Fungus-sourced high-temperature acid B-glucosidase as well as coding gene and application thereof

The present invention relates to the field of genetic engineering, particularly to a fungi-derived high-temperature acid β-glucosidase, coding gene and application thereof.

β-glucosidase (EC3.2.1.21) (β-D-glucoside glucohydrolase) can hydrolyze non-reducing β-D-glycosidic bond binding the end to release β-D-glucose and the corresponding ligands. β-glucosidase can catalyze the final reaction of decomposing cellulose to eliminate inhibition of cellobiose on exoglucanase and endoglucanase as the important component of cellulase system.

The β-glucosidase has important application value in medical treatment, food, and biomass energy conversion, and becomes more important since cellulose is the most widespread carbon source with energy crisis. It can fully hydrolyze straw fiber into glucose so as to be used to produce ethanol in fermentation. In the other hand, β-glucosidase can be applied to food industry by improving food's flavor and nutrition. Furthermore, β-glucosidase is widely used to hydrolyze soy isoflavones in feed industry and medicine.

β-glucosidase can be applied to industrial production of gentiooligsaccharide which can improve the flavor of the food.

The present invention provides a high-temperature acid β-glucosidase having high catalyzing activity and expression, so as to meet the requirement of industrial production, and biomass conversion.

One order of the present invention is to provide a fungi-derived high-temperature acid β-glucosidase.

Another order of the present invention is to provide a gene coding the above high-temperature acid β-glucosidase.

Another order of the present invention is to provide a recombinant vector comprising the above gene.

Another order of the present invention is to provide a recombinant cell comprising the above gene.

Another order of the present invention is to provide a method of preparing above high-temperature acid β-glucosidase.

Another order of the present invention is to provide an use of the above high-temperature acid β-glucosidase.

Thus, in one aspect, the present invention provided a novel high-temperature acid β-glucosidase, BGL3A which was separated from a thermophilic Talaromyces emersonii 12802, and a recombinant yeast highly expressing said β-glucosidase.

According to an embodiment of the present invention, was provided a high-temperature acid β-glucosidase which is selected from:

(a) a polypeptide comprising the amino acid as shown in SEQ ID NO:1 or SEQ ID NO: 2; (b) a polypeptide with β-glucosidase activity which is derived from SEQ ID NO: 1 or SEQ ID NO. 2 by substitution, deletion and/or insertion of one or more amino acid residues.

SEQ ID NO: 1:

According to the embodiment of the present invention, said β-glucosidase comprises 737 amino acids, with a signal peptide of 19 amino acids in N-terminal, as set in forth in SEQ ID NO. 3.

According to the embodiment of the present invention, the mature β-glucosidase protein comprised the amino acid sequence set forth in SEQ ID NO: 2 having molecular weight of 76.3 kDa.

SEQ ID NO: 2

The β-glucosidase of the present invention has high temperature tolerance, high acid tolerance, and high catalytic efficiency. The β-glucosidase of the present invention from Talaromyces emersonii 12802 has the optimal pH value of 4.5 and the optimal temperature of 75° C., is thermostable at 37° C., and maintains over 90% of activity in the optimal condition after being processed at 60° C. for 1 h.

Yet another aspect of the invention is a gene coding the above high-temperature acid β-glucosidase, with the following characteristics:

(a) coding a polypeptide comprising the amino acid as shown in SEQ ID NO. 1 or SEQ ID NO. 2; (b) coding a polypeptide with β-glucosidase activity which is derived from SEQ ID NO: 1 or SEQ ID NO. 2 by substitution, deletion and/or insertion of one or more amino acid residues.

Preferably, the gene coding the above high-temperature acid β-glucosidase according to the embodiment of the present invention is selected from

(a) DNA comprising a nucleotide sequence set in forth in SEQ ID NO.4 or SEQ ID NO.5; or (b) DNA hybridizing under stringent conditions, to a nucleotide sequence set in forth in SEQ ID NO.4 or SEQ ID NO.5, and coding polypeptide with glucosidase activity.

Preferably, said gene has a nucleotide sequence set in forth in SEQ ID NO.4.

agcgccgcactccagtattccggtgatttccagcgacattgatgcggggaa

ggaatcaaggggacatcatccctggaattcctataagatggccgtcaccca

cgcatgaaaaataaaanatgctccttttgatntgcgactcgagtacccaca

gcgacagcgacgatcaccatgcttgctgagcaaatcttcctgagtgttctg

gcagcagccgtcactgtccaggcctatggcttcggcggctctggctgggac

gccgcttatggcagagcaaaggctgcgctgaacaagntcaaccagaccgag

aaggttggtatcgtcaccggtgtcaagtggatgggcggcccttgtgttggc

aacacctacaagcccagttcgattgantacccttctctgtgtttgcaagac

tctcctctcggggtgcgttttgccaaccctgtgactgccttcccggntggt

atcaacgccggcgccacatgggatagatctctcatcaacgcccgtggtgcg

gccatgggcgctgaggccaagggcctcggtgtgaacgtccagcttggcccc

gtcgctggtcctctcggcaagaatcccaatagtggcagaatctgggaaggg

actcgaatgatccctatctcagcggtgagcgatggaggaaaccatcgccgg

aatgcaaggatctggtgtgcaggcctgcgccaaggtacgtggatctcgact

tgcaacatgtacgatctgagagggctgacacgatacctgaatctatagcac

tatattggtaacgagcaagagcacaaccgtgaaaccatcagctccaacatc

gatgaccgcactctgcacgagctctacgtctggccgttcatgaacgccgtc

aaggccaacgtcgcctccgtcatgtgctcgtacaacaaggtcaatggttcc

tggtcctgtgagaatgatgctcactcaacggtctgagaagactgagctcgg

attccccggatacatcatgagcgattggaacgcgcagcacaccacggtcaa

cagcgccaactcgggtctcgatatgaccatgcctggcagtgacttcaacaa

ccctcctggcagcatctgctgggggcccaacctcgaagccgccgtcgccaa

tggctccgaccgcagtcccgtaggacgacatggtcactcgtatccagcgtc

aggcacttggaggccaggatgagggctacccaccggtcgccttcagctcct

ggaatggcggcaaggccaatgttgacgtgacgggcgatcacaagagcgtcg

tcagagctgtggctcgtgactctaccgttcactgaagaacgacaataacgc

tagcctctgcgcaagcccaagagcctcgcgatcatcggccaggatgcaacc

gtcaaccctgccgggcccaacgcttgctctgatcgcggctgcgacactggt

actctcgccatgggaggggcagtggtaccgctcagacccagtgagtcgtcc

cattgcaacttccacaggagcgaccggtgactaacaagcacctagtacatc

gtcggccctctcgatgctatccagtctcaggctgccgctgatggcactaac

atcaccaccagcgcgaccgatgataccaccgcggcagatctgcagccgcct

ccgccggaaccgccatcgtcttcatcaactccgactctggtgaagggtaag

cccgggcgtcaagatcctcgtacagatgggcccgcatcgctaacattctac

agttacatcaccgtcgagggcaacgctggtgaccgcaacaacctcgacccc

tggcacaacggcaacgagctcgtccaggccgttgcggctgcgaacaagaat

gtcattgtcgtcgtccacagcgtcggtcccgtgatcaggagactatccttg

cacagcccaacgtcaaggccattgtgtggcccggtctccctggacaagaga

gcggcaatgccctggtcgatgactgtacggctccacctcccccagcggcaa

gagccctataccattgccaagcagttcagcgactatggctccacctggacg

acctccctggtcgatgacttcaccgagggtctgacattgactaccgccact

agacgagaacaacattactcccagatacgagttcggatacggcagtgttag

tacttcatctctctctcgtagatccatgctgtcatgcaacgacacaaactg

acatgataatagcttacaccaccacaaatactccgacctggacgtcaacgt

ccaggcccgccccggcgcagccgaaggccccatcgtccccggcggcgtcaa

ggaactatcgacaccgtcggcaccgtcaccgtcaccgtccagaacagcggc

aaggagccggcgcggaagttgcccagctgtacatcggccttcccgactctg

ccccgtcgacccctcccaagcagctcagaggattccagaagttgcacctcg

cgcccggccagagagagggcgccactacgaactcacccgccgagacatcag

ctactgggacgttcagcagcagaagtgggttgacctagcggtacgttcaag

gtctatgaggaagctcgagcagggacattagggagcagggatcttgagtac

gagcacatgacggaggcgacgttgaccgtggtgtgctgcgcgaccaatc

According to an embodiment of the present invention, the gene coding β-glucosidase isolated by PCR method, was 2214 bp in length, comprising a nucleotide sequence set in forth in SEQ ID NO.6 coding a signal peptide.

atgcttgctgagcaaatcttcctgagtgttctggcagcagccgtcactgtccaggcc (SEQ ID NO.6).

A gene coding a mature β-glucosidase had a nucleotide sequence set in forth in SEQ ID NO.5.

SEQ ID NO. 5

tatggcttcggcggctctggctgggacgccgcttatggcagagcaaaggct

gcgctgaacaagctcaaccagaccgagaaggaggtatcgtcaccggtgtca

agtggatgggcggcccagtgaggcaacacctacaagcccagttcgattgac

tacccactctgtgatgcaagactctcctctcggggtgcgattgccaaccct

gtgactgccacccggctggtatcaacgccggcgccacatgggatagatctc

tcatcaacgcccgtggtgcggccatgggcgctgaggccaagggcctcggtg

tgaacgtccagcaggccccgtcgctggtcctctcggcaagaatcccaatag

tggcagaatctgggaagggactcgaatgatccctatctcagcggtgagcga

tggaggaaaccatcgccggaatgcaaggatctggtgtgcaggcctgcgcca

agcactatattggtaacgagcaagagcacaaccgtgaaaccatcagctcca

acatcgatgaccgcactctgcacgagctctacgtctggccgttcatgaacg

ccgtcaaggccaacgtcgcctccgtcatgtgctcgtacaacgaggtcaatg

gttcctggtcctgtgagaatgatgctcttctcaacggtctgttgaagactg

agctcggattccccggatacatcatgagcgattggaacgcgcagcacacca

cggtcaacagcgccaactcgggtctcgatatgaccatgcctggcagtgact

tcaacaaccctcctggcagcatctactgggggcccaacctcgaagccgccg

tcgccaatggctccgaccgcagtcccgtttggacgacatggtcactcgtat

ccttgcgtcttggtacttggttggccaggatgagggctacccaccggtcgc

cttcagctcctggaatggcggcaaggccaatgagacgtgacgggcgatcac

aagagcgtcgtcagagctgtggctcgtgactctatcgttcttctgaagaac

gacaataacgctttgcctctgcgcaagcccaagagcctcgcgatcatcggc

caggatgcaactgtcaaccctgccgggcccaacgcttgctctgatcgcggc

tgcgacaccggtactctcgccatgggaggggcagtggtaccgctcagaccc

atacatcgtcggccctctcgatgctatccagtctcaggctgccgctgatgg

cactaacatcaccaccagcacgaccgatgataccaccgcggcagatctgca

gccgcctccgccggaaccgccatcgtatcatcaactccgactctggtgaag

gttacatcaccgtcgagggcaacgctggtgaccgcaacaacctcgacccct

ggcacaacggcaacgagctcgtccaggccgttgcggctgtgaacaagaatg

tcattgtcgagtccacagcgtcggtcccgtgatcaggaggctatccttgca

cagcccaacgtcaaggccattgtgtggcccggtctccctggacaagagagc

ggcaatgccctggtcgatgactgtacggctccacctcccccagcggcaaga

gccctataccattgccaagcagttcagcgactatggcaccacctggacgac

ctccctggtcgatgacttcaccgagggtctgacattgactaccgccaattg

acgagaacaacattactcccagatacgagttcggatacggatgtcttacac

caccacaaatactccgacctggacgtcaacgtccaggcccgccccggcgca

gccgaaggccccatcgtccccggcggcgtcaaggaactatcgacaccgtcg

gcaccgtcaccgtcaccgtccagaacagcggcaaggagccggcgcggaaga

gcccagctgtacatcggccacccgactctgccccgtcgacccctcccaagc

agctcagaggattccagaagagcacctcgcgcccggccagagagagggcgc

cactacgaactcacccgccgagacatcagctactgggacgttcagcagcag

aagtgggttgttcctagcggtacgttcaaggtctatgttggaagctcgagc

agggacattagggagcagggatctttccgtatttga

The molecular mass of the mature protein is 76.3 kDa. Homology searches in GenBank were done using the BLAST server. As a result, the amino acid sequence (SEQ ID NO: 1) showed a homology (83%) with the known β-glucosidase from Neosartorya fischeri NRRL 181, and had a homology (82%) with β-glucosidase from Aspergillus fumigatus Af293. Therefore, it was conformed that the ORF from Talaromyces emersonii 12802 coded a novel β-glucosidase. The amino acid sequence of ORF from Talaromyces emersonii 12802 was named BGL3A like other name of β-glucosidase.

The present invention also provides to an isolated protein comprising the amino acid sequence depicted in SEQ ID NO: 1 or SEQ ID NO: 2. In another embodiment, the present invention relates to a derivate of said protein, which is obtainable from SEQ ID NO: 1 or SEQ ID NO: 2 by substitution, deletion and/or insertion of one or more (e.g., one or several, or a value selected from 1-10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or ranges intermediated to the above-recited values) amino acid residues, and maintains the β-glucosidase activity. For example, a common strategy is conservative amino acid substitutions that is to say, the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, replacement with another amino acid residue from the same side chain of one or more amino acid residue would not substantially change the enzyme activity of said β-glucosidase. Furthermore, it is well known in the art that during the cloning of genes, usually enzyme recognition sites are designed, which would result in one or several non-relating amino acid residues on the ends of target protein without affecting the activity thereof. In addition, in order to construct a fusion protein, to enhance expression of recombinant protein, to obtain an recombinant protein automatically secreted outside the host cell, or to aid in the purification of the recombinant protein, suitable peptide linker, signal peptide, leader peptide, terminal extensions, glutathione S-transferase (GST), maltose E binding protein, protein A, tags such as 6His or Flag, or proteolytic cleavage site for Factor Xa, thrombin or enterokinase are usually introduced into the N- or C-terminus of the recombinant protein or within other suitable regions in the proteins.

In another embodiment, the protein with β-glucosidase activity according to the present invention can comprise an amino acid sequence which is encoded by a nucleotide sequence which hybridizes, e.g., hybridizes under stringent conditions, to a nucleotide sequence of SEQ ID NO: 4 or SEQ ID NO:5 as set forth in the Sequence Listing. As used herein, the term “hybridizes under stringent conditions” is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% homologous to each other typically remain hybridized to each other. Preferably, the conditions are such that sequences at least about 65%, more preferably at least about 70%, and even more preferably at least about 75% or more homologous to each other typically remain hybridized to each other. Such stringent conditions are known to one of ordinary skill in the art and can be found in Current Protocols in Molecular Biology , John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. A preferred, non-limiting example of stringent hybridization conditions are hybridization in 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65° C. A person skilled in the art understands that high stringent condition could be realized by raising the hybridization temperature up to 50° C., 55° C., 60° C. or 65° C.

Besides, it will be appreciated by one of ordinary skill in the art that genetic polymorphism due to natural variation may exist among individuals within a population. Such natural variations can typically result in 1-5% variance in the nucleotide sequence of the (3-glucosidase gene. Any and all such nucleotide variations and resulting amino acid polymorphisms in β-glucosidase that are the result of natural variation and that do not alter the functional activity of β-glucosidase proteins are intended to be within the scope of the invention. Therefore, the present invention also encompasses a polypeptide with β-glucosidase activity encoded by such an allele or natural variant of the polynucleotide as shown in SEQ ID NO: 4 or SEQ ID NO.5.

In a preferred embodiment, a β-glucosidase protein is such a active protein that is at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, more preferably at least about 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, and even more preferably at least about 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more homologous to the entire amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2 of the present invention. Ranges and identity values intermediated to the above-recited values (e.g., 60-90% homologous or 98.1-99.9% identical) are also intended to be included in the present invention.

On the other hand, the present invention provides a novel β-glucosidase gene of SEQ ID NO: 4 or SEQ ID NO:5. The invention further encompasses nucleic acid molecules that differ from one of the nucleotide sequences depicted in SEQ ID NO: 4 or SEQ ID NO: 5 of the invention due to degeneracy of the genetic code and thus encode the same β-glucosidase protein. In another embodiment, an isolated nucleic acid molecule of the invention is a nucleotide sequence which hybridizes, e.g., hybridizes under stringent conditions, to a nucleotide sequence of SEQ ID NO: 4 or SEQ ID NO:5, with the allele or natural variant thereof is preferred. In another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in the SEQ ID NO: 1 or SEQ ID NO:2. In a still further embodiment, the nucleic acid molecule of the invention encodes a full length β-glucosidase protein which is substantially homologous to an amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5, for example, a protein that derived from SEQ ID NO: 1 or SEQ ID NO:2 by substitution, deletion and/or insertion of one or more (e.g., one or several, or a value selected from 1-10) amino acid residues, or one that is at least 99% homologous to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:2. Such a nucleic acid molecule is preferably at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, more preferably at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.7%, 97.8%, 97.9%, or at least about 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, and even more preferably at least about 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more homologous to a nucleotide sequence of SEQ ID NO: 4 or SEQ ID NO:5. Ranges and identity values intermediate to the above-recited values (e.g., 76-97% homologous or 97.8-99.9% identical) are also intended to be included in the present invention.

In yet another embodiment, the present invention relates to a recombinant vector comprising said nucleic acid coding said β-glucosidase, a recombinant host cell (such as Pichia Pastoris , yeast, and E. coli .) having been introduced said vector or said nucleic acid molecule, as well as a method for expressing the enzyme in a host cell. In a preferred embodiment, said β-glucosidase gene was controlled by promoter AOX1 by being inserted between sites of EcoRI and NotI in plasmid pPIC9, so as to obtain the recombinant expression vector pPIC9-bgl3A.

In a preferred embodiment, said recombinant host cell was strain GS115/bgl3A.

The present invention relates to a method of producing the said β-glucosidase, including the steps:

(a) cultivating the host cells to provide the supernatant containing the said β-glucosidase; and (b) recovering the said β-glucosidase.

The recombinant expression vectors of the invention can be designed for expression of β-glucosidase proteins in prokaryotic or eukaryotic cells. For example, β-glucosidase gene can be expressed in bacterial cells such as E. coli , yeast such as Pichia or Aspergillus , insect cells (e.g., Sf9 cell or silkworm cell, using baculovirus expression vectors), or plant cell (such as Arabidopsis , tobacco, corn, and so on, mediated by Agrobacterium tumefaciens ). Thus, the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced, with Pichia preferred. Pichia pastoris is a methylotrophic yeast, capable of metabolizing methanol as its sole carbon source. This system is well-known for its ability to express high levels of heterologous proteins. As an effective expression system, many of β-glucosidase gene have successfully expressed in P. pastoris . The novel β-glucosidase gene also expressed in P. pastoris and had high levels of expression. The extracellular β-glucosidase activity is 33 U/ml for the induced expression in a large flask, and more than 3400 U/ml for the induced expression in tank, respectively. So it will be very easy to mass-produce the β-glucosidase by fermentation, and the cost will be lower than ever.

Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection”, “conjugation” and “transduction” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., linear DNA or RNA (e.g., a linearized vector or a gene construct alone without a vector) or nucleic acid in the form of a vector (e.g., a plasmid, phage, phasmid, phagemid, transposon or other DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, natural competence, chemical-mediated transfer, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. ( Molecular Cloning: A Laboratory Manual. 2 nd, ed., Cold Spring Harbor Laboratory , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals.

A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) a β-glucosidase protein. Accordingly, the invention further provides methods for producing β-glucosidase proteins using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding a β-glucosidase protein has been introduced, or into which genome has been introduced a gene encoding a wild-type or altered β-glucosidase protein) in a suitable medium until β-glucosidase protein is produced. In another embodiment, the method further comprises isolating β-glucosidase proteins from the medium or the host cell.

Yet another aspect of the invention is the β-glucosidase expressed in Pichia pastrois . In order to ascertain the assay of the β-glucosidase, the β-glucosidase was purified by simple approach, such as ammonium sulfate precipitation, dialysis, ultrafiltration and chromatography. After the simple purification, the purity of the β-glucosidase was enough to study the enzyme properties.

Yet another aspect of the invention is the application of said β-glucosidase to the food industry, the feed industry, and the biomass energy.

With the aim to solve the problem of the low activity of the microorganism producing β-glucosidase existing in the art, we had isolated a novel β-glucosidase from Talaromyces emersonii 12802. We had also identified the nucleotide sequence, which codes the protein having β-glucosidase. The β-glucosidase had several advantages: high specific activity, favourable pH-optimum, high stability at higher temperatures, resistance against proteases, easily produce by fermentation. The β-glucosidase was an acidic enzyme showing a high enzyme activity at 60° C.-75° C. and the optimal activity was observed at 75° C. The enzyme activity was very stable between pH 1.0 and pH 10.0, the optimal pH was 4.5. And the enzyme had a high stability at higher temperatures, when the enzyme was left at 60° C. for 1 hour, 90% of the activity was remained.

FIG. 1 shows optimum pH values for novel β-glucosidase.

FIG. 2 shows pH stabilities for novel β-glucosidase.

FIG. 3 shows optimum temperature values for novel β-glucosidase.

FIG. 4 shows heat stability for novel β-glucosidase.

The present invention is further illustrated with reference to the following Examples and the appended drawings, which should by no means be construed as limitations of the present invention.

Test Materials and Reagents

1. Strains and vectors: Talaromyces emersonii 12802; Pichia pastoris strain GS115 (Invitrogen); and vetor pPIC9 (Invitrogen, San Diego, Calif.). 2. Enzymes and other biochemical reagents: restriction endonucleases(TaKaRa); ligase (Invitrogen); and birch xylan(Sigma) 3. Medium:

(1) taking potato dextrose medium as Talaromyces emersonii 12802 Medium, including 1000 mL of potato juice, 10 g of dextrose, and 25 g of arga, natural pH.

(2) E. coli . LB medium: 1% of peptone, 0.5% of yeast extract, and 1% of NaCl, natural pH. (3) BMGY medium: 1% of yeast extract; 2% of peptone; 1.34% of YNB, 0.00004% of Biotin; and 1% of glycerol(V/V). (4) BMMY medium: 1% of yeast extract; 2% of peptone; 1.34% of YNB, 0.00004% of Biotin; and 0.5% of methanol (V/V).

Suitable biology laboratory methods not particularly mentioned in the examples as below can be found in Sambrook, et al. ( Molecular Cloning: A Laboratory Manual. 2 nd, ed., Cold Spring Harbor Laboratory , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other kit laboratory manuals.