# Patent

## Title
Cellulose and ligno-cellulose active proteins

## Abstract

Methods to digest carbohydrates, especially lignocelluloses and hemicelluloses, using fungal proteins previously not recognized as having this activity are described.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a divisional of copending U.S. Ser. No. 13/257,255 having an international filing date of 17 Mar. 2010, which is the national phase of PCT application PCT/NL2010/050138 having an international filing date of 17 Mar. 2010, which claims benefit of European application No. 09155380.0 filed 17 Mar. 2009. The contents of the above patent applications are incorporated by reference herein in their entirety.


REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB
The entire content of the following electronic submission of the sequence listing via the USPTO EFS-WEB server, as authorized and set forth in MPEP §1730 II.B.2(a)(C), is incorporated herein by reference in its entirety for all purposes. The sequence listing is identified on the electronically filed text file as follows:












File Name
Date of Creation
Size (bytes)







313632012110CorrectedSeqList.txt
Jul. 1, 2014
267,079 bytes









FIELD OF THE INVENTION
This invention is related to the field of enzymatic digest of carbohydrate polymers, more specifically enzymatic modification, conversion and degradation of lignocellulose and (hemi-)cellulose containing substrates.
BACKGROUND ART
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 glucans. These complex polymers are often referred to collectively as lignocellulose. Cellulose is a linear polysaccharide composed of glucose residues linked by beta-1,4 bonds. The linear nature of the cellulose fibers, as well as the stoichiometry of the beta-linked glucose (relative to alpha) generates structures more prone to interstrand hydrogen bonding than the highly branched alpha-linked 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 beta-1,4-linked xylose, a five carbon sugar. However, this xylose is often branched as beta-1,3 linkages, and can be substituted with linkages to arabinose, galactose, mannose, glucuronic acid, or by esterification to acetic acid. Hemicellulose can also contain glucan, which is a general term for beta-linked six carbon sugars. The composition, nature of substitution, and degree of branching of hemicellulose is very different in dicot plants as compared to monocot plants. In dicots, hemicellulose is comprised mainly of xyloglucans that are 1,4-beta-linked glucose chains with 1,6-beta-linked xylosyl side chains. In monocots, including most grain crops, the principle components of hemicellulose are heteroxylans. These are primarily comprised of 1,4-beta-linked xylose backbone polymers with 1,3-beta linkages to arabinose, galactose and mannose as well as xylose modified by ester-linked acetic acids. Also present are branched beta glucans comprised of 1,3- and 1,4-beta-linked glucosyl chains. In monocots, cellulose, heteroxylans and beta glucans are present in roughly equal amounts, each comprising about 15-25% of the dry matter of cell walls.
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, and fuels. Current processes to generate soluble sugars from lignocellulose are complex. A key step in the process is referred to as pretreatment. The aim of pretreatment is to increase the accessibility of cellulose to cellulose-degrading enzymes, such as the cellulase mixture derived from fermentation of the fungus Trichoderma reesei. Current pretreatment processes involve steeping lignocellulosic material such as corn stover in strong acids or bases under high temperatures and pressures. Such chemical pretreatments degrade hemicellulose and/or lignin components of lignocellulose to expose cellulose, but also create unwanted by-products such as acetic acid, furfural, hydroxymethyl furfural and gypsum. These products must be removed in additional processes to allow subsequent degradation of cellulose with enzymes or by a co-fermentation process known as simultaneous saccharification and fermentation (SSF). The conditions currently used for chemical pretreatments require expensive reaction vessels, and are energy intensive. Chemical pretreatment occurring at high temperatures and extreme pH conditions (for example 160° C. and 1.1% sulfuric acid at 12 atm. pressure) are not compatible with known cellulose-degrading enzymes. Further, these reactions produce compounds that must be removed before fermentation can proceed. As a result, chemical pretreatment processes currently occur in separate reaction vessels from cellulose degradation, and must occur prior to cellulose degradation.
Thus, methods that are more compatible with the cellulose degradation process, do not require high temperatures and pressures, do not generate toxic waste products, and require less energy, are desirable. For these reasons, efficient methods are needed for biomass conversion.
Filamentous fungi are efficient producers of a large variety of enzymes, and, therefore, they are exploited already for decades for the production of enzymes at industrial scale. Numerous hydrolytic activities have been identified for hydrolysis of starch, (hemi)cellulose and inulin. For many of these enzymes industrial processes have been developed.
Based on extensive research on these carbohydrolytic enzymes, besides catalytic domains also domains involved in substrate binding have been identified. For fungal enzymes in particular, most of the lignocellulose and (hemi-)cellulose degrading enzymes are characterized by having a cellulose binding domain, denominated as CBM1 (see also www.cazy.org/fam/acc_CBM.html. Interestingly, in particular for CBM1, which is unique to fungi, proteins with completely different catalytic activities have been identified. Besides different types of (hemi)cellulases, xylanases, pectinases, esterases, chitinases and lipases amongst others also CBM-1 proteins with unknown activity have been identified. The largest gene family of this latter class is the GH61 protein/gene family. However, there is still need for further enzymes involved in lignocellulose and (hemi-)cellulose degradation.
SUMMARY OF THE INVENTION
The inventors have now discovered two novel gene families of lignocellulose active enzymes, sharing a hitherto unknown domain (sometimes in addition to a CBM1 domain). Therefore the invention comprises a lignocellulose and/or (hemi-)cellulose active protein comprising the domain with the amino acid sequence







(SEQ ID NOS: 1-2)


[DN]-P-[IVL]-[MAIV]-X-[PAF]-[GNQ]-X3−4-[SAP]-X1−2-


 


H-X-H-X3-G-X16−21-C-[ST]-[ST]-X5-D-X-S-[AN]-Y-[YW]-


 


X-[AP]-X-[LVM]-X2−9-G






or a sequence that has an identity of more than 70%, preferably more than 75%, more preferably more than 80%, more preferably more than 85%, more preferably more than 90%, more preferably more than 95%, more preferably more than 98% with said amino acid sequence. Preferably said protein comprises the sequence DPLVFPGAM QSPHVHQIVG GNMFNVTMDP NRHNIGEEAT CTTCTFSEDF SNYWTAILYF RARNGTLIRV PQRPNIDFDG ARGGGMTVYY TATYQNHKPT AFQPGFRMIV GNPMYRTQAE ASRYRQMTFT CLETLSTRTG ETTEMPKQPC REGIMSNVRF PTCWDGKTLD PPDHSSHVAY PSSGTFESGG PCPASHPVRI PQLFYEVLWD TRRFNDRSLW PEDGSQPFVW SYGDYTGYGT HGDYVFGWKG I, (SEQ ID NO:4) or, alternatively, the sequence GAPSVHAVLR FSCSELVTER LDPLVFPGAM QSPHVHQIVG GNMFNVTMDP NRHNIGEEAT CTTCTFSEDF SNYWTAILYF RARNGTLIRV PQRPNIDFDG ARGGGMTVYY TATYQNHKPT AFQPGFRMIV GNPMYRTQAE ASRYRQMTFT CLETLSTRTG ETTEMPKQPC REGIMSNVRF PTCWDGKTLD PPDHSSHVAY PSSGTFESGG PCPASHPVRI PQLFYEVLWD TRRFNDRSLW PEDGSQPFVW SYGDYTGYGT HGDYVFGWKG DSLQRAMDAN CDFYCPQLKT QSIATGNQCR QNQKVAENID1 GPFDRLPGNV EITGPQPGAS (SEQ ID NO:5)
or a sequence that has an identity of more than 70%, preferably more than 75%, more preferably more than 80%, more preferably more than 85%, more preferably more than 90%, more preferably more than 95%, more preferably more than 98% with said amino acid sequences. Said protein is preferably selected from the group consisting of the proteins with the NCBI accession no. XP—001907658.1 (SEQ ID NO:16), XP—001904981.1 (SEQ ID NO:17), XP—001911253.1 (SEQ ID NO:18), XP—001911467.1 (SEQ ID NO:19), XP—001908261.1 (SEQ ID NO:20), XP—001907671.1 (SEQ ID NO:21), XP—001906312.1 (SEQ ID NO:22), XP—001912166.1 (SEQ ID NO:23), XP—001904033.1 (SEQ ID NO:24), XP—001905336.1 (SEQ ID NO:25), XP—001904002.1 (SEQ ID NO:26), XP—001905175.1 (SEQ ID NO:27), XP—001911617.1 (SEQ ID NO:28), XP—001907672.1 (SEQ ID NO:29), XP—001903756.1 (SEQ ID NO:30), XP—001903833.1 (SEQ ID NO:31), XP—001904389.1 (SEQ ID NO:32), XP—001904303.1 (SEQ ID NO:33), XP—001903094.1 (SEQ ID NO:34), XP—001904583.1 (SEQ ID NO:35), XP—001904957.1 (SEQ ID NO:36), XP—001906851.1 (SEQ ID NO:37), XP—001903754.1 (SEQ ID NO:38), XP—001911708.1 (SEQ ID NO:39), XP—001907931.1 (SEQ ID NO:40), and XP—001903118.1 (SEQ ID NO:41) from Podospora anserina, BAE61525.1 (SEQ ID NO:42), BAE54784.1 (SEQ ID NO:43) and BAE66576.1 (SEQ ID NO:44) from Aspergillus oryzae, CAK38435.1 (SEQ ID NO:45) and CAK40357.1 (SEQ ID NO:46) from Aspergillus niger and three proteins from Trichoderma reesei (proteins 108655 (SEQ ID NO:47), 37665 (SEQ ID NO:48) and 102735 (SEQ ID NO:49) from the T. reesei protein database at JTI, genome.jgi-psf.org/Trire2/Trire2.home.html). Also preferred is a protein according to the invention that additionally comprises a CBM1 domain, preferably wherein said CBM1 domain comprises the consensus sequence C-G(2)-X(4-7)-G-X(3)-C-X(4,5)-C-X(3-5)-[NHGS]-X-[FYWMI]-X(2)-Q-C (SEQ ID NO:9), more preferably wherein said protein is the protein from Podospora anserina with the NCBI accession no. CAP68330.1 (SEQ ID NO:81).
In another embodiment, the invention comprises a lignocellulose and/or (hemi-)cellulose active protein comprising the domain with the amino acid sequence [GA]-[ST]-[IV]-[ILV]-W-[DS]-G-[RIFS]-F-[ND]-[DS]-X2-[TS]-X2-D-[LIF]-[ND]-K-W-S-W-[GSA]-N-Q-[IV]-[GP]-[PS]-[YW]-X0-4-Q-[YW]-Y-I-H-G-S-X2-[VT]-X2-Y-[ILV]-X[ILV]-S-X2-[FY]-K-N-P-X5-7-Q-G-X-[KR]-I-T-[LI]-D-X-[ST]-[AS]-X-W-N-G-Q-[NT]-M-X-R-[IST]-E-L-I-P-Q-T-X6-13-G-X-[KLV]-[FY]-Y-H-F-S-[ILV]-X5-N-A-P-X4-E-H-Q-[ILV]-[AC]-F-F-E-X0-13-S-H-F-T-E-[LM]-K-[YST]-G-W-X0-2-G-X6-33-[LF]-X1-24-I-D-F-[ASD]-X3-8-V-[FL]-[FWY]-X-S-[ENT]-G-X2-5-[AP]-L-X2-4-[AV]-[AV]-X-[PAN]-X3-5-[ANS]-[AT]-[AFS]-[ST]-[DN]-[GS]-[AQ]-D-[FW]-H-{FILV}-G-[EIQV]-L-[ERK]-[ILV]-P-X8-18-E-D-[FWY]-[FY]-[FW]-S-G-[IV]-[FY]-[IV]-E (SEQ ID NOS:6-7) or a sequence that has an identity of more than 70%, preferably more than 75%, more preferably more than 80%, more preferably more than 85%, more preferably more than 90%, more preferably more than 95%, more preferably more than 98% with said amino acid sequence. Preferably such a protein comprises the sequence GT ILWDGRFNDM TSSADLNKWS WGNQVGPYQY YIHGSSPVSA YVNLSPDYKN PADTGSRQGA KITLDNTAYW NGQNMRRTEL IPQTTAAINQ GKVYYHFSLM RKDINAPATT REHQIAFFES HFTELKSGWL SGAPGISDTL LRWCIDFAAG TVGFWHSTGS DPLTRKVAPV KTSTSSNGAD WHVGVLELPR SGYPDSNEDF YWSGVYIESG SLTTSVAGPG QPIPGDGG (SEQ ID NO:3) or a sequence that has an identity of more than 70%, preferably more than 75%, more preferably more than 80%, more preferably more than 85%, more preferably more than 90%, more preferably more than 95%, more preferably more than 98% with said amino acid sequence. Preferably said protein is selected from the group consisting of the proteins from Podospora anserina with the NCBI accession numbers XP—001903534.1 (CAP61309.1) (SEQ ID NO:50) and XP—001907960.1 (CAP68633.1) (SEQ ID NO:51), from Aspergillus flavus (EED52126.1 (SEQ ID NO:52) and EED54304.1 (SEQ ID NO:53)), from Aspergillus fumigatus (XP—751054.2 (SEQ ID NO:54), XP—755877.1 (SEQ ID NO:55) and EDP49742.1 (SEQ ID NO:56)), Aspergillus clavatus (XP—001275827.1) (SEQ ID NO:57), Aspergillus oryzae (XP—001825707.1) (SEQ ID NO:58), Aspergillus terreus (XP—001211584.1) (SEQ ID NO:59), Aspergillus nidulans (XP—680867.1) (SEQ ID NO:60), Aspergillus niger (XP—001392581.1) (SEQ ID NO:61), Magnaporthe griseae (XP—362641.1 (SEQ ID NO:62) and XP—001408874.1 (SEQ ID NO:63)), Phaeosphaeria nodorum (XP—001793212.1 (SEQ ID NO:64) and XP—001799980.1 (SEQ ID NO:65)), Neurospra crassa (XP—958348.1 (SEQ ID NO:66) and XP—956768.1 (SEQ ID NO:67)), Pyrenophora tritici-repentis (XP—001932168.1 (SEQ ID NO:68) and XP—001931381.1 (SEQ ID NO:69)), Neosartorya fischeri (XP—001258287.1 (SEQ ID NO:70) and XP—001261005.1 (SEQ ID NO:71)), Chaetomiun globosum (XP—001228503) (SEQ ID NO:72), Botryotinia fuckeliana (XP—001546653.1) (SEQ ID NO:73), Sclerotinia sclerotiorum (XP—001593519.1) (SEQ ID NO:74), Moniliophthora perniciosa (EEB91913.1) (SEQ ID NO:75) and Coprionopsis cinerea (XP—001835742) (SEQ ID NO:76).
In another embodiment, said protein additionally comprises a CBM1 domain, and is preferably selected from the group consisting of the proteins with NCBI accession no. CAP61309.1 (SEQ ID NO:77), BAE64574.1 (SEQ ID NO:78) and CAK45436.1 (SEQ ID NO:79).
In a preferred embodiment the protein from the invention is from fungal origin, preferably wherein the fungus is chosen from the group consisting of Aspergillus, Neurospora, Sclerotina, Gibberella, Coniothyrium, Psiticum, Magnaporthe, Podospora, Chaetomium, Phaeosphaeria, Botryotinia, Neosartorya, Pyrenophora, Panicum, Aureococcus, Trichoderma Penicillium and Chrysosporium. 
Also part of the invention is a nucleic acid encoding a protein according to the invention, a vector comprising said nucleic acid and a host cell capable of expressing a protein according to the invention by harbouring said nucleic acid or vector.
Further, also comprised in the invention is the use of a protein according to the invention in the modification of a raw carbohydrate, preferably lignocellulose and/or (hemi-)cellulose, most preferably non-soluble cellulose, preferably wherein the protein has or enhances cellulase activity.



LEGENDS TO THE FIGURES
FIG. 1. SDS PAGE of proteins produced by A. niger transformants transgenic for CAP68330 and CAP61309. M indicates marker lane.



DETAILED DESCRIPTION
The term “sequence identity,” as used herein, is generally expressed as a percentage and refers to the percent of amino acid residues or nucleotides, as appropriate, that are identical as between two sequences when optimally aligned. For the purposes of this invention, sequence identity means the sequence identity determined using the well-known Basic Local Alignment Search Tool (BLAST), which is publicly available through the National Cancer Institute/National Institutes of Health (Bethesda, Md.) and has been described in printed publications (see, e.g., Altschul et al., J. MoI. Biol, 215(3), 403-10 (1990)). Preferred parameters for amino acid sequences comparison using BLASTP are gap open 11.0, gap extend 1, Blosum 62 matrix. Protein analysis software matches similar sequences using measures of homology assigned to various substitutions, deletions and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
The term “signal peptide” or “signal sequence” as used herein, refers to an amino acid sequence, typically located at the amino terminus of an immature protein or polypeptide (e.g., prior to secretion from a cell and associated processing and cleavage), which directs the secretion of the protein or polypeptide from the cell in which it is produced. The signal peptide typically is removed from an immature protein or polypeptide prior to or during secretion and, thus, is not present in the mature, secreted polypeptide.
As used herein, the term “recombinant nucleic acid molecule” refers to a recombinant DNA molecule or a recombinant RNA molecule. A recombinant nucleic acid molecule is any synthetic nucleic acid construct or nucleic acid molecule containing joined nucleic acid molecules from different original sources or and not naturally occurring or attached together and prepared by using recombinant DNA techniques.
The term “recombinant host cell” as used herein, refers to a host cell strain containing nucleic acid not naturally occurring in that strain and which has been introduced into that strain using recombinant DNA techniques.
The term “nucleic acid” as used herein, includes reference to a deoxyribonucleotide or ribonucleotide polymer, i.e. a polynucleotide, in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues having the essential nature of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides (e.g., peptide nucleic acids). A polynucleotide can be full-length or a subsequence of a native or heterologous structural or regulatory gene. Unless otherwise indicated, the term includes reference to the specified sequence as well as the complementary sequence thereof. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are “polynucleotides” (a polymeric form of nucleotides, either ribonucleotides or deoxyribonucleotides, double- or single-stranded of any length) as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term polynucleotide as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including among other things, simple and complex cells.
Every nucleic acid sequence herein that encodes a polypeptide also, by reference to the genetic code, describes every possible silent variation of the nucleic acid. The term “conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refer to those nucleic acids which encode identical or conservatively modified variants of the amino acid sequences due to the degeneracy of the genetic code.
The term “degeneracy of the genetic code” refers to the fact that a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations” and represent one species of conservatively modified variation.
The term “gene”, as used herein, refers to a nucleic acid sequence containing a template for a nucleic acid polymerase, in eukaryotes, RNA polymerase II. Genes are transcribed into mRNAs that are then translated into protein.
“Expression” refers to the transcription of a gene into structural RNA (rRNA, tRNA) or messenger RNA (mRNA) with subsequent translation into a protein.
The term “complementary”, as used herein, refers to a sequence of nucleotides which forms a hydrogen-bonded duplex with another sequence of nucleotides according to Watson-Crick base-paring rules. For example, the complementary base sequence for 5′-AAGGCT-3′ is 3′-TTCCGA-5′. As used herein, “substantially complementary” means that two nucleic acid sequences have at least about 40, preferably about 50% more preferably at least 55%, more preferably about 60%, more preferably about 70%, more preferably about 80%, even more preferably 90%, and most preferably about 98%, sequence complementarity to each other.
The term “hybridise” refers to the process by which single strands of nucleic acid sequences form double-helical segments through hydrogen bonding between complementary nucleotides.
As used herein, the term “expression control sequence” refers to a nucleic acid sequence that regulates the transcription and translation of a gene to which it is operatively linked. An expression control sequence is “operatively linked” to a gene when the expression control sequence controls and regulates the transcription and, where appropriate, translation of the gene. The term “operatively linked” includes the provision of an appropriate start codon (e.g. ATG), in front of a polypeptide-encoding gene and maintaining the correct reading frame of that gene to permit proper translation of the mRNA.
As used herein, the term “operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. A control sequence “operably linked” to another control sequence and/or to a coding sequence is ligated in such a way that transcription and/or expression of the coding sequence is achieved under conditions compatible with the control sequence. Generally, operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame.
The term “vector” as used herein, includes reference to an autosomal expression vector and to an integration vector used for integration into the chromosome.
The term “expression vector” refers to a DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of interest under the control of (i.e., operably linked to) additional nucleic acid segments that provide for its transcription. Such additional segments may include promoter and terminator sequences, and may optionally include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. In particular an expression vector comprises a nucleotide sequence that comprises in the 5′ to 3′ direction and operably linked: (a) a fungal-recognized transcription and translation initiation region, (b) a coding sequence for a polypeptide of interest, and (c) a fungal-recognized transcription and translation termination region. “Plasmid” refers to autonomously replicating extrachromosomal DNA which is not integrated into a microorganism's genome and is usually circular in nature.
An “integration vector” refers to a DNA molecule, linear or circular, that can be incorporated in a microorganism's genome and provides for stable inheritance of a gene encoding a polypeptide of interest. The integration vector generally comprises one or more segments comprising a gene sequence encoding a polypeptide of interest under the control of (i.e., operably linked to) additional nucleic acid segments that provide for its transcription. Such additional segments may include promoter and terminator sequences, and one or more segments that drive the incorporation of the gene of interest into the genome of the target cell, usually by the process of homologous recombination. Typically, the integration vector will be one which can be transferred into the host cell, but which has a replicon which is nonfunctional in that organism. Integration of the segment comprising the gene of interest may be selected if an appropriate marker is included within that segment.
“Transformation” and “transforming”, as used herein, refers to the insertion of an exogenous polynucleotide into a host cell, irrespective of the method used for the insertion, for example, direct uptake, transduction, f-mating or electroporation. The exogenous polynucleotide may be maintained as a non-integrated vector, for example, a plasmid, or alternatively, may be integrated into the host cell genome.
By “host cell” is meant a cell which contains a vector or recombinant nucleic acid molecule and supports the replication and/or expression of the vector or recombinant nucleic acid molecule. Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, fungus, plant, insect, amphibian, or mammalian cells. Preferably, host cells are fungal cells.
The term “fungus” or “fungi” includes a wide variety of nucleated, spore-bearing organisms which are devoid of chlorophyll. Examples of fungi include yeasts, mildews, molds, rusts and mushrooms. Preferred fungi in aspects of the present invention are organisms of the genera Aspergillus, Neurospora, Sclerotina, Gibberella, Coniothyrium, Psiticum, Magnaporthe, Podospora, Chaetomium, Phaeosphaeria, Botryotinia, Neosartorya, Pyrenophora, Panicum, Aureococcus Penicillium and Chrysospsorium. 
The terms “isolated” or “purified” as used herein refer to a nucleic acid or protein or peptide that is removed from at least one component with which it is naturally associated. In the present invention, an isolated nucleic acid can include a vector comprising the nucleic acid. Purified as used herein to describe a polypeptide produced by cultivation of a recombinant host cell refers to removing that polypeptide from at least one component with which it is naturally associated in the host cell or culture medium.
The CBM1 domain that is present in many of the proteins that are held to have activity on cellulose, chitin, sepharose, xylan has a consensus amino acid sequence that can be denoted as:









(SEQ ID NO: 9)



C-G(2)-X(4−7)-G-X(3)-C-X(4−5)-C-X(3−5)-[NHGS]-X-



 



[FYWMI]-x(2)-Q-C






in which the amino acids between square brackets are alternatives on that position, and Xn denotes a series of n freely chosen amino acids. Alternatively, the CBM1 domain is a sequence that has a high identity with the above consensus sequence. However, no or only very limited catalytic activity has been shown to reside in or be linked to said CBM1 domain.

Now, the inventors have discovered two novel classes of starch active proteins wherein the first class shares a common domain of unknown function (D-U-F), also called the DUF1996 domain, partly represented by the consensus sequence:







(SEQ ID NOS: 1-2)


[DN]-P-[IVL]-[MAIV]-x-[PAF]-[GNQ]-X3−4-[SAP]-X1−2-


 


H-X-H-X3-G-X16−21-C-[ST]-[ST]-X5-D-X-S-[AN]-Y-[YW]-


 


X-[AP]-X-[LVM]-X2−9-G






in which the amino acids between square brackets are alternatives at the same position and Xn denotes a series of n freely chosen amino acids, or an amino acid sequence that has a high degree of identity with said consensus sequence.
An example of such a domain is the sequence:









(SEQ ID NO: 4)



DPLVFPGAM QSPHVHQIVG GNMFNVTMDP NRHNIGEEAT 



 



CTTCTFSEDF SNYWTAILYF RARNGTLIRV PQRPNIDFDG



 



ARGGGMTVYY TATYQNHKPT AFQPGFRMIV GNPMYRTQAE 



 



ASRYRQMTFT CLETLSTRTG ETTEMPKQPC REGIMSNVRF 



 



PTCWDGKTLD PPDHSSHVAY PSSGTFESGG PCPASHPVRI



 



PQLFYEVLWD TRRFNDRSLW PEDGSQPFVW SYGDYTGYGT 



 



HGDYVFGWKG I,






and also the sequence:










(SEQ ID NO: 5)



GAPSVHAVLR FSCSELVTER LDPLVFPGAM QSPHVHQIVG



 



GNMFNVTMDP NRHNIGEEAT CTTCTFSEDF SNYWTAILYF



 



RARNGTLIRV PQRPNIDFDG ARGGGMTVYY TATYQNHKPT



 



AFQPGFRMIV GNPMYRTQAE ASRYRQMTFT CLETLSTRTG



 



ETTEMPKQPC REGIMSNVRF PTCWDGKTLD PPDHSSHVAY



 



PSSGTFESGG PCPASHPVRI PQLFYEVLWD TRRFNDRSLW 



 



PEDGSQPFVW SYGDYTGYGT HGDYVFGWKG DSLQRAMDAN



 



CDFYCPQLKT QSIATGNQCR QNQKVAENID1 GPFDRLPGNV



 



EITGPQPGAS 






or an amino acid sequence that has a high degree of identity with said sequences. A high degree of identity is herein defined as an identity of more than 70%, preferably more than 75%, more preferably more than 80%, more preferably more than 85%, more preferably more than 90%, more preferably more than 95%, more preferably more than 98%. Species of proteins with this new domain are 26 proteins from Podospora anserina (accession numbers XP—001907658.1 (SEQ ID NO:16), XP—001904981.1 (SEQ ID NO:17), XP—001911253.1, (SEQ ID NO:18) XP—001911467.1 (SEQ ID NO:19), XP—001908261.1 (SEQ ID NO:20), XP—001907671.1 (SEQ ID NO:21), XP—001906312.1 (SEQ ID NO:22), XP—001912166.1 (SEQ ID NO:23), XP—001904033.1 (SEQ ID NO:24), XP—001905336.1 (SEQ ID NO:25), XP—001904002.1 (SEQ ID NO:26), XP—001905175.1 (SEQ ID NO:27), XP—001911617.1 (SEQ ID NO:28), XP—001907672.1 (SEQ ID NO:29), XP—001903756.1 (SEQ ID NO:30), XP—001903833.1 (SEQ ID NO:31), XP—001904389.1 (SEQ ID NO:32), XP—001904303.1 (SEQ ID NO:33), XP—001903094.1 (SEQ ID NO:34), XP—001904583.1 (SEQ ID NO:35), XP—001904957.1 (SEQ ID NO:36), XP—001906851.1 (SEQ ID NO:37), XP—001903754.1 (SEQ ID NO:38), XP—001911708.1 (SEQ ID NO:39), XP—001907931.1 (SEQ ID NO:40), XP—001903118.1 (SEQ ID NO:41), BAE61525.1 (SEQ ID NO:42), BAE54784.1 (SEQ ID NO:43) and BAE66576.1 (SEQ ID NO:44) from Aspergillus oryzae, CAK38435.1 (SEQ ID NO:45) and CAK40357.1 (SEQ ID NO:46) from Aspergillus niger and three proteins from Trichoderma reesei (proteins 108655 (SEQ ID NO:47), 37665 (SEQ ID NO:48) and 102735 (SEQ ID NO:49) from the T. reesei protein database at JTI, genome.jgi-psf.org/Trire2/Trire2.home.html, see Martinez, D. et al., 2008, Nature Biotechnology 26, 553-560).

This new class of proteins has been discovered in the search for proteins with CBM1 domains. It appeared that several of the proteins, especially those from fungal origin contained the above conserved DUF1996 domain next to the CBM1 domain. Further search for more proteins that also comprised the conserved DUF1996 domain has led to the proteins of the present invention. It is submitted that for all currently known proteins with said domain that are listed above and/or in the experimental part no function was hitherto known from any of these proteins. A species of a protein with both CBM1 and DUF1996 domains is CAP68330.1 from Podospora anserine (SEQ ID NO:81).
A further new class of proteins concerns proteins that have the domain with the consensus sequence: [GA]-[ST]-[IV]-[ILV]-W-[DS]-G-[RIFS]-F-[ND]-[DS]-X2-[TS]-X2-D-[LIF]-[ND]-K-W-S-W-[GSA]-N-Q-[IV]-[GP]-[PS]-[YW]-X0-4-Q-[YW]-Y-I-H-G-S-X2-[VT]-X2-Y-[ILV]-X[ILV]-S-X2-[FY]-K-N-P-X5-7-Q-G-X-[KR]-I-T-[LI]-D-X-[ST]-[AS]-X-W-N-G-Q-[NT]-M-X-R-[IST]-E-L-I-P-Q-T-X6-13-G-X-[KLV]-[FY]-Y-H-F-S-[ILV]-X5-N-A-P-X4-E-H-Q-[ILV]-[AC]-F-F-E-X0-13-S-H-F-T-E-[LM]-K-[YST]-G-W-X0-2-G-X6-33-[LF]-X1-24-I-D-F-[ASD]-X3-8-V-[FL]-[FWY]-X-S-[ENT]-G-X2-5-[AP]-L-X2-4-[AV]-[AV]-X-[PAN]-X3-5-[ANS]-[AT]-[AFS]-[ST]-[DN]-[GS]-[AQ]-D-[FW]-H-{FILV}-G-[EIQV]-L-[ERK]-[ILV]-P-X8-18-E-D-[FWY]-[FY]-[FW]-S-G-[IV]-[FY]-[IV]-E (SEQ ID NOS:6-7) or an amino acid sequence that has a high degree of identity with this sequence. Particularly, the domain comprises the sequence GT ILWDGRFNDM TSSADLNKWS WGNQVGPYQY YIHGSSPVSA YVNLSPDYKN PADTGSRQGA KITLDNTAYW NGQNMRRTEL IPQTTAAINQ GKVYYHFSLM RKDINAPATT REHQIAFFES HFTELKSGWL SGAPGISDTL LRWCIDFAAG TVGFWHSTGS DPLTRKVAPV KTSTSSNGAD WHVGVLELPR SGYPDSNEDF YWSGVYIESG SLTTSVAGPG QPIPGDGG (SEQ ID NO:3)
or an amino acid sequence that has a high degree of identity therewith. A high degree of identity is herein defined as an identity of more than 70%, preferably more than 75%, more preferably more than 80%, more preferably more than 85%, more preferably more than 90%, more preferably more than 95%, more preferably more than 98%. Species of a protein with this new domain are two proteins from Podospora anserina with accession numbers XP—001903534.1 (CAP61309.1) and XP—001907960.1 (CAP68633.1), wherein CAP61309 comprises the sequence







(SEQ ID NO: 10)









 19
GTILWDGRFNDMTSSADLNKWSWGNQVGPYQYYIHGSSP










VSAYVNLSPDYKNPADTGSRQ


 









 79
GAKITLDNTAYWNGQNMRRTELIPQTTAAINQGKVYYHF










SLMRKDINAPATTREHQIAFF


 









139
ESHFTELKSGWLSGAPGISDTLLRWCIDFAAGTVGFWHS










TGSDPLTRKVAPVKTSTSSNG


 









199 
ADWHVGVLELPRSGYPDSNEDFYWSGVYIESGSLTTSVA 
248









GPGQPIPGDGG






and CAP68633 comprises the sequence








(SEQ ID NO: 11)









 19
GAVLWDGRFNDFTSSADLNKWSWANQVGPYPFTNKEYY










IHGSGTVNRYINLSPDYKNPND


 









 79
TVSKQGARFTLDSTAYWNGQTMRRIELIPQTKAAINRG










KVFYHFSISRRDTNAPSVNKEH


 









139
QICFFESHFTELKYGWISGEQGAANPALQWMTNQRTQW










KLSEWKANVWHNFAYEIDFSGN


 









199
RVGLWYSEGGADLKQVVAPVGGVSTSSNGQDWHLGVLE










LPRSGYPNTNEDYYFSGVFIED


 









259
GAITTKIGGPGE
270






Other examples of this new class of proteins are hitherto hypothetical proteins with unknown function from Aspergillus flavus (EED52126.1 (SEQ ID NO:52) and EED54304.1 (SEQ ID NO:53)), from Aspergillus fumigatus (XP—751054.2 (SEQ ID NO:54), XP—755877.1 (SEQ ID NO:55) and EDP49742.1 (SEQ ID NO:56)), Aspergillus clavatus (XP—001275827.1) (SEQ ID NO:57), Aspergillus oryzae (XP—001825707.1) (SEQ ID NO:58), Aspergillus terreus (XP—001211584.1) (SEQ ID NO:59), Aspergillus nidulans (XP—680867.1) (SEQ ID NO:60), Aspergillus niger (XP—001392581.1) (SEQ ID NO:61), Magnaporthe griseae (XP—362641.1 (SEQ ID NO:62) and XP—001408874.1 (SEQ ID NO:63)), Phaeosphaeria nodorum (XP—001793212.1 (SEQ ID NO:64) and XP—001799980.1 (SEQ ID NO:65)), Neurospra crassa (XP—958348.1 (SEQ ID NO:66) and XP—956768.1 (SEQ ID NO:67)), Pyrenophora tritici-repentis (XP—001932168.1 (SEQ ID NO:68) and XP—001931381.1 (SEQ ID NO:69)), Neosartorya fischeri (XP—001258287.1 (SEQ ID NO:70) and XP—001261005.1 (SEQ ID NO:71)), Chaetomiun globosum (XP—001228503) (SEQ ID NO:72), Botryotinia fuckeliana (XP—001546653.1) (SEQ ID NO:73), Sclerotinia sclerotiorum (XP—001593519.1) (SEQ ID NO:74), Moniliophthora perniciosa (EEB91913.1) (SEQ ID NO:75) and Coprionopsis cinerea (XP—001835742) (SEQ ID NO:76).
This second new class of proteins has also been discovered in the search of proteins with CBM1 domains. It appeared that several of the proteins, especially those from fungal origin contained the above conserved new domain next to the CBM1 domain. Further search for more proteins that also comprised the conserved new domain has led to the proteins of the present invention. It is submitted that all currently known proteins with said domain or a domain which is highly identical thereto are listed in the experimental part and that no function was hitherto known from any of these proteins. Species of the proteins with both domains are CAP61309.1 (SEQ ID NO:77) from Podospora anserina, BAE64574.1 (SEQ ID NO:78) from Aspergillus oryzae, CAK45436.1 (SEQ ID NO:79) from Aspergillus niger and AN7598.2 from Aspergillus nidulans (SEQ ID NO:80).
The proteins of the invention are generally derived from fungi.
Also part of the invention is a nucleotide sequence encoding one or more of the lignocellulose or (hemi-)cellulose active proteins described above. Such a nucleotide sequence can be any nucleotide sequence that encodes said protein(s), but preferably it is the natural coding sequence found in the organisms from which the lignocellulose or (hemi-)cellulose active proteins are derived. However, if these nucleotide sequences are meant for expression in a different host organism, the nucleotide sequence(s) may be adapted to optimize expression is said host organism (codon optimization). For expression purposes, the nucleotide sequence is included in an expression vector that also provides for regulatory sequences, operably linked with the coding nucleotide sequence.
The proteins of the invention can be used in isolated form for addition to a raw carbohydrate (lignocellulose or (hemi-)cellulose) substrate, alone or together with other lignocellulose or (hemi-)cellulose degrading enzymes, such as (hemi)cellulase, xylanase and/or pectinase. The proteins of the invention may yield a lignocellulose or (hemi-)cellulose hydrolytic activity per se, or they increase the accessibility of the lignocellulose or (hemi-)cellulose by other lignocellulose or (hemi-)cellulose degrading enzymes.
In another embodiment, the proteins of the invention can be (over)expressed in a host cell. Overexpression of the proteins of the present invention can be effected in several ways. It can be caused by transforming a host cell with a gene coding for a protein of the invention. Alternatively, another method for effecting overexpression is to provide a stronger promoter in front of and regulating the expression of said gene. This can be achieved by use of a strong heterologous promoter or by providing mutations in the endogenous promoter. An increased expression of the protein can also be caused by removing possible inhibiting regulatory proteins, e.g. that inhibit the expression of such proteins. The person skilled in the art will know other ways of increasing the activity of the above mentioned starch active proteins.
In another aspect of the invention, host cells overexpressing at least one of the above mentioned nucleotide sequences, encoding at least one lignocellulose or (hemi-)cellulose active protein of the invention, are produced and used, for production of said protein(s).
Host cells used in the invention are preferably cells of filamentous fungi, yeasts and/or bacteria, such as, but not limited to, Aspergillus sp., such as the fungi A. terreus, A. itaconicus and A. niger, Aspergillus nidulans, Aspergillus oryzae or Aspergillus fuminagates, Trichoderma, Penicillium Chrysosporium, Ustilago zeae, Ustilago maydis, Ustilago sp., Candida sp., Yarrowia lipolytica, Rhodotorula sp. and Pseudozyma antarctica, the bacterium E. coli and the yeast Saccharomyces cerevisiae. Especially preferred are host cells that also produce one or more lignocellulose degrading enzymes, such as (hemi)cellulase, xylanase or pectinase.
Recombinant host cells described above can be obtained using methods known in the art for providing cells with recombinant nucleic acids. These include transformation, transconjugation, transfection or electroporation of a host cell with a suitable plasmid (also referred to as vector) comprising the nucleic acid construct of interest operationally coupled to a promoter sequence to drive expression. Host cells of the invention are preferably transformed with a nucleic acid construct as further defined below and may comprise a single but preferably comprises multiple copies of the nucleic acid construct. The nucleic acid construct may be maintained episomally and thus comprise a sequence for autonomous replication, such as an ARS sequence. Suitable episomal nucleic acid constructs may e.g. be based on the yeast 2μ or pKD1 (Fleer et al., 1991, Biotechnology 9: 968-975) plasmids. Preferably, however, the nucleic acid construct is integrated in one or more copies into the genome of the host cell. Integration into the host cell's genome may occur at random by illegitimate recombination but preferably the nucleic acid construct is integrated into the host cell's genome by homologous recombination as is well known in the art of fungal molecular genetics (see e.g. WO 90/14423, EP-A-0 481 008, EP-A-0 635 574 and U.S. Pat. No. 6,265,186). Most preferably for homologous recombination the ku70Δ/ku80Δ, technique is used as described for instance in WO 02/052026.
Transformation of host cells with the nucleic acid constructs of the invention and additional genetic modification of the fungal host cells of the invention as described above may be carried out by methods well known in the art. Such methods are e.g. known from standard handbooks, such as Sambrook and Russel (2001) “Molecular Cloning: A Laboratory Manual (3rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, or F. Ausubel et al, eds., “Current protocols in molecular biology”, Green Publishing and Wiley Interscience, New York (1987). Methods for transformation and genetic modification of fungal host cells are known from e.g. EP-A-0 635 574, WO 98/46772, WO 99/60102 and WO 00/37671.
In another aspect the invention relates to a vector comprising a nucleotide sequence encoding a starch active protein as defined above and usable for transformation of a host cell as defined above. In the nucleic acid construct, the coding nucleotide sequences preferably is/are operably linked to a promoter for control and initiation of transcription of the nucleotide sequence in a host cell as defined below. The promoter preferably is capable of causing sufficient expression of the starch active protein described above, in the host cell. Promoters useful in the nucleic acid constructs of the invention include the promoter that in nature provides for expression of the coding genes. Further, both constitutive and inducible natural promoters as well as engineered promoters can be used. Promoters suitable to drive expression of the genes in the hosts of the invention include e.g. promoters from glycolytic genes (e.g. from a glyceraldehyde-3-phosphate dehydrogenase gene), ribosomal protein encoding gene promoters, alcohol dehydrogenase promoters (ADH1, ADH4, and the like), promoters from genes encoding amylo- or cellulolytic enzymes (glucoamylase, TAKA-amylase and cellobiohydrolase). 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 nucleic acid constructs of the present invention may be modified, if desired, to affect their control characteristics. Preferably, the promoter used in the nucleic acid construct for expression of the genes is homologous to the host cell in which genes are expressed.
In the nucleic acid construct, the 3′-end of the coding nucleotide acid sequence(s) preferably is/are operably linked to a transcription terminator sequence. Preferably the terminator sequence is operable in a host cell of choice. In any case the choice of the terminator is not critical; it may e.g. be from any fungal gene, although terminators may sometimes work if from a non-fungal, eukaryotic, gene. The transcription termination sequence further preferably comprises a polyadenylation signal.
Optionally, a selectable marker may be present in the nucleic acid construct. As used herein, the term “marker” refers to a gene encoding a trait or a phenotype which permits the selection of, or the screening for, a host cell containing the marker. A variety of selectable marker genes are available for use in the transformation of fungi. Suitable markers include auxotrophic marker genes involved in amino acid or nucleotide metabolism, such as e.g. genes encoding ornithine-transcarbamylases (argB), orotidine-5′-decarboxylases (pyrG, URA3) or glutamine-amido-transferase indoleglycerol-phosphate-synthase phosphoribosyl-anthranilate isomerases (trpC), or involved in carbon or nitrogen metabolism, such e.g. niaD or facA, and antibiotic resistance markers such as genes providing resistance against phleomycin, bleomycin or neomycin (G418). Preferably, bidirectional selection markers are used for which both a positive and a negative genetic selection is possible. Examples of such bidirectional markers are the pyrG (URA3), facA and amdS genes. Due to their bidirectionality these markers can be deleted from transformed filamentous fungus while leaving the introduced recombinant DNA molecule in place, in order to obtain fungi that do not contain selectable markers. This essence of this MARKER GENE FREETM transformation technology is disclosed in EP-A-0 635 574, which is herein incorporated by reference. Of these selectable markers the use of dominant and bidirectional selectable markers such as acetamidase genes like the amdS genes of A. nidulans, A. niger and P. chrysogenum is most preferred. In addition to their bidirectionality these markers provide the advantage that they are dominant selectable markers that, the use of which does not require mutant (auxotrophic) strains, but which can be used directly in wild type strains.
Optional further elements that may be present in the nucleic acid constructs of the invention include, but are not limited to, one or more leader sequences, enhancers, integration factors, and/or reporter genes, intron sequences, centromers, telomers and/or matrix attachment (MAR) sequences. The nucleic acid constructs of the invention may further comprise a sequence for autonomous replication, such as an ARS sequence. Suitable episomal nucleic acid constructs may e.g. be based on the yeast 2μ or pKD1 (Fleer et al., 1991, Biotechnology 9: 968-975) plasmids. Alternatively the nucleic acid construct may comprise sequences for integration, preferably by homologous recombination (see e.g. WO98/46772). Such sequences may thus be sequences homologous to the target site for integration in the host cell's genome. The nucleic acid constructs of the invention can be provided in a manner known per se, which generally involves techniques such as restricting and linking nucleic acids/nucleic acid sequences, for which reference is made to the standard handbooks, such as Sambrook and Russel (2001) “Molecular Cloning: A Laboratory Manual (3rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, or F. Ausubel et al, eds., “Current protocols in molecular biology”, Green Publishing and Wiley Interscience, New York (1987).
In a further aspect the invention relates to fermentation processes in which the transformed host cells of the invention are used for the conversion of a lignocellulose or (hemi-)cellulose substrate. A preferred fermentation process is an aerobic fermentation process. The fermentation process may either be a submerged or a solid state fermentation process.
In a solid state fermentation process (sometimes referred to as semi-solid state fermentation) the transformed host cells are fermenting on a solid medium that provides anchorage points for the fungus in the absence of any freely flowing substance. The amount of water in the solid medium can be any amount of water. For example, the solid medium could be almost dry, or it could be slushy. A person skilled in the art knows that the terms “solid state fermentation” and “semi-solid state fermentation” are interchangeable. A wide variety of solid state fermentation devices have previously been described (for review see, Larroche et al., “Special Transformation Processes Using Fungal Spores and Immobilized Cells”, Adv. Biochem. Eng. Biotech., (1997), Vol 55, pp. 179; Roussos et al., “Zymotis: A large Scale Solid State Fermenter”, Applied Biochemistry and Biotechnology, (1993), Vol. 42, pp. 37-52; Smits et al., “Solid-State Fermentation-A Mini Review, 1998), Agro-Food-Industry Hi-Tech, March/April, pp. 29-36). These devices fall within two categories, those categories being static systems and agitated systems. In static systems, the solid media is stationary throughout the fermentation process. Examples of static systems used for solid state fermentation include flasks, petri dishes, trays, fixed bed columns, and ovens. Agitated systems provide a means for mixing the solid media during the fermentation process. One example of an agitated system is a rotating drum (Larroche et al., supra). In a submerged fermentation process on the other hand, the transformed fungal host cells are fermenting while being submerged in a liquid medium, usually in a stirred tank fermenter as are well known in the art, although also other types of fermenters such as e.g. airlift-type fermenters may also be applied (see e.g. U.S. Pat. No. 6,746,862).
Preferred in the invention is a submerged fermentation process, which is performed fed-batch. This means that there is a continuous input of feed containing a carbon source and/or other relevant nutrients in order to improve protein yields. The input of the feed can, for example, be at a constant rate or when the concentration of a specific substrate or fermentation parameter falls below some set point.
Further comprised in the invention is the use of an active protein according to the invention for modification of raw carbohydrate substrate (lignocellulose or (hemi-)cellulose). Preferably said use encompasses hydrolysis of the carbohydrate substrate. Also included in this use is the modification of the substrate by the active protein, thereby allowing other carbohydrate substrate hydrolyzing enzymes to approach the substrate more easily and exert their function in resulting in improved hydrolysis.
EXAMPLES
Examples
Expression of Genes Encoding Lignocellulose and/or (Hemi-)Cellulose Active Proteins in Aspergillus niger 
In order to unambiguously establish that the discovered proteins aid to the increased saccharification of beta-glucan containing plant derived substrates, a host naturally not expressing these genes was (co-)transformed with the respective genes under control of a suitable promoter.
(I) Gene Design
Synthetic (codon-optimized) full length gene copies from two selected Podospora anserine genes were generated
CAP 61309: CBM1 Protein
A synthetic gene was designed by back translation from the reannotated protein for further reference called CAP61309, originally deposited under number XM—001903499.1, with codon bias for Aspergillus niger. The start codon of the protein is part of a BspHI site, so as to fit to the NcoI cloning site of vector pAN52-5doubleNotamdS. At the 3′ end of the gene two consecutive stop codons were introduced, followed by a BamHI cloning site.








           KpnI BspHI










   1
GGTACCTCATGAAGTTCCACGTCCTCTCCGGCCTCGTCGCCCAGGTCCTCTCCGTTAGCG



   1
    M K F H V L S G L V A Q V L S


 


  61
CCGGCACCATTCTCTGGGATGGCCGCTTCAACGATATGACCTCCTCCGCCGATCTCAACA


  18
A G T I L W D G R F N D M T S S A D L N


 


 121
AGTGGTCCTGGGGCAACCAGGTCGGCCCCTACCAGTACTATATCCACGGCTCCTCCCCGG


  38
K W S W G N Q V G P Y Q Y Y I H G S S P


 


 181
 TGTCCGCCTACGTCAACCTGTCCCCCGATTACAAGAACCCCGCCGATACCGGCTCCCGCC


  58
V S A Y V N L S P D Y K N P A D T G S R


 


 241
 AGGGCGCCAAGATCACCCTCGATAACACCGCCTACTGGAACGGCCAGAACATGCGCCGCA


  78
Q G A K I T L D N T A Y W N G Q N M R R


 


 301
 CCGAGCTGATCCCCCAGACTACCGCCGCTATCAACCAGGGCAAGGTCTACTACCACTTCA


  98
T E L I P Q T T A A I N Q G K V Y Y H F


 


 361
GCCTCATGCGCAAGGATATCAACGCCCCTGCCACCACCCGCGAGCACCAGATCGCTTTCT


 118
S L M R K D I N A P A T T R E H Q I A F


 


 421
TCGAGTCCCACTTCACCGAGCTGAAGTCCGGCTGGCTCTCCGGCGCTCCCGGCATCTCCG


 138
F E S H F T E L K S G W L S G A P G I S


 


 481
ATACCCTGCTCCGCTGGTGCGTCGGCGGCCAGACCCAGTGGTCCGTCGAGTGGGCCGCTG


 158
D T L L R W C V G G Q T Q W S V E W A A


 


 541
ATGTCTGGCACAACGTCGCCTACGAGATCGATTTCGCCGCTGGCACCGTCGGTTTCTGGC


 178
D V W H N V A Y E I D F A A G T V G F W


 


 601
ACTCCACCGGCTCCGACCCCCTCACCCGCAAGGTCGCCCCCGTCAAGACCAGCACCAGCT


 198
H S T G S D P L T R K V A P V K T S T S


 


 661
CCAACGGTGCTGACTGGCACGTCGGCGTCCTCGAGCTGCCCCGCTCCGGCTACCCCGATT


 218
S N G A D W H V G V L E L P R S G Y P D


 


 721
CCAACGAGGATTTCTACTGGTCCGGCGTCTACATCGAGTCCGGCTCCCTCACCACCTCCG


 238
S N E D F Y W S G V Y I E S G S L T T S


 


 781
TCGCTGGTCCTGGCCAGCCCATCCCTGGTGACGGCGGCTCCTCCAGCTCCAGCTCCTCCT


 258
V A G P G Q P I P G D G G S S S S S S S


 


 841
CCTCCGTCCCTTCCTCCACCTCCACCCGCGTGTCCAGCACCTCCACCCCTGCCCCCGTGT


 278
S S V P S S T S T R V S S T S T P A P V


 


 901
CCTCCACAACCCTCGTTACCAGCACCACTCGCGTCAGCTCCACCTCTACCTCCAGCGCCG


 298
S S T T L V T S T T R V S S T S T S S A


 


 961
CTCCCGTCCAGACCACCCCCTCCGGCTGCACCGCTGGCCAGTACGCCCAGTGCGACGGCA


 318
A P V Q T T P S G C T A G Q Y A Q C D G


 


1021
 TCGGCTTCTCCGGCTGCAAGACCTGCGCCGCTCCCTACACCTGCAAGTACGGCAACGATT


 338
I G F S G C K T C A A P Y T C K Y G N D


 








                  BamHI SacI










1081
GGTACTCCCAGTGCCTCTGATGAGGATCCGAGCTC



 358
W Y S Q C L * *






CAP 68330 DUF1996-CBM1 Protein

A synthetic gene was designed by back translation from the reannotated protein, for further reference called CAP68330, deposited originally under number XM—001907623.1, with codon bias for Aspergillus niger. Since the start of the coding sequence thus obtained (MHSRN . . . ) cannot be comprised in a restriction enzyme recognition site that is compatible with NcoI (that serves as the 5′ cloning site in vector pAN52-5doubleNotamdS) it was decided to include at the 5′ end of the synthetic gene the 3′ end of the Aspergillus specific gpdA promoter sequence (from SalI to NcoI). SalI is the nearest unique site upstream of NcoI in vector pAN52-5doubleNotamdS.
At the 3′ end of the gene two consecutive stop codons were introduced, followed by a BamHI cloning site.








          KpnI SalI










   1
GGTACCGTCGACCCATCCGGTGCTCTGCACTCGACCTGCTGAGGTCCCTCAGTCCCTGGT



 


  61
 AGGCAGCTTTGCCCCGTCTGTCCGCCCGGTGTGTCGGCGGGGTTGACAAGGTCGTTGCGT


 


 121
 CAGTCCAACATTTGTTGCCATATTTTCCTGCTCTCCCCACCAGCTGCTCTTTTCTTTTCT


 


 181
 CTTTCTTTTCCCATCTTCAGTATATTCATCTTCCCATCCAAGAACCTTTATTTCCCCTAA


 


 241
 GTAAGTACTTTGCTACATCCATACTCCATCCTTCCCATCCCTTATTCCTTTGAACCTTTC


 


 301
 AGTTCGAGCTTTCCCACTTCATCGCAGCTTGACTAACAGCTACCCCGCTTGAGCAGACAT


 


 361
CACCATGCACTCCCGCAACGTCCTCGCCGCTGCCGTCGCTCTCGCTGGCGCCCCTTCCGT


   1
 M H S R N V L A A A V A L A G A P S V


 


 421
CCACGCCGTCCTCCGCTTCAGCTGCTCCGAGCTGGTCACCGAGCGCCTCGACCCCCTCGT


  20
H A V L R F S C S E L V T E R L D P L V


 


 481
GTTCCCTGGCGCCATGCAGTCCCCCCACGTCCACCAGATCGTCGGCGGCAACATGTTCAA


  40
F P G A M Q S P H V H Q I V G G N M F N


 


 541
CGTCACTATGGACCCCAACCGCCACAACATCGGCGAGGAAGCCACCTGCACCACCTGTAC


  60
V T M D P N R H N I G E E A T C T T C T


 


 601
CTTCTCCGAGGATTTCTCCAACTACTGGACCGCCATCCTCTACTTCCGCGCTCGCAACGG


  80
F S E D F S N Y W T A I L Y F R A R N G


 


 661
CACCCTCATCCGCGTCCCCCAGCGCCCCAATATCGATTTCGATGGCGCTCGCGGCGGTGG


 100
T L I R V P Q R P N I D F D G A R G G G


 


 721
CATGACCGTCTACTACACCGCCACCTACCAGAACCACAAGCCCACCGCCTTCCAGCCCGG


 120
 M T V Y Y T A T Y Q N H K P T A F Q P G


 


 781
CTTCCGCATGATCGTCGGCAACCCCATGTACCGCACCCAGGCCGAGGCTTCCCGCTACCG


 140
 F R M I V G N P M Y R T Q A E A S R Y R


 


 841
CCAGATGACCTTCACCTGCCTCGAAACCCTCTCCACCCGCACCGGCGAAACCACCGAGAT


 160
 Q M T F T C L E T L S T R T G E T T E M 


 


 901
GCCCAAGCAGCCCTGCCGCGAGGGCATCATGTCCAACGTCCGCTTCCCCACCTGCTGGGA


 180
 P K Q P C R E G I M S N V R F P T C W D 


 


 961
TGGCAAGACCCTCGATCCCCCCGATCACTCCTCCCACGTCGCCTACCCGTCCTCCGGCAC


 200
 G K T L D P P D H S S H V A Y P S S G T


 


1021
CTTCGAGTCCGGCGGTCCCTGCCCTGCTTCCCACCCTGTCCGCATCCCCCAGCTGTTCTA


 220
 F E S G G P C P A S H P V R I P Q L F Y


 


1081
CGAGGTCCTCTGGGATACCCGCCGCTTCAACGATCGCTCCCTCTGGCCCGAGGATGGCTC


 240 
E V L W D T R R F N D R S L W P E D G S


 


1141
CCAGCCCTTCGTCTGGTCCTACGGCGATTACACCGGCTACGGCACCCACGGCGATTACGT


 260 
Q P F V W S Y G D Y T G Y G T H G D Y V 


 


1201
GTTCGGCTGGAAGGGCGATTCCCTCCAGCGCGCTATGGATGCCAACTGCGATTTCTACTG


 280
 F G W K G D S L Q R A M D A N C D F Y C 


 


1261
CCCCCAGCTCAAGACCCAGTCTATCGCCACCGGCAACCAGTGCCGCCAGAACCAGAAGGT


 300
 P Q L K T Q S I A T G N Q C R Q N Q K V


 


1321
CGCCGAGAACATCGATGGCCCCTTCGATCGCCTCCCTGGTAACGTCGAGATCACCGGCCC


 320
 A E N I D G P F D R L P G N V E I T G P


 


1381
TCAGCCTGGCGCCTCCAACCCCAACCCCGGCAATGGCGGTGGCTCTACTCAGACTCCTGT


 360
 Q P G A S N P N P G N G G G S T Q T P V 


 


1441
CCAGCCCACCCCCGTCCCTAACCCTGGCAACGGTGGCGGCTGCTCCGTCCAAAAGTGGGG


 380
 Q P T P V P N P G N G G G C S V Q K W G


 


1501
CCAGTGCGGCGGTCAGGGCTGGTCCGGTTGCACCGTCTGCGCCTCCGGCTCCACCTGCCG


 400
 Q C G G Q G W S G C T V C A S G S T C R


 








                         BamHI SacI










1561
CGCTCAGAACCAGTGGTACTCCCAGTGCCTCTGATGAGGATCCGAGCTC



 420
A Q N Q W Y S Q C L * *






(II) Overexpression of Synthetic Genes Copies

The synthetic gene copies were inserted in an expression vector based on the A. nidulans gpdA promoter, carrying also the amdS selection marker. This Aspergillus expression vector pAN52-4-amdSdoubleNotI was derived by cloning the Aspergillus selection marker amdS and an additional NotI cloning site into the Aspergillus expression vector pAN52-4 (EMBL accession #Z32699).
The resulting expression vectors were introduced in a protease deficient A. niger host strain AB1.13 (Punt et al., 2008). AmdS+ transformants were selected using acrylamide selection.
(III) Protocol MicroTiterPlate Cultivation of Aspergillus 
For cultivation of the strains, standard round bottom 96-well microtiter plates (Corning #3799) were used using a Multitron shaker (Infors) designed for the use with MTP.
Volume: 200 μl MM Aspergillus medium per well (MM+casamino acids+vitamins)

    
    
        Each separate well was inoculated with spores (from colonies on plates), using toothpicks.
        MTP was incubated for 48 hours at 33° C., 850 rpm Good growth was confirmed by visual inspection
        MTP was centrifuged 10 min 3500 rpm to allow biomass separation.

(IV) DNS-CMCase Method in MTP

Reagents:

    
    


Carboxymethyl cellulose sodium salt (CMC), Avicel or non soluble cellulose 3-5, dinitrosalicylic acid, sodium salt (DNS)
Potassium/sodium tartrate (tetrahydrate)
Sodium hydroxide
Glacial acetic acid
Reagent preparation protocol:


    
    
        1. 0.05M NaAc, pH 4.8: Add 2.85 ml of glacial acetic acid to 900 ml of distilled water, adjust the pH to 4.8 with 50% Sodium hydroxide. Bring to total volume of one liter with distilled water.
        2. 1% CMC substrate solution: Add 1 gm CMC to 99 ml NaAc buffer, pH4.8. Keep at 4° C. for at least 1 hour before using. The solution is stable for 3 days at 4° C.
        3. 10.67% (w/v) Sodium hydroxide solution: add 32 gm of sodium hydroxide pellets to 300 ml of distilled water.
        4. 1% 3-5, dinitrosalicylic acid, sodium salt (DNS): suspend 2 gram of DNS in 100 ml of distilled water and gradually add 30 ml of the 10.67% sodium hydroxide solution while mixing. Warm the suspension in water bath set at 50° C. until the solution is clear. Gradually add 60 gm of potassium/sodium artrate (tetrahydrate) to the solution with continuous mixing. Dilute the solution to 200 ml with distilled water. The solution is stable for 2 months. The solution must be clear when used.

Assay Procedure Protocol:

    
    


Making Standard Curve
Choose a lot of cellulase preparation as a standard
Standard Curve: dilute the standard using acetate buffer such that the absorbance (at 540 nm) is between 0.1 and 0.5.
Blank solution: use acetate buffer 0.05M NaAc, pH 4.8 as a blank solution

    
    
        1. Mix 10 μl of each sample with 90 μl buffer 0.05M NaAc, pH 4, 8 using a 1.1 ml volume, 96-deep well Micro Titer Plate (Oxygen; cat. no. P-DW-11-C). At the same time prepare the standard in the same MTP in duplicate.
        2. pre-equilibrate the CMC substrate in a (plastic test plate) in a water bath set at 50° C. for 5 minutes
        3. At 20 second intervals, add 100 μl of the CMC substrate (pre-equilibrated at 50° C.) to the enzyme dilution using a multichannel pipette (12 channel). Mix and incubate at 50° C. for 10 minutes. (Incubation time can be adjusted depending on activity level of parental strain).
        4. at the same time interval as in step 3, add 300 μl of DNS solution and mix
        5. boil the reaction mixture+DNS for exactly 5 minutes by placing the test microtiterplate in a boiling water bath. Cover the tops to prevent evaporation during boiling. As a blanc for remaining glucose in the samples prior to incubation also a duplicate MTP is included in which the reaction is terminated by boiling directly upon addition of the cellulase substrate. All samples, standard and blanks should be boiled together. After boiling, cool the plate in an ice bath
        6. Measure the absorbance of the enzyme samples, standard and blancs at 540 nm in a Tecan Infinite 200 microplate reader
        
            (Measurement range 0-3 OD)

(V) Cellulose Binding Assay

        
        
    
    


For qualitative evaluation of cellulose binding capacity the following assay was used:

    
    
        Incubate 1 ml fermentation samples 1 hour with 10 mg Avicel at 4° C. with gentle mixing.
        Centrifuge 10 min, 3000 g
        Wash the cellulose once with 0.5 ml of 50 mM sodium phosphate pH 7.0
        Elute the bound protein by boiling the cellulose pellet for 10 min in 50 μl of 10% SDS
        Subject 20 μl to SDS-PAGE gel.

(VI) Transformant Screening

    
    


For a number of transformants obtained from each of the two expression vectors described MTP cultures were performed.
For both the CAP68330 and CAP61309 transformants the culture supernatant was used in a DNS-CMCase activity assay to identify the transformants with the highest activity level. For each expression vector transformants with increased CMCase activity were identified.
(VII) Fermentation
Transformants selected from the transformant screening were cultivated in standard fed-batch fermentation and the lignocellulose and/or (hemi-)cellulose active proteins produced were analyzed in various cellulase related assays
VIII Analysis of A. niger Transformants in Controlled Fermentation
Medium samples during the various fermentations were taken and samples at the end of fermentation (around 70-100 h) were analyzed for cellulase related activity using both soluble (CMC) and non-soluble (non-soluble cellulose, avicel) cellulase substrates. In addition, as the produced CAP68330 or CAP61309 proteins could also be non-enzymatic accessory proteins potentiating cellulase activity also an assay was performed in the presence of a fixed amount of a commercial cellulase preparation and samples from the culture fluid of the CAP68330 and CAP61309 strains was added
Results of these assays are shown in the table below












Substrate










non-soluble cellulose
Avicel












Strains
CMC
−cellulase
+cellulase
−cellulase
+cellulase





Blanc
ND
ND
0.07
ND
1.00


cap68330#4
0.25
0.15
0.26
1.92
2.06


cap61309#8
0.22
0.21
0.23
1.90
2.09


CONTROL
0.31
0.17
0.21
1.53
1.86









As shown in the table the activity towards CMC and non-soluble cellulose was not increased compared to the control strain not expressing CAP68330 or CAP61309 protein. The background activity level observed in the Control strain originates from native Aspergillus proteins releasing reducing sugar equivalents from the various substrates.
In contrast as shown in the table, with Avicel as a substrate the cellulase-related activity was higher for the CAP68330/61309 strains than for the control, indicating the presence of cellulase and/or cellulase enhancing activity due to the presence of the CAP68330 or CAP61309 protein.
IX SDS PAGE and Cellulose Binding Analysis
In addition to activity assays also SDS PAGE was carried out with concentrated fermentation samples. In addition cellulose binding analysis followed by SDS PAGE analysis was carried out. As shown in FIG. 1 for CAP61309 protein an additional protein band was observed in SDS PAGE. This band was also identified by binding to Avicel

## Claims

1. A method to degrade a raw carbohydrate which method comprises treating the raw carbohydrate with an effective amount of a protein selected from the group consisting of proteins having the following amino acid sequences
a protein having the amino acid sequence of SEQ ID NO:50
having the amino acid sequence of SEQ ID NO:51,
having the amino acid sequence of SEQ ID NO:52,
having the amino acid sequence of SEQ ID NO:53,
having the amino acid sequence of SEQ ID NO:55,
having the amino acid sequence of SEQ ID NO:56,
having the amino acid sequence of SEQ ID NO:57,
having the amino acid sequence of SEQ ID NO:58,
having the amino acid sequence of SEQ ID NO:59,
having the amino acid sequence of SEQ ID NO:60,
having the amino acid sequence of SEQ ID NO:61,
having the amino acid sequence of SEQ ID NO:62,
having the amino acid sequence of SEQ ID NO:63,
having the amino acid sequence of SEQ ID NO:64,
having the amino acid sequence of SEQ ID NO:65,
having the amino acid sequence of SEQ ID NO:66,
having the amino acid sequence of SEQ ID NO:67,
having the amino acid sequence of SEQ ID NO:68,
having the amino acid sequence of SEQ ID NO:69,
having the amino acid sequence of SEQ ID NO:70,
having the amino acid sequence of SEQ ID NO:71,
having the amino acid sequence of SEQ ID NO:72,
having the amino acid sequence of SEQ ID NO:73,
having the amino acid sequence of SEQ ID NO:74,
having the amino acid sequence of SEQ ID NO:75,
having the amino acid sequence of SEQ ID NO:76,
and a protein having a sequence that has an identity of more than 95% with any of said amino acid sequences that retains lignocellulose and/or hemicellulose degrading activity,
whereby the raw carbohydrate is degraded,
wherein the raw carbohydrate comprises lignocellulose and/or hemicellulose.

2. The method of claim 1, wherein the protein is a protein having the amino acid sequence of SEQ ID NO:58 or having the amino acid sequence of SEQ ID NO:61.