in which the nucleotide sequence according to the invention is expressed. The vectors may be transformed or transfected into a suitable host cell as described above to provide for expression of a polypeptide of the invention. This process may comprise culturing a host cell transformed with an expression vector as described above under conditions to provide for expression by the vector of a coding sequence encoding the polypeptide.

A host cell as defined herein is an organism suitable for genetic manipulation and one which may be cultured at cell densities useful for industrial production of a target product. A suitable organism may be a microorganism, for example one which may be maintained in a fermentation device. A host cell may be a host cell found in nature or a host cell derived from a parent host cell after genetic manipulation or classical mutagenesis.

A host cell may be a prokaryotic, archaebacterial or eukaryotic host cell.

A prokaryotic host cell may, but is not limited to, a bacterial host cell. The term "bacterial cell" includes both Gram-negative and Gram-positive microorganisms.

An eukaryotic host cell may be, but is not limited to, a yeast, a fungus, an amoeba, an algae, an animal, an insect host cell.

An eukaryotic host cell may be a fungal host cell. "Fungi" include all species of the subdivision Eumycotina (Alexopoulos, C. J., 1962, In: Introductory Mycology, John Wiley & Sons, Inc., New York). The term fungus thus includes among others filamentous fungi and yeast.

"Filamentous fungi" are herein defined as eukaryotic microorganisms that include all filamentous forms of the subdivision Eumycotina and Oomycota (as defined by Hawksworth etal., 1995, supra). The filamentous fungi are characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligatory aerobic. Filamentous fungal strains include, but are not limited to, strains of Acremonium, Aspergillus, Agaricus, Aureobasidium, Cryptococcus, Corynascus, Chrysosporium, Filibasidium, Fusarium, Humicola, Magnaporthe, Monascus, Mucor, Myceliophthora, Mortierella, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Phanerochaete Podospora, Pycnoporus, Rhizopus, Schizophyllum, Sordaria, Talaromyces, Rasamsonia, Thermoascus, Thielavia, Tolypocladium, Trametes and Trichoderma. Preferred filamentous fungal strains that may serve as host cells belong to the species Aspergillus niger, Aspergillus oryzae, Aspergillus fumigatus, Penicillium chrysogenum, Penicillium citrinum, Acremonium chrysogenum, Trichoderma reesei, Rasamsonia emersonii (formerly known as Talaromyces emersonii), Aspergillus sojae, Chrysosporium lucknowense, Myceliophtora thermophyla. Reference host cells for the comparison of fermentation characteristics of transformed and untransformed cells, include e.g. Aspergillus niger CBS120.49, CBS 513.88, Aspergillus oryzae ATCC16868, ATCC 20423, IFO 4177, ATCC 101 1 , ATCC 9576, ATCC14488-14491 , ATCC 1 1601 , ATCC12892, Aspergillus fumigatus AF293 (CBS101355), P. chrysogenum CBS 455.95, Penicillium citrinum ATCC 38065, Penicillium chrysogenum P2, Thielavia terrestris NRRL8126, Talaromyces emersonii CBS 124.902, Rasamsonia emersonii CBS393.64, Acremonium chrysogenum ATCC 36225, ATCC 48272, Trichoderma reesei ATCC 26921 , ATCC 56765, ATCC 26921 , Aspergillus sojae ATCC1 1906, Chrysosporium lucknowense ATCC44006 and derivatives of all of these strains. Particularly preferred as filamentous fungal host cell are Aspergillus niger CBS 513.88 and derivatives thereof. An eukaryotic host cell may be a yeast cell. Preferred yeast host cells may be selected from the genera: Saccharomyces (e.g., S. cerevisiae, S. bayanus, S. pastorianus, S. carlsbergensis), Kluyveromyces, Candida (e.g., C. revkaufi, C. pulcherrima, C. tropicalis, C. utilis), Pichia (e.g., P. pastoris), Schizosaccharomyces, Hansenula, Kloeckera, Schwanniomyces, and Yarrowia (e.g., Y. lipolytica (formerly classified as Candida lipolytica)).

Prokaryotic host cells may be bacterial host cells. Bacterial host cell may be Gram negative or Gram positive bacteria. Examples of bacteria include, but are not limited to, bacteria belonging to the genus Bacillus (e.g., B. subtilis, B. amyloliquefaciens, B. licheniformis, B. puntis, B. megaterium, B. halodurans,

B. pumilus,), Acinetobacter, Nocardia, Xanthobacter, Escherichia (e.g., E. coli (e.g., strains DH 1 OB, Stbl2, DH5-alpha, DB3, DB3.1 ), DB4, DB5, JDP682 and ccdA-over (e.g., U.S. Application No. 09/518,188)), Streptomyces, Erwinia, Klebsiella, Serratia (e.g., S. marcessans), Pseudomonas (e.g., P. aeruginosa), Salmonella (e.g., S. typhimurium, S. typhi). Bacteria also include, but are not limited to, photosynthetic bacteria (e.g., green non-sulfur bacteria (e.g., Choroflexus bacteria (e.g.,

C. aurantiacus), Chloronema (e.g., C. gigateum)), green sulfur bacteria (e.g., Chlorobium bacteria (e.g., C. limicola), Pelodictyon (e.g., P. luteolum), purple sulfur bacteria (e.g., Chromatium (e.g., C. okenii)), and purple non-sulfur bacteria (e.g., Rhodospirillum (e.g., R. rubrum), Rhodobacter (e.g. R. sphaeroides, R. capsulatus), and Rhodomicrobium bacteria (e.g., R. vanellii)).

Host Cells may be host cells from non-microbial organisms. Examples of such cells, include, but are not limited to, insect cells (e.g., Drosophila (e.g., D. melanogaster), Spodoptera (e.g., S. frugiperda Sf9 or Sf21 cells) and Trichoplusa (e.g., High-Five cells); nematode cells (e.g., C. elegans cells); avian cells; amphibian cells (e.g., Xenopus laevis cells); reptilian cells; and mammalian cells (e.g., NIH3T3, 293, CHO, COS, VERO, C127, BHK, Per-C6, Bowes melanoma and HeLa cells).

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 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.

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.

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 hdfA (Ku70) and/or hdfB (Ku80) gene. 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 and PCT/EP2013/055051.

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 WO2012/001 169 (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 WO2012/001 169). 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 WO201 1/009700. 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 glaA amplicons (preferably comprising 3, 4, 5, 6, 7 glaA 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 WO201 1/009700 (which is here fully incorporated by reference). An example of these host cells, described in WO201 1/009700, are host cells comprising three glaA amplicons being a BamYW truncated amplicon, a Sa/I truncated amplicon and a BglW truncated amplicon and wherein the BamYW amplicon has been adapted to have enhanced integration preference for a polynucleotide encoding a compound of interest compared to the BamYW 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. Most preferably, the SEC 61 modification is the S376W mutation in which Serine 376 is replaced by Tryptophan.

Host cells according to the invention include plant cells, and the invention therefore extends to transgenic organisms, such as plants and parts thereof, which contain one or more cells of the invention. The cells may heterologously express the polypeptide of the invention or may heterologously contain one or more of the polynucleotides of the invention. The transgenic (or genetically modified) plant may therefore have inserted (e. g. stably) into its genome a sequence encoding one or more of the polypeptides of the invention. The transformation of plant cells can be performed using known techniques, for example using a Ti or a Ri plasmid from Agrobacterium tumefaciens. The plasmid (or vector) may thus contain sequences necessary to infect a plant, and derivatives of the Ti and/or Ri plasmids may be employed.

Alternatively direct infection of a part of a plant, such as a leaf, root or stem can be effected. In this technique the plant to be infected can be wounded, for example by cutting the plant with a razor or puncturing the plant with a needle or rubbing the plant with an abrasive. The wound is then inoculated with the Agrobacterium. The plant or plant part can then be grown on a suitable culture medium and allowed to develop into a mature plant. Regeneration of transformed cells into genetically modified plants can be achieved by using known techniques, for example by selecting transformed shoots using an antibiotic and by sub-culturing the shoots on a medium containing the appropriate nutrients, plant hormones and the like.

The invention also includes cells that have been modified to express the polypeptide of the invention or a variant thereof. Such cells include transient, or preferably stable higher eukaryotic cell lines, such as mammalian cells or insect cells, lower eukaryotic cells, such as yeast and (e. g. filamentous) fungal cells or prokaryotic cells such as bacterial cells.

It is also possible for the proteins of the invention to be transiently expressed in a cell line or on a membrane, such as for example in a baculovirus expression system. Such systems, which are adapted to express the proteins according to the invention, are also included within the scope of the present invention.

According to the present invention, production of the polypeptide of the invention may be performed in an in vitro expression and translation system system. Such systems are known to the person skilled in the art (see Sambrook & Russell; Ausubel, supra), and may e.g. be a rabbit reticulo lysate based system.

According to the present invention, the production of the polypeptide produced using the invention can be effected by the culturing of microbial expression hosts, which have been transformed with one or more polynucleotides of the present invention, in a conventional nutrient fermentation medium.

Polypeptide/Enzyme production

The recombinant host cells according to the invention may be cultured using procedures known in the art. For each combination of a promoter and a host cell, culture conditions are available which are conducive to the expression the DNA sequence encoding the polypeptide. After reaching the desired cell density or titer of the polypeptide the culture is stopped and the polypeptide is recovered using known procedures.

The fermentation medium can comprise a known culture medium containing a carbon source (e. g. glucose, maltose, molasses, starch, cellulose, xylan, pectin, lignocellolytic biomass hydrolysate, etc.), a nitrogen source (e. g. ammonium sulphate, ammonium nitrate, ammonium chloride, etc.), an organic nitrogen source (e. g. yeast extract, malt extract, peptone, etc.) and inorganic nutrient sources (e. g. phosphate, magnesium, potassium, zinc, iron, etc.). Optionally, an inducer (e. g. cellulose, pectin, xylan, maltose, maltodextrin or xylogalacturonan) may be included.

The selection of the appropriate medium may be based on the choice of expression host and/or based on the regulatory requirements of the expression construct. Such media are known to those skilled in the art. The medium may, if desired, contain additional components favoring the transformed expression hosts over other potentially contaminating microorganisms.

The fermentation can be performed over a period of from about 0.5 to about 30 days. It may be a batch, continuous or fed-batch process, suitably at a temperature in the range of 0-100°C or 0-80°C, for example, from about 0 to about 50°C and/or at a pH, for example, from about 2 to about 10. Preferred fermentation conditions are a temperature in the range of from about 20 to about 45°C and/or at a pH of from about 3 to about 9. The appropriate conditions are usually selected based on the choice of the expression host and the protein to be expressed.

After fermentation, if necessary, the cells can be removed from the fermentation broth by means of centrifugation or filtration. After fermentation has stopped or after removal of the cells, the polypeptide of the invention may then be recovered and, if desired, purified and isolated by conventional means.

Polypeptide/enzyme compositions

The invention provides a composition comprising a cellulase and/or a hemicellulase and/or a pectinase and/or ligninase or a lignin-modifying enzyme.

Polypeptides of the composition can be produced by several processes and mixed into the optimal composition, or the polypetides of the composition can be made directly as mixture by one fermentation.

A composition produced using the invention may comprise one, two or three or more classes of cellulase, for example one, two or all of a GH61 , an endo-1 ,4-3- glucanase (EG), an exo-cellobiohydrolase (CBH) and a β-glucosidase (BGL).

A composition of the invention may comprise a polypeptide which has the same enzymatic activity, for example the same type of cellulase, hemicellulase and/or pectinase activity as that provided by a polypeptide of the invention.

A composition of the invention may comprise a polypeptide which has a different type of cellulase activity and/or hemicellulase activity and/or pectinase activity. For example, a composition of the invention may comprise one type of cellulase and/or hemicellulase activity and/or pectinase activity provided by a polypeptide produced using the invention and a second type of cellulase and/or hemicellulase activity and/or pectinase activity provided by an additional hemicellulase/pectinase.

Herein a carbohydrate degrading enzyme and/or carbohydrate hydrolysing enzyme is any polypeptide which is capable of degrading and/or hydrolysing of carbohydrate or enhancing the degradation and/or hydrolysis of carbohydrate. Examples of carbohydrate degrading and/or carbohydrate hydrolysing enzymes are cellulase, hemicellulase and enzymes having cellulase enhancing activity (such as GH61 ) or hemicellulase enhancing activity. The polypeptide of the invention may have a yield increasing effect on top of an enzyme composition designed for (feedstock) hydrolysis such as the compositions produced by TEC-147 or TEC-210 or 4E mix (see Examples). This yield increase is even possible in case of feedstock such as corn stover. This yield increase may be shown as an increase of the amount of glucose released during a fixed hydrolysis period of time compared to the situation without the addition of the present enzyme composition. According to another possibility this yield increase may be shown as an similar amount of glucose production with a lower dosage of the 4E or TEC-210 compared to the situation without the addition of the present enzyme composition to the regular dosage of the 4E or TEC-210.

Herein, a cellulase is any polypeptide which is capable of degrading and/or hydrolysing of cellulose or enhancing the degradation and/or hydrolysis of cellulose. A polypeptide which is capable of degrading cellulose is one which is capable of catalysing the process of breaking down cellulose into smaller units, either partially, for example into cellodextrins, or completely into glucose monomers. A cellulase produced using the invention may give rise to a mixed population of cellodextrins and glucose monomers when contacted with the cellulase. Such degradation will typically take place by way of a hydrolysis reaction.

Herein, a hemicellulase is any polypeptide which is capable of degrading and/or hydrolysing of hemicellulose or enhancing the degradation and/or hydrolysis of hemicellulose. That is to say, a hemicellulase may be capable of degrading or one or more of xylan, glucuronoxylan, arabinoxylan, glucomannan and xyloglucan. A polypeptide which is capable of degrading a hemicellulose is one which is capable of catalysing the process of breaking down the hemicellulose into smaller polysaccharides, either partially, for example into oligosaccharides, or completely into sugar monomers, for example hexose or pentose sugar monomers. A hemicellulase may give rise to a mixed population of oligosaccharides and sugar monomers when contacted with the hemicellulase. Such degradation will typically take place by way of a hydrolysis reaction.

Herein, a pectinase is any polypeptide which is capable of degrading or pectin. A polypeptide which is capable of degrading pectin is one which is capable of catalysing the process of breaking down pectin into smaller units, either partially, for example into oligosaccharides, or completely into sugar monomers. A pectinase produced using the invention may give rise to a mixed population of oligosaccharides and sugar monomers when contacted with the pectinase. Such degradation will typically take place by way of a hydrolysis reaction. Herein, a ligninase or a lignin-modifying enzyme is any polypeptide which is capable of degrading or modifying lignin or degradation components thereof. A polypeptide which is capable of degrading or modifying lignin is one which is capable of catalysing the process of breaking down lignin into smaller units, either partially, for example into monophenolic compounds. A ligninase or a lignin-modifying enzyme produced using the invention may give rise to a mixed population of phenolic compounds when contacted with the lignin. Such degradation will typically take place by way of an oxidation reaction. Herein, a ligninase or a lignin-modifying enzyme may also be any polypeptide which is capable of degrading phenolic degradation products of lignin. A polypeptide which is capable of degrading phenolic degradation products of lignin is one which is capable of catalysing the process of breaking down phenolic degradation products of lignin into even smaller units, for example by catalysing a ring opening reaction of the phenolic ring. A ligninase or a lignin-modifying enzyme produced using the invention may give rise to a mixed population of ring-opened degradation products of phenolic compounds when contacted with the phenolic degradation products of lignin. Such degradation will typically take place by way of an oxidation reaction. The a ligninase or a lignin-modifying enzyme may further be capable of breaking linkages between cellulose or hemicellulose and the lignin or degradation products thereof. Enzymes that can break down lignin include lignin peroxidases, manganese peroxidases, laccases and feruloyl esterases, and other enzymes described in the art known to depolymerize or otherwise break lignin polymers. Also included are enzymes capable of hydrolyzing bonds formed between hemicellulosic sugars (notably arabinose) and lignin. Ligninases include but are not limited to the following group of enzymes: lignin peroxidases (EC 1 .1 1 .14), manganese peroxidases (EC 1.1 1.1.13), laccases (EC 1 .10.3.2) and feruloyl esterases (EC 3.1 .1.73).

Herein, a lignin peroxidase (EC 1.1 1.1.14) catalyzes the H₂0₂ dependent oxidation of lignin-related aromatic compounds including but not limited to vanillic acid, syringic acid and guaiacol and lignin model dimers like diarylpropane and β-aryl ether dimers.

Herein, a laccase (EC 1 .10.3.2) is a multi-copper-containing enzyme that catalyzes the oxidation of phenolic compounds including but not limited to lignin model compounds as vanillyl glycol and syringaldehyde, which generally undergo polymerization via radical coupling. Furthermore laccase oxidizes non-phenolic model compounds and β-1 lignin dimers in the presence of a mediator including but not limited to 2,2'-azinobis-(- ethylbenzthiazoline-6-sulfonate) (ABTS), 1 -hydroxybenzotriazole (HBT) and 3- hyroxyanthranilic acid (HAA).

Herein, a manganese peroxidase (EC 1 .1 1 .1.13) catalyzes the oxidation of Mn(ll) to MN(III). The Mn(lll) on its turn oxidizes all kinds of monomeric phenols and lignin model compounds.

Herein, a feruloyi esterase (EC 3.1 .1 .73) is any polypeptide which is capable of catalyzing a reaction of the form: feruloyl-saccharide + H(2)0 = ferulate + saccharide. The saccharide may be, for example, an oligosaccharide or a polysaccharide. It may typically catalyze the hydrolysis of the 4-hydroxy-3-methoxycinnamoyl (feruloyi) group from an esterified sugar, which is usually arabinose in 'natural' substrates, p-nitrophenol acetate and methyl ferulate are typically poorer substrates. This enzyme may also be referred to as cinnamoyl ester hydrolase, ferulic acid esterase or hydroxycinnamoyl esterase. It may also be referred to as a hemicellulase accessory enzyme, since it may help xylanases and pectinases to break down plant cell wall hemicellulose and pectin.

Accordingly, a composition produced using the invention may comprise any cellulase, for example, a GH61 , a cellobiohydrolase, an endo-3-1 ,4-glucanase, a β- glucosidase or a β-(1 ,3)(1 ,4)-glucanase.

GH61 (glycoside hydrolase family 6 sometimes referred to EGIV) proteins are oxygen-dependent polysaccharide monooxygenases (PMO's) according to the latest literature. Often in literature these proteins are mentioned to enhance the action of cellulases on lignocellulose substrates. GH61 was originally classified as endoglucanase based on measurement of very weak endo-1 ,4-3-d-glucanase activity in one family member. The term "GH61 " as used herein, is to be understood as a family of enzymes, which share common conserved sequence portions and foldings to be classified in family 61 of the well-established CAZY GH classification system (http://www.cazy.org/GH61.html). The glycoside hydrolase family 61 is a member of the family of glycoside hydrolases EC 3.2.1. GH61 is used herein as being part of the cellulases.

Herein, a cellobiohydrolase (EC 3.2.1 .91 ) is any polypeptide which is capable of catalysing the hydrolysis of 1 ,4-3-D-glucosidic linkages in cellulose or cellotetraose, releasing cellobiose from the non-reducing ends of the chains. This enzyme may also be referred to as cellulase 1 ,4-3-cellobiosidase, 1 ,4-3-cellobiohydrolase, 1 ,4-3-D-glucan cellobiohydrolase, avicelase, exo-1 ,4-3-D-glucanase, exocellobiohydrolase or exoglucanase. It may be a have the EC code EC 3.2.1.91.

Herein, an endo-3-1 ,4-glucanase (EC 3.2.1.4) is any polypeptide which is capable of catalysing the endohydrolysis of 1 ,4-3-D-glucosidic linkages in cellulose, lichenin or cereal β-D-glucans. Such a polypeptide may also be capable of hydrolyzing 1 ,4-linkages in β-D-glucans also containing 1 ,3-linkages. This enzyme may also be referred to as cellulase, avicelase, 3-1 ,4-endoglucan hydrolase, 3-1 ,4-glucanase, carboxymethyl cellulase, celludextrinase, endo-1 ,4-3-D-glucanase, endo-1 ,4-β-ϋ- glucanohydrolase, endo-1 ,4^-glucanase or endoglucanase. The endo-glucanase may also catalyze the cleavage of xyloglucan, a backbone of 31→4-linked glucose residues, most of which substituted with 1 -6 linked xylose side chains, and the enzyme is then referred to as a xyloglucan-specific endo-3-1 ,4-glucanase or a xyloglucanase.

Herein, a β-glucosidase (EC 3.2.1 .21 ) is any polypeptide which is capable of catalysing the hydrolysis of terminal, non-reducing β-D-glucose residues with release of β-D-glucose. Such a polypeptide may have a wide specificity for β-D-glucosides and may also hydrolyze one or more of the following: a β-D-galactoside, an oL-arabinoside, a β-D-xyloside or a β-D-fucoside. This enzyme may also be referred to as amygdalase, β-D-glucoside glucohydrolase, cellobiase or gentobiase.

Herein a β-(1 ,3)(1 ,4)-glucanase (EC 3.2.1.73) is any polypeptide which is capable of catalyzing the hydrolysis of 1 ,4^-D-glucosidic linkages in β-D-glucans containing 1 ,3- and 1 ,4-bonds. Such a polypeptide may act on lichenin and cereal β-D- glucans, but not on β-D-glucans containing only 1 ,3- or 1 ,4-bonds. This enzyme may also be referred to as licheninase, 1 ,3-1 ,4^-D-glucan 4-glucanohydrolase, β-glucanase, endo-β-1 ,3-1 ,4 glucanase, lichenase or mixed linkage β-glucanase. An alternative for this type of enzyme is EC 3.2.1.6, which is described as endo-1 ,3(4)-beta-glucanase. This type of enzyme hydrolyses 1 ,3- or 1 ,4-linkages in beta-D-glucans when the glucose residue whose reducing group is involved in the linkage to be hydrolyzed is itself substituted at C-3. Alternative names include endo-1 ,3-beta-glucanase, laminarinase, 1 ,3-(1 ,3;1 ,4)-beta-D-glucan 3 (4) glucanohydrolase; substrates include laminarin, lichenin and cereal beta-D-glucans.

A composition of the invention may comprise any hemicellulase, for example, an endo-xylanase, a β-xylosidase, a a-L-arabionofuranosidase, an a-D-glucuronidase, an cellobiohydrolase, a feruloyl esterase, a coumaroyl esterase, an a-galactosidase, a β- galactosidase, a β-mannanase or a β-mannosidase. Herein, an endoxylanase (EC 3.2.1.8) is any polypeptide which is capable of catalyzing the endo-hydrolysis of 1 ,4-3-D-xylosidic linkages in xylans. This enzyme may also be referred to as endo-1 ,4-3-xylanase or 1 ,4-3-D-xylan xylanohydrolase. An alternative is EC 3.2.1.136, a glucuronoarabinoxylan endoxylanase, an enzyme that is able to hydrolyze 1 ,4 xylosidic linkages in glucuronoarabinoxylans.

Herein, a β-xylosidase (EC 3.2.1 .37) is any polypeptide which is capable of catalyzing the hydrolysis of 1 ,4-3-D-xylans, to remove successive D-xylose residues from the non-reducing termini. Such enzymes may also hydrolyze xylobiose. This enzyme may also be referred to as xylan 1 ,4-3-xylosidase, 1 ,4-3-D-xylan xylohydrolase, exo-1 ,4-3-xylosidase or xylobiase.

Herein, an oL-arabinofuranosidase (EC 3.2.1 .55) is any polypeptide which is capable of acting on oL-arabinofuranosides, oL-arabinans containing (1 ,2) and/or (1 ,3)- and/or (1 ,5)-linkages, arabinoxylans and arabinogalactans. This enzyme may also be referred to as oN-arabinofuranosidase, arabinofuranosidase or arabinosidase.

Herein, an oD-glucuronidase (EC 3.2.1.139) is any polypeptide which is capable of catalyzing a reaction of the following form: alpha-D-glucuronoside + H(2)0 = an alcohol + D-glucuronate. This enzyme may also be referred to as alpha- glucuronidase or alpha-glucosiduronase. These enzymes may also hydrolyze 4-0- methylated glucoronic acid, which can also be present as a substituent in xylans. Alternative is EC 3.2.1.131 : xylan alpha-1 ,2-glucuronosidase, which catalyses the hydrolysis of alpha-1 ,2-(4-0-methyl)glucuronosyl links.

Herein, an acetyl xylan esterase (EC 3.1 .1 .72) is any polypeptide which is capable of catalyzing the deacetylation of xylans and xylo-oligosaccharides. Such a polypeptide may catalyze the hydrolysis of acetyl groups from polymeric xylan, acetylated xylose, acetylated glucose, alpha-napthyl acetate or p-nitrophenyl acetate but, typically, not from triacetylglycerol. Such a polypeptide typically does not act on acetylated mannan or pectin.

Herein, a coumaroyl esterase (EC 3.1 .1 .73) is any polypeptide which is capable of catalyzing a reaction of the form: coumaroyl-saccharide + H(2)0 = coumarate + saccharide. The saccharide may be, for example, an oligosaccharide or a polysaccharide. This enzyme may also be referred to as trans-4-coumaroyl esterase, trans-p-coumaroyl esterase, p-coumaroyl esterase or p-coumaric acid esterase. This enzyme also falls within EC 3.1.1 .73 so may also be referred to as a feruloyl esterase.

Herein, an a-galactosidase (EC 3.2.1 .22) is any polypeptide which is capable of catalyzing the hydrolysis of terminal, non-reducing oD-galactose residues in a-D- galactosides, including galactose oligosaccharides, galactomannans, galactans and arabinogalactans. Such a polypeptide may also be capable of hydrolyzing a-D- fucosides. This enzyme may also be referred to as melibiase.

Herein, a β-galactosidase (EC 3.2.1 .23) is any polypeptide which is capable of catalyzing the hydrolysis of terminal non-reducing β-D-galactose residues in β-D- galactosides. Such a polypeptide may also be capable of hydrolyzing a-L-arabinosides. This enzyme may also be referred to as exo-(1 ->4)-3-D-galactanase or lactase.

Herein, a β-mannanase (EC 3.2.1 .78) is any polypeptide which is capable of catalyzing the random hydrolysis of 1 ,4-3-D-mannosidic linkages in mannans, galactomannans and glucomannans. This enzyme may also be referred to as mannan endo-1 ,4-3-mannosidase or endo-1 ,4-mannanase.

Herein, a β-mannosidase (EC 3.2.1 .25) is any polypeptide which is capable of catalyzing the hydrolysis of terminal, non-reducing β-D-mannose residues in β-D- mannosides. This enzyme may also be referred to as mannanase or mannase.

A composition of the invention may comprise any pectinase, for example an endo polygalacturonase, a pectin methyl esterase, an endo-galactanase, a beta galactosidase, a pectin acetyl esterase, an endo-pectin lyase, pectate lyase, alpha rhamnosidase, an exo-galacturonase, an exo-polygalacturonate lyase, a rhamnogalacturonan hydrolase, a rhamnogalacturonan lyase, a rhamnogalacturonan acetyl esterase, a rhamnogalacturonan galacturonohydrolase or a xylogalacturonase.

Herein, an endo-polygalacturonase (EC 3.2.1.15) is any polypeptide which is capable of catalyzing the random hydrolysis of 1 ,4-oD-galactosiduronic linkages in pectate and other galacturonans. This enzyme may also be referred to as polygalacturonase pectin depolymerase, pectinase, endopolygalacturonase, pectolase, pectin hydrolase, pectin polygalacturonase, poly-a-1 ,4-galacturonide glycanohydrolase, endogalacturonase; endo-D-galacturonase or poly(1 ,4-a-D-galacturonide) glycanohydrolase.

Herein, a pectin methyl esterase (EC 3.1.1 .1 1 ) is any enzyme which is capable of catalyzing the reaction: pectin + n H₂0 = n methanol + pectate. The enzyme may also been known as pectinesterase, pectin demethoxylase, pectin methoxylase, pectin methylesterase, pectase, pectinoesterase or pectin pectylhydrolase.

Herein, an endo-galactanase (EC 3.2.1 .89) is any enzyme capable of catalyzing the endohydrolysis of 1 ,4-3-D-galactosidic linkages in arabinogalactans. The enzyme may also be known as arabinogalactan endo-1 ,4-3-galactosidase, endo-1 ,4-3- galactanase, galactanase, arabinogalactanase or arabinogalactan 4-β-ϋ- galactanohydrolase.

Herein, a pectin acetyl esterase is defined herein as any enzyme which has an acetyl esterase activity which catalyzes the deacetylation of the acetyl groups at the hydroxyl groups of GalUA residues of pectin

Herein, an endo-pectin lyase (EC 4.2.2.10) is any enzyme capable of catalyzing the eliminative cleavage of (1→4)-a-D-galacturonan methyl ester to give oligosaccharides with 4-deoxy-6-0-methyl-a-D-galact-4-enuronosyl groups at their non- reducing ends. The enzyme may also be known as pectin lyase, pectin trans-eliminase; endo-pectin lyase, polymethylgalacturonic transeliminase, pectin methyltranseliminase, pectolyase, PL, PNL or PMGL or (1→4)-6-0-methyl-a-D-galacturonan lyase.

Herein, a pectate lyase (EC 4.2.2.2) is any enzyme capable of catalyzing the eliminative cleavage of (1→4)-a-D-galacturonan to give oligosaccharides with 4-deoxy-o D-galact-4-enuronosyl groups at their non-reducing ends. The enzyme may also be known polygalacturonic transeliminase, pectic acid transeliminase, poygalacturonate lyase, endopectin methyltranseliminase, pectate transeliminase, endogalacturonate transeliminase, pectic acid lyase, pectic lyase, a-1 ,4-D-endopolygalacturonic acid lyase, PGA lyase, PPase-N, endo-a-1 ,4-polygalacturonic acid lyase, polygalacturonic acid lyase, pectin trans-eliminase, polygalacturonic acid trans-eliminase or (1→4)-a-D- galacturonan lyase.

Herein, an alpha rhamnosidase (EC 3.2.1.40) is any polypeptide which is capable of catalyzing the hydrolysis of terminal non-reducing a-L-rhamnose residues in a-L- rhamnosides or alternatively in rhamnogalacturonan. This enzyme may also be known as a-L-rhamnosidase T, a-L-rhamnosidase N or a-L-rhamnoside rhamnohydrolase. Herein, exo-galacturonase (EC 3.2.1 .82) is any polypeptide capable of hydrolysis of pectic acid from the non-reducing end, releasing digalacturonate. The enzyme may also be known as exo-poly-a-galacturonosidase, exopolygalacturonosidase or exopolygalacturanosidase.

Herein, exo-galacturonase (EC 3.2.1.67) is any polypeptide capable of catalyzing: (1 ,4-a-D-galacturonide)_n + H₂0 = (1 ,4-a-D-galacturonide)_n-i + D- galacturonate. The enzyme may also be known as galacturan 1 ,4-ogalacturonidase, exopolygalacturonase, poly(galacturonate) hydrolase, exo-D-galacturonase, exo-D- galacturonanase, exo-poly-D-galacturonase or poly(1 ,4-a-D-galacturonide) galacturonohydrolase.

Herein, exo-polygalacturonate lyase (EC 4.2.2.9) is any polypeptide capable of catalyzing eliminative cleavage of 4-(4-deoxy-a-D-galact-4-enuronosyl)-D-galacturonate from the reducing end of pectate, i.e. de-esterified pectin. This enzyme may be known as pectate disaccharide-lyase, pectate exo-lyase, exopectic acid transeliminase, exo- pectate lyase, exopolygalacturonic acid-trans-eliminase, PATE, exo-PATE, exo-PGL or (1→4)-a-D-galacturonan reducing-end-disaccharide-lyase.

Herein, rhamnogalacturonan hydrolase is any polypeptide which is capable of hydrolyzing the linkage between galactosyluronic acid and rhamnopyranosyl in an endo- fashion in strictly alternating rhamnogalacturonan structures, consisting of the disaccharide [(1 ,2-alpha-L-rhamnoyl-(1 ,4)-alpha-galactosyluronic acid].

Herein, rhamnogalacturonan lyase is any polypeptide which is any polypeptide which is capable of cleaving ol_- hap-(1→4)-a-D-GalpA linkages in an endo-fashion in rhamnogalacturonan by beta-elimination.

Herein, rhamnogalacturonan acetyl esterase is any polypeptide which catalyzes the deacetylation of the backbone of alternating rhamnose and galacturonic acid residues in rhamnogalacturonan.

Herein, rhamnogalacturonan galacturonohydrolase is any polypeptide which is capable of hydrolyzing galacturonic acid from the non-reducing end of strictly alternating rhamnogalacturonan structures in an exo-fashion.

Herein, xylogalacturonase is any polypeptide which acts on xylogalacturonan by cleaving the β-xylose substituted galacturonic acid backbone in an endo-manner. This enzyme may also be known as xylogalacturonan hydrolase.

Herein, an oL-arabinofuranosidase (EC 3.2.1 .55) is any polypeptide which is capable of acting on a-L-arabinofuranosides, oL-arabinans containing (1 ,2) and/or (1 ,3)- and/or (1 ,5)-linkages, arabinoxylans and arabinogalactans. This enzyme may also be referred to as a-N-arabinofuranosidase, arabinofuranosidase or arabinosidase.

Herein, endo-arabinanase (EC 3.2.1 .99) is any polypeptide which is capable of catalyzing endohydrolysis of 1 ,5-a-arabinofuranosidic linkages in 1 ,5-arabinans. The enzyme may also be know as endo-arabinase, arabinan endo-1 ,5-a-L-arabinosidase, endo-1 ,5-a-L-arabinanase, endo-a-1 ,5-arabanase; endo-arabanase or 1 ,5-oL-arabinan 1 ,5-oL-arabinanohydrolase.

A composition of the invention will typically comprise at least one cellulase and/or at least one hemicellulase and/or at least one pectinase. A composition of the invention may comprise a cellobiohydrolase, an endoglucanase and/or a β-glucosidase. Such a composition may also comprise one or more hemicellulases and/or one or more pectinases.

One or more (for example two, three, four or all) of an amylase, a protease, a lipase, a ligninase, a hexosyltransferase or a glucuronidase may be present in a composition of the invention.

"Protease" includes enzymes that hydrolyze peptide bonds (peptidases), as well as enzymes that hydrolyze bonds between peptides and other moieties, such as sugars (glycopeptidases). Many proteases are characterized under EC 3.4, and are suitable for use in the invention incorporated herein by reference. Some specific types of proteases include, cysteine proteases including pepsin, papain and serine proteases including chymotrypsins, carboxypeptidases and metalloendopeptidases.

"Lipase" includes enzymes that hydrolyze lipids, fatty acids, and acylglycerides, including phospoglycerides, lipoproteins, diacylglycerols, and the like. In plants, lipids are used as structural components to limit water loss and pathogen infection. These lipids include waxes derived from fatty acids, as well as cutin and suberin.

"Hexosyltransferase" (2.4.1 -) includes enzymes which are capable of transferring glycosyl groups, more specifically hexosyl groups. In addition to transfer of a glycosyl-group from a glycosyl-containing donor to another glycosyl-containing compound, the acceptor, the enzymes can also transfer the glycosyl-group to water as an acceptor. This reaction is also known as a hydrolysis reaction, instead of a transfer reaction. An example of a hexosyltransferase which may be used in the invention is a β- glucanosyltransferase. Such an enzyme may be able to catalyze degradation of (1 ,3)(1 ,4)glucan and/or cellulose and/or a cellulose degradation product. "Glucuronidase" includes enzymes that catalyze the hydrolysis of a glucoronoside, for example β-glucuronoside to yield an alcohol. Many glucuronidases have been characterized and may be suitable for use in the invention, for example β- glucuronidase (EC 3.2.1.31 ), hyalurono-glucuronidase (EC 3.2.1.36), glucuronosyl- disulfoglucosamine glucuronidase (3.2.1 .56), glycyrrhizinate β-glucuronidase (3.2.1 .128) or a-D-glucuronidase (EC 3.2.1 .139).

A composition of the invention may comprise an expansin or expansin-like protein, such as a swollenin (see Salheimo et al., Eur. J. Biochem. 269, 4202-421 1 , 2002) or a swollenin-like protein.

Expansins are implicated in loosening of the cell wall structure during plant cell growth. Expansins have been proposed to disrupt hydrogen bonding between cellulose and other cell wall polysaccharides without having hydrolytic activity. In this way, they are thought to allow the sliding of cellulose fibers and enlargement of the cell wall. Swollenin, an expansin-like protein contains an N-terminal Carbohydrate Binding Module Family 1 domain (CBD) and a C-terminal expansin-like domain. For the purposes of this invention, an expansin-like protein or swollenin-like protein may comprise one or both of such domains and/or may disrupt the structure of cell walls (such as disrupting cellulose structure), optionally without producing detectable amounts of reducing sugars.

A composition of the invention may comprise the polypeptide product of a cellulose integrating protein, scaffoldin or a scaffoldin-like protein, for example CipA or CipC from Clostridium thermocellum or Clostridium cellulolyticum respectively.

Scaffoldins and cellulose integrating proteins are multi-functional integrating subunits which may organize cellulolytic subunits into a multi-enzyme complex. This is accomplished by the interaction of two complementary classes of domain, i.e. a cohesion domain on scaffoldin and a dockerin domain on each enzymatic unit. The scaffoldin subunit also bears a cellulose-binding module (CBM) that mediates attachment of the cellulosome to its substrate. A scaffoldin or cellulose integrating protein for the purposes of this invention may comprise one or both of such domains.

A composition of the invention may comprise a cellulose induced protein or modulating protein, for example as encoded by cipl or cip2 gene or similar genes from Trichoderma reesei I Hypocrea jacorina (see Foreman et al., J. Biol. Chem. 278(34), 31988-31997, 2003). The polypeptide product of these genes are bimodular proteins, which contain a cellulose binding module and a domain which function or activity can not be related to known glycosyl hydrolase families. Yet, the presence of a cellulose binding module and the co-regulation of the expression of these genes with cellulases components indicates previously unrecognized activities with potential role in biomass degradation.

A composition of the invention may be composed of a member of each of the classes of the polypeptides mentioned above, several members of one polypeptide class, or any combination of these polypeptide classes.

A composition of the invention may be composed of polypeptides, for example enzymes, from (1 ) commercial suppliers; (2) cloned genes expressing polypeptides, for example enzymes; (3) complex broth (such as that resulting from growth of a microbial strain in media, wherein the strains secrete proteins and enzymes into the media; (4) cell lysates of strains grown as in (3); and/or (5) plant material expressing polypeptides, for example enzymes. Different polypeptides, for example enzymes in a composition of the invention may be obtained from different sources.

Use of the polypeptides

The polypeptides and polypeptide compositions according to the invention may be used in many different applications. For instance they may be used to produce fermentable sugars. The fermentable sugars can then, as part of a biofuel process, be converted into biogas or ethanol, butanol, isobutanol, 2 butanol or other suitable substances. So by fermentable sugars is meant sugars which can be consumed by a microorganism or converted by a microorganism in another product. Alternatively the polypeptide produced using the invention may be used as enzyme, for instance in production of food products, in detergent compositions, in the paper and pulp industry and in antibacterial formulations, in pharmaceutical products such as throat lozenges, toothpastes, and mouthwash. Some of the uses will be illustrated in more detail below.

In the uses and methods described below, the components of the compositions described above may be provided concomitantly (i.e. as a single composition per se) or separately or sequentially.

The invention also relates to the use of the polypeptide or composition produced using the invention and compositions in industrial processes.

In principle, a polypeptide or composition produced using the invention may be used in any process which requires the treatment of a material which comprises polysaccharide. Thus, a polypeptide or composition of the invention may be used in the treatment of polysaccharide material. Herein, polysaccharide material is a material which comprises or consists essential of one or, more typically, more than one polysaccharide.

Typically, plants and material derived therefrom comprise significant quantities of non-starch polysaccharide material. Accordingly, a polypeptide produced using the invention may be used in the treatment of a plant or fungal material or a material derived therefrom.

Lignocellulose

An important component of plant non-starch polysaccharide material is lignocellulose (also referred to herein as lignocellulolytic biomass). Lignocellulose is plant material that comprises cellulose and hemicellulose and lignin. The carbohydrate polymers (cellulose and hemicelluloses) are tightly bound to the lignin by hydrogen and covalent bonds. Accordingly, a polypeptide produced using the invention may be used in the treatment of lignocellulolytic material. Herein, lignocellulolytic material is a material which comprises or consists essential of lignocellulose. Thus, in a method of the invention for the treatment of a non-starch polysaccharide, the non-starch polysaccharide may be a lignocellulosic material/biomass.

Accordingly, the invention provides a method of treating a substrate comprising non-starch polysaccharide in which the treatment comprises the degradation and/or hydrolysis and/or modification of cellulose and/or hemicellulose and/or a pectic substance.

Degradation in this context indicates that the treatment results in the generation of hydrolysis products of cellulose and/or hemicellulose and/or a pectic substance, i.e. saccharides of shorter length are present as result of the treatment than are present in a similar untreated non-starch polysaccharide. Thus, degradation in this context may result in the liberation of oligosaccharides and/or sugar monomers.

All plants contain non-starch polysaccharide as do virtually all plant-derived polysaccharide materials. Accordingly, in a method of the invention for the treatment of substrate comprising a non-starch polysaccharide, said substrate may be provided in the form of a plant or a plant derived material or a material comprising a plant or plant derived material, for example a plant pulp, a plant extract, a foodstuff or ingredient therefore, a fabric, a textile or an item of clothing.

Lignocellulolytic biomass suitable for use in the invention includes biomass and can include virgin biomass and/or non-virgin biomass such as agricultural biomass, commercial organics, construction and demolition debris, municipal solid waste, waste paper and yard waste. Common forms of biomass include trees, shrubs and grasses, wheat, wheat straw, sugar cane bagasse, corn, corn husks, corn cobs, corn kernel including fiber from kernels, products and by-products from milling of grains such as corn, wheat and barley (including wet milling and dry milling) often called "bran or fiber" as well as municipal solid waste, waste paper and yard waste. The biomass can also be, but is not limited to, herbaceous material, agricultural residues, forestry residues, municipal solid wastes, waste paper, and pulp and paper mill residues. "Agricultural biomass" includes branches, bushes, canes, corn and corn husks, energy crops, forests, fruits, flowers, grains, grasses, herbaceous crops, leaves, bark, needles, logs, roots, saplings, short rotation woody crops, shrubs, switch grasses, trees, vegetables, fruit peels, vines, sugar beet pulp, wheat middlings, oat hulls, and hard and soft woods (not including woods with deleterious materials). In addition, agricultural biomass includes organic waste materials generated from agricultural processes including farming and forestry activities, specifically including forestry wood waste. Agricultural biomass may be any of the aforestated singularly or in any combination or mixture thereof. Further examples of suitable biomass are orchard primings, chaparral, mill waste, urban wood waste, municipal waste, logging waste, forest thinnings, short- rotation woody crops, industrial waste, wheat straw, oat straw, rice straw, barley straw, rye straw, flax straw, soy hulls, rice hulls, rice straw, corn gluten feed, oat hulls, sugar cane, corn stover, corn stalks, corn cobs, corn husks, prairie grass, gamagrass, foxtail; sugar beet pulp, citrus fruit pulp, seed hulls, cellulosic animal wastes, lawn clippings, cotton, seaweed, trees, shrubs, grasses, wheat, wheat straw, sugar cane bagasse, corn, corn husks, corn hobs, corn kernel, fiber from kernels, products and by-products from wet or dry milling of grains, municipal solid waste, waste paper, yard waste, herbaceous material, agricultural residues, forestry residues, municipal solid waste, waste paper, pulp, paper mill residues, branches, bushes, canes, corn, corn husks, an energy crop, forest, a fruit, a flower, a grain, a grass, a herbaceous crop, a leaf, bark, a needle, a log, a root, a sapling, a shrub, switch grass, a tree, a vegetable, fruit peel, a vine, sugar beet pulp, wheat middlings, oat hulls, hard or soft wood, organic waste material generated from an agricultural process, forestry wood waste, or a combination of any two or more thereof.

Apart from virgin biomass or feedstocks already processed in food and feed or paper and pulping industries, the biomass/feedstock may additionally be pretreated with heat, mechanical and/or chemical modification or any combination of such methods in order to enhance enzymatic degradation.

Pretreatment

Before enzymatic treatment, the feedstock may optionally be pre-treated with heat, mechanical and/or chemical modification or any combination of such methods in order to to enhance the accessibility of the substrate to enzymatic hydrolysis and/or hydrolyse the hemicellulose and/or solubilize the hemicellulose and/or cellulose and/or lignin, in any way known in the art. The pretreatment may comprise exposing the lignocellulosic material to (hot) water, steam (steam explosion), an acid, a base, a solvent, heat, a peroxide, ozone, mechanical shredding, grinding, milling or rapid depressurization, or a combination of any two or more thereof. This chemical pretreatment is often combined with heat-pretreatment, e.g. between 150 and 220 °C for 1 to 30 minutes.

Presaccharifation

After the pretreatment step, a liquefaction/hydrolysis or presaccharification step involving incubation with an enzyme or enzyme mixture can be utilized. The presaccharification step can be performed at many different temperatures but it is preferred that the presaccharification step occur at the temperature best suited to the enzyme mix being tested, or the predicted enzyme optimum of the enzymes to be tested. The temperature of the presaccharification step may range from about 10 °C to about 95 °C, about 20 °C to about 85 °C, about 30 °C to about 70 °C, about 40 °C to about 60 °C, about 37 °C to about 50 °C, preferably about 37 °C to about 80 °C, more preferably about 60-70 °C even more preferably around 65 °C. The pH of the presaccharification mixture may range from about 2.0 to about 10.0, but is preferably about 3.0 to about 7.0, more preferably about 4.0 to about 6.0, even more preferably about 4.0 to about 5.0. Again, the pH may be adjusted to maximize enzyme activity and may be adjusted with the addition of the enzyme. Comparison of the results of the assay results from this test will allow one to modify the method to best suit the enzymes being tested.

The liquefaction/hydrolysis or presaccharification step reaction may occur from several minutes to several hours, such as from about 1 hour to about 120 hours, preferably from about 2 hours to about 48 hours, more preferably from about 2 to about 24 hours, most preferably for from about 2 to about 6 hours. The cellulase treatment may occur from several minutes to several hours, such as from about 6 hours to about 120 hours, preferably about 12 hours to about 72 hours, more preferably about 24 to 48 hours.

Saccharification

The invention provides a method for producing a sugar from a lignocellulosic material which method comprises contacting a polypeptide produced using the invention to a composition of the invention with the lignocellulosic material.

Such a method allows free sugars (monomers) and/or oligosaccharides to be generated from lignocellulosic biomass. These methods involve converting lignocellulosic biomass to free sugars and small oligosaccharides with a polypeptide or composition produced using the invention.

The process of converting a complex carbohydrate such as cellulose or lignocellulose into sugars preferably allows conversion into fermentable sugars. Such a process may be referred to as "saccharification" or "hydrolysis". Accordingly, a method of the invention may result in the liberation of one or more hexose and/or pentose sugars, such as one or more of glucose, xylose, arabinose, galactose, galacturonic acid, glucuronic acid, mannose, rhamnose, ribose and fructose.

Accordingly, another aspect of the invention includes methods that utilize the polypeptide or composition of the invention described above together with further enzymes or physical treatments such as temperature and pH to convert the lignocellulosic plant biomass to sugars and oligosaccharides.

While the composition has been discussed as a single mixture it is recognized that the enzymes may be added sequentially where the temperature, pH, and other conditions may be altered to increase the activity of each individual enzyme. Alternatively, an optimum pH and temperature can be determined for the enzyme mixture.

The enzymes are reacted with substrate under any appropriate conditions. For example, enzymes can be incubated at about 25 °C, about 30 °C, about 35 °C, about 37 °C, about 40 °C, about 45 °C, about 50 °C, about 55 °C, about 60 °C, about 65 °C, about 70 °C, about 75 °C, about 80 °C, about 85 °C, about 90 °C or higher. That is, they can be incubated at a temperature of from about 20 °C to about 95 °C, for example in buffers of low to medium ionic strength and/or from low to neutral pH. By "medium ionic strength" is intended that the buffer has an ion concentration of about 200 millimolar (mM) or less for any single ion component. The pH may range from about pH 2.5, about pH 3.0, about pH 3.5, about pH 4.0, about pH 4.5, about pH 5, about pH 5.5, about pH 6, about pH 6.5, about pH 7, about pH 7.5, about pH 8.0, to about pH 8.5. Generally, the pH range will be from about pH 3.0 to about pH 7. For the production of ethanol an acidic medium is preferred, e.g. pH=4, whereas for the production of biogas neutral pH, e.g. pH=7 is desirable. Incubation of enzyme combinations under these conditions results in release or liberation of substantial amounts of the sugar from the lignocellulose. By substantial amount is intended at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of available sugar.

The polypeptides, such as enzymes, can be produced either exogenously in microorganisms, yeasts, fungi, bacteria or plants, then isolated and added, for example, to lignocellulosic feedstock. Alternatively, the enzymes are produced, but not isolated, and crude cell mass fermentation broth, or plant material (such as corn stover), and the like may be added to, for example, the feedstock. Alternatively, the crude cell mass or enzyme production medium or plant material may be treated to prevent further microbial growth (for example, by heating or addition of antimicrobial agents), then added to, for example, a feedstock. These crude enzyme mixtures may include the organism producing the enzyme. Alternatively, the enzyme may be produced in a fermentation that uses feedstock (such as corn stover) to provide nutrition to an organism that produces an enzyme(s). In this manner, plants that produce the enzymes may themselves serve as a lignocellulosic feedstock and be added into lignocellulosic feedstock.

Fermentation of sugars

The fermentable sugars can be converted to useful value-added fermentation products, non-limiting examples of which include amino acids, vitamins, pharmaceuticals, animal feed supplements, specialty chemicals, chemical feedstocks, plastics, solvents, fuels, or other organic polymers, lactic acid, and ethanol, including fuel ethanol. In particular the sugars may be used as feedstocks for fermentation into chemicals, plastics, such as for instance succinic acid and (bio) fuels, including ethanol, methanol, butanol synthetic liquid fuels and biogas.

For instance, in the method of the invention, an enzyme or combination of enzymes acts on a lignocellulosic substrate or plant biomass, serving as the feedstock, so as to convert this complex substrate to simple sugars and oligosaccharides for the production of ethanol or other useful fermentation products. Sugars released from biomass can be converted to useful fermentation products such a one of those including, but not limited to, amino acids, vitamins, pharmaceuticals, animal feed supplements, specialty chemicals, chemical feedstocks, plastics, and ethanol, including fuel ethanol.

Accordingly, the invention provides a method for the preparation of a fermentation product, which method comprises:

a. degrading lignocellulose using a method as described herein; and

b. fermentation of the resulting material,

thereby to prepare a fermentation product.

The fermentation may be carried out under aerobic or anaerobic conditions. Preferably, the process is carried out under micro-aerophilic or oxygen limited conditions.

An anaerobic fermentation process is herein defined as a fermentation process run in the absence of oxygen or in which substantially no oxygen is consumed, preferably about 5 or less, about 2.5 or less or about 1 mmol/L/h or less, and wherein organic molecules serve as both electron donor and electron acceptors.

An oxygen-limited fermentation process is a process in which the oxygen consumption is limited by the oxygen transfer from the gas to the liquid. The degree of oxygen limitation is determined by the amount and composition of the ingoing gas flow as well as the actual mixing/mass transfer properties of the fermentation equipment used. Preferably, in a process under oxygen-limited conditions, the rate of oxygen consumption is at least about 5.5, more preferably at least about 6 and even more preferably at least about 7 mmol/L/h.

A method for the preparation of a fermentation product may optionally comprise recovery of the fermentation product.

SSF

Fermentation and Saccharification may also be executed in Simultaneous Saccharification and Fermentation (SSF) mode. One of the advantages of this mode is reduction of the sugar inhibition on enzymatic hydrolysis (Sugar inhibition on cellulases is described by Caminal B&B Vol XXVII Pp 1282-1290).

Fermentation products Fermentation products which may be produced according to the invention include amino acids, vitamins, pharmaceuticals, animal feed supplements, specialty chemicals, chemical feedstocks, plastics, solvents, fuels, or other organic polymers, lactic acid, and ethanol, including fuel ethanol (the term "ethanol" being understood to include ethyl alcohol or mixtures of ethyl alcohol and water).

Specific value-added products that may be produced by the methods of the invention include, but not limited to, biofuels (including ethanol and butanol and a biogas); lactic acid; a plastic; a specialty chemical; an organic acid, including citric acid, succinic acid, fumaric acid, itaconic acid and maleic acid; 3-hydoxy-propionic acid, acrylic acid; acetic acid; 1 ,3-propane-diol; ethylene, glycerol; a solvent; an animal feed supplement; a pharmaceutical, such as a β-lactam antibiotic or a cephalosporin; vitamins; an amino acid, such as lysine, methionine, tryptophan, threonine, and aspartic acid; an industrial enzyme, such as a protease, a cellulase, an amylase, a glucanase, a lactase, a lipase, a lyase, an oxidoreductases, a transferase or a xylanase; and a chemical feedstock.

Biogas

The invention also provides use of a polypeptide or composition a described herein in a method for the preparation of biogas. Biogas typically refers to a gas produced by the biological breakdown of organic matter, for example non-starch carbohydrate containing material, in the absence of oxygen. Biogas originates from biogenic material and is a type of biofuel. One type of biogas is produced by anaerobic digestion or fermentation of biodegradable materials such as biomass, manure or sewage, municipal waste, and energy crops. This type of biogas is comprised primarily of methane and carbon dioxide. The gas methane can be combusted or oxidized with oxygen. Air contains 21 % oxygen. This energy release allows biogas to be used as a fuel. Biogas can be used as a low-cost fuel in any country for any heating purpose, such as cooking. It can also be utilized in modern waste management facilities where it can be used to run any type of heat engine, to generate either mechanical or electrical power.

The first step in microbial biogas production consists in the enzymatic degradation of polymers and complex substrates (for example non-starch carbohydrate). Accordingly, the invention provides a method for preparation of a biogas in which a substrate comprising non-starch carbohydrate is contacted with a polypeptide or composition of the invention, thereby to yield fermentable material which may be converted into a biogas by an organism such as a microorganism. In such a method, a polypeptide of the invention may be provided by way of an organism, for example a microorganism which expresses such a polypeptide.

Use of enzymes in food products

The polypeptides and compositions of the invention may be used in a method of processing plant material to degrade or modify the cellulose or hemicellulose or pectic substance constituents of the cell walls of the plant or fungal material. Such methods may be useful in the preparation of food product. Accordingly, the invention provides a method for preparing a food product which method comprises incorporating a polypeptide or composition of the invention during preparation of the food product.

The invention also provides a method of processing a plant material which method comprises contacting the plant material with a polypeptide or composition produced using the invention to degrade or modify the cellulose in the (plant) material. Preferably, the plant material is a plant pulp or plant extract, such as juices.

The present invention also provides a method for reducing the viscosity, clarity and/or filterability of a plant extract which method comprises contacting the plant extract with a polypeptide or composition produced using the invention in an amount effective in degrading cellulose or hemicellulose or pectic substances contained in the plant extract.

Plant and cellulose/hemicellulose/pectic substance-containing materials include plant pulp, parts of plants and plant extracts. In the context of this invention an extract from a plant material is any substance which can be derived from plant material by extraction (mechanical and/or chemical), processing or by other separation techniques. The extract may be juice, nectar, base, or concentrates made thereof. The plant material may comprise or be derived from vegetables, e. g., carrots, celery, onions, legumes or leguminous plants (soy, soybean, peas) or fruit, e. g., pome or seed fruit (apples, pears, quince etc.), grapes, tomatoes, citrus (orange, lemon, lime, mandarin), melons, prunes, cherries, black currants, redcurrants, raspberries, strawberries, cranberries, pineapple and other tropical fruits, trees and parts thereof (e. g. pollen, from pine trees), or cereal (oats, barley, wheat, maize, rice). The material (to be hydrolysed) may also be agricultural residues, such as sugar beet pulp, com cobs, wheat straw, (ground) nutshells, or recyclable materials, e. g. (waste) paper.

The polypeptides produced using the invention can thus be used to treat plant material including plant pulp and plant extracts. They may also be used to treat liquid or solid foodstuffs or edible foodstuff ingredients, or be used in the extraction of coffee, plant oils, starch or as a thickener in foods.

Typically, the polypeptides produced using the invention are used as a composition/enzyme preparation as described above. The composition will generally be added to plant pulp obtainable by, for example mechanical processing such as crushing or milling plant material. Incubation of the composition with the plant will typically be carried out for at time of from 10 minutes to 5 hours, such as 30 minutes to 2 hours, preferably for about 1 hour. The processing temperature is preferably from about 10°C to about 55°C, e. g. from about 15°C to about 25°C, optimally about 20°C and one can use from about 10 g to about 300 g, preferably from about 30 g to about 70 g, optimally about 50 g of enzyme per ton of material to be treated.

All of the enzyme(s) or their compositions used may be added sequentially or at the same time to the plant pulp. Depending on the composition of the enzyme preparation the plant material may first be macerated (e. g. to a pure) or liquefied. Using the polypeptides of the invention processing parameters such as the yield of the extraction, viscosity of the extract and/or quality of the extract can be improved.

Alternatively, or in addition to the above, a polypeptide produced using the invention may be added to the raw juice obtained from pressing or liquefying the plant pulp. Treatment of the raw juice will be carried out in a similar manner to the plant pulp in respect of dosage, temperature and holding time. Again, other enzymes such as those discussed previously may be included. Typical incubation conditions are as described in the previous paragraph.

Once the raw juice has been incubated with the polypeptides produced using the invention, the juice is then centrifuged or (ultra) filtered to produce the final product.

After treatment with the polypeptide produced using the invention the (end) product can be heat treated, e.g. at about 100°C for a time of from about 1 minute to about 1 hour, under conditions to partially or fully inactivate the polypeptide(s) produced using the invention.

A composition containing a polypeptide produced using the invention may also be used during the preparation of fruit or vegetable purees.

The polypeptide of the invention may also be used in brewing, wine making, distilling or baking. It may therefore be used in the preparation of alcoholic beverages such as wine and beer. For example it may improve the filterability or clarity, for example of beers, wort (e.g. containing barley and/or sorghum malt) or wine. Furthermore, a polypeptide or composition of the invention may be used for treatment of brewers spent grain, i.e. residuals from beer wort production containing barley or malted barley or other cereals, so as to improve the utilization of the residuals for, e.g., animal feed.

The protein may assist in the removal of dissolved organic substances from broth or culture media, for example where distillery waste from organic origin is bioconverted into microbial biomass. The polypeptide produced using the invention may improve filterability and/or reduce viscosity in glucose syrups, such as from cereals produced by liquefaction (e.g. with a-amylase).

In baking the polypeptide may improve the dough structure, modify its stickiness or suppleness, improve the loaf volume and/or crumb structure or impart better textural characteristics such as break, shred or crumb quality.

The present invention thus relates to methods for preparing a dough or a cereal-based food product comprising incorporating into the dough a polypeptide or composition produced according to the present invention. This may improve one or more properties of the dough or the cereal-based food product obtained from the dough relative to a dough or a cereal-based food product in which the polypeptide is not incorporated.

The preparation of the cereal-based food product according to the invention further can comprise steps known in the art such as boiling, drying, frying, steaming or baking of the obtained dough.

Products that are made from a dough that is boiled are for example boiled noodles, dumplings, products that are made from fried dough are for example doughnuts, beignets, fried noodles, products that are made for steamed dough are for example steamed buns and steamed noodles, examples of products made from dried dough are pasta and dried noodles and examples of products made from baked dough are bread, cookies and cake.

The term "improved property" is defined herein as any property of a dough and/or a product obtained from the dough, particularly a cereal-based food product, which is improved by the action of the polypeptide according to the invention relative to a dough or product in which the polypeptide according to the invention is not incorporated. The improved property may include, but is not limited to, increased strength of the dough, increased elasticity of the dough, increased stability of the dough, improved machinability of the dough, improved proofing resistance of the dough, reduced stickiness of the dough, improved extensibility of the dough, increased volume of the cereal-based food product, reduced blistering of the cereal-based food product, improved crumb structure of the baked product, improved softness of the cereal-based food product, improved flavour of the cereal-based food product, improved anti-staling of the cereal-based food product. Improved properties related to pasta and noodle type of cereal-based products are for example improved firmness, reduced stickiness, improved cohesiveness and reduced cooking loss.

The improved property may be determined by comparison of a dough and/or a cereal-based food product prepared with and without addition of a polypeptide produced using the present invention. Organoleptic qualities may be evaluated using procedures well established in the baking industry, and may include, for example, the use of a panel of trained taste-testers.

The term "dough" is defined herein as a mixture of cereal flour and other ingredients firm enough to knead or roll. Examples of cereals are wheat, rye, corn, maize, barley, rice, groats, buckwheat and oat. Wheat is I here and hereafter intended to encompass all known species of Triticum genus, for example aestivum, durum and/or spelt. Examples of suitable other ingredients are: the polypeptide composition according to the present invention, additional enzymes, chemical additives and/or processing aids. The dough may be fresh, frozen, pre-pared, or pre-baked. The preparation of dough from the ingredients described above is well known in the art and comprises mixing of said ingredients and processing aids and one or more moulding and optionally fermentation steps. The preparation of frozen dough is described by Kulp and Lorenz in Frozen and Refrigerated Doughs and Batters.

The term "cereal-based food product" is defined herein as any product prepared from a dough, either of a soft or a crisp character. Examples of cereal-based food products, whether of a white, light or dark type, which may be advantageously produced by the present invention are bread (in particular white, whole-meal or rye bread), typically in the form of loaves or rolls, French baguette-type bread, pasta, noodles, doughnuts, bagels, cake, pita bread, tortillas, tacos, cakes, pancakes, biscuits, cookies, pie crusts, steamed bread, and crisp bread, and the like.

The term "baked product" is defined herein as any cereal-based food product prepared by baking the dough.

Non-starch polysaccharides (NSP) can increase the viscosity of the digesta which can, in turn, decrease nutrient availability and animal performance. Adding specific nutrients to feed improves animal digestion and thereby reduces feed costs. A lot of feed additives are being currently used and new concepts are continuously developed. Use of specific enzymes like non-starch carbohydrate degrading enzymes could breakdown the fibre releasing energy as well as increasing the protein digestibility due to better accessibility of the protein when the fibre gets broken down. In this way the feed cost could come down as well as the protein levels in the feed also could be reduced.

Non-starch polysaccharides (NSPs) are also present in virtually all feed ingredients of plant origin. NSPs are poorly utilized and can, when solubilized, exert adverse effects on digestion. Exogenous enzymes can contribute to a better utilization of these NSPs and as a consequence reduce any anti-nutritional effects. Non-starch enzyme compositions of the present invention can be used for this purpose in cereal- based diets for poultry and, to a lesser extent, for pigs and other species.

A non-starch polypeptide/enzyme of the invention (of a composition comprising the polypeptide/enzyme produced using the invention) may be used in the detergent industry, for example for removal from laundry of carbohydrate-based stains. A detergent composition may comprise a polypeptide/enzyme produced using the invention and, in addition, one or more of a cellulase, a hemicellulase, a pectinase, a protease, a lipase, a cutinase, an amylase or a carbohydrase.

Use of enzymes in detergent compositions

A detergent composition comprising a polypeptide or composition of the invention may be in any convenient form, for example a paste, a gel, a powder or a liquid. A liquid detergent may be aqueous, typically containing up to about 70% water and from about 0 to about 30% organic solvent or non-aqueous material.

Such a detergent composition may, for example, be formulated as a hand or machine laundry detergent composition including a laundry additive composition suitable for pre-treatment of stained fabrics and a rinse added fabric softener composition, or be formulated as a detergent composition for use in general household hard surface cleaning operations, or be formulated for hand or machine dish washing operations.

In general, the properties of the enzyme should be compatible with the aselected detergent (for example, pH-optimum, compatibility with other enzymatic and/or non- enzymatic ingredients, etc.) and the enzyme(s) should be present in an effective amount. A detergent composition may comprise a surfactant, for example an anionic or non- ionic surfactant, a detergent builder or complexing agent, one or more polymers, a bleaching system (for example an H₂0₂ source) or an enzyme stabilizer. A detergent composition may also comprise any other conventional detergent ingredient such as, for example, a conditioner including a clay, a foam booster, a sud suppressor, an anti- corrosion agent, a soil-suspending agent, an an-soil redeposition agent, a dye, a bactericide, an optical brightener, a hydrotropes, a tarnish inhibitor or a perfume.

Use of enzymes in paper and pulp processing

A polypeptide or composition of the present invention may be used in the paper and pulp industry, inter alia in the bleaching process to enhance the brightness of bleached pulps whereby the amount of chlorine used in the bleaching stages may be reduced, and to increase the freeness of pulps in the recycled paper process (Eriksson, K. E. L, Wood Science and Technology 24 (1990)79-101 ; Paice, et al., Biotechnol. and Bioeng. 32 (1988):235-239 and Pommier et al., Tappi Journal (1989):187-191 ). Furthermore, a polypeptide or composition produced using the invention may be used for treatment of lignocellulosic pulp so as to improve the bleachability thereof. Thereby the amount of chlorine need to obtain a satisfactory bleaching of the pulp may be reduced.

A polypeptide or composition of the invention may be used in a method of reducing the rate at which cellulose-containing fabrics become harsh or of reducing the harshness of cellulose-containing fabrics, the method comprising treating cellulose- containing fabrics with a polypeptide or composition as described above. The present invention further relates to a method providing colour clarification of coloured cellulose- containing fabrics, the method comprising treating coloured cellulose-containing fabrics with a polypeptide or composition as described above, and a method of providing a localized variation in colour of coloured cellulose-containing fabrics, the method comprising treating coloured cellulose-containing fabrics with a polypeptide or composition as described above. The methods of the invention may be carried out by treating cellulose-containing fabrics during washing. However, if desired, treatment of the fabrics may also be carried out during soaking or rinsing or simply by adding the polypeptide or composition as described above to water in which the fabrics are or will be immersed.

Other enzyme uses In addition, a polypeptide or composition of the present invention can also be used in antibacterial formulation as well as in pharmaceutical products such as throat lozenges, toothpastes, and mouthwash.

The following Examples illustrate the invention:

EXAMPLES

Experimental information

Strains

Aspergillus niger strain was deposited at the CENTRAAL BUREAU VOOR SCHIMMELCULTURES, Uppsalalaan 8, P.O. Box 85167, NL-3508 AD Utrecht, The Netherlands on 10 August 1988 under the deposit number CBS 513.88.

Rasamsonia (Talaromyces) emersonii strain was deposited at CENTRAAL BUREAU VOOR SCHIMMELCULTURES, Uppsalalaan 8, P.O. Box 85167, NL-3508 AD Utrecht, The Netherlands in December 1964 having the Accession Number CBS 393.64.

Other suitable strains can be equally used in the present examples to show the effect and advantages of the invention. For example TEC-101 , TEC-147, TEC-192, TEC- 201 or TEC-210 are suitable Rasamsonia strains wich are described in WO201 1/000949. The "4E mix", "basic 4E mix" or "4E composition" was used containing CBHI, CBHII, EG4 and BG (30wt%, 25wt%, 28wt% and 8wt%, respectively, as described in WO201 1/098577, wt% on dry matter protein.

Rasamsonia (Talaromyces) emersonii strain TEC-101 (also designated as FBG 101 ) was deposited at CENTRAAL BUREAU VOOR SCHIMMELCULTURES, Uppsalalaan 8, P.O. Box 85167, NL-3508 AD Utrecht, The Netherlands on 30^th June 2010 having the Accession Number CBS 127450.

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, NY, 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 W0199846772, W0199932617, WO2001 121779, WO2005095624, WO2006040312, EP 635574B, WO2005100573, WO201 1009700, WO2012001 169 and WO201 1054899. All gene replacement vectors comprise approximately 1 - 2 kb flanking regions of the respective Open Reading Frame (ORF) sequences, to target for homologous recombination at the predestined genomic loci. In addition, A.niger vectors contain the A. nidulans bidirectional 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.

Media and solutions:

Potato dextrose agar, PDA, (Fluka, Cat. No. 70139): per litre: Potato extract 4 g; Dextrose 20 g; Bacto agar 15 g; pH 5.4; Sterilize 20 min at 120°C.

Rasamsonia agar medium: per litre: Salt fraction no.3 15 g; Cellulose 30 g; Bacto peptone 7.5 g; Grain flour 15 g; KH2P04 5 g; CaCI2.2aq 1 g; Bacto agar 20 g; pH 6.0; Sterilize 20 min at 120°C.

Salt fraction composition: The "salt fraction no.3" was fitting the disclosure of W098/37179, Table 1 . Deviations from the composition of this Table were CaCI2.2aq 1 .0 g/l, KCI 1.8 g/L, citric acid 1 aq 0.45 g/L (chelating agent).

Shake flask media for Rasamsonia

Rasamsonia medium 1 : per litre: Glucose 20 g; Yeast extract (Difco) 20 g; Clerol FBA3107 (AF) 4 drops; MES 30 g; pH 6.0; Sterilize 20 min at 120°C.

Rasamsonia medium 2: per litre: Salt fraction no.3 10 g; glucose 10 g; KH2P04 5 g; NaH2P04 2 g; (NH4)2S04 5 g; MES 30 g; pH 5.4; Sterilize 20 min at 120°C.

Rasamsonia medium 3: per litre: Salt fraction no.3 10 g; cellulose 20 g; KH2P04 5 g; NaH2P04 2 g; (NH4)2S04 5 g; MES 30 g; pH 5.4; Sterilize 20 min at 120°C. Rasamsonia medium 4: per litre: Salt fraction no.3 10 g; cellulose 5 g; glucose 5 g; KH2P04 5 g; NaH2P04 2 g; (NH4)2S04 5 g; MES 30 g; pH 5.4; Sterilize 20 min at 120°C.

Rasamsonia medium 5: per litre: Salt fraction no.3 10 g; Xylose 10 g; KH2P04 5 g; NaH2P04 2 g; (NH4)2S04 5 g; MES 30 g; pH 5.4; Sterilize 20 min at 120°C.

Rasamsonia medium 6: per litre: Salt fraction no.3 10 g; cellulose 1 g; Xylose 9 g; KH2P04 5 g; NaH2P04 2 g; (NH4)2S04 5 g; MES 30 g; pH 5.4; Sterilize 20 min at 120°C.

Spore batch preparation for Rasamsonia

Strains were grown from stocks on Rasamsonia agar medium in 10 cm diameter Petri dishes for 5-7 days at 40°C. For MTP fermentations, strains were grown in 96-well plates containing Rasamsonia agar medium. Strain stocks were stored at -80°C in 10% glycerol.

Chromosomal DNA isolation

Strains were grown in YGG medium (per liter: 8 g KCI, 16 g glucose. H20, 20 ml of 10% yeast extract, 10 ml of 100x pen/strep, 6.66 g YNB+amino acids, 1.5 g citric acid, and 6 g K2HP04) for 16 hours at 42°C, 250 rpm, and chromosomal DNA was isolated using the DNeasy plant mini kit (Qiagen, Hilden, Germany).

Shake flask growth protocol of Rasamsonia

Spores were inoculated into 100 ml shake flasks containing 20 ml of Rasamsonia medium 1 and incubated at 45°C at 250 rpm in an incubator shaker for 1 day (preculture 1 ) and 3 ml of biomass from preculture 1 was transferred to 500 ml shake flasks containing 30 ml of Rasamsonia medium 2 and grown under conditions as described above for 1 day (preculture 2). Subsequently, 3 ml of biomass from preculture 2 was transferred to 500 ml shake flasks containing 30 ml of Rasamsonia medium 3 or 4 and grown under conditions described above for 2 days. 2 ml supernatants were taken every 20 hours . Subsequently, the samples were centrifuged for 10 minutes at 13000 rpm in an Eppendorf centrifuge and 1 ml of the supernatants was transferred to clean tube.

Protein analysis using SDS PAGE 50 μΙ of each protein sample was transferred into an eppendorf tube containing 50 μΙ of a 20% (w/v) trichloro acetic acid solution in water and mixed thoroughly. Subsequently, the tubes were incubated on ice water for one hour and centrifuged for 30 minutes, at 4°C and 13000 rpm. The supernatant was discarded and pellets were washed with 200 μΙ ethanol. The tubes were centrifuged for 10 minutes, at 4°C and 13000 rpm the pelled dried by inverting the tubes on a tissue and letting them stand for 30 minutes at room temperature..Subsequently the pellet was dissolved in 50 μΙ protein sample buffer (Invitrogen, Breda, The Netherlands) according to manufacturer's instructions. Protein samples were separated under reducing conditions on NuPAGE 4- 12% Bis-Tris gel (Invitrogen, Breda, The Netherlands) and stained. Gels were stained with either InstantBlue (Expedeon, Cambridge, United Kingdom), SimplyBlue safestain (Invitrogen, Breda, The Netherlands) or Sypro Ruby (Invitrogen, Breda, The Netherlands) according to manufacturer's instructions.

Total protein concentration determination with TCA-biuret method

Concentrated protein samples (supernatants) were diluted with water to a concentration between 2 and 8 mg/ml. Bovine serum albumin (BSA) dilutions (0, 1 , 2, 5, 8 and 10 mg/ml) were made and included as samples to generate a calibration curve. 1 ml of each diluted protein sample was transferred into a 10 ml tube containing 1 ml of a 20% (w/v) trichloro acetic acid solution in water and mixed thoroughly. Subsequently, the tubes were incubated on ice water for one hour and centrifuged for 30 minutes, at 4°C and 6000 rpm. The supernatant was discarded and pellets were dried by inverting the tubes on a tissue and letting them stand for 30 minutes at room temperature. Next, 4 ml BioQuant Biuret reagent mix was added to the pellet in the tube and the pellet was solubilised upon mixing. Next, 1 ml water was added to the tube, the tube was mixed thoroughly and incubated at room temperature for 30 minutes. The absorption of the mixture was measured at 546 nm with a water sample used as a blank measurement and the protein concentration was calculated via the BSA calibration line.

Cellulase activity assays

In order to measure cellulase activity corn stover activity assays are performed. Cellulase activity is measured in supernatants (the liquid part of the broth wherein the cells were cultured) of an empty strain and the transformant:

Preparation of pre-treated, corn stover substrate. Dilute-acid pre-treated corn stover was obtained as described in Schell, D.J., Applied Biochemistry and Biotechnology (2003), vol. 105-108, pp 69-85. A pilot scale pretreatment reactor was used operating at steady state conditions of 190°C, 1 min residence time and an effective H2S04 acid concentration of 1.45% (w/w) in the liquid phase.

Assay 1 : Microtiter plate (MTP) 2% unwashed acid pretreated corn-stover sugar-release assay in which supernatants are spiked on top of TEC-210 or 4E mix

For each (hemi-)cellulase assay, the stored samples were analyzed twice; 100 μΙ_ of sample (e.g. shake flask supernatant) and 100 μΙ of a (hemi-)cellulase base mix [3.5 mg/g DM TEC-210 or a 4 enzyme mix at a total dosage of 3.5 mg/g DM consisting of 0,3 mg/g DM BG (9% of total protein 4E mix), 1 mg/g DM CBHI (30% of total protein 4E mix), 0,9 mg/g DM CBHII (25% of total protein 4E mix) and 1 .3 mg/g DM GH61 (36% of total protein 4E mix)] was transferred to two suitable vials: one vial containing 800 μΙ_ 2.5 % (w/ w) dry matter of the acid pre treated corn stover in a 50 mM citrate buffer, buffered at pH 4.5. The other vial consisted of a blank, where the 800 μΙ_ 2.5 % (w/ w) dry matter, acid pre treated corn stover was replaced by 800 μΙ_ 50 mM citrate buffer, buffered at pH 4.5. The assay samples were incubated for 72 hrs at 65 °C. After incubation of the assay samples, a fixed volume of D20 (with 0.5 g/L DSS) containing an internal standard, maleic acid (20 g/L) and EDTA (40 g/L), was added. The amount of sugar released, is based on the signal between 4.65 - 4.61 ppm, relative to DSS, and is determined by means of 1 D 1 H NMR operating at a proton frequency of 500 MHz, using a pulseprogram with water suppression, at a temperature of 27°C.

The (hemi)-cellulase enzyme solution may contain residual sugars. Therefore, the results of the assay are corrected for the sugar content measured after incubation of the enzyme solution.

Assay 2: Dose-response 2% unwashed acid pretreated corn stover sugar-release assay in MTP

Since glucose release by cellulases is not a linear function of the quantity of enzyme in the composition, in other words, twice the amount of enzyme does not automatically result in twice the amount of glucose at a fixed time point. Therefore the activity of the cellulose enzyme mixture has been assessed in a dose response based assay, in which the dosage is based on equal amount of protein per cellulose mixture tested.

Overall cellulase activity of the mixture measured with unwashed acid pretreated corn stover as substrate. The frozen enzyme samples were thawed and a series of 6 dilutions was made ranging from undiluted in steps of two-fold up to 32-fold in 50 mM citrate buffer pH 4.5.

200 μΙ of sample was transferred to a vial containing 800 μΙ_ of 2.5 % (w/w) dry matter of the acid pretreated corn stover in 50 mM citrate buffer, buffered at pH 4.5. Another 200 μΙ sample was transferred to a vial, referred to as blank, containing 800 μΙ of 50 mM citrate buffer, buffered at pH 4.5. In addition, a sugar background of corn stover was determined by incubating 800 μΙ_ 2.5 % (w/w) dry matter of the acid pretreated corn stover in 50 mM citrate buffer, buffered at pH 4.5 with 200 μΙ of 50 mM citrate buffer. All vials were incubated for 72 hr at 65 °C.

After incubation, 100 μΙ of internal standard solution (20 g/L maleic acid, 40 g/L EDTA in D20) was added to the vials. All vials containing pretreated corn stover were centrifuged for 30 minutes at 5300g and, subsequently, 600 μΙ of the supernatant was transferred to a new vial containing 400 μΙ of H20/D20 9:1.

The 1 D 1 H-NMR spectra were recorded on an Avance III Bruker operating at a proton frequency of 500 MHz, using a pulse program with water suppression, at a temperature of 27°C. Glucose quantification (arbitrary units) was performed based on the signal at 5.20 ppm, relative to 4,4-Dimethyl-4-silapentane sulfonic acid with relation to the internal standard signal at 6.30 ppm. The relative glucose release (AGIc) was calculated by correcting the glucose measured in the samples by the residual sugar present in the enzyme solution (measured from the blank) and the residual sugar present in the acid pretreated corn stover.

Since the protein concentration of the samples was known the sugar release can be depicted as a function of protein mg/ml of the tested diluted sample versus the relative glucose release at time point 72 hours.

Filter paper assay.

Cellulase activity was measured in term of "filter paper units" (FPU) per milliliter in a Filter Paper Unit assay (FPU assay) of original (undiluted) enzyme solution, based on the analytical procedure of Adney and Baker (1996, MoA Laboratory Analytical Procedures-006 entitled: Measurement of cellulase activities by Adney and Baker (1996) www.nrel.gov/biomass/analvtical procedures.html), which is based on International Union of Pure and applied chemistry (lUPAC) guidelines. For quantitative results the enzyme compositions were compared on the basis of significant and equal conversions. The value of 2.0 mg of reducing sugar as glucose from 50 mg of filter paper (4 % conversion) in 60 minutes was designated as the intercept for calculation filter paper units (FPU) by lUCA. In this procedure, reducing sugar yield is not a linear function of the quantity of enzyme in the assay mixture, twice the amount of enzyme will not be expected to yield twice the amount of reducing sugar in equal time. The assay procedure therefore involved finding a dilution of the original enzyme stock such that a 0.5 ml aliquot of the dilution will catalyze 4% conversion in 60 minutes, and then calculating the activity (in FPU/ml) of the original stock from the dilution required.

FPU was calculated using the following formula:

Filter paper activity = 0.37 / ([Enzyme] releasing 2.0 mg glucose) [Units/ml]

[Enzyme] represents the proportion of the original enzyme solution present in the directly tested enzyme dilution.

Example 1. Inactivation of the Ku80 gene in Rasamsonia emersonii strain TEC-210

In order to improve the targeting efficiency at a defined locus in Rasamsonia emersonii strain TEC-210 the Ku80 gene was deleted as previously described for strain TEC-142S (WO2013/35732). For this linear DNA of the deletion constructs pEBA1001 and pEBA1002 was isolated and used to transform Rasamsonia emersonii strain TEC- 210 using method as described earlier in WO201 1\054899. Transformants were selected on phleomycin media and colony purified and tested according to procedures as described in WO201 1\054899. Transformants were selected on phleomycin media and colony purified and tested according to procedures as described in WO201 1\054899. Growing colonies were diagnosed by PCR for integration at the Ku80 locus using a primer in the gpdA promoter of the deletion cassette and a primer directed against the genomic sequence directly upstream of the 5' targeting region. Subsequently, 3 candidate Ku80 knock out strains were transformed with pEBA513 to remove the ble selection marker by transient expression of the ere recombinase according to procedures as described in (WO2013/35732). Removal of the ble marker was tested phenotypically by growing the transformants on media with and without 10 μg ml of phleomycin and removal of the pEBA513 construct in ble-negative strains was subsequently diagnosed phenotypically by growing the transformants on media with and without 50 μg ml of hygromycin. Candidate marker-free knock-out strains were tested by Southern analysis for deletion of the Ku80 gene as described in (WO2013/35732). Strain TEC-221 was selected as a representative TEC-210 strain with the Ku80 gene inactivated.

Example 2: Constructs for overexpression of the sugar transporter genes.

This example describes the construction of expression constructs for overexpression of TEMER01209, TEMER09077, TEMER061 18, TEMER03835 in R. emersonii. The expression cassettes were targeted integrated into the PepA locus as described in PCT/EP2013/055051 .

Two vectors were constructed according to routine cloning procedures for targeting into the PepA locus. The insert fragments of both vectors together were applied in the so-called "bipartite gene-targeting" method (Nielsen et al., 2006, Efficient PCR- based gene targeting with a recyclable marker for Aspergillus nidulans. Fungal Genet Biol 43(1 ):54-64). In our constructs we used two non-functional DNA fragments of the phleomycin selection marker which are overlapping (see also WO20081 13847 for further details of the bipartite method) together with sequences which allow targeting of the gene to the PepA locus. Upon correct homologous recombination the selection marker becomes functional by integration at the PepA locus. As also detailed in WO 20081 13847, two different deletion vectors, Te pep.bbn and pEBA1006 (figure 2), were designed and constructed to be able to provide the two overlapping DNA molecules for bipartite gene-targeting. Te pep.bbn and pEBA1006 are described in PCT/EP2013/055051.

The ccdB gene in vector Te pep.bbn was replaced by either the TEMER01209, TEMER09077, TEMER061 18 or TEMER03835 expression cassettes according to routine cloning procedures. R. emersonii promoter 2, represented by SEQ ID NO: 19, was cloned upstream of the R. emersonii TEMER01209, TEMER09077, TEMER061 18 or TEMER03835 coding region with A. nidulans amdS terminator, generating constructs pEBA01209, pEBA09077, pEBA061 18 or pEBA03835, respectively. The A.nidulans amdS terminator sequence is represented by SEQ ID NO: 20. A schematic representation of the overexpression vectors is shown in Figure 3. Example 3: Transformation of overexpression constructs to Rasamsonia emersonii

Linear DNA of the overexpression constructs pEBA01209, TEMER09077, TEMER061 18, TEMER03835 and pEBA1006 was obtained by digesting the plasmids with the restriction enzyme Not1 and isolating the appropriate fragments. This DNA was purified using a PCR purification kit (Machery Nagel, 96 well PCR clean up,

MACHEREY-NAGEL GmbH & Co. KG, Neumann-Neander-StraBe 6-8, 52355 Duren, Germany ) according the manufacturer's recommendations and used to transform Rasamsonia emersonii TEC-221 using method as described earlier in WO201 1054899. These linear DNAs can integrate into the genome at the PepA locus, thus substituting the ORF encoding the PEPA protein by the overexpression cassette and the phleomycin resistance marker as depicted in Figure 1. Transformants were selected on phleomycin media and colony purified and tested according to procedures as described in

WO201 1054899.

Growing colonies were diagnosed by PCR for absence of the PepA locus by using the primers SEQ ID NO: 21 and SEQ ID NO: 22 whereas the presence of the phleomycin resistance marker was checked using primers SEQ ID NO: 23 and SEQ ID NO: 24.

From sugar-transporter overexpressing strains chromosomal DNA was isolated in order to confirm the integration of the overexpressing construct at the PepA locus using the same primers as used for the colony PCR.

Sugar transporter overexpressing strains were fermented in shake flask as described in Example 4.

Example 4: Shake flask analysis of TEMER01209, TEMER09077, TEMER06118 or TEMER03835 overexpressing strains

Wild-type strain TEC-221 was fermented in shake flask with strains

overexpressing either TEMER01209 (TEC-221 -EBA01209), TEMER03835 (TEC-221 - EBA03835), TEMER061 18 (TEC-221 -EBA061 18) or TEMER09077 (TEC-221 - EBA09077 in medium 4. Supernatant of all strains was harvested after 20 hours and equal amounts of supernatants was analysed by SDS-PAGE.

The gel was stained using InstantBlue and on On the different gels it is clear that in supernatants of TEC-221 -EBA01209 (Figure 4), TEC-221 -EBA03835 (Figure 5), TEC- 221 -EBA061 18 (Figure 6) and TEC-221 -EBA09077 (Figure 6) more extracellular proteins are produced as compared to the wild-type strain TEC-221 . The most abundant protein runs a little bit above 55 kD Marker protein (lane M) which is at the same height as the CBH1 protein. This shows that under these conditions more cellulases are produced in sugar-transporter overexpressing strains as compared to the wild-type TEC-221 strain.

Claims

1 . A polypeptide which comprises the amino acid sequence set out in SEQ ID NO: 4, 8, 9, 14 or 18 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1 , 2, 3, 5, 6, 7, 1 1 , 12, 13, 15, 16 or 17 or a variant polypeptide , wherein the variant polypeptide (i) has at least 70% sequence identity with the sequence set out in SEQ ID NO: 4, 8, 9, 14 or 18 (ii) has an amino acid sequence that differs in 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 or 12 amino acids from the amino acid sequence of SEQ ID NO: 4, 8, 9, 14 or 18.

2. A polypeptide according to claim 1 which is a polypeptide having saccharide transporter or sugar transporter activity, such as a saccharide transporter or sugar transporter. .

3. A polynucleotide having a nucleic acid sequence coding for a polypeptide, whereby the nucleic acid sequence is selected from the group consisting of:

4. A polynucleoide of claim 3 having a nucleic acid sequence coding for a polypeptide which is a polypeptide having saccharide transporter or sugar transporter activity, such as a saccharide transporter or sugar transporter.

5. A nucleic acid construct or vector comprising the polynucleotide according to claims 3 or 4.

6. A recombinant cell comprising the polynucleotide according to claims 3 or 4 or a nucleic acid construct or vector according to claim 5

7. A recombinant cell wherein the polynucleotide according to claim 3 or 4 is mutated or deleted from the genome to obtain lower or no expression of the polypeptide encoded by said polynucleotide compared to the cell wherein the polynucleotide according to claim 3 or 4 is not mutated or deleted from the genome.

8. A recombinant cell according to claim 6 or 7 wherein the cell is a fungal cell, preferably a fungal cell selected from the group consisting of the genera Acremonium, Agaricus, Aspergillus, Aureobasidium, Chrysosporium, Coprinus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Panerochaete, Pleurotus, Schizophyllum, Talaromyces, Rasamsonia, Saccharomyces, Thermoascus, Thielavia, Tolypocladium, and Trichoderma.

9. A recombinant cell wherein the polynucleotide according to claim 3 or 4 is overexpressed compared to the native or starting cell, preferably by altering the promoter or by introducing one or more copies of polynucleotide according to the invention in the cell.

10. A recombinant cell according to claim 1 1 wherein the polypeptide having saccharide transporter or sugar transporter activity, preferably TEMER01209, TEMER09077, TEMER061 18 or TEMER03835 is overexpressed compared to the native or starting cell.

1 1 . A method of producing an enzyme or enzyme composition comprising the fermentation of a cell according any of claims 6 to 12 and optionally recovery of the enzyme or enzyme composition.