Conversion of lignocellulose to bioethanol. Field

The present invention relates to the production of bioethanol. In particular, the invention relates to the use of an enzyme in a method of producing bioethanol from lignocellulosic material. The invention also relates to a method of producing bioethanol.

Background

In general terms, bioconversion is the conversion of organic matter, such as plant dry matter, into a biofuel by a microorganism. Lignocellulose, or plant biomass, is commonly used as a low-cost raw material in the production of biofuels, such as ethanol, which is the most widely used biofuel in the world. While in recent years much progress has been made in developing methods for biofuel production, efficient and cost effective production of bioethanol remains a challenge. Industry and academics alike are continually seeking to improve the efficacy and processing costs associated with the production of bioethanol from lignocellulosic material. The main components of lignocellulose are cellulose and hemicellulose, which are carbohydrate polymers, and lignin, which is an organic polymer. Hemicellulose is the second most abundant component of lignocellulose and comprises a heterogenous class of polymers comprised of both pentose (xylose and arabinose) and hexose (mannose, galactose and glucose) sugars. Galactose residues are a key component of plant hemicelluloses, particularly in xyloglucan, arabinogalactan, galactoglucomannans and glucomannans. Generally speaking, lignocellulose components play a role in plant stability. In plant cell walls, hemicellulose binds cellulose fibrils to form microfibrils, while also cross-linking with lignin, to create a complex web of bonds or macrofibrils that mediate structural stability in the plant cell wall. It is this complex entanglement macrofibrils that makes plant biomass recalcitrant to microbial decomposition as the acting microorganism is unable to access metabolically-useful sugars, such as galactose, that are needed for survival.

The current strategy for the production of bioethanol from lignocellulose is largely based on a pre-treatment step, usually effected by chemical addition, followed by enzymatic hydrolysis. Microbe mediated Consolidated Bioprocessing (CBP) offers an eco-friendly and low cost alternative to this type of bioethanol production.

The process of CBP comprises four microorganism-mediated transformations involving saccharolytic enzyme production, enzymatic hydrolysis of carbohydrate components and fermentation of released hexose and pentose sugars, in a single step to produce ethanol. The l process can be mediated by a single microorganism or by a consortium of different microorganisms. During CBP the fermenting microbe(s) is faced with numerous challenges which can impede efficient conversion, including a limited supply of available nutrients and exposure to various plant defence mechanisms ranging from physical barriers to anti-microbial chemicals/proteins.

Much research has been undertaken to find a suitable microbe for use as an effective CBP agent. Potential microbial candidates for use in the CBP process include Fusarium oxysporum, Phlebia sp., Rhizopus oryzae, Trametes hirsute, Mucor hiemalis and Neurospora crassa. In particular, the pathogenic filamentous fungus, F. oxysporum, has shown promise as an effective CBP agent due to its ability to infiltrate the plant's lignin barrier, degrade complex carbohydrates and produce ethanol from various lignocellulose substrates. Nevertheless, drawbacks such as low level ethanol production and accumulation of acetic acid by-product have prevented it from wide spread exploitation.

Thus, at present, the microbial agents used in CBP are largely ineffective at co-fermenting the variety of sugars found in lignocellulose, particularly the hemicellulosic C5 sugars and to date no suitable microbe has been identified that is equipped with all the necessary enzymatic machinery to adequately convert hemicellulose. As hemicellulose accounts for 20% - 40% of lignocellulose biomass and represents the major polysaccharide fraction wasted in most cellulosic bioethanol pilot and demonstration plants, an efficient and cost effective means to ferment these sugars is desirable and would allow bioethanol to be available at a commercially reasonable price.

The nucleotide diphopho sugar-epimerase (NDPSE) gene is 954 base pairs in length and encodes a protein of 317 amino acids with a predicted molecular mass of 35.36kDa. Computer-assisted analysis of the NDPSE protein sequence identified the presence of classical SDR signature residues including a conserved glycine-rich NAD+ cofactor binding domain 'GXXGXXG', the presence of a critical catalytic tyrosine residue (Tyr-147) serving as the active site base along with an upstream serine (Ser-1 12) and asparagine (Asn-1 19) contributing to the active site.

The current invention serves to alleviate the above problems by providing an efficient and cost effective means to produce bioethanol from lignocellulose, in particular the hemicellulose component of lignocellulose. Summary of the Invention

A first aspect of the current invention provides use of nucleotide diphopho sugar-epimerase (NDPSE) enzyme or an enzyme substantially similar thereto in the production of bioethanol from lignocellulose or parts thereof, wherein said enzyme is provided as an isolated enzyme or by a microorganism that has been genetically modified to overexpress NDPSE compared to a non-modified microorganism.

Preferably, the NDPSE enzyme has the amino acid sequence comprising (or consisting of) SEQ ID NO. 1 , or a variant thereof or fragment thereof.

The variant or fragment thereof are functional variants or functional fragments thereof. The functional variants and functional fragments are as described herein.

Typically, said variant comprises a sequence having from about 70% to about 99% sequence identity to SEQ ID N0.1 .

Preferably, the lignocellulose or parts thereof comprises hemicellulose material.

Typically, the lignocellulose or parts thereof consists of hemicellulose material. Suitably, the hemicellulose material is galactose.

A further aspect of the current invention provides an isolated protein, peptide or polypeptide comprising (or consisting of) amino acid sequence of SEQ ID NO. 1 or a variant thereof or fragment thereof

The variant or fragment thereof are functional variants or functional fragments thereof. The functional variants and functional fragments are as described herein.

A still further aspect of the current invention provides an isolated nucleic acid molecule comprising (or consisting of) a nucleotide sequence of SEQ ID NO. 2, or a variant thereof or fragment thereof.

The variant or fragment thereof are functional variants or functional fragments thereof. The functional variants and functional fragments are as described herein.

Typically, said variant comprises a sequence having from about 70% to about 99% sequence identity to SEQ ID NO.2.

Preferably, the variant is SEQ ID NO. 3.

The isolated protein, peptide or polypeptide or isolated nucleotide is for producing bioethanol from lignocellulose. Another aspect of the current invention provides a method for producing bioethanol from lignocellulose, comprising contacting said lignocellulose with an effective amount of NDPSE enzyme or an enzyme substantially similar thereto, wherein said enzyme is provided as an isolated enzyme or by a microorganism that has been genetically modified to overexpress NDPSE compared to a non-modified microorganism.

Suitably, the lignocellulose is dried prior to being contacted with said NDPSE enzyme.

Alternatively, or in addition, the lignocellulose is processed by grinding or milling prior to being contacted with said NDPSE enzyme.

Another aspect of the invention provides a transformation vector comprising the nucleotide sequence of the invention and described herein.

Another aspect of the invention provides a host cell transformed with the vector of the invention and described herein.

According to a further aspect of the current invention there is provided a host cell comprising a recombinant nucleic acid molecule according the invention. Conveniently, the host cell is a yeast, a fungus, bacterium or other microorganism, or is a mammalian, plant or other cell culture.

Preferably, the host cell expresses NDPSE at a higher or increased level compared with a nonmodified host cell.

Typically, said host cell is F. oxysporum. A composition comprising (NDPSE) enzyme or an enzyme substantially similar thereto is also provided. Preferably, said enzyme is provided as an isolated enzyme or by a microorganism that has been genetically modified to overexpress NDPSE compared to a non-modified microorganism.

Use of NDPSE in a method of converting galactose to glucose is also provided, wherein said enzyme is provided as an isolated enzyme or by a microorganism that has been genetically modified to overexpress NDPSE compared to a non-modified microorganism.

Another aspect of the current invention provides a method for converting galactose to glucose, comprising contacting lignocellulose comprising galactose with an effective amount of NDPSE enzyme or an enzyme substantially similar thereto, wherein said enzyme is provided as an isolated enzyme or by a microorganism that has been genetically modified to overexpress NDPSE compared to a non-modified microorganism. Definitions

Where used herein and unless specifically indicated otherwise, the following terms are intended to have the following meanings in addition to any broader (or narrower) meanings the terms might enjoy in the art: Unless otherwise required by context, the use herein of the singular is to be read to include the plural and vice versa. The term "a" or "an" used in relation to an entity is to be read to refer to one or more of that entity. As such, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.

In the specification, the terms "comprise, comprises, comprised and comprising" or any variation thereof and the terms "include, includes, included and including" or any variation thereof are considered to be totally interchangeable and they should all be afforded the widest possible interpretation and vice versa.

"Nucleotide diphopho sugar-epimerase enzyme" or "NDPSE" when used herein means an enzyme that is capable of converting galactose to glucose and that has uridine diphosphogalactase-4-epimerase activity when tested using, for example, an NADH-couple reaction. NDPSE is encoded by the amino acid sequence of SEQ ID No. 1 or a variant thereof. NDPSE is the NDPSE obtained/obtainable from a bacterial or fungal species. NDPSE is the NDPSE obtained/obtainable from a Fusarium species. Typically, NDPSE is NDPSE obtained/obtainable from Fusarium oxysporum. However, it will be appreciated that NDPSE may be NDPSE obtained/obtainable from Fusarium species including but not limited to F. avenaceum, F. bubigeum, F. culmorum, F. graminearum, F. langsethiae, F. oxysporum, F. poae, F. solani, F. sporotrichioides, F. tricinctum, F. verticillioides, F. virguliforme.

"Substantially similar thereto" as used herein, includes identical sequences, as well as deletions, substitutions or additions to a polynucleotide or polypeptide sequence that maintains any biologically active portion and possesses any of the conserved motifs thereof.

As used herein the term "variant thereof" should be understood to mean a sequence which is substantially identical to a given sequence, but which is altered in respect of one or more amino acid residues or nucleotide residues compared to the given sequence, in such a way so as not to significantly alter the claimed function. Generally, the variant is a (nucleotide or amino acid) sequence having from 70% to 99% sequence identity, preferably 70, 75, 80, 85, 86, 88, 87, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99%, sequence identity with a given sequence and which is typically capable of producing bioethanol from lignocellulose, in particular which is capable of producing glucose from galactose. The variant is a functional variant. Such alterations include, insertion, addition, deletion and/or substitution of an amino acid residue(s), or a nucleotide residue(s). It will be appreciated that such variants may be naturally occurring variants or may be a non-natural variant. The term variant also includes a fragment of a sequence. In relation to a variant of a peptide, the insertion, addition and substitution with natural and modified amino acids are envisaged. The variant may have conservative amino acid changes, wherein the amino acid being introduced is similar structurally, chemically, or functionally to that being substituted.

The term variant is also taken to encompass the term "fragment" and as such means a segment of a given sequence. Typically, the fragment has from about 10 to about 316 contiguous amino acids, preferably, 50, 100, 150, 200, 250, 300, amino acids. Typically, the fragment has from 10 to about 953 contiguous nucleotides, preferably, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 nucleotides. The fragment is one which is capable of producing bioethanol from lignocellulose, in particular which is capable of producing glucose from galactose, i.e. the fragment is a functional fragment. In this specification, the term "sequence identity" should be understand to comprise both sequence identity and similarity, i.e. a variant (or homolog) that shares 70% sequence identity with a reference sequence is one in which any 70% of aligned residues of the variant (or homolog) are identical to, or conservative substitutions of, the corresponding residues in the reference sequence across the entire length of the sequence. Sequence identity is the amount of characters which match exactly between two different sequences. For sequence comparison, one sequence acts as a reference sequence, to which test sequences are compared. Hereby, gaps may not be counted. The measurement may be relational to the shorter of the two sequences.

In terms of "sequence homology", the term should be understood to mean that a variant (or homolog) which shares a defined percent similarity or identity with a reference sequence when the percentage of aligned residues of the variant (or homolog) are either identical to, or conservative substitutions of, the corresponding residues in the reference sequence and here the variant (or homolog) shares the same function as the reference sequence.

This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example, one alignment program is BLAST, using default parameters. Details of these programs can be found at the following Internet address: http://www.ncbi.nIm.nih.gov/bIast/BIast.cgi.

As used herein, the term "genetically modified" as applied to a cell, including a microorganism, means genetically engineered using recombinant DNA technology, and generally involves the step of synthesis of a suitable expression vector (see below) and then transfecting (i.e. stably or transiently) the expression vector into a host cell (generally stable transfection).

As used herein, the term "recombinant cell" or "transformed cell" refers to a cell comprising an exogenous nucleic acid stably integrated into the cellular genome that comprises a nucleotide sequence coding for NDPSE. In another embodiment, it may be a cell comprising a non- integrated (i.e., episomal) exogenous nucleic acid, such as a plasmid, cosmid, phagemid, or linear expression element, which comprises a sequence coding suitable for expression of NDPSE. In other embodiments, the present invention provides a cell line produced by stably transfecting a host cell with a plasmid comprising an expression vector of the invention. In one embodiment, the cell is engineered for heterologous expression of NDPSE.

The term "non-modified" when used herein refers to a cell which has not been subjected to genetic engineering to overexpress NDPSE.

As used herein, "expression" refers to the process by which polynucleotides are transcribed into mRNA and/or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. Expression may be constitutive.

The term "overexpression" refers to expression of a gene or protein in an increased quantity relative to the wild-type. In one embodiment, the expression may be enhanced by transfection of an expression vector containing the necessary machinery to express NDPSE into a host cell. The expression may be enhanced by a promoter to produce multiple copies of mRNA and large quantities of the selected product NDPSE. The host cell may already express endogenous NDPSE.

The term "encode" as it is applied to nucleotide sequences refers to a nucleotide which is said to "encode" a polypeptide or peptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and/or translated to produce the mRNA for the polypeptide and/or a fragment thereof.

The term "isolated" when used herein refers to an NDPSE that has been removed from the components which exist around it when naturally occurring.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms also apply to amino acid polymers in which one or more amino acid residues is a modified residue, or a non-naturally occurring residue, such as an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The peptide may or may not be "isolated", that is to say removed from the components which exist around it when naturally occurring. "Expression control sequences" when used herein refer to sequences that are engineered to control and drive the transcription of genes of interest, and subsequent expression of proteins in various cell systems. Plasmids combine an expressible gene of interest with expression control sequences (i.e. expression cassettes) that comprise desirable elements such as, for example, promoters, enhancers, selectable markers, operators, etc. In an expression vector of the invention, NDPSE machinery-encoding nucleic acid molecules may comprise or be associated with any suitable promoter, enhancer, selectable marker, operator, repressor protein, polyA termination sequences and other expression-facilitating elements.

"Promoter" as used herein indicates a DNA sequence sufficient to direct transcription of a DNA sequence to which it is operably linked, i.e., linked in such a way as to permit transcription of the NDPSE encoding nucleotide sequence when the appropriate signals are present. The expression NDPSE may be placed under control of any promoter or enhancer element known in the art. The promotor may be gpdA promoter. Examples of such elements include strong expression promoters (e.g., human CMV IE promoter/enhancer or CMV major IE (CMV-MIE) promoter, as well as RSV, SV40 late promoter, SL3-3, MMTV, ubiquitin (Ubi), ubiquitin C (UbC), and HIV LTR promoters). In some embodiments, the vector comprises a promoter selected from the group consisting of SV40, CMV, CMV-IE, CMV-MIE, RSV, SL3-3, MMTV, Ubi, UbC and HIV LTR.

The term "amino acid" as used herein refers to naturally occurring and synthetic amino acids, as well as amino acid analogues and amino acid mimetics that have a function that is similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those modified after translation in cells (e.g. hydroxyproline, gammacarboxyglutamate, and O-phosphoserine).

The phrase "amino acid analogue" refers to compounds that have the same basic chemical structure (an alpha carbon bound to a hydrogen, a carboxy group, an amino group, and an R group) as a naturally occurring amino acid but have a modified R group or modified backbones (e.g. homoserine, norleucine, methionine sulfoxide, methionine methyl sulphonium). The phrase "amino acid mimetic" refers to chemical compounds that have different structures from, but similar functions to, naturally occurring amino acids. It is to be appreciated that, owing to the degeneracy of the genetic code, nucleic acid molecules encoding a particular polypeptide may have a range of polynucleotide sequences. For example, the codons GCA, GCC, GCG and GCT all encode the amino acid alanine.

The term "nucleic acid molecule" when used herein to include unmodified DNA or RNA or modified DNA or RNA. For example, the nucleic acid molecules or polynucleotides of the disclosure can be composed of single- and double stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is a mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically double-stranded or a mixture of single- and double-stranded regions. In addition, the nucleic acid molecules can be composed of triplestranded regions comprising RNA or DNA or both RNA and DNA. The nucleic acid molecules of the disclosure may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. "Modified" bases include, for example, tritiated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus "nucleic acid molecule" embraces chemically, enzymatically, or metabolically modified forms. The term "polynucleotide" shall have a corresponding meaning.

The term "hemicellulose material" when used herein is taken to mean a plant fibre material comprising a mixture of polysaccharides, of smaller molecular weight than cellulose including xylan, glucuronoxylan, arabinoxylan, glucomannan and xloglucan.

Brief Description of the Drawings The invention will be more clearly understood from the following description of an embodiment thereof, given by way of example only, with reference to the accompanying drawings, in which:

Figure 1 illustrates NDPSE accumulation during saccharification of wheat straw/bran by Fusarium oxysporum strains 1 1 C and 7E.

Figure 2(A) illustrates the putative crystal structure of the Fusarium oxysporum uridine-5'- diphosphogalactose 4-epimerase (NDPSE).

Figure 2(B) displays sequence similarity with other UDP-galactose-4-epimerase proteins from F. oxysporum.

Figure 3 displays phylogenetic tree depicting the sequence similarity among the putative UDPgalactose-4-epimerase NDPSE protein from Fusarium oxysporum and 27 other UDPgalactose-4-epimerase proteins from fungi, mammals and plants aligned using the CLUSTALW algorithm.

Figure 4 (A) illustrates PCR detection of hyg gene from the pSilentl backbone. Figure 4 (B) illustrates Southern blot analysis to confirm plasmid integration. Figure 5 (A) illustrates PCR detection the bar gene from the pBARGPEI backbone. Figure 5 (B) illustrates Southern blot analysis to confirm plasmid integration. Figure 6 (A) displays accumulation of transcript encoding a putative UDP-galactose-4 epimerase (NDPSE) in wild type and gene-silenced mutants of Fusarium oxysporum strain 1 1 C.

Figure 6 (B) displays accumulation of transcript encoding a putative UDP-galactose-4 epimerase (NDPSE) in wild type and overexpression mutants of Fusarium oxysporum strain 1 1 C.

Figure 7 (A) illustrates the effect of silencing a UDP-galactose-4-epimerase (NDPSE) gene on the ability of Fusarium oxysporum strain 1 1 C to colonise and release bioethanol from a straw/bran mix (10:1 ratio). Figure 7 (B) illustrates the effect of overexpressing a UDP-galactose-4-epimerase (NDPSE) gene on the ability of Fusarium oxysporum strain 1 1 C to colonise and release bioethanol from a straw/bran mix (10:1 ratio).

Figure 8 (A) is a graph illustrating the effect of overexpressing UDP-galactose-4 epimerase (NDPSE) on the ability of Fusarium oxysporum strain 1 1 C grown in minimal media shake flask cultures supplemented with 1 % (w/v) glucose to ferment hexose and pentose sugars to bioethanol.

Figure 8 (B) is a graph illustrating the effect of overexpressing UDP-galactose-4-epimerase (NDPSE) on the ability of Fusarium oxysporum strain 1 1 C grown in minimal media shake flask cultures supplemented with cellulose to ferment hexose and pentose sugars to bioethanol. Figure 8(C) is a graph illustrating the effect of overexpressing UDP-galactose-4-epimerase (NDPSE) on the ability of Fusarium oxysporum strain 1 1 C grown in minimal media shake flask cultures supplemented with xylose to ferment hexose and pentose sugars to bioethanol.

Figure 9 is a graph illustrating the effect of over expressing UDP-galactose-4-epimerase (NDPSE) gene on the ability of Fusarium oxysporum strain 1 1 C to ferment galactose to bioethanol.

Figure 10 (A) displays the results of SDS-PAGE of purified GST-NDPSE fusion protein.

Figure 10 (B) illustrates a western blot image of purified GST-NDPSE fusion protein.

Figure 11 is a graph illustrating the uridine-diphosphogalactose-4-epimerase activity of the Fusarium oxysporum NDPSE protein. Figure 12 shows the silencing vector pSilent-NDPSE.

Figure 13 shows the expression vector pBARGEI -NDPSE. Detailed Description of the Drawings

Currently used methods to convert lignocellulose to bioethanol are largely inefficient. Broadly speaking, the method of the current invention provides a means to utilise the hemicellulosic fraction of lignocellulose, or plant biomass, to produce bioethanol. In this regard, the current inventors have surprisingly found that NDPSE is a hemicellulosic fermentation enzyme that converts hemicellulose sugar, specifically galactose, into metabolically useful glucose. The microbe can then ferment these sugars into bioethanol. In this manner, the invention provides a more efficient means of producing bioethanol, thereby providing a significant reduction in the operational cost of bioethanol production making it an attractive and cost competitive option.

The current invention provides the use of NDPSE in the production of bioethanol from lignocellulose. The source of lignocellulose utilised as a starting material may be any type of plant biomass, such as, but not limited to, any type of plant, trees, crops, grasses, including by products and waste materials such as agricultural residues and forestry waste, or a combination thereof.

The lignocellulose may be the hemicellulosic material fraction of lignocellulose. The lignocellulose may consist of hemicellulose material. The hemicelllulose fraction may be galactose.

The NDPSE enzyme is provided as an isolated enzyme or by a microorganism that is genetically modified to overexpress the NDPSE compared to non-modified microorganisms.

The genetically modified microorganism may express endogenous NDPSE. It will be appreciated that any means known to the person skilled in the art may be used to modify the microorganism to overexpress NDPSE compared to non-modified microorganism. A transformation vector may be used to express NDPSE in a host microorganisms. The microorganism may be modified to express at least from 1 to 100-fold increase in the NDPSE transcript levels compared to the wild type or non-modified microorganism. The microorganism may be modified to express at least from 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 65, 70, 75, 80, 85, 90, 95-fold increase in the NDPSE transcript levels compared to the wild type or non-modified microorganism. The microorganism may be modified to express at least 30-fold increase in the NDPSE transcript levels compared to the wild type or non-modified microorganism.

The microorganism may be a yeast, a fungus, bacterium or other microorganism, or is a mammalian, plant or other cell culture. Preferably, the microorganism is from the genus Fusarium, such as a species selected from the group comprising F, avenaceum, F. bubigeum, F. graminearum, F. langsethiae, F. oxysporum, F. poae, F. sporotrichioides, F.tricinctum, F. verticillioides and F. virguliforme.

The NDPSE enzyme has an amino acid sequence comprising (or consisting of) SEQ ID NO. 1 or a variant thereof. SEQ ID N0.1 has the following sequence:

MPKYVLTGADGNLGRIAASFATEIAKPGDELVFTTYKDEAIPADLRKSWTDKGAKVV TATYD DVESLKAAFQGAEAVSLI STWLFGEGRRRQAQTVVDAAKACGVKRVCYTSFCGAGIEAENEE DIPFLPRDHHFIEKI IYASGLEY IQRNYLYAD IPTLFAPSWKFCGDRWLVNTHGKAGAYV AREDCGNVLAALLLGRGEPNKVYEITGPKAVTNEEVFKWMCEQTGYSGGIVDLPDEELKR WW LDHGLPTDFFGDFSKLPMKLCIGDLLCCGEMVAREFMAETNDNVEKLTGRKPIPYQEALL QY KDFFPKP

The variant may comprise a sequence having from 70% to 99% sequence identify to SEQ ID NO. 1 . The variant is a functional variant. In some embodiments, the variant may have a sequence of at least 70%, 75%, 80%, 85%, 90%, 95% sequence identity to SEQ ID NO. 1 . The variant may have a sequence of 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO. 1 . The variant is a functional or bioactive variant that is capable of producing bioethanol from lignocellulose. More specifically the functional or bioactive variant is capable of producing glucose from galactose. The glucose can then be further converted or fermented into bioethanol.

The peptide of the invention comprises (or consists of) an amino acid sequence SEQ ID NO. 1 , or a variant thereof.

In one embodiment, the variant has 1 to 100 amino acid changes compared to SEQUENCE ID NO: 1 . In one embodiment, the variant has 1 to 90 amino acid changes compared to SEQUENCE ID NO: 1 . In one embodiment, the variant has 1 to 80 amino acid changes compared to SEQUENCE ID NO: 1 . In one embodiment, the variant has 1 to 70 amino acid changes compared to SEQUENCE ID NO: 1 . In one embodiment, the variant has 1 to 60 amino acid changes compared to SEQUENCE ID NO: 1 . In one embodiment, the variant has 1 to 50 amino acid changes compared to SEQUENCE ID NO: 1 . In one embodiment, the variant has 1 to 40 amino acid changes compared to SEQUENCE ID NO: 1 . In one embodiment, the variant has 1 to 30 amino acid changes compared to SEQUENCE ID NO: 1 . In one embodiment, the variant has 1 to 20 amino acid changes compared to SEQUENCE ID NO: 1 . In one embodiment, the variant has 1 to 10 amino acid changes compared to SEQUENCE ID NO: 1 . In one embodiment, the variant has 1 to 5 amino acid changes compared to SEQUENCE ID NO: 1 . In one embodiment, the amino acid change is an amino acid substitution. In one embodiment, the amino acid substitution is a conservative substitution. In one embodiment, the amino acid change is an amino acid addition. In one embodiment, the amino acid change is an amino acid deletion. The nucleic acid molecule of the invention comprises (or consists of) a nucleotide sequence of SEQ ID NO. 2, or a variant thereof or fragment thereof. The nucleic acid molecule encodes the peptide of the invention or variant thereof.

SEQ ID NO.2 has the following sequence:

AIGCCAAAGTACGTTCTCACAGGCGCAGACGGTAATCTCGGCCGCATCGCAGCAAGC TTCGC CACAGAAATCGCCAAACCTGGCGATGAACTCGTCTTCACCACATACAAAGACGAAGCAAT CC CCGCTGACCTCCGCAAATCATGGACCGACAAAGGCGCAAAAGTCGTAACAGCCACTTACG AC GATGTTGAAAGTCTCAAAGCGGCTTTTCAGGGCGCTGAAGCCGTGTCTCTCATCTCAACT TG GCTCTTCGGTGAAGGTCGACGTCGTCAGGCCCAAACTGTCGTTGACGCTGCCAAGGCATG TG GTGTCAAGAGAGTCTGCTATACTTCGTTCTGTGGAGCTGGTATAGAGGCTGAGAATGAGG AG GATATTCCTTTCCTCCCCCGAGATCATCACTTCATTGAGAAGATTATCTACGCTTCTGGC TT AGAGTACAATATCCAGCGTAATTATCTTTACGCTGATAATATTCCAACTCTCTTTGCACC AT CGTGGAAATTCTGCGGTGATCGCTGACTCGTCAACACGCACGGCAAAGCAGGCGCATACG TT GCCCGCGAAGACTGCGGCAACGTCCTAGCCGCTCTGCTCCTAGGCCGCGGCGAACCTAAC AA AGTGTACGAGATCACTGGCCCTAAAGCCGTCACCAACGAGGAGGTTTTCAAGTGGATGTG CG AGCAAACAGGGTACAGTGGAGAAATTGTCGACCTTCCTGATGAAGAGTTGAAGAGATGGT GG TTAGATCATGGATTGCCTACGGACTTCTTTGGTGACTTTTCAAAGTTGCCGATGAAGTTG TG TATTGGCGATCTTTTGTGCTGTGGAGAGATGGTTGCGAGGGAGTTCATGGCTGAGACGAA TG ATAATGTTGAGAAGTTGACGGGAAGGAAGCCAATTCCGTATCAGGAGGCTTTGTTGCAGT AT AAGGACTTCTTCCCGAAGCCATAG The variant may comprise a sequence having from 70% to 99% sequence identify to SEQ ID NO. 2. The variant is a functional variant. In some embodiments, the variant may have a sequence of at least 70%, 75%, 80%, 85%, 90%, 95% sequence identity to SEQ ID NO. 2. The variant may have a sequence of 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sequence identity to SEQ ID NO. 2.

In one embodiment, the variant has 1 to 300 nucleotide changes compared to SEQUENCE ID NO: 2. In one embodiment, the variant has 1 to 250 nucleotide changes compared to SEQUENCE ID NO: 2. In one embodiment, the variant has 1 to 200 nucleotide changes compared to SEQUENCE ID NO: 2. In one embodiment, the variant has 1 to 150 nucleotide changes compared to SEQUENCE ID NO: 2. In one embodiment, the variant has 1 to 100 nucleotide changes compared to SEQUENCE ID NO: 2. In one embodiment, the variant has 1 to 75 nucleotide changes compared to SEQUENCE ID NO: 2. In one embodiment, the variant has 1 to 50 nucleotide changes compared to SEQUENCE ID NO: 2. In one embodiment, the variant has 1 to 25 nucleotide changes compared to SEQUENCE ID NO: 2. 5 In one embodiment, the variant has 1 to 20 nucleotide changes compared to SEQUENCE ID NO: 2. In one embodiment, the variant has 1 to 10 nucleotide changes compared to SEQUENCE ID NO: 2. In one embodiment, the variant has 1 to 5 nucleotide changes compared to SEQUENCE ID NO: 2. Peptides (including variants and fragments thereof) of and for use in the invention may be generated wholly or partly by chemical synthesis or by expression from nucleic acid. Also provided is an enzyme encoded by the amino acid sequence of the invention.

Also provided is a nucleotide sequence encoding an amino acid sequence according to SEQUENCE ID NO. 2, or a functional variant of said amino acid sequence or functional fragment thereof. The functional variant may have from about 70% to about 99.9% sequence identity with SEQUENCE ID NO. 2. An enzyme encoded by the amino acid sequence is provided. The enzyme is for producing bioethanol from lignocellulose.

The invention also provides a vector comprising nucleic acid molecule of the invention. In an embodiment, the vector is an expression vector. It will be understood that the vector may be any vector suitable for replicating in a host microorganism. The vector may be a plasmid, a virus (including bacteriophage), a cosmid, an artificial chromosome or a transposable element. The vector may comprise regulatory machinery, for example promoters, terminators, and/or enhancers. The nucleotide may be under the control of a promoter region. The promoter may be a constitutive fungal-specific promoter or an inducible fungal-specific promotor. The promoter may be the gpdA promoter. The promoter may be such that multiple copies of the NDPSE product are produced. In some alternative embodiments, the vector is a virus, such as a bacteriophage and comprises, in addition to the nucleic acid sequence of the invention, nucleic acid sequences for replication of the bacteriophage, such as structural proteins, promoters, transcription activators and the like.

In an embodiment of the invention, the vector of the invention and described herein may be used to transfect or transform host cells to produce a recombinant cell in order to synthesize the NDPSE protein. A recombinant host cell comprising a vector as described herein is also provided by the current invention. The host cell may be any biological cell which can be cultured in medium and used for the expression of a recombinant gene. Such host cells may be eukaryotic, such as yeast cells, or mammalian or plant cell lines or prokaryotic and may be a microorganism such as a bacterial cell, e.g. E. Coli, or may be a cell from a cell line (such as an immortal mammalian cell line). The host cell may be a fungus. It will be appreciated that the host cell may be a yeast, bacterium or other microorganism, or is a mammalian, plant or other type of cell culture. In some embodiments, the host cell is F. oxysorum. It will be appreciated that the host cell may be any suitable fungal or yeast species. The host cell may be from the genus Fusarium, such as a species selected from the group comprising F, avenaceum, F. bubigeum, F. graminearum, F. langsethiae, F. oxysporum, F. poae, F. sporotrichioides, F.tricinctum, F. verticillioides and F. virguliforme. Host cells are transfected or transformed using techniques known in the art such as electroporation; calcium phosphate base methods; a biolistic technique or by use of a viral vector. After transfection, the nucleotide of the invention is transcribed as necessary and translated. In some embodiments, the synthesized protein is allowed to remain in the host cell and cultures of the recombinant host cell are subsequently used. In other embodiments, the synthesized NDPSE enzyme is extracted from the host cell, either by virtue of its being secreted from the cell due to, for example, the presence of secretion signal in the vector, or by lysis of the host cell and purification of the enzyme.

The NDPSE enzyme may be provided as an isolated enzyme. It will be appreciated that methods of isolation/extraction are known to the person skilled in the art. An exemplary method for isolating the enzyme is described in the Examples.

In an embodiment of the invention, the host cell expresses NDPSE gene and/or enzyme at a higher or increased level compared to the level of expression of an unmodified host cell. In other words, the host cell overexpresses NDPSE compared to unmodified host cell. As detailed by the examples herein, a F.oxysporum mutant with NDPSE overexpression showed an increased ability to ferment galactose to ethanol and also wheat straw/bran to ethanol compared with wild-type and genetically silenced F. oxysporum.

The unmodified host cell may be unmodified or wild type F. oxysporum.

The invention also provides a composition comprising isolated NDPSE. The enzyme may be present together with the microbe that is expressing or producing it. It will be appreciated that the enzyme may be present without the microbe that produced it. The enzyme may be added directly to a substrate of lignocellulose or hemicellulose material. It is to be understood that the enzyme may be the only enzyme used in the method. Alternatively, the enzyme may be used in combination with other enzymes or active agents or other microorganisms. Other enzymes may be selected from cellulase, hemicellulase, glucanase and xylanse. The enzyme may be one that is capable of fermenting glucose. The method of producing bioethanol from lignocellulose or parts thereof comprises contacting NDPSE enzyme, a nucleotide, a peptide, a vector or a host cell according to the invention with said lignocellulose substrate under conditions such that the substrate is converted to ethanol.

In one embodiment, the method may be Consolidated Bioprocessing. The NDPSE enzyme may be the only enzyme used in the method. Alternatively, the method comprises the addition of one or more enzymes, or active agents.

EXAMPLES

MATERIALS AND METHODS

Strains Fusarium oxysporum strains 1 1 C and 7E were used. F. oxysporum strains were isolated from agricultural soil, peat and plant samples collected from different sites around Ireland between October 2007 to February 2008 (see Table 1 ) using Komada's selective medium (Komada, 10 1975) as described by AN et al. (2012). Strain 1 1 C has a high capacity for bioethanol production from straw via CBP, as compared to strain 7E. Fungal isolates were submersed in 30% glycerol (vv-1 ), flash-frozen in liquid nitrogen and stored at -70 Ό. Prior to use, fungal isolates were subcultured onto PDA plates and incubated at 25°C for 5 days. Fungal conidial inoculants were produced in mung bean broth as described by Brennan et al. (2005) and were re-suspended in minimal medium (Mishra et al. 1984) at a concentration of 106 conidia ml-1 .

Table 1: Code and origin of Fusarium oxysporum strains

Code Sample Origin IMI accession number lie Potato rhizosphere soil Bulked samples from 501118

various Irish locations

7E Potato rhizosphere soil Raphoe, Co. Donegal, 501116

Ireland

Solid-state cultivation (SSC)

The carbon substrate used for SSC experiments was based on non-alkali-treated wheat straw (Triticum aestivum L. cultivar Einstein) blended with wheat bran (Odiums, Ireland). Dry wheat straw was ground in a coffee grinder (Model 203C, KRUPS, Mexico), passed through a sieve (2mm pore size) and blended with unprocessed wheat bran (particle size < 3mm) (10:1 ratio of straw to bran). One gram of the straw/bran blend was mixed with 5ml minimal medium (see below; excluding a C-source) and autoclaved (121 °C for 15min) in a 100ml Erlenmeyer flask.

Except where otherwise stated below, the cultures were grown in the minimal medium described by Mishra et al. (1984) (pH 5); they contained 91 % initial moisture (vw ) and were maintained at 25 Ό. Flasks were supplemented with 4ml of either fungal conidial suspension (see previous section) or minimal medium (negative controls). For studies which analysed bioethanol production by mutant and wild type fungal strains, mycelia were allowed to grow aerobically on untreated wheat straw/bran blend (9:1 ) for 96h for biomass and saccharolytic enzyme production, followed by 96h incubation under oxygen-limiting conditions growth to facilitate the fermentation of released sugars into ethanol. For the aerobic growth period, Erlenmeyer flasks were plugged with non-absorbent cotton covered with aluminium foil. For the subsequent oxygen-limited incubation period, the cotton plugs were replaced with cork and sealed with parafilm for 96h prior to bioethanol analysis. The experiment was based on two replica trials, each including three technical replica flasks per fungal strain/mutant. For the gene expression experiment, samples were harvested at 24-96h post-inoculation under aerobic conditions for follow up RNA isolation and RT-PCR analysis. This experiment was based on two replica trials, each including three technical replica flasks per fungal strain/mutant.

Fermentation Assays The ability of wild type and mutant strains of F. oxysporum to ferment glucose and other pentose sugars was determined using shake flask cultures and oxygen-limiting conditions. Flasks (100ml) contained 30ml of the minimal media described by Leung et at. (1995) supplemented with 1 % wv ^~1 carbon (glucose/cellulose/xylose/galactose). For the initial aerobic growth phase, medium was inoculated with fungal spores (105 ml ¹ ), plugged with sterile cotton wool and incubated at 30 °C for 24h at 150rpm in the dark. Thereafter, flasks were plugged with cork and sealed with parafilm and incubated at 50rpm, 30 ^ for 24-192h prior to bioethanol analysis, except in the case of galactose where flasks were incubated at 50rpm, 30 'Ό for 144h prior to bioethanol analysis. The experiment was based on two replica trials, each including three technical replica flasks per fungal strain/mutant. Bioethanol and fungal biomass estimations

Following fungal culture, flasks were incubated at 4 ^< C for one hour to condense any alcohol produced. Flasks were then supplemented with 10ml of sterile cold water, plugged with cork and incubated at 150rpm, 25°C for 1 h. All bioethanol extraction procedures thereafter were performed in a cold room (4°C). Flasks were incubated for 1 h and two sub-samples (2ml) of liquid were removed to sterile tubes, and centrifuged at l OOOOrcf at 4 ^< C for 20min. The supernatant was decanted and stored at -70 °C until bioethanol estimation. Bioethanol (mgg substrate) was determined for two subsamples per flask using the QuantiChromTM Ethanol Assay Kit (DIET-500) (BioAssay Systems, USA) according to manufacturer's instructions.

Fungal biomass levels in the solid component of cultures was determined based on the chitin derived glucosamine content. Chitin was hydrolysed into N-acetyl glucosamine as previously described by Scotti et al. (2001 ), which was then assayed by the modified colorimetric method described by Ride & Drysdale (1972). In the case of galactose, Gas Chromotography - Mass

Spectrometry (GC-MS) (Agilent Technologies, USA) was used to quantify ethanol content using a capillary column with Helium as the mobile phase and a pump speed 0.5ml/min and the detector temperature set to 260 °C.

RNA isolation and real time RT-PCR

Mycelial samples were flash-frozen with liquid nitrogen, freeze-dried and homogenised in a mixer mill (Tissue Lyser II, The Netherlands) at 30Hz for 1 min with two 2.3mm steel beads. RNA was isolated as previously described by Chang et al. (1993). RNA was DNased-treated using the TURBO DNA-free kit (Ambion, USA), according to the manufacturers' recommendations. RNA integrity was validated by visualising RNA following agarose gel electrophoresis and yields were quantified using a NanoDrop® ND-1000 Spectrophotometer, all as described previously (Walter et al. 2008).

In the case of real time RT-PCR analysis, NDPSE and /3-fUib-specific primers were used. The protocol for reverse transcription (RT) of total RNA was conducted as described by (Ansari et al. 2007), except that the primer used was oligo dTi ₂ -is (Invitrogen). The housekeeping gene used for normalisation of real-time RT-PCR data was β -tubulin (Gl: 758212039). Real-time PCR quantification of target gene and of the housekeeping gene was performed in separate reactions. RT products were diluted to 200μΙ and 2.5μΙ was PCR-amplified in a 25μΙ reaction volume containing 12.5μΙ Premix Ex Taq™ (Perfect Real Time, Takara,) and 100nM each of either the NDPSE- or /Hub-specific primers. PCR reactions were conducted in a Stratagene Mx3000TM real-time PCR machine (Stratagene) and the programme consisted of 1 cycle of 95 ^< C for 10s, 40 cycles of 95 °C for 5s, 60°C for 30s and 1 cycle of 95 °C for 60s. Data were analysed using Stratagene Mx3000TM software. The threshold cycle (CT) values obtained by real-time RT-PCR were used to calculate the accumulation of target gene (relative mRNA accumulation), relative to β-tub transcript, by the 2 ^Λ~ΔΔ01 method, (Livak & Schmittgen, 2001 ). Results were based on the average obtained for two replicate RT-PCR reactions per sample.

Rapid Amplification of cDNA Ends (RACE)

30 5'- and 3'-RACE was conducted in order to clone the full-length mRNA sequence of the NDPSE gene from F. oxysporum strain 1 1 C. RACE analysis was conducted using the Clontech SMARTer™ RACE kit (Clontech Laboratories Inc., USA), according to the manufacturers' protocols and gene-specific primers (RACE-NDPSE-MF: CAGGGCGCTGAAGCCGTGT (SEQ ID NO. 4) and RACE-NDPSE-MR: GAGTCTGCTATACTTC (SEQ ID NO. 5)). RACE products were gel-purified using the same SMARTer™ RACE kit, cloned using the pGEM-T Easy "TA" cloning kit (Promega, USA) according to the manufacturers' protocol and sequenced (5 Macrogen, Korea).

The ORF was determined using NCBI ORF finder (http://www.ncbi.nlm.nih.gov/projects/gorf/) and was used to query the deduced amino acid sequence (http://web.expasy.org/translate) against protein sequences in the Saccharomyces Genome Database (SGD) (http://www. yeastgenome.org/) and the Fusarium comparative genomics database (FCGD)

(http: ^' wA^ group MultiHome.html)

Crystal structure

The putative crystal structure of the deduced NDPSE ORF was generated using SWISSMODEL Workspace (http://swissmodeI.expasy.org) (Arnold et al. 2006). The crystal structure is illustrated by Figure 2.

Vector construction

Post-transcriptional gene silencing (PTGS) was used to generate mutants of F. oxysporum strain 1 1 C silenced in the NDPSE gene function. The silencing vector pSilent-1 -/VDPS£ was constructed using the pSilent-1 vector (Nakayashiki et al. 2005), which contains Aspergillus nidulans trpC promoter and terminator flanking two MCS that are separated by an intron from a Magnaporthe grisea cutinase gene and hyg as a selectable marker gene which provides resistance to hygromycin. A fragment of the NDPSE gene with appropriate overhanging restriction sites was inserted into each of the two MCS in the sense (upstream of the intron) or antisense (downstream of the intron) direction. The gene fragment is a 418 base pair fragment of NDPSE having a sequence of SEQ ID NO. 3 as follows:

AGGCGCAGACGGTAATCTCGGCCGCATCGCAGCAAGCTTCGCCACAGAAATCGCCAA ACCTG

GCGATGAACTCGTCTTCACCACATACAAAGACGAAGCAATCCCCGCTGACCTCCGCA AATCA TGGACCGACAAAGGCGCAAAAGTCGTAACAGCCACTTACGACGATGTTGAAAGTCTCAAA GC GGCTTTTCAGGGCGCTGAAGCCGTGTCTCTCATCTCAACTTGGCTCTTCGGTGAAGGTCG AC GTCGTCAGGCCCAAACTGTCGTTGACGCTGCCAAGGCATGTGGTGTCAAGAGAGTCTGCT AT ACTTCGTTCTGTGGAGCTGGTATAGAGGCTGAGAATGAGGAGGATATTCCTTTCCTCCCC CG AG AT CAT C AC T T C AT T G AG AAG ATTATCTACGCTTCTGGCTTAGAG [nucleotides in bold added restriction sites for cloning]. Primers Si_NDPSE-L-F2/R2 and Si_NDPSE-R-F2/R2 were respectively used to amplify the sense and antisense direction inserts The PCR amplification reaction (20μΙ) contained 100ng plasmid (from the representative SSH clone), 1 ^χ PCR buffer, 1 unit Hifi-Taq DNA polymerase (Invitrogen), 2mM each of dNTPs, 2pM each of the forward and reverse primers and 0.2mM MgS04. Amplifications were performed in a Peltier Thermal Cycler (PTC) -200 DNA Engine (MJ Research) with the following conditions: initial denaturation for 30s at 95°C was followed by 30 cycles of 94 ^< C for 30s, 60 °C for 45s, 72 °C for 30s with a final extension of 5min at 72 °C. Products and plasmid were digested with Sphl and Hindi 11 (New England Biolabs, USA), ligated (T4 DNA ligase, Promega, USA) and the correct alignment of sense and antisense segments were confirmed by partial sequencing of the plasmid using ACpSi-F/R primers designed to anneal to the end of trpC promoter and the beginning of trpC terminator, respectively.

The NDPSE overexpression vector pBARGPEI -NDPSE was constructed using the pBARGPEI vector (Pall & Brunelli, 1993) which contains A. nidulans gpdA promoter and trpC terminator flanking a MCS and bar as a selectable marker gene which provides resistance to Basta (active ingredient = phosphinothricin). NDPSE-specific primers FL NDPSE-F/R (Table 2) were used to amplify the NDPSE ORF with appropriate overhanging restriction sites.

The PCR amplification reaction (50μΙ) contained 5μΙ 5'-RACE ready cDNA from F. oxysporum strain 1 1 C, 5μΙ 10χΙ_Α PCR Buffer (Mg2+ plus), 0.5μΙ (2.5 unit) TaKaRa LA Taq DNA polymerase (Takara, Japan), 8μΙ (2.5mM each) dNTPs mix and 0.2μΜ each of the forward and reverse primers. Amplifications were performed in a Peltier Thermal Cycler (PTC)-200 DNA Engine (MJ Research) with the following conditions: initial denaturation for 60s at 94 °C was followed by 38 cycles of 98 °C for 10s, 60 °C for 30s, 68 ^< C for 105s with a final extension of 10min at 72 ^< C. Products and plasmid were digested with EcoRV and Smal (New England Biolabs, USA), ligated (T4 DNA ligase, Promega, USA) and the correct alignment of the gene was confirmed by partial sequencing of the plasmid using ACpBg- F/ACpSi-/R primers (Table 2) designed to anneal to the end of gpdA promoter and the beginning of the trpC terminator, respectively.

Table 2: Primers

Table 2: Primers

Protoplast production

Fungal conidia from a 5-day-old Mung bean culture were collected by centrifugation ( ^" l OOOxg, 5 20 ^, 15min), washed with sterile phosphate-buffered saline (PBS), and again collected by centrifugation as above. Spores were resuspended in the lytic enzyme 5 solution [100mg zymolase, ^" l OOmg lysing enzyme (Sigma, Germany), 200mg drisilase (Sigma, Germany), 200mg BSA, 30μΙ β-mercaptoethanol and 20μΙ 1 M DTT in 20ml 0.7M NaCI] and incubated at 30^, 50rpm. After 3h, the solution was filtered through mira-cloth (Merck Millipore, Germany) 10 and diluted to 50ml using 0.7M NaCI. Protoplasts were collected, washed and resuspended in STC buffer (1 .2M sorbitol, 10mM Tris-HCL, 10mM CaCI2) (Salch & Beremand, 1993) to a concentration of 4x108 protoplasts ml-1 as described by Doohan ei a/.(1998).

Generation and selection of fungal mutants

F. oxysporum strain 1 1 C was transformed with pSilent-1 -NDPSE and pBARGPEI -NDPSE in 15 order to respectively silence and overexpress the NDPSE gene. Fungal transformation was performed using the polyethylene glycol (PEG)-mediated method as described by Doohan et al. (1998). Protoplasts were transformed with pSilent-1 - NDPSE and pBARGPEI - NDPSE. Putative transformants were identified following selection on PDA (Oxoid, UK) or minimal medium, (Leung et al. 1995) containing 60μgml ^~ hygromycin (Sigma, Germany) (Doohan et 20 20 al. 1998) for silencing or 1000μgml ^" phosphinothricin (Sigma, Germany) (Leung et al.

1995), for overexpression studies. Putative transformants were subcultured five times on the same selective medium, then four times on non-selective medium and finally, transformant stability was verified by growing on the selective PDA/minimal medium. Fungal mycelium generated from a single spore was subcultured on PDA and transferred to a 15% vv glycerol solution for storage at -70 °C. Transformation was confirmed by both PCR and southern blot analysis. The PCR target/Southern blot probe was a fragment of the hyg gene for silencing mutants and of the bar gene for overexpression mutants. Wild type/mutants were cultured in PDB (Oxoid, UK) and mycelia were harvested by centrifugation at 3200rcf for 15min and flash- frozen in liquid nitrogen followed by freeze-drying. Dry mycelium was ground into fine powder in a mixer mill (Retsch MM400, Germany) at 30Hz for 1 min with two 2.3mm steel beads and total genomic DNA was isolated by adding fungal DNA extraction buffer as described by Edel et at. (2000). DNA was purified by phenol-chloroform treatment and subjected to ethanol precipitation and re-suspended in Tris-EDTA buffer (pH 7.4). For putative silencing mutants, PCR was conducted using 7yg-specific primers (Hyg-F1 /R1 ; see Appendix III, Table A3.5), which were used to amplify a 747bp hyg gene fragment from either 100ng gDNA of putative mutants or 50ng pSilent-1 plasmid DNA (as a positive control and to produce probe for southern blot analysis). For the putative overexpression mutants, PCR was conducted using bar-specific primers (Bar-F1 /R1 ), which were used to amplify a 433bp bar gene fragment from 100ng gDNA of putative mutants or 50ng pBARGPEI plasmid DNA (as a positive control and to produce probe for southern blot analysis). The PCR reaction components and the conditions were as described above. PCR products (1 ΟμΙ) were electrophoresed through 1 % (wt vol ¹ TAE) agarose gels containing 0.5 μg ml ^-1 ethidium bromide and visualised using Imagemaster VDS and Liscap software (Pharmacia Biotech, USA).

For southern blot analysis of mutants, genomic DNA (10μg) from wild type F. oxysporum strain 1 1 C and putative mutants was digested overnight with restriction enzymes (New England Biolabs, USA). Blots of putative gene-silenced mutants were prepared using DNA digested with either Sacl (single digest) or Sacl plus Kpnl (double digest) to confirm mutant copy number by cleaving the hyg probe. Likewise, blots of putative overexpression mutants were prepared using DNA digested with either Smal (single digest) or Smal plus Pmll (double digest) both of which cleave the bar probe. Digests were electrophoresed through 0.8% (wt 30 vol ¹ TAE) agarose gels (at 30v; overnight) and blotted onto reinforced nitrocellulose membrane (Optitran BA-S85, Schleich & Schuell, Germany) as described by Sambrook et al. (2001 ). A 747bp fragment of the hyg gene and a 433bp fragment of the bar gene (amplified by PCR as described above) were respectively used as probes for Southern blot analysis of putative gene silenced and overexpression mutants. The AlkPhos Direct Labeling and Detection System with CDP-Star (GE Healthcare, USA) were used for labelling and detecting the 747bp hyg and bar probes. The hybridisation and detection procedures were performed according to the manufacturer's instructions.

Gene cloning and 5 protein expression

The glutathione S-transferase (GST) Gene Fusion System (GE Healthcare, UK) and pGex- 6P1 expression vector equipped with a glutathione S-transferase (GST) tag was used to purify the NDPSE protein as per the manufacturer's instructions. The 954bp fragment containing the complete NDPSE gene was amplified as described above and was PCR purified (Qiagen, Netherlands). Products and plasmid (pGex-6P1 ) were digested with restriction endonucleases BamHI and EcoRI (New England Biolabs, USA), ligated (T4 DNA ligase, Promega, USA) and the correct alignment and the absence of any mutation of the gene was confirmed by sequencing of the PCR product using gene-specific primers FL NDPSE-F/R (see Table 2). The pGex-6p1 -NDPSE expression vector was cloned into E. coli strain BL21 using the pGEM- T Easy "TA" cloning kit (Promega, USA) according to the manufacturers' protocol. Colonies containing the pGex-6p1 -NDPSE vector were identified following selection on LB media (Oxoid, UK) containing ^" l OC^gml ^"1 ampicillin. Isolated colonies were subsequently grown in 100ml of LB media (37 ^< C, 200rpm) containing 100μgml ^~ ampicillin until such time as the A600 reached 0.6-0.8. For induced cultures, expression of the GSJ-NDPSE fusion protein was induced by20 addition of isopropyl-1 -thio-B-D-galactopyranoside (IPTG) (0.5mM) and the culture was incubated for a further 3h as above. Bacterial cells were pelleted via centrifugation and the cytosolic fraction was obtained by using the Bacterial Protein Extraction Reagent (Thermoscientific,USA). Fusion proteins were purified using GST-GraviTrap (GE Healthcare, UK) columns as per the manufacturer's instructions.

Analysis of enzyme activity

The deduced NDPSE protein was tested for uridine diphosphogalactose-4-epimerase activity using an NADH-coupled reaction previously described by Wilson & Hogness (1964). Uridine diphosphoglucose, the product of epimerization is instantly converted to UDP-glucuronic acid by coupling the reaction with NAD+ (Product no. BIB301 1 ; Apollo Scientific, UK) and UDP30 glucose dehydrogenase (Cat no. U6885-1 VL Sigma, Germany). The enzyme reaction mixture contained 0.1 M glycylglycine buffer (pH 8.8), 0.25mM NAD+, 0.16 units of UDP-glucose dehydrogenase and 0^g of epimerase. Purified UDP-Gal-4-epimearse (Cat no. U3251 ; Sigma, Germany) was used in the case of the positive control. The reaction was performed at 25 ^< C and initiated by adding 0.35mM UDP-galactose (Cat no. 94333; Sigma, Germany) and samples were taken every minute for 5 minutes. The reaction was stopped by putting the samples on ice and the increase in optical density due to the production of NADH was quantified at 340nm on a Spectramax 340PC (Molecular Devices, USA) 5 spectrophotometer. One unit of enzyme is defined as that amount which catalyses the formation of 1 μηιοΐβ of UDP-glucose per minute in this assay solution. This experiment was based on one replica trial, including one technical replica reaction per purified protein per time point.

Statistical analysis All RT-PCR data and bioethanol yield data from galactose experiments were non-normally distributed, as determined using the Ryan Joiner test (Ryan & Joiner, 1983) within Minitab (Minitab release 16©, 2000 Minitab Inc.). All other data sets were normally distributed. All data except the temporal analysis of NDPSE transcript accumulation (24h & 96h time points), could be transformed to fit a normal distribution using the Johnson transformation (Ryan & Joiner, 1983) or using a Box-Cox transformation in the case of bioethanol yield data from galactose within Minitab (Minitab release 16©, 2000 Minitab Inc.). The Kruskal-Wallis test was employed to analyse non-normally distributed data that could not be transformed to fit a normal distribution (temporal analysis of NDPSE transcript accumulation (24 & 96h timepoints). The homogeneity of data sets across replicate experiments was confirmed by two tailed correlation analysis (non-normal data: Spearman Rank; normal data: Pearson product moment) conducted within the Statistical Package for the Social Sciences (SPSS 1 1 .0, SPSS Inc.) (r≥0.588; P =0.05) (Snedecor & Cochran, 1980). Therefore, data sets from the replicate experiments were pooled for the purposes of further statistical analysis. The significance of treatment effects was analysed within the Statistical Package for the Social Sciences (SPSS 1 1 .0, SPSS Inc.) by either (i) normally distributed data - oneway ANOVA with Post Hoc pair wise Tukey's procedure comparisons (P = 0.05), or (ii) non-normally-distributed data - the Kruskal-Wallis H test (Snedecor & Cochran, 1980). Correlations between mean values from different normally-distributed data sets were calculated using Pearson product moment analysis. Method for isolating NDPSE enzyme

The conserved domain sequence of NDPSE is cloned into the pGEX vector as a GST fusion protein. The vector is transformed into Escherichia coli using the pGEX protein expression. Recombinant E. coli is cultured in medium (YTA) and lysates are analysed for protein expression via western blot analysis using anti-GST antibody. Protein is then purified by affinity chromatography using GST trap columns. Purified protein is visualized via western blot analysis. GST is cleaved from the purified protein by enzymatic cleavage. NDPSE protein is then quantified and the enzyme activity (NDPSE activity) is determined as per the fungal protein extracts. RESULTS

Cloning and characterization of the F. oxysporum NDPSE gene

Based on sequence analysis of the full-length NDPSE mRNA, the ORF was determined to be 954 nucleotides. NCBI domain analysis indicated that the protein contained a NADB Rossman superfamily conserved domain and NADH(P) binding domain. The crystal 5 structure of the deduced amino acid sequence highlighted 9 helixes (Figure 2). The deduced amino acid sequences showed homology with only one different hypothetical protein (FOXG_04372) encoded within the F. oxysporum f. sp. lycopersici (strain 4287) genome ( h tt p : ^{' '} www . b ro ad i n s t i t u te . o rg ^' ) . There were no characterised homologs of this protein within the wider Fusarium sp. genome. A global sequence similarity search of the F. oxysporum

NDPSE protein sequence against the NCBI protein dataset indicated that the homologs of this protein sequence are only present in bacteria and fungi. Phylogenetic tree analysis did not place NDPSE amongst other known UDP-galactose-4-epimerases, instead NDPSE was shown to be phylogenetically distinct (Fig. 3). Temporal accumulation of NDPSE transcript during CBP of wheat straw/bran

The expression levels of the NDPSE transcript were analysed in F. oxysporum strain 1 1 C and 7E during CBP of wheat straw/bran. The NDPSE transcript was identified via Real Time PCR as being up-regulated in F. oxysporum strain 1 1 C when compared to 7E during the CBP of wheat straw/bran. Real time RT-PCR was used to analyse the temporal accumulation of the NDPSE transcript in these two strains of F. oxysporum during the aerobic growth phase of the CBP (24-96h post-inoculation) of wheat straw/bran, relative to that of the housekeeping gene β-tubulin.

Transcript levels peaked at 48-72h post-inoculation, declining greatly by 96h. NDPSE transcription was highly up-regulated in F. oxysporum strain 1 1 C as compared to 7E at all 25 times, as determined by RT-PCR analysis (P<0.05). This is illustrated by Figure 1 . Expression in strain 1 1 C varied from 73.7 to 9.6 fold higher than in strain 7E (at 48h and 96h, respectively).

Gene silenced and overexpression mutants

Post transcriptional gene silencing (PTGS) and gene overexpression via fungal transformation were used to respectively silence and up-regulate NDPSE in F. oxysporum strain 1 1 C. Transformation with empty vector pSilent-1 or with pSilent-1 -NDPSE (Figure 12) was confirmed by PCR analysis of the hyg gene and by Southern analysis using a PCR-amplified segment of the hyg gene as a probe. This is illustrated by Figure 4 A and 4B, respectively. Southern hybridisation confirmed that all the silencing mutants contained a single copy of the vector integrated into the genomic DNA. Real time RT-PCR analysis of NDPSE transcript levels in fungi cultured for 24h on straw/bran under aerobic conditions confirmed the efficacy of gene silencing (Fig. 6A). There was a 15 & 7-fold decrease in the expression level of this gene in the two Λ/DPSE-silenced mutants (pSilent-1 -NDPSE-1 and pSilent-1 -NDPSE-5) as compared to the wild type strain 1 1 C and the mutant (pSilent-1 -1 ) transformed with the empty vector.

Transformation with empty over expression vector pBARGPEI or with pBARGPEI -NDPSE (Figure 13) was confirmed by PCR analysis of the bar gene (Fig. 5A) and southern blot analysis (Fig 5B). Southern hybridisation confirmed that all the mutants contained a single copy of the vector integrated into the genomic DNA. The efficacy of gene over expression was verified by real time RT-PCR analysis of the NDPSE transcript levels in fungi cultured for 24h on straw/bran under aerobic conditions. With regards to the overexpression mutants, there was almost a 30-fold increase in the NDPSE transcript levels in mutant's pBARGPEI - NDPSE-6 and pBARGPEI -NDPSE-13 as compared to the wild type strain 1 1 C or the mutant (pBARGPEI -1 ) transformed with the empty vector (Fig 6B).

The contribution of the NDPSE gene to lignocellulose bioconversion by F. oxysporum

Silencing of NDPSE resulted in significant reductions in the amount of bioethanol produced by strain 1 1 C following CBP of a straw/bran mix (P<0.05) (Fig. 7A). PTGS mutants pSilent-1 - NDPSE-1 and pSilent-1 -NDPSE-5 yielded 25 and 21 .3% less bioethanol respectively from straw/bran when compared to the wild type strain 1 1 C or a mutant strain transformed with the empty vector (P≤0.05). There was a high correlation between the level of NDPSE transcript accumulation and bioethanol productivity (r=0.613; n=6, P<0.05). Gene silencing did not show any significant effect on fungal biomass (P≥0.05). Gene over expression via fungal transformation was used to up-regulate the function of NDPSE in F. oxysporum strain 1 1 C. Over expression of the NDPSE gene increased the bioethanol yield by the fungus following CBP of a straw/bran mix by almost 18.2 to 19.1 % in the case of the 30 two mutants, pBARGPEI -NDPSE-3 and pBARGPEI -NDPSE -9, compared to the wild type strain 1 1 C or a mutant strain transformed with the empty vector (Fig. 7B). Like the silencing mutants, there was also a correlation between the level of transcript accumulation and bioethanol production (r = 0.588; n=6; P<0.05).

Effect of NDPSE on bioethanol yield from C5 and C6 sugars

The effect of NDPSE on F. oxysporum's capacity to ferment hexose and pentose sugars to bioethanol was evaluated in silenced and overexpressing mutants, relative to wild type fungus and control mutants transformed with the empty silencing (pSilentl -1 ) and overexpressing (pBARGPEM ) vector. The overexpression mutant, pBARGPE1 -/VDPS£-3 and produced a significantly higher yield of bioethanol from galactose (P<0.05) (Fig. 9), but not from glucose (P> 0.05) (Fig. 8A), cellulose (P>0.05) (Fig. 8B) or xylose (P≥0.05) (Fig. 8C) when compared to either the wild type strain 1 1 C or the mutant transformed with the empty vector (pBARGPEI - 1 )-

Purification of the GST-NDPSE fusion protein and western blotting

The GST-NDPSE fusion protein and GST-tag fusion protein were generated via the glutathione S-transferase (GST) Gene Fusion System (GE Healthcare, UK) and purified using the GST15 GraviTrap system. Subsequent western blotting was used to confirm the purification of the GST-NDPSE fusion protein (Fig. 10). A band of 60kDa which corresponds to the GST-NDPSE fusion protein was detected in the GST-NDPSE induced sample only. A band of 45.3kDa was detected which may be the homodimeric form of the GST-NDPSE fusion protein. The intensity of the band corresponding to the full size GST-NDPSE (60kDa) is faint with a number of lower molecular weight bands below it suggesting possible degradation of the GST-NDPSE fusion protein, a known undesirable feature of GST-tagged fusion proteins (ThermoFisher Scientific, 2015). The 26kDa GST tag was detected in all samples.

Determining if NDPSE has uridine-diphosphogalactose-4-epimerase activity through a specific enzyme activity assay The deduced NDPSE protein was tested for uridine diphosphogalactose-4-epimerase activity using an NADH-coupled reaction previously described by Wilson & Hogness (1964). The GST- NDPSE fusion protein's activity exhibited a similar trend to that of the UDP galactose-4- epimerase positive control, peaking in activity at 4 and 3 minutes respectively after addition of UDP-galactose and both showing a sudden rapid decline in activity at 5 minutes (Fig. 1 1 ). The highest level of enzyme activity per mg of protein recorded for the GST-NDPSE fusion protein was 385.33μηιοΙ min ^_ mg ^"1 occurred after 4 minutes. Conversely, the purified pGex-6p1 empty vector (EV) purified protein (negative control) showed little or no enzyme activity throughout.

DISCUSSION The current inventors have shown that NDPSE influences the CBP efficacy of Fusarium oxysporum. PGTS mutants with the NDPSE gene silenced showed a significant decline in their ability to produce bioethanol from a wheat straw/bran mix. Overexpression mutants showed a greater capacity to metabolize plant-derived galactose in lignocellulosic substrate. Overexpression of the NDPSE protein improved the maximum theoretical yield of bioethanol from 38 to 89% in the case of galactose. This yield is higher than other maximum theoretical yields that have been reported for various mutants of the most widely used microorganism for industrial bioethanol production Saccharomyces cerevisiae. Thus, NDPSE protein enables a more efficient and productive method of producing bioethanol.

The invention is not limited to the embodiments hereinbefore described but may be varied in both construction and detail.