WO2019169855A1 - 一种用丝状真菌宿主细胞表达的重组草酸脱羧酶 - Google Patents

一种用丝状真菌宿主细胞表达的重组草酸脱羧酶 Download PDF

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WO2019169855A1
WO2019169855A1 PCT/CN2018/107053 CN2018107053W WO2019169855A1 WO 2019169855 A1 WO2019169855 A1 WO 2019169855A1 CN 2018107053 W CN2018107053 W CN 2018107053W WO 2019169855 A1 WO2019169855 A1 WO 2019169855A1
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recombinant
host cell
seq
decarboxylase
filamentous fungal
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PCT/CN2018/107053
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English (en)
French (fr)
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汪卫
汪小锋
刘艳红
黄荷
陈火晴
陈先桥
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武汉康复得生物科技股份有限公司
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Priority to US16/978,763 priority Critical patent/US11613745B2/en
Priority to JP2020570612A priority patent/JP2021514679A/ja
Priority to EP18909228.1A priority patent/EP3674403A4/en
Publication of WO2019169855A1 publication Critical patent/WO2019169855A1/zh
Priority to JP2022081863A priority patent/JP2022110110A/ja

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/01—Carboxy-lyases (4.1.1)
    • C12Y401/01002—Oxalate decarboxylase (4.1.1.2)

Definitions

  • the invention relates to the technical field of genetic engineering, in particular to a recombinant filamentous fungal host cell capable of efficiently expressing oxalic acid decarboxylase, and a recombinant oxalic acid decarboxylase, a production method and application of the recombinant oxalic acid decarboxylase.
  • Oxalic acid also known as oxalic acid, is a metabolite of organisms and is widely found in plants, animals and fungi in the form of oxalates. Many foods of humans and other mammals, such as spinach, strawberries, beets, cocoa, taro, sweet potato, rhubarb and tea, have higher oxalate content. In humans and other mammals, oxalate is the end product of metabolism due to the absence of enzymes that degrade oxalate in the body, exogenous oxalate absorbed from food and endogenous by physiological metabolism. Oxalate is mainly excreted into the urine through the kidneys.
  • oxalate When humans and other mammals consume high levels of oxalate foods, the concentration of oxalate is likely to increase in the blood and urine of the human body. When combined with calcium ions, calcium oxalate is formed, and calcium oxalate is urinary. The main component of the stone. In addition, high concentrations of oxalate are associated with a variety of other pathologies, such as hyperoxaluria, cardiac conduction disorders, Crohn's disease, and other intestinal disease states. Therefore, the decomposition of oxalate derived from food in an in vitro or in vivo environment, reducing the absorption of oxalate in the body, can reduce the risk of occurrence of related diseases including urinary calculi.
  • Enzymes that are known to exist in the biological world and have oxalic acid-decomposing functions include oxalate decarboxylase, oxalate oxidase and oxalyl CoA decarboxylase.
  • Oxalate decarboxylase is an enzyme whose active center contains manganese ions and can catalyze the degradation of oxalic acid to produce formic acid and carbon dioxide.
  • the oxalic acid decarboxylase found so far is mainly found in some plants, bacteria and fungi, such as Aspergillus niger, Coniothyrium minitans, Flammulina velutipes, T. versicolor, Agaricus bisporus, brown rot fungus, Bacillus subtilis Bacillus subtilis, Agrobacterium tumefaciens, and the like.
  • oxalate decarboxylase has very low yields and yields in the various natural sources mentioned above, resulting in high production costs and high prices, making it difficult to commercialize and market.
  • bacterial-derived oxalate decarboxylase recombinant expression such as Bacillus subtilis YvrK gene-derived oxalate decarboxylase, has been achieved in prokaryotic cells, bacterial-derived oxalate decarboxylase is unstable at low pH (below pH 3.0).
  • Oxthera has prepared a formulation in another formulation, which is mixed with an acid-insoluble polymer and spray-dried to make microparticles (Oxazyme, Oxthera). Clinical trials have shown that Oxazyme does not reduce urinary acid. .
  • the fungal-derived oxalic acid decarboxylase has good stability under low pH conditions and good resistance to pepsin, and is therefore very suitable for degrading oxalic acid as an oral enzyme preparation.
  • the fungal-derived oxalate decarboxylase did not obtain good recombinant expression results whether using prokaryotic expression systems or eukaryotic expression systems.
  • the present invention has undergone a long-term trial and effort in a long period of time, and has tried various expression systems and applied various biological techniques.
  • the prokaryotic expression system the inventors performed various expression attempts, expression elements and expression in prokaryotic cells such as different types of E. coli expression cells, Bacillus subtilis cells, Bacillus licheniformis cells, Bacillus pumilus cells, and Lactobacillus cells. The strategy was optimized, but no effective recombinant expression was obtained.
  • the inventors performed transient expression and stable transformation expression in tobacco and pea plants, and used tobacco cells for suspension culture expression.
  • Yeast cells such as insect cells, Saccharomyces cerevisiae cells and Pichia pastoris cells are optimized for expression and expression elements and expression strategies. The results obtained are either no enzyme activity detected, or the expression level is extremely low, and there is no possibility of industrial production.
  • the inventors finally obtained efficient recombinant expression in filamentous fungi by combining and optimizing each link.
  • the object of the present invention is to provide a recombinant oxalic acid decarboxylase which is recombinantly expressed by a filamentous fungal host cell, such that the glycosylation modified form and degree of the recombinant oxalate decarboxylase are different from the oxalic acid decarboxylation expressed by the original host cell.
  • the enzyme, the recombinant oxalate decarboxylase has a glycosylation modified form and extent characteristic of a filamentous fungal host cell.
  • the recombinant oxalic acid decarboxylase maintains all or part of its enzyme activity at a pH of 1.5-7.0, and maintains not less than 10% of its enzyme activity under optimal pH conditions at a pH of 1.5-2.5. Maintaining no less than 50% of the enzyme activity at the optimum pH under the conditions of pH 2.5-4.5, and maintaining the enzyme activity at pH 4.5-7.0 below its optimum pH. %.
  • the recombinant oxalic acid decarboxylase has an optimum pH of from 2.5 to 3.5.
  • the recombinant oxalate decarboxylase encoding gene is derived from a eukaryote, which is a tea tree mushroom, a poplar mushroom, a mushroom, a cloud mushroom, a brown rot fungus, an Aspergillus niger, an agaric mushroom or a gold. Fungus such as mushrooms.
  • the amino acid sequence of the recombinant oxalate decarboxylase is the amino acid sequence of amino acids 20 to 470 of SEQ ID NO. 1 or SEQ ID NO. 5, or amino acid sequences 25 to 472 of SEQ ID NO. Or the amino acid sequence of amino acids 20 to 455 of SEQ ID NO. 3, or the amino acid sequence of positions 21 to 447 of SEQ ID NO. 4, or the amino acid sequence of 21 to 455 of SEQ ID NO. 6, or SEQ ID NO.
  • the amino acid sequence of amino acids 25 to 440 of 7 and the amino acid sequence of positions 24 to 472 of SEQ ID NO. 8 have at least 60% identity, preferably at least 65% identity, at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity.
  • the amino acid sequence of the recombinant oxalate decarboxylase is from amino acid sequence 20 to 470 of SEQ ID NO. 1 or SEQ ID NO. 5, or amino acid sequence 25 to 472 of SEQ ID NO. 2, or SEQ ID
  • Another object of the present invention is to provide a novel method for expressing oxalic acid decarboxylase, which can express oxalic acid decarboxylase gene efficiently, and the expression amount and enzyme activity are far superior to the existing expression methods, and the practical application value is achieved.
  • a recombinant filamentous fungal host cell comprising a gene sequence encoding the recombinant oxalate decarboxylase of any of the above, is provided in the chromosomal DNA of the recombinant filamentous fungal host cell .
  • the oxalate decarboxylase expression cassette integrated in its genome, the oxalate decarboxylase expression cassette comprising a promoter, a signal peptide coding sequence, an oxalate decarboxylase encoding gene and a terminator.
  • the inventors have found through extensive research work that oxalic acid decarboxylase can efficiently recombine and express in filamentous fungal host cells.
  • the oxalic acid decarboxylase recombinantly expressed in a filamentous fungal host cell is capable of various post-translational processing, such as glycosylation modification, etc., and the recombinantly expressed oxalate decarboxylase has similar enzymatic properties as the oxalate decarboxylase prepared by the natural host cell.
  • the recombinantly expressed oxalic acid decarboxylase can be efficiently secreted into the culture substrate, which facilitates subsequent separation and purification and reduces production costs.
  • the signal peptide coding sequence refers to a signal peptide coding region capable of guiding oxalate decarboxylase into a specific compartment or secretory channel of a cell, which may be obtained from, but not limited to, an oxalate decarboxylase original signal peptide, a Trichoderma cellobiohydrolase I.
  • Trichoderma cellobiohydrolase II Trichoderma endoglucanase I, Trichoderma endoglucanase II, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicola insolens endoglucanase V, Humicola lanuginosa lipase, Rhizomucor miehei aspartic protease, etc.
  • the signal peptide coding sequence of the gene any signal peptide coding region that directs the oxalate decarboxylase to the secretory pathway of the filamentous fungal host cell, can be used in the present invention.
  • a preferred signal peptide coding sequence is a T. reesei cellobiohydrolase I gene (cbh1) signal sequence.
  • the promoter refers to a transcriptional and translational control sequence involved in binding RNA polymerase that mediates expression of an oxalate decarboxylase gene.
  • the promoter may be any nucleotide sequence that is transcriptionally active in a selected host cell and may be derived from a gene encoding a protein either homologous or heterologous to the host cell.
  • the promoter may be an inducible promoter or a constitutive promoter.
  • promoters of the present invention for mediating transcription of an oxalate decarboxylase expression cassette in a filamentous fungal host cell are promoters of genes derived from, but not limited to, the following enzymes: SV40, hCMV, CaMV 35S, Aspergillus nidulans acetamide Enzyme, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus oryzae TAKA amylase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamyl starch Enzyme (glaA), Rhizomucor miehei lipase, Trichoderma pyruvate decarboxylase, Trichoderma ⁇ -glucosidase, Trichoderma cellobiohydr
  • the promoter is derived from a T. reesei cellobiohydrolase I gene promoter (Pcbh1). In some preferred embodiments, the promoter is derived from T. reesei pyruvate decarboxylation. Enzyme gene promoter (Ppdc).
  • the terminator refers to a sequence that can be recognized by a filamentous fungal host cell to terminate transcription. Any terminator that functions in the host cell can be used in the present invention.
  • terminators for mediating transcription termination of oxalate decarboxylase expression cassettes in filamentous fungal host cells are those derived from, but not limited to, the genes of the following enzymes: Aspergillus nidulans acetamidase, Aspergillus oryzae alkaline Protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus oryzae TAKA amylase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), rice Rhizopus arrhizus lipase, Trichoderma pyruvate decarboxylase, Trichoderma
  • the terminator is derived from a T. reesei cellobiohydrolase I gene terminator (Tcbh1), and in some preferred embodiments, the terminator is derived from T. reesei pyruvate decarboxylation Enzyme gene terminator (Tpdc).
  • Tcbh1 T. reesei cellobiohydrolase I gene terminator
  • Tpdc T. reesei pyruvate decarboxylation Enzyme gene terminator
  • the filamentous fungus is Aspergillus, Clostridium, Mucor, White Rot, Acremonium, Cryptococcus, Rhizoma Phytophthora, Humicola, genus Mycelium, Aureobasidium, Trametes, Pleurotus, Neurospora, Penicillium, Paecilomyces, Pseudomonas, Tobacco, A genus of genus, a genus, a genus, a genus, a genus, a genus, a genus, a genus, a genus, a genus, a genus, a genus, a genus, a genus, a genus, a genus, a genus, a genus, a genus, a genus, a genus, a genus, a genus, a genus, a genus
  • the filamentous fungus is Aspergillus niger, Aspergillus nidulans, Aspergillus oryzae or Aspergillus awamori strain.
  • the filamentous fungus is a Trichoderma harzian, a Trichoderma koningii, a Trichoderma reesei, a Trichoderma longum or a Trichoderma viride strain.
  • the filamentous fungal host cell is a T. reesei cell, including but not limited to ATCC No. 56765, ATCC NO. 13631, ATCC NO. 26921, ATCC NO. 56764, ATCC NO. 56767, and NRRLNO. 15709, etc. Trichoderma reesei strain cells.
  • the filamentous fungal host cell is a Trichoderma reesei strain Rut-C30 cell.
  • the filamentous fungal host cell can be a variant cell of the T. reesei strain Rut-C30, including a variety of natural genes, including the encoding orotate, that are genetically engineered to knock out T.
  • the recombinant filamentous fungal host cell at least 10% of the nucleotide sequence of the oxalate decarboxylase gene is codon optimized according to the codon preference of the filamentous fungal host cell.
  • the optimized gene encodes or at least partially encodes an oxalate decarboxylase protein.
  • the partial encoding refers to deletion of a partial amino acid sequence but also has an oxalic acid decarboxylase function.
  • the above nucleotide sequence is selected from the group consisting of SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. ID NO. 15 and the nucleic acid sequence of SEQ ID NO. 16; or at least 50% identical, preferably at least 60% identical, at least 70% identical, at least 80% identical to any of SEQ ID NO. 9-16 Sequence of sex or at least 90% identity.
  • an oxalic acid decarboxylase gene expression vector is constructed, and the expression vector contains an oxalic acid decarboxylase gene expression cassette, and also contains a coding selectivity. Labeled gene expression cassette.
  • the selectable marker refers to a marker gene that provides a simple selection of transformed host cells.
  • suitable selectable markers include, but are not limited to, resistance genes such as hygromycin and bar.
  • a nutritional selection marker can also be used, for example, acetamidase (amdS), ornithine carbamoyltransferase (argB), orotic acid nucleoside-5'-phosphate decarboxylase, and the like.
  • a 350-500 bp forward repeat nucleotide sequence flanking the 5' flanking and 3' flanking of the selectable marker expression cassette can be excised by spontaneous intramolecular homologous recombination under reverse selection pressure.
  • the selectable marker is the pyr4 gene and the auxotrophic screening marker orotic acid nucleoside-5'-phosphate decarboxylase is a key enzyme in the synthesis of uracil nucleotides, the deletion of which results in a uridine nucleoside Acid synthesis is hindered, so auxotrophic strains lacking the enzyme require the addition of uracil/uridine to grow.
  • 5-fluoroorotic acid is an analogue of uracil-producing precursors, which produces toxic substances to cells under the action of pyr4, so wild-type strains are at 5 '-FOA cannot grow, and when it becomes a pyr4 gene-deficient strain, it exhibits 5'-FOA resistance, thereby achieving reverse screening (see Jeffrey L. Smith et al. Curr Genet, 1991, 19: 27-23). A simple choice to provide transformed host cells.
  • the above oxalic acid decarboxylase gene expression vector comprises a random integration expression vector and a site-specific integration expression vector.
  • the expression cassette of oxalate decarboxylase was randomly integrated into the T. reesei genome, and its position and copy in the genome were analyzed by Tail-PCR. number.
  • the transformed strains with different integration sites and copy numbers can be obtained by two rounds of transformation and screening, and the enzyme production conditions are compared by shake flask fermentation, a series of engineering strains are screened, and the copy number thereof is analyzed and Integration sites in the Trichoderma genome.
  • the site-specific integrated expression vector contains a DNA sequence identical to the specific site of the T. reesei at both ends of the oxalate decarboxylase expression cassette as a 5' homology arm and a 3' homology arm, through a site-specific integration expression vector
  • the gene at these specific sites can also be knocked out while introducing the oxalate decarboxylase expression cassette to a specific site.
  • several cellulase genes (CBH1, CBH2, EG1, and EG2), which are the major components of the T. reesei extracellular secretory protein, are selected as site-specific integration sites for integration into oxalate decarboxylase expression.
  • these gene coding cassettes were knocked out, and a 4-copy expression strain was constructed. Under the fermentation conditions, the recombinantly secreted oxalate decarboxylase could reach more than 90% in total extracellular protein.
  • a second aspect of the invention provides a method of constructing a recombinant filamentous fungal host cell according to any of the above (random integration method), wherein the recombinant filamentous fungal host cell comprises one or more copies of the integration thereof
  • An oxalate decarboxylase expression cassette in the genome the oxalate decarboxylase expression cassette comprising a promoter, a signal peptide coding sequence, an oxalate decarboxylase encoding gene and a terminator, the method comprising the steps of:
  • S1 Construction of at least one integrative expression vector comprising a selectable marker gene expression cassette and an oxalate decarboxylase expression cassette.
  • S2 Integral Expression Vector After transformation of a filamentous fungal host cell, a recombinant filamentous fungal host cell containing one copy or multiple copies of an oxalate decarboxylase expression cassette is obtained by screening.
  • step S2 wherein the filamentous fungal host cell described in step S2 is an artificially constructed auxotrophic cell, and the integrated expression vector is integrated into the genome of the filamentous fungal host cell Can repair this type of auxotrophy.
  • the integrative expression vector is randomly integrated into the filamentous fungal host cell genome by non-homologous recombination.
  • the integrated expression vector comprises a 5'-end homology arm and a 3' end homologous to a nucleotide sequence of a specific length in a genome of a filamentous fungal host cell.
  • a source arm such that the transformed expression vector can be integrated into a genome-specific site by homologous recombination after transformation of the filamentous fungal host cell; preferably integrated into a gene encoding an extracellular protein; more preferably integrated into a coding cell
  • the exoprotease or the gene encoding the extracellular glycoside hydrolase is most preferably integrated into the CBH1, CBH2, EG1 or EG2 gene.
  • the starting strain is an artificially constructed pyr4 gene-deficient T. reesei, comprising the following steps:
  • a medium for culturing a host cell prepared by the above method (random integration method), comprising: glucose 3-8 g/L, microcrystalline cellulose 10-25 g/L, Corn flour powder 5-15g / L, (NH 4 ) 2 SO 4 0.5-5g / L, MgSO 4 ⁇ 7H 2 O 1.56g / L, CaCl 2 0.5g / L, KH 2 PO 4 2-8g / L, Urea 0-1 g / L, bran powder 0.2-2 g / L, Mandels trace element (1000X) 1 ml, MnCl 2 0.5-5 mM, pH 3.0-4.5.
  • a recombinant filamentous fungal host cell of any of the above site-specific integration method
  • the method comprising the steps of:
  • the mus53 and pyr4 genes were repaired to the strain obtained in the step (2) by the pyr4 and mus53 gene repair vectors; the successfully repaired strain was the target host cell.
  • the repair of the pyr4 gene does not require the addition of uracil or uridine in the medium during fermentation.
  • the host cell can preserve the inherent metabolic balance without increasing the cost of fermentation; the repair of the mus53 gene allows the host cell to preserve its inherent stability. Elimination of genomic instability caused by mus53 gene deletion.
  • a fifth aspect of the present invention there is provided another medium suitable for the culture of a host cell prepared by the above method (site-specific integration method), comprising: glucose 3-6 g/L, lactose 30-40 g/ L, corn syrup powder 7-10g / L, (NH 4 ) 2 SO 4 0.5-1g / L, MgSO 4 ⁇ 7H 2 O1.56g / L, CaCl 2 0.5g / L, KH 2 PO 4 2-4g / L, urea 0-1 g/L, bran powder 10-20 g/L, Mandels trace element (1000X) 1 ml, MnCl 2 0.5-5 mM, pH 3.5-4.0.
  • site-specific integration method comprising: glucose 3-6 g/L, lactose 30-40 g/ L, corn syrup powder 7-10g / L, (NH 4 ) 2 SO 4 0.5-1g / L, MgSO 4 ⁇ 7H 2 O1.56g / L, CaCl 2 0.5g
  • a method for producing an oxalate decarboxylase comprising constructing an oxalate decarboxylase expression cassette comprising a promoter, a signal peptide coding sequence, an oxalate decarboxylase-encoding gene and a terminator, is transformed into a filament by an expression vector.
  • the fungal host cell integrates one or more oxalate decarboxylase expression cassettes in the host cell genome, cultures the host cell to express oxalate decarboxylase, and finally separates and purifies the expression product from the host cell culture substrate.
  • the use of the oxalic acid decarboxylase secreted and expressed by the recombinant oxalic acid decarboxylase or the recombinant filamentous fungal host cell cultured in any one of the above is provided for the preparation of a medicament or a food.
  • the medicament is a medicament for preventing and/or treating urinary calculi.
  • a pharmaceutical composition for preventing or treating a disease of urinary oxalate comprising the oxalic acid decarboxylase prepared by the above method.
  • the present invention has the following beneficial effects:
  • the invention overcomes the technical problem that the fungal-derived oxalate decarboxylase can not be effectively recombinantly expressed.
  • the oxalic acid decarboxylase can be subjected to various post-translational processing by recombinant expression of the filamentous fungal host cell, and the highly secreted oxalic acid decarboxylase has the same preparation with the natural host cell. Similar enzymatic properties of oxalic acid decarboxylase.
  • the host cell is simple to culture, the oxalic acid decarboxylase is secreted in a large amount and the enzyme activity is high; the two medium formulations of the present invention are respectively applicable to two recombinant filamentous fungal host cells, which can effectively increase the yield; the production of oxalate decarboxylase is expressed by
  • the construction of the cassette, the construction of the vector, the construction of the host cell and the adjustment of the final medium formula greatly increase the yield and the enzyme activity of the product, effectively solving the prior art that the artificial production of oxalate decarboxylase cannot be industrialized on a large scale, and the enzymatic properties are Unstable, high production costs.
  • Figure 1 shows the map of the vector pMDT05.
  • Figure 2 is a map showing the construction of the pyrT gene knockout vector pMDT05-pyr4KO of the Trichoderma reesei Rut-C30 strain.
  • Figure 3 is a map showing the construction of a random integration-inducible expression vector pMGU-cbh1-TRA2 of T. reesei.
  • Figure 4 is a map of the constructed recombinant expression vector pDGU-pdc-TRA2 of T. reesei.
  • Figure 5 is a map of the pMDT05-pyr4KI repair vector of the Pyribacterium mirabilis transformant pyr4 gene.
  • Figure 6 is a SDS-PAGE diagram of the fermentation broth of 144h and 168h fermentation, and the arrow is the recombinant oxalate decarboxylase band.
  • Figure 7 is a map of the construction of the mus53 gene knockout vector.
  • Figure 8 is a map of the construction of a knock-in vector at a CBH1 site.
  • Figure 9 is a map of the construction of a knock-in vector at the CBH2 site.
  • Figure 10 is a map of the construction of a knock-in vector at the EG1 site.
  • Figure 11 is a map of the construction of a knock-in vector at the EG2 site.
  • Figure 12 is a map showing the construction of the mus53 gene repair vector.
  • Figure 13 is a graph showing the change in fermentation viability of a 7L fermentor.
  • Figure 14 is a 10-fold SDS-PAGE of the 136h and 160h samples of the 7L fermenter fermentation.
  • Figure 15 is a plot of 200-fold and 500-fold Western Blot analysis of 160 h sample dilution.
  • Figure 16 shows the relative viability of oxalate decarboxylase at a pH of 1.5-7.0.
  • Figure 17 is a SDA-PAGE diagram of oxalic acid decarboxylase in three expression system tables, wherein lanes 1 and 2 are recombinant oxalate decarboxylase expressed by T. reesei; lanes 3 and 4 are oxalic acid decarboxylase expressed by natural host tea tree; 5 and 6 are prokaryotic expression of oxalic acid decarboxylase.
  • Figure 18 is a MALDI-TOF-MS map of recombinant oxalate decarboxylase expressed by T. reesei.
  • Figure 19 is a MALDI-TOF-MS spectrum of recombinant oxalate decarboxylase expressed by T. reesei after trypsinization.
  • Figure 20 is a MALDI-TOF-MS spectrum of oxalic acid decarboxylase expressed by the natural host tea tree mushroom after trypsin digestion.
  • Figure 21 is a MALDI-TOF-MS spectrum of prokaryotic expression of oxalate decarboxylase after trypsinization.
  • the present invention is illustrated by the specific expression of the oxalic acid decolorizing enzyme derived from the tea tree mushroom in the filamentous fungus T. reesei, so that those skilled in the art can better understand the present invention and can implement it.
  • the examples are not intended to limit the invention.
  • Trichoderma reesei Rut-C30 (ATCC 56765) used in the present invention was purchased from the Guangdong Microbial Culture Collection.
  • the Aspergillus niger CICC 2439 used in the present invention was purchased from the China Industrial Microbial Culture Collection.
  • Example 1 Codon optimization and artificial synthesis of oxalic acid decarboxylase (OXDC) gene
  • the filamentous fungal expression system can be used to express oxalic acid decarboxylase derived from eukaryotes, preferably from tea tree mushroom, poplar mushroom, enoki mushroom, Yunzhi, brown rot fungus, Aspergillus niger, and double spore.
  • Oxalic acid decarboxylase of fungi such as mushrooms and mushroom.
  • the gene encoding oxalate decarboxylase may be derived from Agrocybe aegerita, and the amino acid sequence of the oxalate decarboxylase is the amino acid sequence shown in SEQ ID NO. 1, wherein the signal peptide sequence of the oxalate decarboxylase is 1 shown in SEQ ID NO.
  • the amino acid sequence at position -19, the mature peptide sequence is the amino acid sequence of amino acid 20-470 shown in SEQ ID NO.
  • the OXDC gene derived from Agrocybe aegerita was optimized according to the T. reesei codon preference (see Codon Usage Database: Hypocrea jecorina), and the DNA coding sequence of the new oxalate decarboxylase mature peptide was designed and synthesized. Compared with before optimization, the optimized OXDC sequence changed from 0.51 to 0.99 for CAI (Codon Adaptation Index), and the content of G+C changed from 53.09% to 69.23%. The optimized sequence is shown in SEQ ID NO. Shown. The DNA coding sequence of the optimized OXDC gene mature peptide of Agrocybe aegerita was renamed as TRA2.
  • the filamentous fungal host cell used for eukaryotic expression of eukaryotic OXDC is selected from the group consisting of Aspergillus, Trichosporon, Mucor, White Rot, Acrium, Cryptococcus, Fusarium, Humicola Genus, genus Mycelium, Aureobasidium, Trametes, Pleurotus, Neurospora, Penicillium, Paecilomyces, Pseudomonas, Fusarium, Pseudomonas, Clostridium Genus, genus, genus, genus, genus, genus, genus, genus, genus, genus, genus, genus, genus, genus, genus, genus, genus, or genus Sexual or synonymous cells, but are not limited to this.
  • Trichoderma host cells are Trichoderma harzianum, Trichoderma koningii, Trichoderma reesei, Trichoderma longum and Trichoderma viride, preferably Trichoderma reesei and Trichoderma viride.
  • the present invention will be described below by taking Trichoderma reesei as an example.
  • Trichoderma reesei Rut-C30 (ATCC 56765) was inoculated on PDA medium and cultured at 28 ° C for 7 days until the spores matured.
  • the spores were eluted with sterile water, and an appropriate amount of spore suspension was prepared and inoculated into 20 ml of liquid medium, and cultured at 28 ° C, 170 rpm for 36-48 h.
  • the mycelium was collected by vacuum suction filtration, washed twice with deionized water, the mycelium was ground to a fine powder under liquid nitrogen freezing, and genomic DNA was isolated using a Sangon Biotech Ezup column fungal genomic DNA extraction kit.
  • the above PDA medium formula is: peeled potato slices 200g, 1000ml water is boiled for 30min, 8 layers of gauze are filtered, the filtrate is added with 20g of glucose, and the water is added to 1L, natural pH, 2% agar powder. Sterilize at 115 ° C for 30 min.
  • the liquid medium formulation is: mixing 15 g of glucose, 20 g of yeast extract, 2.5 g of ammonium sulfate, 0.8 g of magnesium sulfate heptahydrate, and 1.0 g of anhydrous calcium chloride, and then dissolving in distilled water and diluting to 1 L to adjust the pH to 4.8.
  • the pCAMBIA1300 plasmid was used as a template, and PCR amplification was carried out using the primers pMDT05-F1 and pMDT05-R1 in Table 1 below.
  • the PCR product was separated by 1% agarose gel electrophoresis, and a fragment of about 6.8 kb was excised from the gel.
  • the recombinant plasmid was recovered according to the omega gel extraction kit method, and the purified fragment was digested with restriction endonucleases XhoI and XbaI for 1 hour, and then purified and recovered according to the omega PCR product recovery kit method after digestion.
  • the promoter Pgpd (about 1.4 kb) was amplified using the primers Hyg-Pgpd-F and pMDT05-R2 in Table 1.
  • the hygromycin gene (about 1 kb) was amplified using the primers pMDT05-F2 and Pgpd-Hyg-R in Table 1.
  • the amplified promoter Pgpd fragment and the hygromycin gene were mixed as a template, and primers pMDT05-F2 and pMDT05-R2 were used for upstream and downstream primers for SOE-PCR amplification (amplification conditions were 94 ° C, 10 min). ; 98 ° C, 10 s, 60 ° C, 30 s, 68 ° C, 1 min 20 s, 30 cycles; 68 ° C, 10 min) to obtain a fusion fragment of about 2.4 kb, the fusion fragment was separated by 1% agarose gel electrophoresis, the fragment of about 2.4 kb was The gel was excised and recovered according to the omega gel recovery kit method. The purified fragment was digested with restriction endonucleases XhoI and XbaI for 1 h, and purified and recovered according to the omega PCR product recovery kit method after digestion.
  • Trichoderma reesei pyr4 gene encoding orotate-5'-monophosphate decarboxylase
  • BLASTN program Locus sequence information at the location of the pyr4 gene in the Trichoderma genomic database (http://genome.jgi-psf.org/Trire2/Trire2.home.html). Using the above extracted T.
  • the primers pyr4-3F/pyr4-3R and pyr4-5F/pyr4-5R in Table 2 were used to amplify a homologous arm fragment of about 1.3 kb upstream of the pyr4 gene.
  • a homologous arm fragment downstream of the 1.3 kb pyr4 gene was mixed in a molar ratio of 1:1 as a template, pyr4-3F and pyr4-5R were used as upstream and downstream primers, and SOE-PCR amplification was about 2.6 Kb. Pyr4 gene knockout box.
  • the pMDT05 vector and the above 2.6 kb pyr4 knockout cassette were digested with restriction endonucleases XbaI and BglII for 1 h, and the digested fragments were separately recovered by the omega gel extraction kit, and the gel purified XbaI/BglII was used.
  • the digested pMDT05 vector was mixed with the digested 2.6 kb fragment at a molar ratio of 1:3, added with T4 DNA ligase and ligation buffer, ligated at 22 °C for 3 h, transformed into E. coli TOP10 competent cells, and the correct vector for verification and sequencing was named pMDT05-pyr4KO, as shown in Fig. 2 is a map of the pMDT05-pyr4KO pyrome gene knockdown vector pMDT05-pyr4KO of the T. reesei Rut-C30 strain.
  • the above knockout vector pMDT05-pyr4KO was transferred into Agrobacterium tumefaciens AGL-1 competent cells by freeze-thaw method (see, in particular, An. G. et. al Binary vectors, in Plant Molecular Biology Manual, 1988), at 28 After activation for 3-4 h at °C, an appropriate amount of the bacterial solution was applied to an LB plate medium containing kan (50 ⁇ g/mL) + Gent (50 ⁇ g/mL), and cultured at 28 ° C for 48-72 h, and then picked up for monoclonal inoculation.
  • kan 50 ⁇ g/mL
  • Gent 50 ⁇ g/mL
  • the cells were cultured in an LB liquid medium containing kan (50 ⁇ g/mL) + Gent (50 ⁇ g/mL) at a shaking temperature of 220 rpm for 24 hours at 28 ° C, and a small amount of the bacterial liquid was taken for colony PCR verification to screen positive transformants.
  • Trichoderma reesei transformation receptor Spores of Trichoderma reesei were washed from 4-7 days of PDA plates with 4-5 ml of sterile water, cotton was filtered to obtain spore suspension, and spores were collected by centrifugation, using IM medium. The cells were washed twice and resuspended in IM medium and adjusted to a spore concentration of 10 7 /ml, and germinated at 28 ° C for 3-4 h.
  • Co-cultivation of Agrobacterium tumefaciens and Trichoderma reesei 100 ⁇ l of prepared Agrobacterium tumefaciens solution was mixed with 100 ⁇ l of germinated spore suspension, coated on cellophane of IM solid medium plate, and cultured at 24 ° C for 36 h. . The cellophane was peeled off, and spread on a solid MM primary sieve medium plate containing 5 mg/ml 5-FOA, 300 ⁇ g/mL cephalosporin and 10 mM uridine, and cultured at 28 ° C for 4-6 days until the transformants grew.
  • Transformant rescreening The transformants were spotted on a PDA solid plate containing 100 ⁇ g/mL hygromycin and a solid MM medium plate containing 5 mg/ml 5-FOA and 10 mM uridine, and cultured at 28 ° C for 2-3 d.
  • the transformant genome which was normally grown on a solid MM medium plate containing 5 mg/ml 5-FOA and 10 mM uridine was picked up in a PDA solid plate containing 100 ⁇ g/mL hygromycin, and the reconstituted transformant genome was extracted.
  • PCR was performed using primers pyr4-CX-F and pyr4-CX-R (sequences shown in Table 2) of the upstream homologous arm and the outer genome of the downstream homologous arm, if the pyr4 gene was knocked out, The added fragment should be about 2.8 kb, and if it is not knocked out, the amplified fragment should be about 4.2 kb.
  • a total of 23 transformants (No. 1#-23#) were screened, and all of the rescreened transformants were verified by PCR to amplify only a fragment of about 2.8 kb, which contained both in the presence of 100 ⁇ g/mL.
  • the normal growth of PDA solid plate of mycin was also able to grow normally on a solid MM medium plate containing 5 mg/ml 5-FOA and 10 mM uridine, indicating that the transformant also occurred at the same time as homologous recombination occurred.
  • the random integration insert was used to knock out the transformant, so the effective knockout rate of the pyr4 gene reached 95.6%.
  • Monospores were isolated and purified: The mycelium numbered 8# knockout strain of the above 23 transformants was picked and inoculated into a PDA medium plate containing 10 mM uridine, and cultured at 28 ° C for 7 days until the spores matured. The mature spores were washed with 4-5 ml of sterile water, diluted with sterile water, plated on a PDA medium plate containing 10 mM uridine and 0.1% Triton-100, and cultured at 28 ° C for 3 days, picking up the separated Monospora colonies were re-inoculated into PDA medium plates containing 10 mM uridine, and spore culture was carried out at 28 °C. The isolated monospora colony and the strain still positive after PCR detection were uracil auxotrophs, and named Rut-C30 (pyr4 - ).
  • Example 3 Construction of a random integration recombinant expression vector for oxalic acid decarboxylase
  • the vector backbone fragment of about 6.6 kb was amplified by PCR using the primers F1 and R1 in Table 3 as upstream and downstream primers, and the restriction endonuclease DpnI was used after recovery using a gel. Digestion for 3 h, recovery of the target fragment, spare.
  • the Aspergillus niger CICC2439 genomic DNA was extracted, and the genomic DNA was used as a template to amplify about 2.9 kb of Aspergillus niger pyrG using the primers pyrG-F and pyrG-R in Table 3.
  • the gene expression cassette, the gel gel recovered the target fragment, and was used.
  • the primers Pcbh-DR-F and Pcbh-DR-R in Table 2 were used to amplify a 0.4 kb fragment of the CBH1 gene promoter Pcbh1, which was recovered by gelation.
  • the 2.9 kb Aspergillus niger pyrG gene expression cassette and the 0.4 kb fragment of Pcbh1 were mixed as a template, and the primers Pcbh-DR-F and pyrG-R were used as primers for upstream and downstream primers, and the amplification conditions were amplified by SOE-PCR. It was: 94 ° C, 10 min; 98 ° C, 10 s, 60 ° C, 30 s, 68 ° C, 1 min 50 s, 30 cycles; 68 ° C, 10 min to obtain a fusion fragment of about 3.3 kb, and the target fragment was recovered by gel.
  • Trichoderma reesei as a template, using the primers Pcbh1-F and Pcbh1-R in Table 3 as the upstream and downstream primers, PCR-amplification of the CBH1 gene promoter and CBH1 gene signal peptide coding sequence Pcbh1-sig; Trichoderma genomic DNA was used as a template, and the terminator sequence Tcbh1 of the CBH1 gene was PCR-amplified using the primers Tcbh1-F and Tcbh1-R in Table 3 as upstream and downstream primers.
  • the Pcbh1-sig fragment and the Tcbh1 fragment were mixed with 1:1 as a template, and the primers Pcbh1-F and Tcbh1-R were used as the upstream and downstream primers to amplify the fusion fragment of about 3.3 kb Pcbh1-sig-Tcbh1, and the target fragment was recovered.
  • the fragment was digested with EcoRI and PstI, and the gel was recovered for use.
  • the plasmid pUC19 was also digested with restriction endonucleases EcoRI and PstI for 3 h, and the vector backbone fragment was recovered by gelation, and the Pcbh1-sig-Tcbh1 fragment after digestion and digestion was ligated with T4 DNA ligase to transform E. coli TOP10.
  • the correct vector for verification and sequencing was named pUC19-Pcbh1-sig-Tcbh1.
  • the 5.8 kb vector backbone fragment was amplified by PCR using the primers WF-CBH-R and WF-CBH-F in Table 3 as upstream and downstream primers, and digested by DpnI for 3 hours. The glue is recycled and ready for use.
  • PCR-amplified TRA2 gene mature peptide coding using plasmid pUC57-TRA2 (provided by Gene Synthesis) containing the synthetic TRA2 gene as a template and primers WF-TRA2-F and WF-TRA2-R in Table 3 as upstream and downstream primers sequence.
  • the vector pMGU prepared above was digested with restriction endonucleases EcoRI and XbaI for 3 hours, and the vector backbone fragment was recovered by gelation.
  • the primers F2 and R2 in Table 3 were used as primers for upstream and downstream primers, and the Pcbh1-sig-TRA2-Tcbh1 fragment was PCR amplified, and the target fragment was recovered by gel. .
  • the vector backbone and Pcbh1-sig-TRA2-Tcbh1 fragment recovered after digestion of pMGU with restriction enzymes EcoRI and XbaI were followed.
  • the method of the one-step cloning kit was assembled and transformed into E. coli TOP10 competent cells.
  • the correct vector for verification and sequencing was named pMGU-cbh1-TRA2, and the construction of T. reesei random integration inducible expression vector pMGU-cbh1-TRA2 was constructed.
  • the map is shown in Figure 3.
  • the 6.6 kb vector backbone was amplified by PCR using primers F1 and R1 in Table 3. The fragment was gel-recovered and digested with restriction endonuclease DpnI for 3 hours, and the target fragment was recovered for use.
  • the expression cassette of the 2.9 kb Aspergillus niger pyrG gene was amplified by the primers pdcDR-pyrG-F and pyrG-R in Table 3.
  • the target fragment was recovered by gel and used.
  • the 5'-end 0.4 kb fragment of the pdc gene promoter Ppdc was amplified using the primers Ppdc-DR-F and pyrG-pdcDR-R in Table 3, and the gel was recovered and used.
  • the 2.9 kb Aspergillus niger pyrG gene expression cassette and the 5'-end 0.4 kb fragment of Ppdc were mixed in a molar ratio of 1:1 as a template, and primers Ppdc-DR-F and pyrG-R were used as primers for upstream and downstream, SOE-PCR amplification. A 3.3 kb fragment was obtained, and the target fragment was recovered by gel.
  • the amplification conditions are: 94 ° C, 10 min; 98 ° C, 10 s, 60 ° C, 30 s, 68 ° C, 1 min 50 s , 30 cycles; 68 ° C, 10 min obtained and recovered 3.3 kb fragments were assembled, transformed into E. coli TOP10 competent cells, coated with plates, and the correct vector for verification and sequencing was named pDGU.
  • the Ppdc fragment obtained above and the Tpdc fragment were mixed as a template, and the primers NdeI-Pdc-F and PstI-Tpdc-R were used as the upstream and downstream primers to PCR-amplify the fusion fragment Ppdc-Tpdc of about 2.5 kb.
  • the fragment of interest was recovered and the fragment was digested with NdeI and PstI and the gel was recovered for later use.
  • the plasmid pUC19 was also digested with NdeI and PstI, and the vector backbone was gel-recovered and ligated with the Ppdc-Tpdc fragment after digestion and transformed into E. coli TOP10 competent cells.
  • the correct vector for verification and sequencing was named pUC19-Ppdc. -Tpdc.
  • the proximal and downstream primers of the primers WF-pdc-R and WF-pdc-F in Table 3 were used to PCR-amplify the vector backbone fragment of about 5.0 kb, and the DpnI was digested for 3 hours. use.
  • primers WF-TRA2-F2 and WF-TRA2-R2 in Table 3 were used as upstream and downstream primers, and a sig-TRA2 sequence of about 1.4 kb was amplified by PCR.
  • the approximately 5.0 kb vector backbone fragment recovered by digestion and the sig-TRA2 fragment were The one-step cloning kit was assembled and transformed into E. coli TOP10 competent cells, and the correct vector for verification and sequencing was named pUC19-Ppdc-sig-TRA2-Tpdc.
  • the vector pDGU prepared above was digested with restriction endonuclease XbaI for 3 h, and then digested with restriction endonuclease EcoRI for 5 min for incomplete digestion, and the larger vector backbone fragment was recovered by gel.
  • the primers F3 and R3 in Table 3 were used as primers for upstream and downstream primers, and the Ppdc-sig-TRA2-Tpdc fragment was PCR-amplified, and the target fragment was recovered by gel.
  • the vector backbone fragment obtained by enzymatic cleavage of the above pDGU and the Ppdc-sig-TRA2-Tpdc fragment are The method of the one-step cloning kit was assembled and transformed into E. coli TOP10 competent cells.
  • the correct vector for verification and sequencing was named pDGU-pdc-TRA2, which was constructed by random integration of constitutive expression vector pDGU-pdc-TRA2.
  • the map is shown in Figure 4.
  • Example 4 Construction of a T. reesei oxalate decarboxylase expression strain by random integration, and screening and molecular identification of transformants
  • the two randomly integrated recombinant expression vectors pMGU-cbh1-TRA2 and pDGU-pdc-TRA2 in the above Example 3 were separately transferred into Agrobacterium tumefaciens AGL-1 competent cells by freeze-thaw method.
  • the positive clones which were verified by PCR were prepared in accordance with the method of Example 2 to prepare Agrobacterium tumefaciens for transformation.
  • T. reesei transformation receptor Washing spores of Trichoderma reesei Rut-C30 (pyr4 - ) strain from cotton culturing 6-7d PDA (containing 10 mM uridine) plate with 4-5 ml of sterile water, cotton The spore suspension was filtered, the spores were collected by centrifugation, washed twice with IM medium, resuspended in IM medium (added to a final concentration of 10 mM uridine) and adjusted to a spore concentration of 10 7 /ml, and germinated at 28 ° C. 3-4h.
  • Co-cultivation of Agrobacterium tumefaciens and Trichoderma reesei 100 ⁇ l of prepared Agrobacterium tumefaciens solution was mixed with 100 ⁇ l of germinated spore suspension, coated on cellophane of solid IM medium plate, and cultured at 24 ° C for 36 h. . The cellophane was peeled off, back-plated onto a plate containing 300 ⁇ g/mL cephalosporin solid MM primary screening medium, and cultured at 28 ° C for 4-6 days until the transformants grew. In this example, three batches of recombinant expression vector pMGU-cbh1-TRA2 were transformed into the T.
  • Transformant rescreening The obtained transformants were spotted on a solid MM medium plate containing 300 ⁇ g/mL cephalosporin, incubated at 28 ° C for 2-3 d, and the transformants with normal growth rate and morphology were picked and transferred to PDA. Spore culture was carried out on the plate at 28 ° C for 7 days.
  • the spores were washed out with sterile water to prepare a spore suspension, which was subjected to gradient dilution, and coated on a PDA medium containing 0.1% Triton-100 to isolate the monospore strain, and cultured at 28 ° C for 3 days until the single spore After the growth of the bacteria, 3 single spore isolates were picked and transferred to PDA medium for 3 days at 28 ° C. A small amount of hyphae was picked into 20 ⁇ l of sterile water, heated at 98 ° C for 10 minutes, and the supernatant was centrifuged for TRA2. -F and TRA2-R were identified by PCR, and the single spore isolates identified as positive transformants by PCR were continuously cultured until the 7th spore maturation.
  • TRA2-F ATGTATCGGAAGTTGGCCGTCATC
  • TRA2-R TTAGGCAGGGCCGACGACAATAGG
  • the above IM medium formulation is: K 2 HPO 4 10 mmol/L, KH 2 PO 4 10 mmol/L, NaCl 2.5 mmol/L, MgSO 4 ⁇ 7H 2 O 2 mmol/L, CaCl 2 0.7 mmol/L, (NH 4 ) 2 SO 4 4 mmol/L, Glucose 10 mmol/L, Clycerol 0.5%, AS 200 ⁇ mol/L, Mandels trace element (1000X) 1 ml, pH 5.3.
  • the above MM medium formula is (g/L): glucose 20, peptone 2, (NH 4 ) 2 SO 4 5, MgSO 4 ⁇ 7H 2 O 0.6, CaCl 2 0.6, KH 2 PO 4 15, Mandels trace elements (1000X) ) 1 ml / L, pH 4.5-5.5.
  • Example 5 T. reesei random integration transformant shake flask fermentation expression screening
  • the mature spores of the isolates of the above Example 4 were washed with 4-5 ml of sterile water, and inoculated into the liquid seed culture medium of Trichoderma reesei according to the inoculation amount of 1%, and cultured for 24 hours, and then transferred according to the inoculation amount of 10%.
  • the activity of oxalic acid decarboxylase in the supernatant of the fermentation broth was analyzed by culturing for 168 hours at 170 rpm in T. reesei transformant expression medium for different promoter types.
  • T. reesei liquid seed medium formula: glucose 15g / L, peptone 2g / L, (NH 4 ) 2 SO 4 2.5g / L, MgSO 4 ⁇ 7H 2 O 0.8g / L, CaCl 2 1.0g / L 50 mM citrate buffer (pH 4.5), urea 0.3 g/L, KH 2 PO 4 2 g/L, Mandels trace element (1000X) 1 ml/L, 1-2 g/L Tween 80.
  • the expression medium of the T. reesei transformant expressing the oxalate decarboxylase by the inducible promoter is: lactose 18 g/L, microcrystalline cellulose 10 g/L, corn syrup powder 12 g/L, (NH) 4 ) 2 SO 4 0.5g/L, MgSO 4 ⁇ 7H 2 O 1g/L, CaCl 2 1.0g/L, KH 2 PO 4 6g/L, bran powder 2g/L, Mandels trace element (1000X) 1ml, MnCl 2 5 mM, pH 4.5.
  • the expression medium of the T. reesei transformant expressing the oxalate decarboxylase by the constitutive promoter is: glucose 50 g/L, peptone 4.5 g/L, (NH 4 ) 2 SO 4 1.4 g/L. MgSO 4 ⁇ 7H 2 O 0.3g/L, CaCl 2 0.4g/L, 50mM citrate buffer (pH 4.5), urea 0.3g/L, KH 2 PO 4 2g/L, Mandels trace element (1000X) 1ml , 1-2 g / L Tween 80, pH 4.5.
  • the amount of enzyme required to degrade 1 ⁇ mol of oxalic acid per minute or 1 ⁇ mol of formic acid per minute under specific conditions was defined as one unit of activity (IU).
  • All the transformants were screened by shake flask fermentation, and the highest enzyme activity at the 168 h fermentation of the T. reesei transformant expressing the oxalate decarboxylase by the inducible promoter reached 17940 IU/L.
  • the highest enzyme activity of the T. reesei transformant expressing oxalic acid decarboxylase with a constitutive promoter at 168 h reached 8800 IU/L.
  • composition Lactose 18g / L, microcrystalline cellulose 10g / L, corn syrup 12g / L, (NH 4 ) 2 SO 4 0.5g / L, MgSO 4 ⁇ 7H 2 O 1.56g / L, CaCl 2 0.5g / L, KH 2 PO 4 6g / L, bran powder 2g / L, Mandels trace element (1000X) 1ml, MnCl 2 5mM, pH 4.0), oxalic acid decarboxylase activity in the fermentation broth of fermentation 168h is about 3000IU / L.
  • the optimized medium had an initial glucose concentration of 8 g/L and microcrystalline cellulose at 23 g/L. After 168 hours of shake flask fermentation, the oxalic acid decarboxylase activity in the supernatant fermentation broth reached 50876 IU/L.
  • the preferred medium composition is: glucose 3-8g/L, microcrystalline cellulose 10-25g/L, corn syrup powder 5-15g/L, (NH 4 ) 2 SO 4 0.5-5g/L, MgSO 4 ⁇ 7H 2 O 1.56g/L, CaCl 2 0.5g/L, KH 2 PO 4 2-8g/L, urea 0-1g/L, bran powder 0.2-2g/L, Mandels trace element (1000X) 1ml, MnCl 2 0.5-5 mM, pH 3.0-4.5.
  • the genomic DNA of the T. reesei transformant strain was extracted according to the method of Example 2, and the TD-TAIL PCR (Touchdown TAIL-PCR) method of Song Gao et al. (Analytical Biochemistry, 59 (2016) 79-81) was used to analyze the Richter scale.
  • the Trichoderma transformant T-DNA i.e., the transfer DNA fragment containing the oxalate decarboxylase expression cassette
  • the random primers (LAD1-LAD5) and specific primers AC1, RB-1, RB-2, and Tail-CX-F used in this example (see Table 4).
  • degenerate primer V represents A/G/C
  • N represents A/G/C/T
  • B represents G/C/T
  • D represents A/G/T
  • H represents A/C/T.
  • the genomic DNA of Trichoderma reesei was diluted to 20-30 ng/ ⁇ l with ddH 2 O as a template for pre-amplification, using LAD1-LAD5 as a random primer and a specific primer RB-, respectively.
  • 1 Pre-amplification PCR then dilute the product of the pre-amplification PCR reaction 50 times as a template for Touchdown PCR, and perform 1% agarose gel electrophoresis on the Touchdown PCR amplification product. The gel recovery is relatively simple and bright.
  • the bands were analyzed by Sail-CX-F primers in Table 3. The sequence of the sequencing results was searched in the T. reesei genomic database using the BLASTN program to analyze the inserted genomic locus.
  • LAD1 ACGATGGACTCCAGAGCGGCCGCVNVNNNGGAA
  • LAD2 ACGATGGACTCCAGAGCGGCCGCBNBNNNGGTT
  • LAD3 ACGATGGACTCCAGAGCGGCCGCVVNVNNNCCAA
  • LAD4 ACGATGGACTCCAGAGCGGCCGCBDNBNNNCGGT
  • LAD5 ACGATGGACTCCAGAGCGGCCGCBHNDNNNGACC
  • AC1 ACGATGGACTCCAGAG RB-1 GGCCGTCGTTTTACAACGTCGTGAC RB-2
  • GCGTAATAGCGAAGAGGCCCGCACC Tail-CX-F GCGTAATAGCGAAGAGGCCCGCACC
  • Pre-amplification amplification reaction system (20 ⁇ l): 20-30 ng of genomic DNA, LAD primer 1.0 ⁇ M, RB-1 primer 0.3 ⁇ M, dNTP 2 ⁇ l, 10 ⁇ buffer 2 ⁇ l, Taq enzyme 0.5 U, and ddH 2 O to 20 ⁇ l.
  • Touchdown PCR reaction system 50 ⁇ l: The pre-amplified PCR reaction product was diluted 50-fold, AC1 primer 0.3 ⁇ M, RB-1 primer 0.3 ⁇ M, dNTP 5 ⁇ l, 10 ⁇ buffer 5 ⁇ l, Taq enzyme 1 U, and ddH 2 O to 50 ⁇ l.
  • a total of 35 F. reesei transformants expressing an enzyme activity between 25000-65000 IU/L in a shake flask were selected for flanking sequence analysis of the T-DNA insertion site, and all obtained T-DNA flanking sequences were obtained. Six of them identified only about 0.5 kb of the vector sequence outside the RB border, and the insertion position on the genome was not identified, and 42 of them identified the position of the T-DNA flanking sequence corresponding to the genome. Eight of the 42 T-DNA flanking sequences retained the complete RB border sequence, and 34 T-DNA right border sequences were deleted.
  • the enzyme activity of the two-copy transformant shake flask fermentation was 60%-100% higher than that of the single copy, showing a good gene dose relationship.
  • the enzyme activity of the two-copy transformant shake flask fermentation was 60%-100% higher than that of the single copy, showing a good gene dose relationship.
  • the activity of the single spore-fermentation-expressing enzyme isolated from the transformant with 2 copies at the same site and in the inverted repeat form was very parallel, and the enzyme activity expressed by the mother was equivalent under the same fermentation conditions.
  • B4-6 One of the isolated strains with higher fermentation activity (higher than 50,000 IU/L) and 2 copies at the same site and present in inverted repeats was designated B4-6.
  • the insertion site of isolate B4-6 was analyzed between Trire2
  • Example 7 The B4-6 strain of Example 7 was inoculated into PDA medium (containing 10 mM uridine), cultured at 28 ° C for 7 days until the spores matured, and the spores were washed with 4-5 ml of sterile water to prepare a spore suspension.
  • the spore suspension was spread on PDA medium containing 5 mg/ml 5-FOA, 0.1% Trinton-100 and 10 mM uridine, and cultured at 28 ° C for 4-5 days to grow a single colony. Approximately 100 isolates of 5-FOA resistance were obtained. Five 5-FOA resistant isolates were transferred to PDA medium containing 10 mM uridine and cultured at 28 ° C for 7 days until the spores matured.
  • the primers pyrG-F2 and pyrG-R2 were used for PCR to identify colonies in which the homologous recombination of the pyrG expression cassette was generated. The results showed that the pyrG expression cassettes were excised from the five spore isolates.
  • B4-6 pyr4 - ).
  • Example 9 Random integration of recombinant expression vector pMGU-cbh1-TRA2 into T. reesei B4-6 (pyr4 - ) strain to construct multi-copy transformants
  • the expression vector pMGU-cbh1-TRA2 was transformed into the T. reesei B4-6 (pyr4 - ) strain by Agrobacterium-mediated transformation according to the methods and procedures described in Example 4. 42 transformants were obtained, and each transformant was separately transferred to a solid MM medium plate containing 300 ⁇ g/mL cephalosporin, and incubated at 28 ° C for 3 d, and 39 rescreened transformants were selected and transferred to a PDA plate. Incubate at 28 ° C for 7 days.
  • a small amount of mycelium was extracted from the PDA plate cultured to the third day into 20 ⁇ l of sterile water, heated at 98 ° C for 10 minutes, and the supernatant primers pyrG-F3 and WF-CBH-R were centrifuged for PCR. It was identified that a positive transformant capable of amplifying a fragment of about 2.3 kb was obtained.
  • the above primer sequence is pyrG-F3: 5'-TTACTTGTGGTGTTCTCAGCTTG-3'; the sequence of the primer WF-CBH-R is shown in Table 2.
  • the new copy insertion site of the highest transformant was analyzed by the method of Example 7. After sequencing analysis, the transformant was inserted into a new copy at two different sites, respectively, and the insertion sites were respectively Trire2
  • the selection marker gene pyrG was excised by the method of Example 8, and the transformant was named HH03-26-8 (pyr4 - ).
  • primers pyr4-F1 and pyr4-R1 were used as primers to amplify the complete expression cassette of pyr4 and homologous arm fragments on both sides of pyr4 gene.
  • PCR amplification products After separation by 1% agarose gel electrophoresis, the band of about 4.0 kb was excised, purified by gel extraction kit, digested with restriction endonucleases BglII and XbaI for 1 h, and the target fragment was recovered by PCR product recovery kit. spare.
  • the vector pMDT05 was also digested with the restriction enzymes BglII and XbaI for 3 hours, and the purified vector fragment was recovered by a gel extraction kit, which was mixed with the 4.0 kb fragment of the digestion and recovered by a molar ratio of 1:3, and T4 DNA ligase was added. After ligation at 22 °C for 3 h, the ligated product was transformed into E. coli TOP10 competent cells, and positive clones were screened by PCR and verified by sequencing. The correct vector for sequencing verification was named pMDT05-pyr4 KI, and its map is shown in Fig. 5.
  • the above primer sequences are:
  • pyr4-F1 5'-TCAGATCTAGTGTTTGATGCTCACGCTCGGAT-3';
  • pyr4-R1 5'-TTTCTAGATGAACAGTAAGGTGTCAGCA-3'.
  • the expression vector pMDT05-pyr4KI was transformed into the T. reesei HH03-26-8 (pyr4 - ) strain by Agrobacterium-mediated transformation. 153 transformants were obtained, and the transformants were spotted on solid MM medium and cultured at 28 ° C for 48 h until the mycelium grew outward to a plaque having a diameter of about 1 cm. All transformants on the MM plate were numbered, and some mycelium spots were picked up onto a PDA solid plate containing 100 ⁇ g/mL hygromycin, and cultured at 28 ° C for 48 h.
  • the above primer sequence is: pyr4-F2: 5'-CAAACGAACACATCACTTTCAAAG-3'; pyr4-R2: 5'-GTGGGCTTCCTTGTTTCTCGACC-3'.
  • the PCR amplification band was approximately 4.2 kb, and when no homologous recombination occurred, the amplified band was approximately 2.7 kb.
  • 28 transformants were able to amplify a fragment of about 4.2 kb
  • 7 transformants were able to amplify a fragment of about 2.7 kb. It is speculated that these 7 transformants are located outside the pyr4 locus. Random insertion occurred while losing hygromycin resistance.
  • Example 11 Mus53 gene knockout in Trichoderma reesei Rut-C30 (pyr4 - ) strain
  • the mus53 gene (homologous to the human Lig4 gene) is required for non-homologous end joining (NHEJ) function, The disruption of function can bring nearly 100% homologous recombination efficiency.
  • NHEJ non-homologous end joining
  • the mus53 gene in the T. reesei Rut-C30 (pyr4-) strain was knocked out, which laid the foundation for subsequent site-integrated knock-in examples.
  • the primers mus53-3F/mus53-3R and mus53-5F/mus53-5R in Table 5 were used to amplify the homologous Arm Up fragment of about 1.4 kb upstream of the mus53 gene and about 1.3 kb.
  • the Down fragment of the homologous arm downstream of the mus53 gene was amplified with the primer mus53-mid-F/mus53-mid-R to a small fragment of about 1.3 kb of the mus53 locus.
  • a 1.5 kb pyr4 gene coding region and its terminator were amplified with the primer pyr4-TprC-F/pyr4-R.
  • the plasmid pBARGPE1 was used as a template and the primer pyr4-F/pyr4- was used.
  • TrpC-R amplifies the 386 bp PtrpC promoter.
  • the above 5-stage PCR amplified fragments were recovered according to the omega gel recovery kit method, and were collected and mixed in an equimolar ratio as a PCR amplification template, using the primer mus53-3R/mus53-mid-F as the upstream and downstream primer SOE. - Approximately 6.1 kb of the fusion fragment was amplified by PCR and the fragment of interest was recovered according to the omega gel recovery kit method.
  • the plasmid pMDT05 was digested with restriction endonucleases EcoRI and XbaI for 3 h, and the vector fragment was recovered by gelation and the recovered 6.1 kb fragment was used.
  • the method of the one-step cloning kit was assembled and transformed into E. coli TOP10 competent cells.
  • the correct vector for verification and sequencing was named pMDT05-mus53KO, and the vector construction map is shown in Fig. 7.
  • the mus53 gene knockout vector pMDT05-mus53KO was transformed into the Trichoderma reesei Rut-C30 (pyr4 - ) strain by Agrobacterium-mediated transformation, and 294 transformants were obtained. Each transformant was plated into solid MM medium (300 ⁇ g/mL cephalosporin and 200 ⁇ g/mL hygromycin) plate and solid MM medium (300 ⁇ g/mL cephalosporin) plate, and cultured at 28 ° C for 3 days. Forty-four transformants without hygromycin resistance were obtained, and 31 transformants were selected and transferred to a PDA plate, and cultured at 28 ° C for 7 d.
  • solid MM medium 300 ⁇ g/mL cephalosporin and 200 ⁇ g/mL hygromycin
  • solid MM medium 300 ⁇ g/mL cephalosporin
  • a small amount of mycelium was extracted from the PDA plate cultured to the third day into 20 ⁇ l of sterile water, heated at 98 ° C for 10 minutes, and the supernatant was centrifuged as a template, using primer MUS-F.
  • /TrpC-CX-F and pyr4-LB-R/MUS-R can be amplified to about 3.1 kb and 1.6 kb, respectively, indicating that the expected form of homologous recombination occurred in the corresponding region, while using the primer RB-YZ-F And the primer RB-YZ-R could not amplify the 425 bp fragment, indicating that no random integration occurred.
  • the corresponding DNA sequence information of the CBH1 (Cel7A) locus was obtained by keyword search in the T. reesei genomic database (http://genome.jgi-psf.org/Trire2/Trire2.home.html).
  • primers CBH1-F1 and CBH1-R1 were used to amplify a Pcbh1-TRA2-Tcbh1 fragment containing a part of Pcbh1, and a 1115 bp part of Pcbh1 was used as a 5'-end homology arm sequence.
  • a 500 bp sequence after amplification of the Tcbh1 terminator was amplified using the primers CBH1-F2 and CBH1-R2 as a repeat (DR).
  • the pyr4 expression cassette was amplified using primers CBH1-F3 and CBH1-R3.
  • the 1041 bp sequence after the Tcbh1 terminator was amplified using the primers CBH1-F4 and CBH1-R4 as the 3'-end homology arm sequence.
  • All PCR amplified fragments were recovered according to the omega gel recovery kit method.
  • the recovered fragments were mixed in an equimolar ratio as a PCR amplification template, and the primers CBH1-F1 and CBH1-R4 were used as the upstream and downstream primers SOE-PCR.
  • a fusion fragment of about 7 kb was added.
  • the linearization vector pMDT-05 was amplified using primers pMDT-SpeI-R and pMDT-XbaI-F, and the amplified product was digested with DpnI for 3 h.
  • the above 2 fragments were recovered according to the omega gel recovery kit method, and the recovered target fragments were The method of the one-step cloning kit was assembled and transformed into E. coli TOP10 competent cells.
  • the correct vector for verification and sequencing was named pMDT05-CBH1-TRA2(KI), and the map is shown in Fig. 8.
  • the primer sequences are shown in Table 6.
  • the corresponding DNA sequence information of the CBH2 (Cel6A) locus was obtained by keyword search in the T. reesei genomic database (http://genome.jgi-psf.org/Trire2/Trire2.home.html).
  • the 5'-end homology arm sequence (1087 bp) was amplified using primers CBH2-F1 and EcoRI-CBH2-UR.
  • the plasmid pMDT05-mus53KO was used as a template, and the pyr4 expression cassette was amplified using the primers EcoRI-CBH2-TrpC-F and CBH2-D-TU-R.
  • the 3'-end homology arm sequence (1187 bp) was amplified using primers Tpyr4-CBH2-D-F and CBH2-R3.
  • All PCR amplified fragments were recovered according to the omega gel recovery kit method.
  • the recovered fragments were mixed in an equimolar ratio as a PCR amplification template, and the primers CBH2-F1 and CBH2-R3 were used as the upstream and downstream primers SOE-PCR.
  • a fusion fragment of about 4.2 kb was added.
  • the linearization vector pMDT-05 was amplified using primers pMDT-SpeI-R and pMDT-XbaI-F, and the amplified product was digested with DpnI for 3 h.
  • the above 2 fragments were recovered according to the omega gel recovery kit method, and the recovered target fragments were The method of the one-step cloning kit was assembled, transformed into E. coli TOP10 competent cells, and the correct vector for verification and sequencing was named pMDT05-CBH2-pyr4, see FIG.
  • the expression cassette Pcbh1-TRA2-Tcbh1 of about 4.7 kb was amplified using primers E-CBH2-PCBH-F and CBH2-DR-R2.
  • primers CBH-DR-F and E-CBH2-DR-R were used to amplify the 437 bp sequence as a repeat (DR).
  • the above two PCR fragments were recovered according to the omega gel recovery kit method, and the recovered fragments were mixed in an equimolar ratio as a PCR amplification template using primers E-CBH2-PCBH-F and E-CBH2-DR-R.
  • a fusion fragment of about 5.1 kb was amplified by SOE-PCR for upstream and downstream primers, and the fragment of interest was recovered according to the omega gel recovery kit method.
  • the recovered fragment was ligated with the vector pMDT05-CBH2-pyr4 (EcoRI) digested with EcoRI.
  • the method of the one-step cloning kit was assembled and transformed into E. coli TOP10 competent cells, and the correct vector for verification and sequencing was named pMDT05-CBH2-TRA2 (KI).
  • the primer sequences are shown in Table 6.
  • the corresponding DNA sequence information of the EG1 (Cel7B) locus was obtained by keyword search in the T. reesei genomic database (http://genome.jgi-psf.org/Trire2/Trire2.home.html).
  • the 5'-end homologous arm sequence (1149 bp) was amplified using the T. reesei genomic DNA as a template.
  • the pyr4 expression cassette was amplified using primers EG1-pyr4-F and CBH2-R6.
  • primers CBH2-F5 and EG1-TRA2-R were used to amplify the 501 bp sequence as a repeat (DR).
  • the 3'-end homology arm sequence (1211 bp) was amplified using primers EG1-DW-F and EG1-DW-R.
  • PCR amplified fragments were recovered according to the omega gel recovery kit method, and the recovered fragments were mixed in an equimolar ratio as a PCR amplification template, using primers WF-EG1-UF1 and EG1-DW-R as upstream and downstream primers.
  • a fusion fragment of about 4.8 kb was amplified by SOE-PCR.
  • the linearization vector pMDT-05 was amplified using primers pMDT-SpeI-R and pMDT-XbaI-F, and the amplified product was digested with DpnI for 3 h.
  • the above 2 fragments were recovered according to the omega gel recovery kit method, and the recovered target fragments were The method of the one-step cloning kit was assembled and transformed into E. coli TOP10 competent cells, and the correct vector for verification and sequencing was named pMDT05-EG1-pyr4.
  • the expression cassette Pcbh1-TRA2-Tcbh1 of about 4.7 kb was amplified using primers EG1-TRA2-F and CBH2-R22.
  • the plasmid pMDT05-EG1-pyr4 was used as a template, and the primers CBH2-F66 and P-EG1-R were used to linearize the amplified vector, and the amplified product was digested with DpnI for 3 hours.
  • the above two fragments were recovered according to the omega gel recovery kit method, and the recovered fragments were The method of the one-step cloning kit was assembled and transformed into E. coli TOP10 competent cells, and the correct vector for verification and sequencing was named pMDT05-EG1-TRA2(KI), see FIG.
  • the primer sequences are shown in Table 6.
  • the corresponding DNA sequence information of the EG2 (Cel5B) locus was obtained by keyword search in the T. reesei genomic database (http://genome.jgi-psf.org/Trire2/Trire2.home.html).
  • the 5'-end homology arm sequence (1100 bp) was amplified using primers WF-EG2-UF1 and P-EG2-R.
  • the pyr4 expression cassette was amplified using primers EG2-pyr4-F and CBH2-R6.
  • primers CBH2-F5 and EG2-TRA2-R were used to amplify the 501 bp sequence as a repeat (DR).
  • the 3'-end homology arm sequence (1098 bp) was amplified using primers EG2-DW-F and EG2-DW-R.
  • PCR amplified fragments were recovered according to the omega gel recovery kit method, and the recovered fragments were mixed in an equimolar ratio as a PCR amplification template, using primers WF-EG2-UF1 and EG2-DW-R as upstream and downstream primers.
  • a fusion fragment of about 4.6 kb was amplified by SOE-PCR.
  • the linearization vector pMDT-05 was amplified using primers pMDT-SpeI-R and pMDT-XbaI-F, and the amplified product was digested with DpnI for 3 h.
  • the above 2 fragments were recovered according to the omega gel recovery kit method, and the recovered target fragments were The method of the one-step cloning kit was assembled and transformed into E. coli TOP10 competent cells, and the correct vector for verification and sequencing was named pMDT05-EG2-pyr4.
  • the expression cassette Pcbh1-TRA2-Tcbh1 of about 4.7 kb was amplified using primers EG2-TRA2-F and CBH2-R22.
  • the plasmid pMDT05-EG2-pyr4 was used as a template, and the primers CBH2-F66 and P-EG2-R were used to linearize the amplified vector, and the amplified product was digested with DpnI for 3 hours.
  • the above two fragments were recovered according to the omega gel recovery kit method, and the recovered fragments were The method of the one-step cloning kit was assembled and transformed into E. coli TOP10 competent cells, and the correct vector for verification and sequencing was named pMDT05-EG2-TRA2(KI), see FIG.
  • the primer sequences are shown in Table 6.
  • CBH1, CBH2, EG1 and EG2 in the extracellular cellulase system accounted for more than 75% of total extracellular protein.
  • the inducible promoter Pcbh1 also induced the expression of a large number of cellulase genes while inducing the expression of the target gene TRA2, so that there were more cellulase components in the supernatant of the fermentation broth as heteroproteins, which were downstream.
  • the treatment brings disadvantages, and at the same time, some of the raw materials are consumed to synthesize the components of these cellulase systems.
  • the CBH1 site-specific integrated expression vector pMDT05-CBH1-TRA2(KI) was transformed into Trichoderma reesei Rut-C30 (pyr4 - , mus53 - by Agrobacterium-mediated transformation) . ), pick 36 transformant plate to the solid MM medium (300 ⁇ g / mL cephalosporin) plate and sift on the plate, culture at 28 ° C for 3d, pick 20 of which will grow well transformed transformant hyphae to The PDA plate was cultured at 28 ° C for 7 days.
  • transformants were screened by primers NdeI-Pcbh1-F2/TRA2-CX-R1 and pyr4-LB-R/CBH-down-R to determine whether the 5'-end homology arm was passed at the CBH1 locus. Homologous recombination occurs in the 3'-end homology arm Transformants were screened by PCR amplification using primers RB-YZ-F and primer RB-YZ-R (see Table 5) to determine if random integration occurred outside the CBH1 gene locus.
  • a small amount of mycelium was extracted from a PDA plate cultured to day 3 into 20 ⁇ l of sterile water, heated at 98 ° C for 10 minutes, and the supernatant was centrifuged as a template, using primer NdeI-Pcbh1.
  • -F2/TRA2-CX-R1 and pyr4-LB-R/CBH-down-R can be amplified to about 2.7 kb and 1.3 kb fragments, respectively, indicating that the expected form of homologous recombination occurred in the corresponding region, while using primer RB -YZ-F and the primer RB-YZ-R could not amplify a fragment of 425 bp, indicating that no random integration occurred, and in this example, 14 positive transformants satisfying these conditions were screened.
  • the strain was named LYH-D1 (pyr4 - , mus53 - ).
  • the primer sequences are shown in Table 7.
  • the CBH2 site was integrated into the expression vector pMDT05-CBH2-TRA2(KI) by Agrobacterium
  • the method of transforming transformed the T. reesei LYH-D1 (pyr4 - , mus53 - ) strain, and obtained 2 copies of the site-directed integrated strain LYH-D2 (pyr4 - , mus53 - ).
  • the transformants were screened by primers CBH2-F/Pcbh1-CX and pyr4-LB-R/CBH2-R to determine whether the 5'-end homology arm and the 3' end were passed at the CBH2 gene locus. Homologous recombination occurs in the homology arms Transformants were screened by PCR amplification using primers RB-YZ-F and primer RB-YZ-R (see Table 5) to determine if random integration occurred outside the CBH2 gene locus. Whether or not the pyr4 gene expression cassette was deleted was confirmed by the primers Tcbh1-CX-F and CBH2-R2. The primer sequences are shown in Table 7.
  • the EG1 site was integrated into the expression vector pMDT05-EG1-TRA2(KI) by Agrobacterium
  • the method of transforming transformed the T. reesei LYH-D2 (pyr4 - , mus53 - ) strain, and obtained 3 copies of the site-directed integrated strain LYH-D3 (pyr4 - , mus53 - ).
  • transformants were screened by primers EG1-UF1/Pcbh1-CX and pyr4-LB-R/EG1-R to determine whether the 5'-end homology arm and the 3' end were passed at the EG1 locus. Homologous recombination occurs in the homology arms Transformants were screened by PCR amplification using primers RB-YZ-F and primer RB-YZ-R (see Table 5) to determine if random integration occurred outside the EG1 gene locus. Whether or not the pyr4 gene expression cassette was deleted was confirmed by the primers Tcbh1-CX-F and EG1-DR1. The primer sequences are shown in Table 7.
  • the EG2 site was integrated into the expression vector pMDT05-EG2-TRA2(KI) by Agrobacterium
  • the method of transforming transformed the T. reesei LYH-D3 (pyr4 - , mus53 - ) strain, and obtained 4 copies of the site-directed integrated strain LYH-D4 (pyr4 - , mus53 - ).
  • the transformants were screened by primers EG2-UF1/Pcbh1-CX and pyr4-LB-R/EG22-R to determine whether the 5'-end homology arm and the 3' end were passed at the EG2 locus. Homologous recombination occurs in the homology arms Transformants were screened by PCR amplification using primers RB-YZ-F and primer RB-YZ-R (see Table 5) to determine if random integration occurred outside the EG1 gene locus. Whether or not the pyr4 gene expression cassette was deleted was confirmed by the primers Tcbh1-CX-F and EG2-DR1. The primer sequences are shown in Table 7.
  • a fragment containing the 5'-end homologous arm sequence and the repeat (DR) (2209 bp) was amplified using the primer mus53-up-F and mus53-up-R using the T. reesei genomic DNA as a template.
  • the plasmid pMDT05-mus53KO was used as a template, and the pyr4 expression cassette was amplified using the primers mus53-pyr4-F and mus53-pyr4-R.
  • the above two PCR amplified fragments were recovered according to the omega gel recovery kit method, and the recovered fragments were mixed in an equimolar ratio as a PCR amplification template using primers mus53-up-F and mus53-pyr4-R.
  • the downstream primer SOE-PCR amplifies a fusion fragment of about 4.0 kb.
  • the linearization vector pMDT-05 was amplified using primers pMDT-SpeI-R and pMDT-XbaI-F, and the amplified product was digested with DpnI for 3 h.
  • the above 2 fragments were recovered according to the omega gel recovery kit method, and the recovered target fragments were The method of the one-step cloning kit was assembled and transformed into E. coli TOP10 competent cells, and the correct vector for verification and sequencing was named pMDT05-mus53-pyr4.
  • a fragment containing the 3'-end homology arm and the mus53 repair region (4343 bp) was amplified using mus53-down-F and mus53-down-R using T. reesei genomic DNA as a template.
  • the vector pMDT05-mus53-pyr43h was digested with restriction endonuclease EcoRI.
  • the above PCR amplified fragment and the restriction enzyme linearized pMDT05-mus53-pyr4 (EcoRI) vector were recovered according to the omega gel recovery kit method, and the recovered target fragment was The method of the one-step cloning kit was assembled and transformed into E. coli TOP10 competent cells.
  • the correct vector for verification and sequencing was named pMDT05-mus53 (KI), and the construction map is shown in Fig. 12.
  • Example 15 Repair of mus53 and pyr4 genes in 4 copies of strain LYH-D4 (pyr4 - , mus53 - )
  • the T. reesei mus53 gene was repaired in the T. reesei LYH-D4 (pyr4 - , mus53 - ) strain, and the mus53 gene repair vector pMDT05-mus53 (KI) was prepared according to the method and procedure described in Example 4.
  • the T. reesei LYH-D4 (pyr4 - , mus53 - ) strain was transformed by Agrobacterium-mediated transformation, and 27 transformant platelets were picked and sieved onto solid MM medium (300 ⁇ g/mL cephalosporin). The cells were cultured at 28 ° C for 3 days, and 15 of the transformant hyphae which were well-grown were picked to the PDA plate, and cultured at 28 ° C for 7 days.
  • the pyr4 gene expression cassette was deleted according to the method of Example 11, and the primers mus3-YZ-F and MUS-YZ-R2 were used to verify whether the pyr4 gene expression cassette was deleted, and the positive strain for repairing the mus53 gene was named LYH-D4 (pyr4 - ). Verify the primer sequence (see Table 5 for some):
  • MUS-YZ-F2 GTGCTGGGAGACGATGTGATG
  • mus3-YZ-F CAGCAGCGACGCGATTCCTTC
  • LYH-D4 pyr4 gene in LYH-D4 (pyr4 - ) was repaired by the pyr4 gene repair vector pMDT05-pyr4KI according to the method and procedure of Example 10, and the resulting positive strain was named LYH-D4.
  • Example 16 Optimized fermentation of fixed-point integration 4-copy strain LYH-D4 shake flask
  • Example 6 Since the four major cellulase genes of the T. reesei LYH-D4 strain were knocked out and microcrystalline cellulose could not be used as an inducer and a carbon source, the culture for random integration of transformed strains in Example 6 was optimized. The base is not suitable for the T. reesei LYH-D4 strain.
  • composition lactose 30g / L, corn syrup 12g / L, (NH 4 ) 2 SO 4 0.5g / L, MgSO 4 ⁇ 7H 2 O 1.56g / L, CaCl 2 0.5g / L, KH 2 PO 4 6g /L, bran powder 2g / L, Mandels trace element (1000X) 1ml, MnCl 2 5mM, pH 4.0), oxalic acid decarboxylase activity in the fermentation broth of fermentation 168h is about 6800IU / L. Using optimized medium, after 168 hours of fermentation in shake flask fermentation, the activity of oxalate decarboxylase in the supernatant fermentation broth reached 26500 IU/L.
  • the preferred medium composition is: glucose 3-6g/L, lactose 30-40g/L, corn syrup powder 7-10g/L, (NH 4 ) 2 SO 4 0.5-1g/L, MgSO 4 ⁇ 7H 2 O 1.56 g/L, CaCl 2 0.5g/L, KH 2 PO 4 2-4g/L, urea 0-1g/L, bran powder 10-20g/L, Mandels trace element (1000X) 1ml, MnCl 2 0.5-5mM , pH 3.5-4.0.
  • Example 17 Site-integrated 4-copy strain LYH-D4 fermentor fermentation
  • the mycelium of recombinant T. reesei LYH-D4 was inoculated into a plurality of PDA solid slant culture medium, cultured at 28 ° C for 7 d, and after the conidia grew green, the spore suspension was collected by washing with sterile water, and the spores were adjusted.
  • the concentration is about 1.0*10 8 /ml, inoculated in 500mL MM liquid medium with 1% inoculum, and cultured at 28 °C for 24 to 36 hours in the dark (170 rpm), as the seed of 7L fermenter fermentation. liquid.
  • the whole fermentation process of Trichoderma reesei is divided into the following two stages: the first stage is the mycelial growth stage (0-72h): 4.5L basic fermentation is added to the 7L fermenter (Shanghai Baoxing Biological Equipment Engineering Co., Ltd.) Medium (glucose 20g/L, corn syrup 7g/L, KH 2 PO 4 4g/L, urea 1g/L, ammonium sulfate 2g/L, MgSO 4 ⁇ 7H 2 O 0.5g/L, CaCl 2 1g/L , MnCl 2 1 mM, Mandels trace element (1000X) 1ml / L, pH 4.0), inoculated in a 10% ratio of prepared T.
  • the 7L fermenter Sthanghai Baoxing Biological Equipment Engineering Co., Ltd.
  • Medium glucose 20g/L, corn syrup 7g/L, KH 2 PO 4 4g/L, urea 1g/L, ammonium s
  • reesei seed solution 28 ° C aeration and stirring culture for 72 hours, dissolved oxygen maintained at 30 Above 100%, the stirring speed is adjusted according to the amount of dissolved oxygen, generally controlled at 250-500 rpm, and the pH is maintained at about 3.5-4.0.
  • the glucose is generally consumed in about 24-28 h, and 250 g/L lactose solution is added at a rate of 12 ml/h.
  • the dry weight of the cells up to 72 h was 15-18 g/L.
  • the second stage is the induction expression stage (72-168h): at the 72h of the start of fermentation, a 250g/L lactose solution is added by peristaltic pump flow so that the working concentration is never more than 2g/l, and the dissolved oxygen is always greater than 20%.
  • the mixture was agitated and cultured at 28 ° C for about 168 hours after inoculation, and the pH was maintained at about 4.0.
  • the activity of oxalic acid decarboxylase in the fermentation broth was sampled every 24 hours, and the supernatant activity of the fermentation broth reached 271756 U/L after about 160 hours of fermentation (Fig. 13).
  • the supernatant of the fermentation broth of 136 h and 160 h was diluted 10 times for SDS-PAGE, and the target protein band with a molecular weight of about 60 kDa was clearly observed (Fig. 14).
  • the 160 h fermentation broth samples were diluted 200-fold and 500-fold for Western Blot analysis (Figure 15).
  • Example 18 Recovery and extraction of recombinant oxalic acid decarboxylation
  • the fermentation broth was centrifuged at 5000 rpm for 15 minutes at room temperature, and the supernatant was taken.
  • the supernatant of the fermentation broth was filtered through an inorganic ceramic membrane (Sida Film Environmental Technology Co., Ltd.) having a pore size of 100 nm, and the permeate was collected.
  • the clarified permeate was added with a concentration of 10% of tannic acid while stirring to make tannin.
  • the final concentration of the acid was about 1%, and it was allowed to stand at room temperature for 1 hour, and then centrifuged at 8000 rpm for 15 minutes at normal temperature to collect a complex of oxalic acid decarboxylase and tannic acid.
  • the complex precipitate was thoroughly resuspended in sterile water of 1/2 volume of permeate, centrifuged at 8000 rpm for 15 minutes, and the complex precipitate was taken, and this was repeated once.
  • a clear liquid volume of polyethylene glycol solution polyethylene glycol is used in an amount of 0.3-0.5% of the volume of the clear liquid
  • the mixture is continuously stirred at normal temperature for 4 hours, using polyethylene glycol and tannin.
  • the tannic acid polyethylene glycol polymer was removed by centrifugation at 8000 rpm for 15 minutes at room temperature, and the supernatant was taken, which was a concentrated enzyme solution concentrated 2.5 times.
  • the sugar was added to the concentrated enzyme solution by 2% decolorization with activated carbon to obtain a pale yellow oxalic acid decarboxylase enzyme solution, and the enzyme recovery rate was 90-95%.
  • the decolorized oxalic acid decarboxylase enzyme solution was concentrated 10-30 times with an ultrafiltration membrane having a molecular weight cut off of 10 KDa, concentrated, and spray dried to obtain an oxalic acid decarboxylase enzyme powder.
  • Example 19 Properties and comparative analysis of recombinant oxalic acid decarboxylase
  • the relative oxalate decarboxylase expressed by the T. reesei filamentous fungal host cell and the oxalate decarboxylase induced by the natural host Agrocybe aegerita were assayed for relative enzyme activity at a pH of 1.5-7.0. The results are shown in FIG. Under different pH conditions, the recombinant oxalate decarboxylase has similar relative enzyme activity retention to the natural oxalic acid decarboxylase induced by the tea tree.
  • the recombinant oxalic acid decarboxylase maintains all or part of its enzyme activity at a pH of 1.5-7.0, and maintains at a pH of 1.5-2.5 at a pH of not less than 10% of its enzyme activity at the optimum pH, at pH
  • the value of 2.5-4.5 is maintained at not less than 50% of its enzyme activity at the optimum pH, and maintained at a pH of 4.5-7.0 at a temperature not lower than 25% of its enzyme activity at the optimum pH.
  • the optimum pH is 2.5-3.5.
  • the recombinant oxalate decarboxylase expressed by the T. reesei filamentous fungal host cell, the oxalic acid decarboxylase induced by the natural host Agrocybe aegerita, and the oxalic acid decarboxylase expressed in prokaryotic cells were subjected to SDS-PAGE analysis, and the results are shown in FIG.
  • the recombinant oxalate decarboxylase expressed by Trichoderma and the oxalate decarboxylase expressed by Agrocybe aegerita have different apparent molecular weights due to the different forms and degrees of glycosylation modification.
  • the molecular weight of oxalic acid decarboxylase expressed by the natural host Agrocybe aegerita is about 70kDa.
  • the molecular weight of the recombinant oxalic acid decarboxylase expressed by Trichoderma reesei is about 60 kDa, but both of them have a larger molecular weight than the oxalate decarboxylase expressed by the prokaryotic expression without aglycosylation, and the molecular weight of the oxalate decarboxylase modified by prokaryotic expression without aglycosylation At around 50kDa.
  • the recombinant oxalic acid decarboxylase expressed by T. reesei was subjected to molecular weight analysis by MALDI-TOF-MS, as shown in Fig. 18, and its true molecular weight was 57.1 kDa.
  • the oxalic acid decarboxylase expressed by the above three expression systems was digested with TPCK-treated trypsin, and then subjected to MALDI-TOF-MS analysis.
  • the peak maps are shown in Figures 19, 20 and 21, and the three expression systems are shown.
  • the expressed oxalate decarboxylase differs in the mass spectrometry peak after trypsin digestion due to the different forms and degrees of glycosylation modification, and this difference is host cell specific.
  • the single-copy recombinant expression of the sequences of SEQ ID NO. 10-16 of the present invention in T. reesei can be similar to that of SEQ ID NO.

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Abstract

提供了一种用丝状真菌宿主细胞表达的重组草酸脱羧酶，重组菌株的构建方法，重组酶的生产方法及应用，还提供了针对不同重组丝状真菌宿主细胞的培养基配方。

Description

一种用丝状真菌宿主细胞表达的重组草酸脱羧酶
本申请要求在2017年3月7日提交中国专利局，申请号为201710130999.5，发明名称为“用于真核微生物表达草酸脱羧酶的表达盒、菌株、方法及应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及基因工程技术领域，具体涉及能够高效表达草酸脱羧酶的重组丝状真菌宿主细胞，以及重组草酸脱羧酶，重组草酸脱羧酶的生产方法及应用。
背景技术
草酸(oxalic acid)又名乙二酸，是生物体的一种代谢产物，以草酸盐的形式广泛存在于植物、动物和真菌体中。人类和其他哺乳动物的许多食物，如菠菜、草莓、甜菜、可可、芋头、甘薯、大黄和茶叶等，草酸盐含量都比较高。在人体和其他哺乳动物中，由于体内没有降解草酸盐的相关酶类，导致草酸盐是代谢的终产物，从食物中吸收的外源性草酸盐和由生理代谢产生的内源性草酸盐主要通过肾脏排泄到尿中。人类和其他哺乳动物在食用高含量草酸盐的食物时，在人体的血液和尿液中容易发生草酸盐浓度的升高，当与钙离子结合会生成不溶性的草酸钙，草酸钙是泌尿系结石的主要成分。另外高浓度的草酸盐还与其他多种病理有关，例如，高草酸尿症，心脏传导障碍、克罗恩病(Crohn’s disease)以及其他肠道疾病状态等。因此，在体外或者体内环境中分解来源于食物中的草酸盐，减少草酸盐在体内的吸收，能降低发生包括尿路结石在内的相关疾病的风险。
近年来，研究酶法降解草酸从而预防治疗草酸钙结石症等相关疾病成为研究热点。目前生物界已知存在的具有分解草酸功能的酶，包括草酸脱羧酶、草酸氧化酶和草酰CoA脱羧酶。草酸脱羧酶是一种活性中心含有锰离子，能催化降解草酸生产甲酸和二氧化碳的酶。目前发现的草酸脱羧酶主要存在于一些植物、细菌和真菌中，如黑曲霉、盾壳霉(Coniothyrium minitans)、金针菇、白腐菌(T.versicolor)、双孢蘑菇、褐腐菌、枯草芽孢杆菌(Bacillus subtilis)、根癌农杆菌(Agrobacterium tumefaciens)等。然而，草酸脱羧酶在上述各种天然来源的物种中产率和产量非常低，导致生产成本和价格高昂，难以进行商业化应用和推广。
因此将草酸脱羧酶进行重组表达生产，降低生产成本使其能进行商业化应用成为一种必然的选择。目前虽然在原核细胞中实现了细菌来源的草酸脱羧酶重组表达，如枯草芽孢杆菌YvrK基因来源草酸脱羧酶，但由于细菌来源的草酸脱羧酶在低pH条件下(低于pH值3.0)不稳定且没有活力，而人胃中的pH常常低于3.0，并且细菌来源的草酸脱羧酶在胃蛋白酶的作用容易被消化而失去活性，因此应用范围、领域和有效性受到极大限制。为了改善细菌来源的草酸脱羧酶的使用性能，Allena医药公司将草酸脱羧酶(PCT/US2007/075091)制成蛋白晶体再用戊二醛交联提高其稳定性，然后将这些晶体制成口服药剂用于降解胃肠中的草酸盐。临床试验表明，在严重高尿草酸的病人中，口服高剂量的该酶制剂也只能降低13％的尿草酸。Oxthera公司制备了另一种制剂形式的制剂，将该草酸脱羧酶与酸不溶聚合物混合，经喷雾干燥而制成微颗粒(Oxazyme，Oxthera公司)，临床试验表明，Oxazyme没有降低尿草酸的效果。
真菌来源的草酸脱羧酶在低pH值条件下具有良好的稳定性以及对胃蛋白酶也具有良好的抵抗性，因此非常适合作为口服酶制剂降解草酸。尽管人们做了很多努力和尝试,但从目前的公开报道来看，真菌来源的草酸脱羧酶不管是用原核表达系统还是用真核表达系统，都未获得良好重组表达结果，其中研究的最多是来源于金针菇的草酸脱羧酶，Meenu等(参见文献：Meenu Kesarwani，et.al，Oxalate Decarboxylase from Collybia velutipes，THE JOURNAL OF BIOLOGICAL CHEMISTRY，2000)将其通过烟草进行重组表达，表达结果能检测显示有活力，但表达量极低，同时也进行了原核(E.coli)表达但无酶活力。Mohammad等(Mohammad Azam，et.al，A Secretion Signal Is Present in the Collybia velutipesOxalate Decarboxylase Gene，doi:10.1006/bbrc.2001.6049)将其在酿酒酵母和粟酒裂殖酵母进行重组表达，其中在酿酒酵母未检测到酶活力，在粟酒裂殖酵母虽然检测到了酶活力，但表达量很低，无法进行商业化应用。
发明内容
为了解决现有技术中真菌来源的草酸脱羧酶无法进行有效的重组表达这一技术问题，本发明前期经过了长期的大量的试验和努力，尝试了多种表达系统，运用了多种生物技术手段，以期能够实现真菌来源的草酸脱羧酶的有效重组表达。在原核表达系统中，发明人在不同种类的大肠杆菌表达细胞、枯草芽孢杆菌细胞、地衣芽孢杆菌细胞、短小芽孢杆菌细胞、乳酸杆菌细胞等原核细胞中进行了各种表达尝试 以及表达元件及表达策略的优化工作，但均未获得有效的重组表达；在真核表达系统中，发明人在烟草和豌豆植物中进行过瞬时表达和稳定转化表达，运用过烟草细胞进行悬浮培养表达，也运用过昆虫细胞、酿酒酵母细胞和毕赤酵母细胞等酵母属细胞进行表达和表达元件及表达策略的优化，获得的结果要么是没有检测到酶活力，要么是表达量极低，没有产业化生产可能。在经过长期而艰辛的探索和研究后，组合与优化各个环节，发明人最终在丝状真菌中获得了高效的重组表达。
本发明的目的在于提供一种重组草酸脱羧酶，所述重组草酸脱羧酶通过丝状真菌宿主细胞重组表达，致使重组草酸脱羧酶的糖基化修饰形式和程度不同于原始宿主细胞表达的草酸脱羧酶，所述重组草酸脱羧酶具有丝状真菌宿主细胞特有的糖基化修饰形式和程度。
所述重组草酸脱羧酶在pH值1.5-7.0的条件下保持其全部或者部分酶活力，且在pH值1.5-2.5的条件下保持不低于其在最适pH条件下酶活力的10％，在pH值2.5-4.5的条件下保持不低于其在最适pH条件下酶活力的50％，在pH值4.5-7.0的条件下保持不低于其在最适pH条件下酶活力的25％。
可选或优选地，所述重组草酸脱羧酶的最适pH值为2.5-3.5。
可选或优选地，所述重组草酸脱羧酶编码基因来源于真核生物，所述真核生物为茶树菇、杨树菇、金针菇、云芝，褐腐菌、琉球曲霉、双孢蘑菇或金福菇等真菌。
可选或优选地，所述重组草酸脱羧酶的氨基酸序列与SEQID NO.1或SEQ ID NO.5的第20～470位氨基酸序列、或SEQ ID NO.2的第25～472位氨基酸序列、或SEQ ID NO.3的第20～455位氨基酸序列、或SEQ ID NO.4的第21～447位氨基酸序列、或SEQ ID NO.6的第21～455的氨基酸序列、或SEQ ID NO.7的第25～440位氨基酸序列、SEQ ID NO.8的第24～472位氨基酸序列具有至少60％同一性，优选至少65％同一性、至少70％同一性、至少75％同一性、至少80％同一性、至少85％同一性、至少90％同一性、或至少95％同一性。
优选地，所述重组草酸脱羧酶的氨基酸序列由SEQID NO.1或SEQ ID NO.5的第20～470位氨基酸序列、或SEQ ID NO.2的第25～472位氨基酸序列、或SEQ ID NO.3的第20～455位氨基酸序列、或SEQ ID NO.4的第21～447位氨基酸序列、或SEQ ID NO.6的第21～455的氨基酸序列、或SEQ ID NO.7的第25～440位氨基酸序列、SEQ ID NO.8的第24～472位氨基酸序列构成。
本发明的另一个目的在于提供一种新的表达草酸脱羧酶的方法，能够高效表达草酸脱羧酶基因，表达量和酶活力均远优于现有的表达方法，达到实际应用价值。
为了实现上述目的，本发明的第一方面，提供了一种重组丝状真菌宿主细胞，所述重组丝状真菌宿主细胞的染色体DNA中包含编码以上任一所述的重组草酸脱羧酶的基因序列。
具体来说，其包含一个或多个拷贝的整合在其基因组中的草酸脱羧酶表达盒，所述草酸脱羧酶表达盒包括启动子、信号肽编码序列、草酸脱羧酶编码基因和终止子。
发明人经过大量的研究工作发现，草酸脱羧酶能在丝状真菌宿主细胞中高效地重组分泌表达。在丝状真菌宿主细胞重组表达的草酸脱羧酶能够进行各种翻译后加工，如糖基化修饰等，重组表达的草酸脱羧酶具有与天然宿主细胞制备的草酸脱羧酶相似的酶学性质。通过在草酸脱羧酶5’端添加编码引导分泌的信号肽序列，能将重组表达的草酸脱羧酶有效分泌到培养基质中，有利于后续的分离纯化，降低生产成本。
所述信号肽编码序列，是指能引导草酸脱羧酶进入细胞特定区室或分泌通道的信号肽编码区，其可以得自但不限于草酸脱羧酶原始信号肽、木霉纤维二糖水解酶I、木霉纤维二糖水解酶II、木霉内切葡聚糖酶I、木霉内切葡聚糖酶II、黑曲霉中性淀粉酶、黑曲霉葡糖淀粉酶、米曲霉TAKA淀粉酶、特异腐质霉(Humicola insolens)纤维素酶、特异腐质霉内切葡聚糖酶V、柔毛腐质霉(Humicola lanuginosa)脂肪酶和米黑根毛霉(Rhizomucor miehei)天冬氨酸蛋白酶等基因的信号肽编码序列，任何能将草酸脱羧酶引导至丝状真菌宿主细胞分泌途径的信号肽编码区都可用于本发明中。在一些实施方案中，优选的信号肽编码序列是里氏木霉纤维二糖水解酶I基因(cbh1)信号序列。
所述启动子，是指涉及结合RNA聚合酶含有可介导草酸脱羧酶基因表达的转录和翻译控制序列。启动子可以是在选定的宿主细胞中具有转录活性的任何核苷酸序列，可以源自编码与宿主细胞同源或异源的蛋白质的基因。启动子可以是诱导型启动子或组成型启动子。
本发明用于介导草酸脱羧酶表达盒在丝状真菌宿主细胞中转录的启动子的实例有得自但不限于以下酶的基因的启动子：SV40，hCMV，CaMV 35S、构巢曲霉乙酰胺酶、米曲霉碱性蛋白酶、米曲霉磷酸丙糖异构酶、米曲霉TAKA淀粉酶、黑曲霉中性α-淀粉酶、黑曲霉酸稳定性α-淀粉酶、黑曲霉或泡盛曲霉葡糖淀粉酶(glaA)、米黑根毛霉脂肪酶、木霉丙酮酸脱羧酶、木霉β-葡糖苷酶、木霉纤维二糖水解酶I、木霉纤维二糖水解酶II、木霉内切葡聚糖酶I、木霉内切葡聚糖酶II、木霉内切葡聚糖酶III、木霉内切葡聚糖酶IV、木霉内切葡聚糖酶V、木霉木聚糖酶I、木霉木聚糖酶II、或木霉β-木糖苷酶等，以及前述基因启动子经过突变、截短或杂合后的同功能序列。
在一些优选的实施方案中，启动子是源自里氏木霉纤维二糖水解酶I基因启动子(Pcbh1)，在一些 优选的实施方案中，启动子是源自里氏木霉丙酮酸脱羧酶基因启动子(Ppdc)。
所述终止子，是指可被丝状真菌宿主细胞识别而终止转录的一段序列。在宿主细胞中起作用的任何终止子均可用于本发明。本发明用于介导草酸脱羧酶表达盒在丝状真菌宿主细胞中转录终止的终止子的实例有得自但不限于以下酶的基因的终止子：构巢曲霉乙酰胺酶、米曲霉碱性蛋白酶、米曲霉磷酸丙糖异构酶、米曲霉TAKA淀粉酶、黑曲霉中性α-淀粉酶、黑曲霉酸稳定性α-淀粉酶、黑曲霉或泡盛曲霉葡糖淀粉酶(glaA)、米黑根毛霉脂肪酶、木霉丙酮酸脱羧酶、木霉β-葡糖苷酶、木霉纤维二糖水解酶I、木霉纤维二糖水解酶II、木霉内切葡聚糖酶I、木霉内切葡聚糖酶II、木霉内切葡聚糖酶III、木霉内切葡聚糖酶IV、木霉内切葡聚糖酶V、木霉木聚糖酶I、木霉木聚糖酶II、木霉β-木糖苷酶等。
在一些优选的实施方案中，终止子是源自里氏木霉纤维二糖水解酶I基因终止子(Tcbh1)，在一些优选的实施方案中，终止子是源自里氏木霉丙酮酸脱羧酶基因终止子(Tpdc)。
可选或优选地，所述的重组丝状真菌宿主细胞中，所述丝状真菌为曲霉属、革盖菌属、毛霉属、白腐菌属、枝顶孢属、隐球菌属、镰孢霉属、腐质霉属、毁丝霉属、短梗霉属、栓菌属、侧耳属、脉孢菌属、青霉属、拟青霉属、平革菌属、烟管菌属、拟蜡菌属、梭孢壳属、金孢子菌属、裂褶菌属、鬼伞属、稻瘟菌属、新美鞭菌属、弯颈霉属踝节菌属、嗜热子囊菌属或木霉属等真菌。
可选或优选地，上述重组丝状真菌宿主细胞中，所述丝状真菌为曲霉属的黑曲霉、构巢曲霉、米曲霉或泡盛曲霉菌株。
可选或优选地，上述重组丝状真菌宿主细胞中，所述丝状真菌为木霉属的哈茨木霉、康宁木霉、里氏木霉、长梗木霉或绿色木霉菌株。
更优选地，丝状真菌宿主细胞是里氏木霉细胞，包括但不限制于ATCC NO.56765、ATCC NO.13631、ATCC NO.26921、ATCC NO.56764、ATCC NO.56767和NRRLNO.15709等里氏木霉菌株细胞。在一些实施方案中，所述丝状真菌宿主细胞是里氏木霉菌株Rut-C30细胞。在一些实施方案中，丝状真菌宿主细胞可以是里氏木霉菌株Rut-C30的变体细胞，包括通过遗传改造，敲除里氏木霉宿主细胞的多种天然基因，包括编码乳清酸核苷-5’-磷酸脱羧酶的基因(pyr4)和参与非同源重组过程的基因(mus53)。敲除所述编码乳清酸核苷-5’-磷酸脱羧酶基因的菌株为尿嘧啶营养缺陷型菌株(pyr4 -)，基于pyr4基因的营养缺陷筛选标记已被证明是十分行之有效的，并已在多种真核微生物中成功应用(Long et al.2008；Weidner et al.1998)；敲除所述参与非同源重组过程的基因，能显著降里氏木霉宿主细胞的非同源重组频率，有利于提高其同源重组的筛选。
可选或优选地，上述重组丝状真菌宿主细胞中，所述草酸脱羧酶基因的核苷酸序列中至少10％的碱基根据丝状真菌宿主细胞密码子偏爱性进行了密码子优化。优化后的基因编码或至少部分编码草酸脱羧酶蛋白。所述部分编码，是指删除部分氨基酸序列但还具有草酸脱羧酶功能。
可选或优选地，上述核苷酸序列选自SEQ ID NO.9、SEQ ID NO.10、SEQ ID NO.11、SEQ ID NO.12、SEQ ID NO.13、SEQ ID NO.14、SEQ ID NO.15和SEQ ID NO.16的核酸序列；或与SEQ ID NO.9-16中任一具有至少50％同一性，优选至少60％同一性、至少70％同一性、至少80％同一性或至少90％同一性的序列。
当然地，作为本领域技术人员应该知晓的内容，上述重组丝状真菌宿主细胞在制备过程中，先构建草酸脱羧酶基因表达载体，表达载体除了含有草酸脱羧酶基因表达盒，还含有编码选择性标记的基因表达盒。
所述选择性标记，是指可以提供已转化宿主细胞的简单选择的标记基因。合适的选择性标记的实例包括但不限于潮霉素和bar等抗性基因。还可以使用营养选择标记，例如，乙酰胺酶(amdS)、鸟氨酸氨甲酰基转移酶(argB)和乳清酸核苷-5’-磷酸脱羧酶等。
在编码选择性标记的表达盒的5’侧翼和3’侧翼有350-500bp的正向重复核苷酸序列，在反向选择压力下能通过自发的DNA分子内同源重组而切除。在一个实施方案中，选择标记是pyr4基因，营养缺陷筛选标记乳清酸核苷-5’-磷酸脱羧酶是尿嘧啶核苷酸合成的一个关键酶，该基因的缺失将导致尿嘧啶核苷酸合成受阻，因此缺乏该酶的营养缺陷型菌株需要添加尿嘧啶/尿苷才能生长。当pyr4基因成功转化进pyr4基因缺失菌株后，该基因的表达使受体菌能够自身合成尿嘧啶/尿苷，从而不依赖尿嘧啶/尿苷生长，起到正向筛选的作用。另一方面，5-氟乳清酸(5′-fluorooroticacid，5′-FOA)是尿嘧啶合成前体的类似物，在pyr4的作用下会产生对细胞有毒害物质，所以野生型菌株在5′-FOA下不能生长，而变成pyr4基因缺失菌时就呈现5′-FOA抗性，从而实现反向筛选(参见Jeffrey L.Smith等.Curr Genet,1991,19:27-23)。用来提供已转化宿主细胞的简单选择。
上述的草酸脱羧酶基因表达载体，包括随机整合型表达载体和位点特异性整合型表达载体。例如，随机整合型表达载体通过农杆菌介导转化里氏木霉后，草酸脱羧酶的表达盒随机整合到里氏木霉基因组中，通过Tail-PCR方法分析其整合在基因组中的位置及拷贝数。在一个实施例中，通过2轮转化和筛选可获得不同整合位点及拷贝数的转化菌株，通过摇瓶发酵比较产酶情况，筛选了一系列工程菌株并分析 了其拷贝数及其在里氏木霉基因组中的整合位点。位点特异性整合表达载体在草酸脱羧酶表达盒两端含有与里氏木霉特定位点相同的一段DNA序列作为5’同源臂和3’同源臂，通过位点特异性整合表达载体，在导入草酸脱羧酶表达盒到特定位点的同时还可以敲除这些特定位点上的基因。在一个实施例中，选择里氏木霉胞外分泌蛋白中占主要成分的几个纤维素酶基因(CBH1、CBH2、EG1和EG2)作为位点特异性整合位点，在整合进草酸脱羧酶表达盒的同时敲除了这些基因编码盒，构建了4拷贝表达菌株，该菌株在发酵条件下，重组分泌表达的草酸脱羧酶在胞外总蛋白中含量能达到90％以上。
本发明的第二方面，提供了一种构建以上任一所述的重组丝状真菌宿主细胞的方法(随机整合法)，所述重组丝状真菌宿主细胞包含一个或多个拷贝的整合在其基因组中的草酸脱羧酶表达盒，所述草酸脱羧酶表达盒包括启动子、信号肽编码序列、草酸脱羧酶编码基因和终止子，所述方法包括如下步骤：
S1：构建至少一个整合型表达载体，所述整合型表达载体包括选择标记基因表达盒和草酸脱羧酶表达盒。
S2:整合型表达载体在转化丝状真菌宿主细胞后，通过筛选获得含有一个拷贝或多个拷贝草酸脱羧酶表达盒的重组丝状真菌宿主细胞。
可选或优选地，上述方法中，其中步骤S2中所述的丝状真菌宿主细胞为人工构建的营养缺陷型细胞，所述整合型表达载体在整合进所述丝状真菌宿主细胞基因组中时能修复该类型营养缺陷。
可选或优选地，上述方法中，在转化丝状真菌宿主细胞后，所述整合型表达载体通过非同源重组而随机整合进丝状真菌宿主细胞基因组中。
可选或优选地，上述方法中，所述的整合型表达载体包含与丝状真菌宿主细胞基因组中特异性基因座一定长度核苷酸序列同源的5’端同源臂和3’端同源臂，从而在转化丝状真菌宿主细胞后所述整合型表达载体能通过同源重组的方式整合进基因组特定的位点；优选整合进编码胞外蛋白的基因中；更优选整合进编码胞外蛋白酶类或编码胞外糖苷水解酶类的基因中，最优选地整合进CBH1、CBH2、EG1或EG2基因中。
在一个实施例(随机整合法)中，其出发菌株为人工构建的pyr4基因缺失型里氏木霉，包括如下步骤：
构建至少一个随机整合表达载体，通过冻融法转入根癌农杆菌AGL-1感受态细胞，获得含有上述随机整合表达载体的根癌农杆菌AGL-1细胞，将该细胞制备成侵染液与pyr4基因缺失的里氏木霉共培养，筛选获得含有一个拷贝或多个拷贝草酸脱羧酶表达盒的里氏木霉菌株，即为目标宿主细胞。
本发明的第三方面，提供了一种培养基，适用上述方法(随机整合法)制备的宿主细胞的培养，其组成为：葡萄糖3-8g/L，微晶纤维素10-25g/L，玉米浆粉5-15g/L，(NH 4) 2SO 40.5-5g/L，MgSO 4·7H 2O 1.56g/L、CaCl 20.5g/L，KH 2PO 42-8g/L，尿素0-1g/L，麸皮粉0.2-2g/L，Mandels微量元素(1000X)1ml，MnCl 20.5-5mM，pH 3.0-4.5。
本发明的第四方面，提供了另外一种构建以上任一所述的重组丝状真菌宿主细胞的方法(位点特异性整合法)，在一个实施例中，该方法包括如下步骤：
(1)分别构建针对里氏木霉CBH1、CBH2、EG1、EG2四个基因座位点的草酸脱羧酶表达载体；
(2)在pyr4和mus53基因缺失的里氏木霉菌株中，通过位点特异性整合敲入的方式在CBH1，CBH2，EG1和EG2位点基因组上整合步骤(1)中各位点对应的表达载体中的表达盒；定点整合到这几个位点后，胞外分泌总蛋白中杂蛋白含量非常少，目的蛋白占绝大部分，用该方法生产的草酸脱羧酶发酵液后处理更简单更经济。mus53基因敲除后定点整合的概率大大提高，有利于后续位点特异性整合菌株的筛选。
(3)通过pyr4和mus53基因修复载体，对步骤(2)获得的菌株进行mus53和pyr4基因修复；修复成功的菌株即为目标宿主细胞。pyr4基因的修复，发酵过程中培养基中就不需要额外加入尿嘧啶或者尿苷，宿主细胞能保存固有的代谢平衡，不提高发酵成本；mus53基因的修复，宿主细胞能保存固有的稳定性，消除mus53基因缺失带来基因组不稳定的因素。
本发明的第五方面，提供了另外一种培养基，适用于上述方法(位点特异性整合法)制备的宿主细胞的培养，其组成为：葡萄糖3-6g/L，乳糖30-40g/L，玉米浆粉7-10g/L，(NH 4) 2SO 40.5-1g/L，MgSO 4·7H 2O1.56g/L、CaCl 20.5g/L，KH 2PO 42-4g/L，尿素0-1g/L，麸皮粉10-20g/L，Mandels微量元素(1000X)1ml，MnCl 20.5-5mM，pH 3.5-4.0。
本发明的第六方面，提供了一种生产草酸脱羧酶的方法，构建含有启动子、信号肽编码序列、草酸脱羧酶编码基因和终止子的草酸脱羧酶表达盒，通过表达载体转入丝状真菌宿主细胞，使宿主细胞基因组中整合一个或多个草酸脱羧酶表达盒，培养宿主细胞表达草酸脱羧酶，最后从宿主细胞培养基质中分离纯化表达产物。
本发明的第七方面，提供了以上任一所述的重组草酸脱羧酶或重组丝状真菌宿主细胞培养后分泌表达的草酸脱羧酶在制备药物、食品中的应用。
可选或优选地，上述应用中，所述药物为预防和/或治疗泌尿系结石的药物。
本发明的第八方面，提供了一种药物组合物，用于预防或治疗尿草酸过多的疾病，其包含有上述方法制备的草酸脱羧酶。
与现有技术相比，本发明具有如下有益效果：
本发明克服了真菌来源草酸脱羧酶无法得到有效重组表达的技术难题，草酸脱羧酶通过丝状真菌宿主细胞重组表达能够进行各种翻译后加工，高效分泌表达的草酸脱羧酶具有与天然宿主细胞制备的草酸脱羧酶相似的酶学性质。该宿主细胞培养简单，草酸脱羧酶分泌量大而且酶活高；本发明的两种培养基配方分别适用于两种重组丝状真菌宿主细胞，能够有效提高产量；草酸脱羧酶的生产，通过表达盒的构建、载体构建、宿主细胞的构建以及最终培养基配方的调整，使得产率和产品酶活都大大提高，有效解决了现有技术中草酸脱羧酶人工生产无法大规模工业化，酶学特性不稳定，生产成本高昂的难题。
附图说明
图1为载体pMDT05构建图谱。
图2为里氏木霉Rut-C30菌株pyr4基因敲除载体pMDT05-pyr4KO构建图谱。
图3为里氏木霉随机整合诱导型表达载体pMGU-cbh1-TRA2构建图谱。
图4为里氏木霉随机整合组成型表达载体pDGU-pdc-TRA2构建图谱。
图5为里氏木霉转化体pyr4基因修复载体pMDT05-pyr4KI构建图谱。
图6为发酵144h和168h发酵液上清SDS-PAGE图，箭头处为重组草酸脱羧酶条带。
图7为mus53基因敲除载体构建图谱。
图8为在CBH1位点定点整合敲入载体构建图谱。
图9为在CBH2位点定点整合敲入载体构建图谱。
图10为在EG1位点定点整合敲入载体构建图谱。
图11为在EG2位点定点整合敲入载体构建图谱。
图12为mus53基因修复载体构建图谱。
图13为7L发酵罐发酵活力变化图。
图14为7L发酵罐发酵第136h和160h样品稀释10倍SDS-PAGE图。
图15为160h样品稀释200倍和500倍Western Blot检测分析图。
图16为pH值在1.5-7.0条件下草酸脱羧酶的相对活力。
图17为三种表达系统表的草酸脱羧酶SDA-PAGE图，其中泳道1和2为里氏木霉表达的重组草酸脱羧酶；泳道3和4为天然宿主茶树菇表达的草酸脱羧酶；泳道5和6为原核表达的草酸脱羧酶。
图18为里氏木霉表达的重组草酸脱羧酶MALDI-TOF-MS图谱。
图19为里氏木霉表达的重组草酸脱羧酶经胰蛋白酶消化后的MALDI-TOF-MS图谱。
图20为天然宿主茶树菇表达的草酸脱羧酶经胰蛋白酶消化后的MALDI-TOF-MS图谱。
图21为原核表达的草酸脱羧酶经胰蛋白酶消化后的MALDI-TOF-MS图谱。
具体实施方式
本发明以来源于茶树菇的草酸脱色酶在丝状真菌里氏木霉中重组表达为具体实施例进行说明，以使本领域的技术人员可以更好的理解本发明并能予以实施，但所举实施例不作为对本发明的限定。
除特别指明以外，所用技术术语均为本领域中的普通技术人员常用术语；本说明书中未注明具体条件的实验方法是按常规实验方法；本说明书中所用的试验材料、试剂如无特别说明均为市售购买产品，各种试剂及培养基的成分和配制方法可参见常规实验手册中的操作。
本发明中用到的里氏木霉Rut-C30(ATCC 56765)购买自广东微生物菌种保藏中心。
本发明中用到的的黑曲霉CICC2439购买自中国工业微生物菌种保藏中心。
实施例1：草酸脱羧酶(OXDC)基因密码子优化及人工合成
本发明人经大量实验研究发现，可用于丝状真菌表达系统表达来源于真核生物的草酸脱羧酶，优选来自茶树菇、杨树菇、金针菇、云芝，褐腐菌、琉球曲霉、双孢蘑菇和金福菇等真菌的草酸脱羧酶。
编码草酸脱羧酶的基因可来自于茶树菇，所述草酸脱羧酶氨基酸序列为SEQ ID NO.1所示氨基酸序列，其中所述草酸脱羧酶的信号肽序列为SEQ ID NO.1所示的1-19位氨基酸序列，成熟肽序列为SEQ ID NO.1所示的20-470位氨基酸序列。
将来源于茶树菇的OXDC基因分别按照里氏木霉密码子偏好性(见Codon Usage Database：Hypocrea jecorina)进行优化，人工设计和合成了新的草酸脱羧酶成熟肽的DNA编码序列。优化后的OXDC序列与优化前相比，CAI(Codon Adaptation Index)由原来的0.51变为0.99，G+C的含量由原来的53.09％变为69.23％，优化后的序列见SEQ ID NO.17所示。将优化后的茶树菇的OXDC基 因成熟肽的DNA编码序列重新命名为TRA2。
实施例2：里氏木霉Rut-C30(pyr4 -)营养缺陷株构建
真核系统表达真核的OXDC所用丝状真菌宿主细胞选自曲霉属、革盖菌属、毛霉属、白腐菌属、枝顶孢属、隐球菌属、镰孢霉属、腐质霉属、毁丝霉属、短梗霉属、栓菌属、侧耳属、脉孢菌属、青霉属、拟青霉属、平革菌属、烟管菌属、拟蜡菌属、梭孢壳属、金孢子菌属、裂褶菌属、鬼伞属、稻瘟菌属、新美鞭菌属、弯颈霉属踝节菌属、嗜热子囊菌属或木霉属细胞或其有性型或同物异名型的细胞，但不限制于此。
木霉属宿主细胞是哈茨木霉、康宁木霉、里氏木霉、长梗木霉和绿色木霉，优选为里氏木霉和绿色木霉。下面以里氏木霉为例来说明本发明。
1、里氏木霉基因组提取
将里氏木霉Rut-C30(ATCC 56765)接种于PDA培养基上，于28℃恒温培养7d至孢子成熟。用无菌水洗脱孢子，制备适量孢子悬液并接种于20ml液体培养基中，于28℃，170rpm条件下培养36-48h。使用真空抽滤收集菌丝体，并用去离子水洗涤2次，在液氮冷冻下将菌丝体研磨成细粉，并使用Sangon Biotech Ezup柱式真菌基因组DNA抽提试剂盒分离基因组DNA。
其中，上述PDA培养基配方为：去皮土豆薄片200g，加1000ml水煮沸30min，8层纱布过滤，滤液添加葡萄糖20g，并补足水分至1L，自然pH，2％琼脂粉。115℃灭菌30min。
上述液体培养基配方为：将葡萄糖15g、酵母提取物20g、硫酸铵2.5g、七水硫酸镁0.8g、无水氯化钙1.0g混合后，用蒸馏水溶解并定容至1L，调节pH到4.8。
2、载体pMDT05的构建
以pCAMBIA1300质粒为模板，使用下表1中的引物pMDT05-F1和pMDT05-R1进行PCR扩增，PCR产物用1％琼脂糖凝胶电泳进行分离，将约6.8kb的片段从凝胶中切出，按照omega公司凝胶提取试剂盒方法进行回收，回收纯化的片段用限制性内切酶XhoI和XbaI消化1h，消化完成后按照omega公司PCR产物回收试剂盒方法进行纯化回收。
以上述提取的里氏木霉基因组DNA为模板，使用表1中的引物Hyg-Pgpd-F和pMDT05-R2扩增启动子Pgpd(约1.4kb)。以pCAMBIA1300质粒为模板，使用表1中的引物pMDT05-F2和Pgpd-Hyg-R扩增hygromycin基因(约1kb)。将上述扩增的启动子Pgpd片段和hygromycin基因按摩尔比1:1混合作为模板，使用引物pMDT05-F2和pMDT05-R2为上下游引物进行SOE-PCR扩增(扩增条件为94℃，10min；98℃，10s，60℃，30s，68℃，1min20s，30cycles；68℃，10min)得到约2.4kb的融合片段，融合片段经过1％琼脂糖凝胶电泳分离，将约2.4kb的片段从凝胶中切出，按照omega公司凝胶回收试剂盒方法进行回收，回收纯化的片段用限制性内切酶XhoI和XbaI消化1h，消化完成后按照omega公司PCR产物回收试剂盒方法进行纯化回收。
将上述消化后的6.8kb和2.4kb片段按照摩尔比1:3混合，加入T4 DNA连接酶和连接缓冲液，在22℃下连接3h，将连接产物转化大肠杆菌TOP10感受态细胞，涂布在50μg/mL卡那霉素抗性平板上筛选克隆子，用引物pMDT05-F2和pMDT05-R2进行PCR验证和测序验证，测序验证正确的质粒载体命名pMDT05，如图1为载体pMDT05构建图谱。
表1.所用引物的序列
Figure PCTCN2018107053-appb-000001
3、里氏木霉Rut-C30菌株pyr4基因敲除载体构建
参照公开文献中提供的里氏木霉pyr4基因(编码乳清酸-5′-单磷酸脱羧酶)信息(Jeffrey L.Smith，Curr Genet,1991,19:27-33)，使用BLASTN程序检索里氏木霉基因组数据库中pyr4基因所在位置的基因座序列信息(http://genome.jgi-psf.org/Trire2/Trire2.home.html)。以上述提取的里氏木霉基因组DNA为模板，利用表2中的引物pyr4-3F/pyr4-3R和pyr4-5F/pyr4-5R分别扩增得到约1.3kb的pyr4基因上游同源臂片段和约1.3kb的pyr4基因下游同源臂片段。将pyr4基因上游同源臂片段和pyr4基因下游同源臂片段按摩尔比1:1的比例混合作为模板，以pyr4-3F和pyr4-5R为上下游引物，SOE-PCR扩增得到约2.6Kb的pyr4基因敲除盒。
将pMDT05载体和上述2.6kb的pyr4基因敲除盒用限制性内切酶XbaI和BglII消化1h，将消化后的 片段分别用omega公司凝胶提取试剂盒回收，将凝胶纯化的经过XbaI/BglII消化的pMDT05载体与消化的2.6kb片段按摩尔比1:3混合，加入T4 DNA连接酶和连接缓冲液，22℃连接3h，转化大肠杆菌TOP10感受态细胞，将验证和测序正确的载体命名为pMDT05-pyr4KO，如图2为里氏木霉Rut-C30菌株pyr4基因敲除载体pMDT05-pyr4KO构建图谱。
4、根癌农杆菌介导法构建里氏木霉pyr4基因缺失突变株
将上述的敲除载体pMDT05-pyr4KO通过冻融法(具体参见An,G.et.al Binary vectors,in Plant Molecular Biology Manual，1988)转入根癌农杆菌AGL-1感受态细胞中，在28℃活化3-4h后取适量的菌液涂布在含有kan(50μg/mL)+Gent(50μg/mL)的LB平板培养基中，于28℃倒置培养48-72h之后，挑取单克隆接种于含有kan(50μg/mL)+Gent(50μg/mL)的LB液体培养基中，于28℃摇床220rpm培养24h，取少量菌液作菌落PCR验证筛选阳性转化体。
用于转化的根癌农杆菌制备：将上述验证的阳性转化体接种到含有kan(50μg/mL)+Gent(50μg/mL)的LB液体培养基中，于28℃摇床220rpm培养20-24h，收集菌体，用IM培养基洗涤2次，并用IM培养基稀释至OD600＝0.15-0.20，添加乙酰丁香酮至终浓度为200μmol/L，于28℃下220rpm培养约6-10h至OD600＝0.6-0.8。
里氏木霉转化受体的制备：用4-5ml无菌水从培养6-7d的PDA平板上洗下里氏木霉的孢子，棉花过滤得到孢子悬液，离心收集孢子，用IM培养基洗涤2次，并用IM培养基重悬并调整至孢子浓度为10 7个/ml，于28℃萌发培养3-4h。
根癌农杆菌和里氏木霉共培养：取制备好的根癌农杆菌菌液100μl与100μl萌发处理的孢子悬液混合，涂布在IM固体培养基平板的玻璃纸上，24℃暗培养36h。将玻璃纸揭下，反铺到含有5mg/ml 5-FOA，300μg/mL头孢霉素和10mM尿苷的固体MM初筛培养基平板上，28℃培养4-6d至转化体长出。
转化体复筛：将转化体分别点在含有100μg/mL潮霉素的PDA固体平板和含有5mg/ml 5-FOA和10mM尿苷的固体MM培养基平板上，于28℃培养2-3d，挑取在含有100μg/mL潮霉素的PDA固体平板不能生长而在含有5mg/ml 5-FOA和10mM尿苷的固体MM培养基平板上能正常生长的转化体，提取复筛的转化体基因组DNA，用上游同源臂和下游同源臂两端外侧基因组的引物pyr4-CX-F和pyr4-CX-R(序列如表2中所示)进行PCR验证，如果pyr4基因被敲除，扩增出的片段应为约2.8kb，如果没有被敲除扩增出的片段应该为约4.2kb。
本实施例一共筛选了23个转化体(编号1#-23#)，经PCR验证所有复筛的转化体都只能扩增出约2.8kb的片段，其中包含既能在含有100μg/mL潮霉素的PDA固体平板正常生长也能在含有5mg/ml 5-FOA和10mM尿苷的固体MM培养基平板上正常生长的转化体1个，说明该转化体在发生同源重组的同时也发生了随机整合插入，剔除该转化体，因此pyr4基因有效敲除率达到95.6％。
单孢子分离纯化：挑取上述23个转化体中编号为8#敲除菌株的菌丝体接种到含有10mM尿苷的PDA培养基平板中，28℃培养7d至孢子成熟。将成熟的孢子用4-5ml无菌水洗下来，并用无菌水梯度稀释，涂布在含有10mM尿苷和0.1％Triton-100的PDA培养基平板中，于28℃培养3d，挑取分离的单孢子菌落，重新接种到含有10mM尿苷的PDA培养基平板中，于28℃进行生孢子培养。将上述分离的单孢子菌落和PCR检测后仍为阳性的菌株为尿嘧啶营养缺陷株，命名为Rut-C30(pyr4 -)。
表2.构建pyr4基因敲除载体引物
引物名称 引物序列(5’-3’)
pyr4-3F GCTCTAGATGAACAGTAAGGTGTCAGCATGC
pyr4-3R TAAATGCCTTTCTTTCGAGGCGAGGGAGTTGCTTTAATG
pyr4-5F CTCCCTCGCCTCGAAAGAAAGGCATTTAGCAAGAAGG
pyr4-5R GAAGATCTAGTGTTTGATGCTCACGCTCGGAT
pyr4-CX-F CGCCTCTTCTTTGTGCTTTTCTC
pyr4-CX-R GTGGGCTTCCTTGTTTCTCGACC
实施例3：草酸脱羧酶随机整合重组表达载体的构建
1、随机整合诱导型表达载体pMGU-cbh1-TRA2的构建
载体pMGU的构建：
以实施例2中制备的质粒载体pMDT05为模板，用表3中的引物F1和R1为上下游引物PCR扩增约6.6kb的载体骨架片段，使用凝凝胶回收后用限制性内切酶DpnI消化3h，回收目的片段，备用。
参照实施例2中里氏木霉基因组提取的方法，提取黑曲霉CICC2439基因组DNA，以该基因组DNA为模板，用表3中的引物pyrG-F和pyrG-R扩增约2.9kb的黑曲霉pyrG基因表达框，凝凝胶回收目的片段，备用。以里氏木霉基因组DNA为模板，用表2中的引物Pcbh-DR-F和Pcbh-DR-R扩增CBH1基因启动子Pcbh1大小为0.4kb片段，凝凝胶回收后备用。将2.9kb的黑曲霉pyrG基因表达框和Pcbh1的0.4kb 片段按摩尔比1:1混合作为模板，用引物Pcbh-DR-F和pyrG-R为上下游引物，SOE-PCR扩增扩增条件为：94℃，10min；98℃，10s，60℃，30s，68℃，1min50s，30cycles；68℃，10min得到约3.3kb的融合片段，凝胶回收该目的片段备用。
按照
Figure PCTCN2018107053-appb-000002
II一步法克隆试剂盒的方法，将上述消化后的载体骨架片段和SOE-PCR扩增得到并回收的3.3kb的片段进行组装，转化大肠杆菌TOP10感受态细胞，涂布平板，将验证和测序正确的载体命名为pMGU。
诱导型表达框pUC19-Pcbh1-sig-TRA2-Tcbh1构建：
以里氏木霉基因组DNA为模板，使用表3中的引物Pcbh1-F和Pcbh1-R为上下游引物，PCR扩增CBH1基因的启动子及CBH1基因信号肽编码序列Pcbh1-sig；以里氏木霉基因组DNA为模板，使用表3中的引物Tcbh1-F和Tcbh1-R为上下游引物PCR扩增CBH1基因的终止子序列Tcbh1。将Pcbh1-sig片段和Tcbh1片段按摩尔比1:1混合作为模板，用引物Pcbh1-F和Tcbh1-R作为上下游引物，PCR扩增约3.3kb的融合片段Pcbh1-sig-Tcbh1，回收目的片段并用EcoRI和PstI消化该片段，凝凝胶回收备用。将质粒pUC19同样用限制性内切酶EcoRI和PstI消化3h，凝凝胶回收载体骨架片段，并将其与消化回收后的Pcbh1-sig-Tcbh1片段用T4 DNA连接酶连接，转化大肠杆菌TOP10感受态细胞，将验证和测序正确的载体命名为pUC19-Pcbh1-sig-Tcbh1。
以质粒pUC19-Pcbh1-sig-Tcbh1为模板，用表3中引物WF-CBH-R和WF-CBH-F为上下游引物PCR扩增约5.8kb的载体骨架片段，DpnI消化3小时后凝凝胶回收后备用。以含有合成的TRA2基因的质粒pUC57-TRA2(基因合成公司提供)为模板，用表3中的引物WF-TRA2-F和WF-TRA2-R为上下游引物，PCR扩增TRA2基因成熟肽编码序列。将消化回收的载体骨架片段与TRA2片段按照
Figure PCTCN2018107053-appb-000003
II一步法克隆试剂盒的方法进行组装，转化大肠杆菌TOP10感受态细胞，将验证和测序正确的载体命名为pUC19-Pcbh1-sig-TRA2-Tcbh1。
随机整合诱导型表达载体pMGU-cbh1-TRA2的构建：
将上述制备的载体pMGU用限制性内切酶EcoRI和XbaI消化3h，凝凝胶回收载体骨架片段备用。以上述制备的载体pUC19-Pcbh1-sig-TRA2-Tcbh1为模板，用表3中的引物F2和R2为上下游引物，PCR扩增Pcbh1-sig-TRA2-Tcbh1片段，凝胶回收该目的片段备用。将pMGU用限制性内切酶EcoRI和XbaI消化后回收的载体骨架与Pcbh1-sig-TRA2-Tcbh1片段按照
Figure PCTCN2018107053-appb-000004
II一步法克隆试剂盒的方法组装，转化大肠杆菌TOP10感受态细胞，将验证和测序正确的载体命名为pMGU-cbh1-TRA2，里氏木霉随机整合诱导型表达载体pMGU-cbh1-TRA2的构建图谱如图3所示。
2、随机整合组成型表达载体pDGU-pdc-TRA2的构建
载体pDGU的构建：
以实施例2中制备的载体pMDT05为模板，用表3中的引物F1和R1PCR扩增6.6kb的载体骨架，凝胶回收该片段后用限制性内切酶DpnI消化3h，回收目的片段备用。
以黑曲霉CICC2439基因组DNA为模板，用表3中的引物pdcDR-pyrG-F和pyrG-R扩增获得2.9kb的黑曲霉pyrG基因的表达框，凝胶回收目的片段，备用。以里氏木霉基因组DNA为模板，用表3中的引物Ppdc-DR-F和pyrG-pdcDR-R扩增pdc基因启动子Ppdc的5’端0.4kb片段，凝胶回收后备用。将2.9kb的黑曲霉pyrG基因表达框和Ppdc的5’端0.4kb片段按摩尔比1:1混合作为模板，用引物Ppdc-DR-F和pyrG-R为上下游引物，SOE-PCR扩增得到3.3kb的片段，凝胶回收该目的片段备用。
按照
Figure PCTCN2018107053-appb-000005
II一步法克隆试剂盒的方法，将上述消化后的6.6kbp载体骨架片段和SOE-PCR扩增，扩增条件为：94℃，10min；98℃，10s，60℃，30s，68℃，1min50s，30cycles；68℃，10min得到并回收的3.3kb的片段进行组装，转化大肠杆菌TOP10感受态细胞，涂布平板，将验证和测序正确的载体命名为pDGU。
组成型表达框构建pUC19-Ppdc-sig-TRA2-Tpdc构建：
以里氏木霉基因组DNA为模板，使用表3中的引物NdeI-Pdc-F和Ppdc-R为上下游引物，PCR扩增约1.4kb的PDC基因的启动子序列Ppdc；以里氏木霉基因组DNA为模板，使用表2中的引物Tpdc-F和PstI-Tpdc-R为上下游引物PCR扩增约1.0kb的PDC基因的终止子序列Tpdc。将上述获得的Ppdc片段和Tpdc片段按摩尔比1:1混合作为模板，用引物NdeI-Pdc-F和PstI-Tpdc-R作为上下游引物，PCR扩增约2.5kb的融合片段Ppdc-Tpdc，回收目的片段并用NdeI和PstI消化该片段，凝胶回收备用。将质粒pUC19同样用NdeI和PstI消化，凝胶回收载体骨架，并将其与消化回收后的Ppdc-Tpdc片段连接，转化大肠杆菌TOP10感受态细胞，将验证和测序正确的载体命名为pUC19-Ppdc-Tpdc。
以质粒pUC19-Ppdc-Tpdc为模板，用表3中引物WF-pdc-R和WF-pdc-F为上下游引物PCR扩增约5.0kb的载体骨架片段，DpnI消化3小时后凝胶回收后备用。以质粒pUC19-Pcbh1-sig-TRA2-Tcbh1为模板，用表3中的引物WF-TRA2-F2和WF-TRA2-R2为上下游引物，PCR扩增约1.4kb的sig-TRA2序列。将消化回收的约5.0kb的载体骨架片段与sig-TRA2片段按照
Figure PCTCN2018107053-appb-000006
II一步法克隆试剂盒的方法进 行组装，转化大肠杆菌TOP10感受态细胞，将验证和测序正确的载体命名为pUC19-Ppdc-sig-TRA2-Tpdc。
随机整合组成型表达载体pDGU-pdc-TRA2的构建：
将上述制备的载体pDGU用限制性内切酶XbaI酶切消化3h，然后加入限制性内切酶EcoRI酶切消化5min进行不完全酶切，凝胶回收较大的载体骨架片段备用。以质粒pUC19-Ppdc-sig-TRA2-Tpdc为模板，用表3中的引物F3和R3为上下游引物，PCR扩增Ppdc-sig-TRA2-Tpdc片段，凝胶回收目的片段。将上述pDGU用酶切回收后的载体骨架片段与Ppdc-sig-TRA2-Tpdc片段按照
Figure PCTCN2018107053-appb-000007
II一步法克隆试剂盒的方法组装，转化大肠杆菌TOP10感受态细胞，将验证和测序正确的载体命名为pDGU-pdc-TRA2，即里氏木霉随机整合组成型表达载体pDGU-pdc-TRA2构建图谱如图4所示。
表3.诱导型表达载体构建引物
引物名称 引物序列(5’-3’)
F1 GAATTCAGATCTGGTACCTCTAGAAAGCTTAC
R1 GGATCCCGAATTAATTCGGCGTTAATTCAGTAC
pyrG-F TTCCCTTCCTCTAGGCCGCTGGTCAATGTTATCTGG
pyrG-R ACCAGATCTGAATTCCTTCCTAATACCGCCTAGTCATAGC
Pcbh-DR-F ATTAATTCGGGATCCTCACGGTGAATGTAGGCCTTTTGTA
Pcbh-DR-R CATTGACCAGCGGCCTAGAGGAAGGGAAAAGAATGGCAC
Pcbh1-F CGGAATTCTCACGGTGAATGTAGG
Pcbh1-R CTTTCGCACGGAGCTAGCACGAGCTGTGGCCAAGAAG
Tcbh1-F CCACAGCTCGTGCTAGCTCCGTGCGAAAGCCTGACGCACC
Tcbh1-R AACTGCAGCATCGTAACCGAGAATCCAGAGCTG
WF-CBH-R AGCACGAGCTGTGGCCAAGAAG
WF-CBH-F AGCTCCGTGCGAAAGCCTGACGCACC
WF-TRA2-F GCCACAGCTCGTGCTGCCCCCGCCCCCAGCAGCGCCGCC
WF-TRA2-R CTTTCGCACGGAGCTTTAGGCAGGGCCGACGACAATAGG
F2 ATTAGGAAGGAATTCTCACGGTGAATGTAGGCCTTTTG
R2 AGTAAGCTTTCTAGACATCGTAACCGAGAATCCAGAGC
Ppdc-DR-F ATTAATTCGGGATCCAGGACTTCCAGGGCTACTTGGCGC
pdcDR-pyrG-F TCAATGGTACGAGGTGCCGCTGGTCAATGTTATCTGG
pyrG-pdcDR-R CATTGACCAGCGGCACCTCGTACCATTGACTCTGTCTG
NdeI-Pdc-F ACCATATGAGGACTTCCAGGGCTACTTGG
Ppdc-R GACTTCATGCCGGGGATTGTGCTGTAGCTGCGCTG
Tpdc-F GCTACAGCACAATCCCCGGCATGAAGTCTGACC
PstI-Tpdc-R GCCTGCAGTGGACGCCTCGATGTCTTCCTC
WF-TRA2-F2 AGCTACAGCACAATCATGTATCGGAAGTTGGCCGTCATC
WF-TRA2-R2 AGACTTCATGCCGGGTTAGGCAGGGCCGACGACAATAGG
WF-pdc-R GATTGTGCTGTAGCTGCGCTG
WF-pdc-F CCCGGCATGAAGTCTGACC
F3 ATTAGGAAGGAATTCAGGACTTCCAGGGCTACTTGGC
R3 AGTAAGCTTTCTAGATGGACGCCTCGATGTCTTCCTC
实施例4：通过随机整合构建里氏木霉草酸脱羧酶表达菌株，以及转化体的筛选和分子鉴定
将上述实施例3中的两种随机整合重组表达载体pMGU-cbh1-TRA2和pDGU-pdc-TRA2通过冻融法分别转入根癌农杆菌AGL-1感受态细胞。将PCR验证正确的阳性克隆按照实施例2中的方法制备用于转化的根癌农杆菌菌液。
里氏木霉转化受体的制备：用4-5ml无菌水从培养6-7d的PDA(含有10mM尿苷)平板上洗下里氏木霉Rut-C30(pyr4 -)菌株的孢子，棉花过滤得到孢子悬液，离心收集孢子，用IM培养基洗涤2次，并用IM培养基(加入终浓度为10mM尿苷)重悬并调整至孢子浓度为10 7个/ml，于28℃萌发培养3-4h。
根癌农杆菌和里氏木霉共培养：取制备好的根癌农杆菌菌液100μl与100μl萌发处理的孢子悬液混合，涂布在固体IM培养基平板的玻璃纸上，24℃暗培养36h。将玻璃纸揭下，反铺到含有300μg/mL头孢霉素固体MM初筛培养基平板上，28℃培养4-6d至转化体长出。本实施例进行了3批次重组表达载体pMGU-cbh1-TRA2转化入实施例2制备的里氏木霉Rut-C30(pyr4 -)菌株，获得了230个转化株，一批次重组表达载体pDGU-pdc-TRA2转化里氏木霉Rut-C30(pyr4 -)菌株获得了73个转化株。
转化体复筛：将获得的转化株点在含有300μg/mL头孢霉素的固体MM培养基平板上，于28℃培养2-3d，挑取生长速度和形态正常的转化体，转接到PDA平板上于28℃进行生孢子培养7d。孢子成熟后，用无菌水将孢子洗出制备成孢子悬液，进行梯度稀释，涂布在含有0.1％Triton-100的PDA培养基上进行 分离单孢子菌株，28℃培养3d，待单孢子长成菌落后，各挑取3个单孢子分离株转接到PDA培养基上28℃培养3d，挑取少量菌丝到20μl无菌水中，98℃加热10分钟，离心取上清用引物TRA2-F和TRA2-R作PCR鉴定，将各个转化体PCR鉴定为阳性转化体的单孢子分离株继续培养到第7d孢子成熟。
PCR鉴定引物序列(5’-3’)：
TRA2-F：ATGTATCGGAAGTTGGCCGTCATC
TRA2-R：TTAGGCAGGGCCGACGACAATAGG
上述IM培养基配方为：K 2HPO 410mmol/L，KH 2PO 410mmol/L,，NaCl 2.5mmol/L，MgSO 4·7H 2O2mmol/L，CaCl 20.7mmol/L，(NH 4) 2SO 44mmol/L，Glucose 10mmol/L，Clycerol 0.5％，AS 200μmol/L，Mandels微量元素(1000X)1ml，pH5.3。
上述MM培养基配方为(g/L)：葡萄糖20，蛋白胨2，(NH 4) 2SO 45,MgSO 4·7H 2O 0.6,CaCl 20.6，KH 2PO 415，Mandels微量元素(1000X)1ml/L，pH4.5-5.5。
上述Mandels微量元素(1000X)配方：FeSO 4·7H 2O 5g/L，MnSO 41.6g/L，ZnSO 4·7H 2O 1.7g/L，CoCl·6H 2O 3.7g/L。
实施例5：里氏木霉随机整合转化体摇瓶发酵表达筛选
将上述实施例4中分离株的成熟孢子，用4-5ml无菌水洗下，按照1％的接种量接种于里氏木霉液体种子培养基中，培养24h后按10％的接种量转接到用于不同启动子类型的里氏木霉转化体表达培养基中，于28℃下，170rpm培养168h后分析发酵液上清中的草酸脱羧酶活力。
上述里氏木霉液体种子培养基配方：葡萄糖15g/L,蛋白胨2g/L,(NH 4) 2SO 42.5g/L,MgSO 4·7H 2O 0.8g/L,CaCl 21.0g/L，50mM柠檬酸缓冲液(pH 4.5)，尿素0.3g/L，KH 2PO 42g/L，Mandels微量元素(1000X)1ml/L，1-2g/L吐温80。
在本实施例中，以诱导型启动子表达草酸脱羧酶的里氏木霉转化体表达培养基配方为：乳糖18g/L，微晶纤维素10g/L，玉米浆粉12g/L，(NH 4) 2SO 40.5g/L，MgSO 4·7H 2O 1g/L、CaCl 21.0g/L，KH 2PO 46g/L，麸皮粉2g/L，Mandels微量元素(1000X)1ml，MnCl 25mM，pH 4.5。
上述Mandels微量元素(1000X)配方：FeSO 4·7H 2O 5g/L，MnSO 41.6g/L，ZnSO 4·7H 2O 1.7g/L，CoCl·6H 2O 3.7g/L。
在本实施例中，以组成型启动子表达草酸脱羧酶的里氏木霉转化体表达培养基配方为：葡萄糖50g/L、蛋白胨4.5g/L、(NH 4) 2SO 41.4g/L、MgSO 4·7H 2O 0.3g/L、CaCl 20.4g/L，50mM柠檬酸缓冲液(pH 4.5)，尿素0.3g/L，KH 2PO 42g/L，Mandels微量元素(1000X)1ml，1-2g/L吐温80，pH 4.5。
以草酸为底物，将在特定条件(37℃，pH3.0)下中每分钟降解1μmol草酸或者每分钟生成1μmol甲酸所需酶量定义为一个活力单位(IU)。经过摇瓶发酵筛选所有的转化体，以诱导型启动子表达草酸脱羧酶的里氏木霉转化体发酵168h最高酶活力到达17940IU/L。以组成型启动子表达草酸脱羧酶的里氏木霉转化体发酵168h最高酶活力达到8800IU/L。
实施例6：里氏木霉转化体摇瓶发酵条件优化
本实施例通过优化初始培养基中的不同碳源成分、初始浓度以及氮源成分、初始浓度对诱导型重组菌株表达草酸脱羧酶的影响，发酵结果表明，使用优化前的发酵培养基(组成：乳糖18g/L，微晶纤维素10g/L，玉米浆12g/L，(NH 4) 2SO 40.5g/L，MgSO 4·7H 2O 1.56g/L、CaCl 20.5g/L，KH 2PO 46g/L，麸皮粉2g/L，Mandels微量元素(1000X)1ml，MnCl 25mM，pH 4.0)，发酵168h发酵液上清中草酸脱羧酶活力在3000IU/L左右。使用优化后的培养基，其葡糖初始浓度8g/L，微晶纤维素23g/L时表达效果最佳。摇瓶发酵表达168h后上清发酵液中草酸脱羧酶活力到达50876IU/L。优选的培养基组成为：葡萄糖3-8g/L，微晶纤维素10-25g/L，玉米浆粉5-15g/L，(NH 4) 2SO 40.5-5g/L，MgSO 4·7H 2O 1.56g/L、CaCl 20.5g/L，KH 2PO 42-8g/L，尿素0-1g/L，麸皮粉0.2-2g/L，Mandels微量元素(1000X)1ml，MnCl 20.5-5mM，pH 3.0-4.5。
实施例7：里氏木霉随机整合转化体插入位点侧翼序列分析
根据实施例2中的方法提取里氏木霉转化体菌株的基因组DNA，采用Song Gao等(Analytical Biochemistry，59(2016)79-81)的TD-TAIL PCR(Touchdown TAIL-PCR)方法分析里氏木霉转化体T-DNA(即含有草酸脱羧酶表达盒的转移DNA片段)插入位点侧翼序列。本实施例采用的随机引物(LAD1-LAD5)和特异引物AC1、RB-1、RB-2、Tail-CX-F(见表4)。其中简并引物中V代表A/G/C，N代表A/G/C/T，B代表G/C/T，D代表A/G/T，H代表A/C/T。
首先，将里氏木霉基因组DNA用ddH 2O稀释成20-30ng/μl，以此作为预扩增PCR反应(Pre-amplification)的模板，分别以LAD1-LAD5为随机引物和特异引物RB-1进行预扩增PCR，然后将预扩增PCR反应的产物稀释50倍作为Touchdown PCR的模板，将Touchdown PCR扩增产物进行1％的琼脂糖凝胶电泳，切凝胶回收比较单一和明亮的条带并用表3中的Tail-CX-F引物测序分析。将测序结果 序列在里氏木霉基因组数据库中，使用BLASTN程序检索，分析其插入的基因组位点。
表4.TD-TAIL PCR中所采用的LAD引物序列和特异引物序列
引物名称 引物序列(5’-3’)
LAD1 ACGATGGACTCCAGAGCGGCCGCVNVNNNGGAA
LAD2 ACGATGGACTCCAGAGCGGCCGCBNBNNNGGTT
LAD3 ACGATGGACTCCAGAGCGGCCGCVVNVNNNCCAA
LAD4 ACGATGGACTCCAGAGCGGCCGCBDNBNNNCGGT
LAD5 ACGATGGACTCCAGAGCGGCCGCBHNDNNNGACC
AC1 ACGATGGACTCCAGAG
RB-1 GGCCGTCGTTTTACAACGTCGTGAC
RB-2 GCGTAATAGCGAAGAGGCCCGCACC
Tail-CX-F GCGTAATAGCGAAGAGGCCCGCACC
Pre-amplification扩增反应体系(20μl)：20-30ng基因组DNA，LAD引物1.0μM，RB-1引物0.3μM，dNTP 2μl，10×buffer2μl，Taq酶0.5U，补ddH 2O至20μl。
预扩增PCR反应条件：
93℃，120s；
95℃，60s；
94℃，30s；60℃，60s；72℃，180s；10个循环；
94℃，30s；25℃，120s；150s内温度均匀增至72℃；72℃，180s；
94℃，20s；58℃，60s；72℃，120s；25个循环；
最后72℃，300s。
Touchdown PCR反应体系(50μl)：预扩增PCR反应产物稀释50倍，AC1引物0.3μM，RB-1引物0.3μM，dNTP 5μl，10×buffer 5μl，Taq酶1U，补ddH 2O至50μl。
Touchdown PCR反应条件：
94℃，120s；
94℃，20s；68℃(-1℃/cycle)，60s；72℃，180s；15个循环；
94℃，20s；53℃，60s；72℃，180s；15个循环；
最后72℃，300s。
本实施例一共选取了摇瓶发酵表达酶活力在25000-65000IU/L之间的35株里氏木霉转化体作T-DNA插入位点的侧翼序列分析，在所有获得的T-DNA侧翼序列中有6条只鉴定出了RB边界外的约0.5kb载体序列，未鉴定出其基因组上的插入位置，有42条鉴定出了T-DNA侧翼序列对应在基因组上的位置。在42条T-DNA侧翼序列中有8条保存着完整的RB边界序列，34条T-DNA右边界序列发生了缺失现象。
在35株里氏木霉转化体中，经过多次PCR进一步分析，25株推断为单一拷贝T-DNA插入，5株推断为在同一位点存在2个拷贝并以正向重复形式存在，3株推断为在同一位点存在2个拷贝并以反向重复形式存在，2株推断为在2个不同位点各存在一个拷贝插入。
在选取的35株里氏木霉转化体中，2拷贝的转化体摇瓶发酵的酶活力比单一拷贝的高出60％-100％，体现出了很好的基因剂量关系。在后续分离纯化单孢子和平行发酵实验中，发现同一位点存在2个拷贝并以正向重复形式存在的菌株不稳定，同一转化体分离出的单孢子间发酵表达酶活力相差较大，相同发酵条件下，大多数比母体表达的酶活力低。同一位点存在2拷贝并以反向重复形式存在的转化体分离出的单孢子间发酵表达酶活力平行性很好，相同发酵条件下与母体表达的酶活力相当。
将其中一个发酵表达酶活力较高(高于50000IU/L)的且在同一位点存在2个拷贝并以反向重复形式存在的分离菌株命名为B4-6。分离株B4-6的插入位点经分析在Trire2|scaffold_12:102924-105333之间。
实施例8：里氏木霉转化体筛选标记基因pyrG删除
将实施例7中B4-6菌株，接种到PDA培养基(含有10mM尿苷)上，于28℃下培养7d至孢子成熟，用4-5ml无菌水洗下孢子制成孢子悬液，取适量孢子悬液涂布在含有5mg/ml 5-FOA，0.1％Trinton-100和10mM尿苷的PDA培养基上，28℃培养4-5d至长出单菌落。获得约100个5-FOA抗性的分离株。选取5个5-FOA抗性的分离株转接到含有10mM尿苷的PDA培养基上，于28℃培养7d至孢子成熟。用引物pyrG-F2和pyrG-R2做PCR鉴定发生同源重组切除pyrG表达框的菌落，结果表明5个孢子分离株均已切除pyrG表达框。上述引物序列：pyrG-F2：5’-TTATAGTATTAGTTTTCCGCCGAC-3’，pyrG-R2：5’-ATGTCCTCCAAGTCGCGATTGAC-3’。将其中一个已切除pyrG表达框的分量株命名为B4-6(pyr4 -)。
实施例9：随机整合重组表达载体pMGU-cbh1-TRA2转化里氏木霉B4-6(pyr4 -)株构建多拷贝转化体
根据实施例4中所述的方法和步骤，将表达载体pMGU-cbh1-TRA2通过农杆菌介导转化的方法转化里氏木霉B4-6(pyr4 -)株。得到42个转化体，将各个转化体分别转到含有300μg/mL头孢霉素的固体MM培养基平板上复筛，28℃培养3d，挑选其中39个复筛的转化体转接到PDA平板，于28℃下培养7d。
通过引物pyrG-F3和引物WF-CBH-R做PCR筛选所有39个转化体，以确认在原来拷贝数的基础上有新增的拷贝。
对于各个转化体，通过从培养到第3天的PDA平板上提取少量菌丝体到20μl无菌水中，98℃加热10分钟，离心取上清用引物pyrG-F3和WF-CBH-R作PCR鉴定，能扩增得到约2.3kb片段的为阳性转化体。上述引物序列为pyrG-F3：5’-TTACTTGTGGTGTTCTCAGCTTG-3’；引物WF-CBH-R的序列见表2。
使用实施例6中优化的培养基进行发酵筛选所有的转化体，从72h开始测定酶活力，每隔24h测定一次，并一直持续到168h发酵结束，结果发现，26#转化体活力最高，发酵168h酶活力可以达到103951IU/L。将144h和168h发酵液上清样品各稀释5倍，进行SDS-PAGE电泳检测，电泳结果如图6所示，泳道1和2分别为发酵144h和168h的发酵液上清，上样量为10μl发酵液上清。采用实施例7中的方法分析最高转化体的新拷贝插入位点，经过测序分析，该转化体在2个不同的位点分别插入一个新的拷贝，插入位点分别为Trire2|scaffold_7:1288320-1288321和Trire2|scaffold_1:1129134-1129157。采用实施例8中的方法切除筛选标记基因pyrG，并命名该转化体为HH03-26-8(pyr4 -)。
实施例10：里氏木霉转化体HH03-26-8(pyr4 -)中pyr4基因的修复
以里氏木霉Rut-C30的基因组DNA为模板，用引物pyr4-F1和pyr4-R1为上下游引物PCR扩增pyr4的完整表达盒以及pyr4基因两侧的同源臂片段，PCR扩增产物经过1％琼脂糖凝胶电泳分离，切除约4.0kb的条带，用凝胶提取试剂盒回收纯化，用限制性内切酶BglII和XbaI消化1h后，用PCR产物回收试剂盒回收目的片段，备用。将载体pMDT05同样用限制性内切酶BglII和XbaI消化3h后，凝胶提取试剂盒回收纯化载体片段，将其与消化回收的4.0kb的片段按摩尔比1:3混合，加入T4 DNA连接酶于22℃条件下连接3h，连接产物转化大肠杆菌TOP10感受态细胞，PCR筛选阳性克隆，并测序验证。将测序验证正确的载体命名为pMDT05-pyr4 KI，其图谱如图5所示。上述引物序列为：
pyr4-F1：5’-TCAGATCTAGTGTTTGATGCTCACGCTCGGAT-3’；
pyr4-R1：5’-TTTCTAGATGAACAGTAAGGTGTCAGCA-3’。
根据实施例4中所述的方法和步骤，将表达载体pMDT05-pyr4KI通过农杆菌介导转化的方法转化里氏木霉HH03-26-8(pyr4 -)株。获得了153个转化体，将转化体点到固体MM培养基上，于28℃培养48h，待菌丝体向外生长到直径为1厘米左右菌斑。将MM平板上的所有转化体做好编号，分别挑取部分菌丝体点到含有100μg/mL潮霉素的PDA固体平板上，于28℃培养48h。记录下在含100μg/mL潮霉素的PDA固体平板上不能生长的转化体的编号，获得了35个该转化体。从MM固体平板上挑取相对应的编号的转化体菌丝体转接到PDA平板上，于28℃下培养，在培养到第3天时，挑取少量菌丝体到20μl无菌水中，98℃加热10分钟，离心取上清用引物pyr4-F2和pyr4-R2做PCR验证。上述引物序列为：pyr4-F2：5’-CAAACGAACACATCACTTTCAAAG-3’；pyr4-R2：5’-GTGGGCTTCCTTGTTTCTCGACC-3’。当在pyr4基因位点发生同源重组修复了pyr4表达盒时，PCR扩增条带约为4.2kb，当未发生同源重组是扩增的条带约为2.7kb。通过PCR分析，在35个转化体中，28个转化体能扩增到约4.2kb的片段，7个转化体能扩增出约为2.7kb片段，推测这7个转化体在pyr4基因座外其他位置发生了随机插入同时丢失了潮霉素抗性。
实施例11：里氏木霉Rut-C30(pyr4 -)菌株中mus53基因敲除
根据公开文献报道(Matthias G.Steiger，APPLIED AND ENVIRONMENTALMICROBIOLOGY,Jan.2011,p.114–121)mus53基因(与人类Lig4基因同源)为非同源性末端结合(NHEJ)功能所必需的，其功能的破坏，能够带来将近100％的同源重组效率。在本实施例中，将里氏木霉Rut-C30(pyr4-)菌株中的mus53基因进行敲除，为后续定点整合敲入实施例的进行奠定基础。
1、构建mus53基因敲除载体pMDT05-mus53KO
参照公开文献中提供的里氏木霉mus53基因(Protein Id:58509)信息(Matthias G.Steiger，APPLIED AND ENVIRONMENTAL MICROBIOLOGY,Jan.2011,p.114–121)，检索里氏木霉基因组数据库中mus53基因所在位置的基因座序列信息(http://genome.jgi-psf.org/Trire2/Trire2.home.html)。
以里氏木霉基因组DNA为模板，利用表5中的引物mus53-3F/mus53-3R和mus53-5F/mus53-5R分别扩增得到约1.4kb的mus53基因上游同源臂Up片段和约1.3kb的mus53基因下游同源臂Down片段，用引物mus53-mid-F/mus53-mid-R扩增mus53基因座约1.3kb的Middle片段。
以里氏木霉基因组DNA为模板，用引物pyr4-TprC-F/pyr4-R扩增约1.5kb的pyr4基因编码区和其终 止子，以质粒pBARGPE1为模板，用引物pyr4-F/pyr4-TrpC-R扩增386bp的PtrpC启动子。
将上述5段PCR扩增的片段按照omega公司凝胶回收试剂盒方法进行回收，回收后按照等摩尔比混合作为PCR扩增模板，用引物mus53-3R/mus53-mid-F为上下游引物SOE-PCR扩增约6.1kb的融合片段，并按照omega公司凝胶回收试剂盒方法进行回收目的片段。
将质粒pMDT05用限制性内切酶EcoRI和XbaI消化3h，凝凝胶回收载体骨架片段，并与回收的6.1kb片段按照
Figure PCTCN2018107053-appb-000008
II一步法克隆试剂盒的方法组装，转化大肠杆菌TOP10感受态细胞，将验证和测序正确的载体命名为pMDT05-mus53KO，载体构建图谱参见图7。
2、里氏木霉Rut-C30(pyr4 -)中mus53基因敲除
根据实施例4中所述的方法和步骤，将mus53基因敲除载体pMDT05-mus53KO通过农杆菌介导转化的方法转化里氏木霉Rut-C30(pyr4 -)株，得到294个转化体，将各个转化体分别点板到固体MM培养基(300μg/mL头孢霉素和200μg/mL潮霉素)平板和固体MM培养基(300μg/mL头孢霉素)平板上复筛，28℃培养3d，得到44个没有潮霉素抗性的转化体，挑选其中的31个转化体转接到PDA平板，于28℃下培养7d。
通过引物MUS-F/TrpC-CX-F和pyr4-LB-R/MUS-R做PCR筛选所有31个转化体，以确定是否在mus53基因座位点通过Up与Middle区域发生同源重组。通过引物RB-YZ-F和引物RB-YZ-R做PCR扩增筛选转化体，以确定是否在mus53基因座位点外发生随机整合。
本实施例中，对于各个转化体，通过从培养到第3天的PDA平板上提取少量菌丝体到20μl无菌水中，98℃加热10分钟，离心取上清作为模板，用引物MUS-F/TrpC-CX-F和pyr4-LB-R/MUS-R能分别扩增到约3.1kb和1.6kb片段的说明在相应区域发生了预期形式的同源重组，同时用引物RB-YZ-F和引物RB-YZ-R不能扩增到425bp的片段，说明没有发生随机整合，本实施例中筛选到同时满足这些条件的阳性转化子15株。将其中一个阳性转化子接种到PDA培养基(含有10mM尿苷)上，于28℃下培养7d至孢子成熟，用4-5ml无菌水洗下孢子制成孢子悬液，取适量孢子悬液涂布在含有5mg/ml 5-FOA，0.1％Trinton-100和10mM尿苷的PDA培养基上，28℃培养4-5d至长出单菌落，选择其中3个菌落转接到含有10mM尿苷的PDA培养基上，于28℃培养7d至孢子成熟。用引物MUS-F/MUS-R做PCR鉴定发生同源重组切除pyr4表达框的菌落，发生同源重组切除pyr4的能扩增到约2.9kb的片段，结果表明3个菌落均已切除pyr4表达框。并将验证的阳性菌株命名为Rut-C30(pyr4 -，mus53 -)。引物序列见表5。
表5.构建mus53基因敲除载体引物
引物名称 引物序列(5’-3’)
mus53-3R TCCTTCTTCTGCGTCGAATTCTCCGTATTTCAGCAGTAACCCCCTG
mus53-3F ACCCTTGCATATGCTCCTTGAAAGGACCTTGACAGAACGGAG
mus53-5R CCGTTCTGTCAAGGTCCTTTCAAGGAGCATATGCAAGGGTATC
mus53-5F TCAATATCATCTTCTGTCGATCATTGTCATGACGCTACAGAAGC
mus53-mid-R GGATGGTTTGGATGCAGTTGAAGGTGGGCGCTACCGAGAAG
mus53-mid-F GCCACTAGTAAGCTTTCTAGAGCTTTGAGTTCCGATTCTACCCTC
pyr4-R CTGTAGCGTCATGACAATGATCGACAGAAGATGATATTGAAGGAGC
pyr4-F GTCTTCTCGGTAGCGCCCACCTTCAACTGCATCCAAACCATCCTAC
pyr4-TprC-F GATTAGGAAGTAACCATGGCACCACACCCGACGCTCAAG
pyr4-TrpC-R GGGTGTGGTGCCATGGTTACTTCCTAATCGAAGCTTTGC
MUS-F GAACCCGGACGTTGAATCTGC
TrpC-CX-F GCATTCATTGTTGACCTCCACTAGC
Pyr4-LB-R GCATTTGCTTTTGCGCGTGGAG
MUS-R GTGGATCAACGTCAATGGGCTCAG
RB-YZ-F GTGGATTCGGCCAAAGGACTCCG
RB-YZ-R GTTTAAACTGAAGGCGGGAAACGAC
实施例12:定点整合表达载体构建
1、CBH1位点定点整合表达载体构建pMDT05-CBH1-TRA2(KI)
通过关键词搜索在里氏木霉基因组数据库中(http://genome.jgi-psf.org/Trire2/Trire2.home.html)获取CBH1(Cel7A)基因座相应DNA序列信息。
以质粒pMGU-cbh1-TRA2为模板，使用引物CBH1-F1和CBH1-R1扩增包含部分Pcbh1在内的Pcbh1-TRA2-Tcbh1片段，其中的1115bp的部分Pcbh1作为5’端同源臂序列。以里氏木霉基因组DNA为模板，使用引物CBH1-F2和CBH1-R2扩增Tcbh1终止子后一段500bp的序列作为重复序列(DR)。以质粒pMDT05-mus53KO为模板，使用引物CBH1-F3和CBH1-R3扩增pyr4表达框。以里氏木霉基因组DNA为模板，使用引物CBH1-F4和CBH1-R4扩增Tcbh1终止子后1041bp序列作为3’端同源臂序列。
所有PCR扩增的片段按照omega公司凝胶回收试剂盒方法进行回收，回收后的片段按照等摩尔比混合作为PCR扩增模板，用引物CBH1-F1和CBH1-R4为上下游引物SOE-PCR扩增约7kb的融合片段。使用引物pMDT-SpeI-R和pMDT-XbaI-F扩增线性化载体pMDT-05，扩增产物用DpnI消化3h。将上述2片段并按照omega公司凝胶回收试剂盒方法进行回收，回收后的目的片段按照
Figure PCTCN2018107053-appb-000009
II一步法克隆试剂盒的方法组装，转化大肠杆菌TOP10感受态细胞，将验证和测序正确的载体命名为pMDT05-CBH1-TRA2(KI)，图谱参见图8。引物序列见表6。
2、CBH2位点定点整合表达载体构建pMDT05-CBH2-TRA2(KI)
通过关键词搜索在里氏木霉基因组数据库中(http://genome.jgi-psf.org/Trire2/Trire2.home.html)获取CBH2(Cel6A)基因座相应DNA序列信息。
以里氏木霉基因组DNA为模板，使用引物CBH2-F1和EcoRI-CBH2-UR扩增其5’端同源臂序列(1087bp)。以质粒pMDT05-mus53KO为模板，使用引物EcoRI-CBH2-TrpC-F和CBH2-D-TU-R扩增pyr4表达框。以里氏木霉基因组DNA为模板，使用引物Tpyr4-CBH2-D-F和CBH2-R3扩增其3’端同源臂序列(1187bp)。
所有PCR扩增的片段按照omega公司凝胶回收试剂盒方法进行回收，回收后的片段按照等摩尔比混合作为PCR扩增模板，用引物CBH2-F1和CBH2-R3为上下游引物SOE-PCR扩增约4.2kb的融合片段。使用引物pMDT-SpeI-R和pMDT-XbaI-F扩增线性化载体pMDT-05，扩增产物用DpnI消化3h。将上述2片段并按照omega公司凝胶回收试剂盒方法进行回收，回收后的目的片段按照
Figure PCTCN2018107053-appb-000010
II一步法克隆试剂盒的方法组装，转化大肠杆菌TOP10感受态细胞，将验证和测序正确的载体命名为pMDT05-CBH2-pyr4，参见图9。
以质粒pMGU-cbh1-TRA2为模板，使用引物E-CBH2-PCBH-F和CBH2-DR-R2扩增约4.7kb的表达框Pcbh1-TRA2-Tcbh1。以里氏木霉基因组DNA为模板，使用引物CBH-DR-F和E-CBH2-DR-R扩增437bp序列作为重复序列(DR)。
将上述2个PCR片段按照omega公司凝胶回收试剂盒方法进行回收，回收后的片段按照等摩尔比混合作为PCR扩增模板，用引物E-CBH2-PCBH-F和E-CBH2-DR-R为上下游引物SOE-PCR扩增约5.1kb的融合片段，并按照omega公司凝胶回收试剂盒方法进行回收目的片段。回收后的片段与用EcoRI酶切的载体pMDT05-CBH2-pyr4(EcoRI)按照
Figure PCTCN2018107053-appb-000011
II一步法克隆试剂盒的方法组装，转化大肠杆菌TOP10感受态细胞，将验证和测序正确的载体命名为pMDT05-CBH2-TRA2(KI)。引物序列见表6。
3、EG1位点定点整合表达载体构建pMDT05-EG1-TRA2(KI)
通过关键词搜索在里氏木霉基因组数据库中(http://genome.jgi-psf.org/Trire2/Trire2.home.html)获取EG1(Cel7B)基因座相应DNA序列信息。
以里氏木霉基因组DNA为模板，使用引物WF-EG1-UF1和P-EG1-R扩增其5’端同源臂序列(1149bp)。以质粒pMDT05-mus53KO为模板，使用引物EG1-pyr4-F和CBH2-R6扩增pyr4表达框。以里氏木霉基因组DNA为模板，使用引物CBH2-F5和EG1-TRA2-R扩增501bp序列作为重复序列(DR)。以里氏木霉基因组DNA为模板，使用引物EG1-DW-F和EG1-DW-R扩增其3’端同源臂序列(1211bp)。
所有PCR扩增的片段按照omega公司凝胶回收试剂盒方法进行回收，回收后的片段按照等摩尔比混合作为PCR扩增模板，使用引物WF-EG1-UF1和EG1-DW-R为上下游引物SOE-PCR扩增约4.8kb的融合片段。使用引物pMDT-SpeI-R和pMDT-XbaI-F扩增线性化载体pMDT-05，扩增产物用DpnI消化3h。将上述2片段并按照omega公司凝胶回收试剂盒方法进行回收，回收后的目的片段按照
Figure PCTCN2018107053-appb-000012
II一步法克隆试剂盒的方法组装，转化大肠杆菌TOP10感受态细胞，将验证和测序正确的载体命名为pMDT05-EG1-pyr4。
以质粒pMGU-cbh1-TRA2为模板，使用引物EG1-TRA2-F和CBH2-R22扩增约4.7kb的表达框Pcbh1-TRA2-Tcbh1。以质粒pMDT05-EG1-pyr4为模板，使用引物CBH2-F66和P-EG1-R扩增载体线性化，扩增后的产物用DpnI消化3h。将上述两个片段按照omega公司凝胶回收试剂盒方法进行回收，回收的片段按照
Figure PCTCN2018107053-appb-000013
II一步法克隆试剂盒的方法组装，转化大肠杆菌TOP10感受态细胞，将验证和测序正确的载体命名为pMDT05-EG1-TRA2(KI)，参见图10。引物序列见表6。
4、EG2位点定点整合表达载体构建pMDT05-EG2-TRA2(KI)
通过关键词搜索在里氏木霉基因组数据库中(http://genome.jgi-psf.org/Trire2/Trire2.home.html)获取EG2(Cel5B)基因座相应DNA序列信息。
以里氏木霉基因组DNA为模板，使用引物WF-EG2-UF1和P-EG2-R扩增其5’端同源臂序列(1100bp)。以质粒pMDT05-mus53KO为模板，使用引物EG2-pyr4-F和CBH2-R6扩增pyr4表达框。以里氏木霉基因组DNA为模板，使用引物CBH2-F5和EG2-TRA2-R扩增501bp序列作为重复序列(DR)。以里氏木霉基因组DNA为模板，使用引物EG2-DW-F和EG2-DW-R扩增其3’端同源臂序列(1098bp)。
所有PCR扩增的片段按照omega公司凝胶回收试剂盒方法进行回收，回收后的片段按照等摩尔比 混合作为PCR扩增模板，使用引物WF-EG2-UF1和EG2-DW-R为上下游引物SOE-PCR扩增约4.6kb的融合片段。使用引物pMDT-SpeI-R和pMDT-XbaI-F扩增线性化载体pMDT-05，扩增产物用DpnI消化3h。将上述2片段并按照omega公司凝胶回收试剂盒方法进行回收，回收后的目的片段按照
Figure PCTCN2018107053-appb-000014
II一步法克隆试剂盒的方法组装，转化大肠杆菌TOP10感受态细胞，将验证和测序正确的载体命名为pMDT05-EG2-pyr4。
以质粒pMGU-cbh1-TRA2为模板，使用引物EG2-TRA2-F和CBH2-R22扩增约4.7kb的表达框Pcbh1-TRA2-Tcbh1。以质粒pMDT05-EG2-pyr4为模板，使用引物CBH2-F66和P-EG2-R扩增载体线性化，扩增后的产物用DpnI消化3h。将上述两个片段按照omega公司凝胶回收试剂盒方法进行回收，回收的片段按照
Figure PCTCN2018107053-appb-000015
II一步法克隆试剂盒的方法组装，转化大肠杆菌TOP10感受态细胞，将验证和测序正确的载体命名为pMDT05-EG2-TRA2(KI)，参见图11。引物序列见表6。
表6.构建定点整合载体引物
Figure PCTCN2018107053-appb-000016
Figure PCTCN2018107053-appb-000017
实施例13:4拷贝定点整合表达菌株构建
在里氏木霉纤维素酶诱导表达条件下，其胞外纤维素酶系中CBH1，CBH2，EG1和EG2占胞外总蛋白的75％以上。诱导型启动子Pcbh1在诱导目的基因TRA2表达的同时，也诱导了大量纤维素酶系基因的表达，这样在发酵液上清中会有比较多的纤维素酶系成分作为杂蛋白存在，给下游处理带来不利因素，同时也消耗部分原料合成这些纤维素酶系的成分。本实施例在Rut-C30(pyr4 -，mus53 -)菌株基础上通过定点整合敲入的方式在CBH1，CBH2，EG1和EG2位点基因组上一共整合4个拷贝的目的基因表达框。
1、CBH1位点定点整合表达株构建
根据实施例4中所述的方法和步骤，将CBH1位点定点整合表达载体pMDT05-CBH1-TRA2(KI)通过农杆菌介导转化的方法转化里氏木霉Rut-C30(pyr4 -，mus53 -)株，挑取36个转化体点板到固体MM培养基(300μg/mL头孢霉素)平板上复筛，28℃培养3d，挑取其中20个将生长良好的转化体菌丝点板至PDA平板，28℃生孢培养7d。
通过引物NdeI-Pcbh1-F2/TRA2-CX-R1和pyr4-LB-R/CBH-down-R做PCR筛选这20个转化体，以确定是否在CBH1基因座位点通过5’端同源臂和3’端同源臂发生同源重组。通过引物RB-YZ-F和引物RB-YZ-R(见表5)做PCR扩增筛选转化体，以确定是否在CBH1基因座位点外发生随机整合。本实施例中，对于各个转化体，通过从培养到第3天的PDA平板上提取少量菌丝体到20μl无菌水中，98℃加热10分钟，离心取上清作为模板，用引物NdeI-Pcbh1-F2/TRA2-CX-R1和pyr4-LB-R/CBH-down-R能分别扩增到约2.7kb和1.3kb片段的说明在相应区域发生了预期形式的同源重组，同时用引物RB-YZ-F和引物RB-YZ-R不能扩增到425bp的片段，说明没有发生随机整合，本实施例中筛选到同时满足这些条件的阳性转化子14株。选取其中一个阳性转化子，按照实施例11中方法删除pyr4基因表达框，并使用引物HC2-JD-F2和CBH1-JD-R2进行验证，发生删除的能扩增到698bp片段，将验证阳性的菌株命名为LYH-D1(pyr4 -，mus53 -)。引物序列见表7。
2、CBH2位点定点整合表达株构建
在菌株LYH-D1(pyr4 -，mus53 -)的基础上，根据上述CBH1位点定点整合敲入的方法和步骤，将CBH2位点定点整合表达载体pMDT05-CBH2-TRA2(KI)通过农杆菌介导转化的方法转化里氏木霉LYH-D1(pyr4 -，mus53 -)株，获得了2拷贝的定点整合菌株LYH-D2(pyr4 -，mus53 -)。
在本实施例中，通过引物CBH2-F/Pcbh1-CX和pyr4-LB-R/CBH2-R做PCR筛选转化体，以确定是否在CBH2基因座位点通过5’端同源臂和3’端同源臂发生同源重组。通过引物RB-YZ-F和引物RB-YZ-R(见表5)做PCR扩增筛选转化体，以确定是否在CBH2基因座位点外发生随机整合。通过引物Tcbh1-CX-F和CBH2-R2验证是否删除pyr4基因表达框。引物序列见表7。
3、EG1位点定点整合表达株构建
在菌株LYH-D2(pyr4 -，mus53 -)的基础上，根据上述CBH1位点定点整合敲入的方法和步骤，将EG1位点定点整合表达载体pMDT05-EG1-TRA2(KI)通过农杆菌介导转化的方法转化里氏木霉LYH-D2(pyr4 -，mus53 -)株，获得了3拷贝的定点整合菌株LYH-D3(pyr4 -，mus53 -)。
在本实施例中，通过引物EG1-UF1/Pcbh1-CX和pyr4-LB-R/EG1-R做PCR筛选转化体，以确定是否在EG1基因座位点通过5’端同源臂和3’端同源臂发生同源重组。通过引物RB-YZ-F和引物RB-YZ-R(见表5)做PCR扩增筛选转化体，以确定是否在EG1基因座位点外发生随机整合。通过引物Tcbh1-CX-F和EG1-DR1验证是否删除pyr4基因表达框。引物序列见表7。
4、EG2位点定点整合表达株构建
在菌株LYH-D3(pyr4 -，mus53 -)的基础上，根据上述CBH1位点定点整合敲入的方法和步骤，将EG2位点定点整合表达载体pMDT05-EG2-TRA2(KI)通过农杆菌介导转化的方法转化里氏木霉LYH-D3(pyr4 -，mus53 -)株，获得了4拷贝的定点整合菌株LYH-D4(pyr4 -，mus53 -)。
在本实施例中，通过引物EG2-UF1/Pcbh1-CX和pyr4-LB-R/EG22-R做PCR筛选转化体，以确定是否在EG2基因座位点通过5’端同源臂和3’端同源臂发生同源重组。通过引物RB-YZ-F和引物RB-YZ-R(见表5)做PCR扩增筛选转化体，以确定是否在EG1基因座位点外发生随机整合。通过引物Tcbh1-CX-F和EG2-DR1验证是否删除pyr4基因表达框。引物序列见表7。
表7.定点整合敲入验证引物
引物名称 引物序列(5’-3’)
NdeI-Pcbh1-F2 AATTCTGGAGACGGCTTGTTGAATC
TRA2-CX-R1 CGAGTCGCATGTTGACAGAGG
pyr4-LB-R GCATTTGCTTTTGCGCGTGGAG
CBH-down-R AGTAAGCTTTCTAGAGATCGGTGAACAGTTGTCGACC
HC2-JD-F2 GAATGTGCTGCCTCCAAAATCCTGCG
CBH1-JD-R2 CAAAGCGGCTCGTCTTGGCCAGG
CBH2-F GGTGCTGAGAGCTGGACAATG
CBH2-R GCGACCAGGTTCCCACGAACTAC
CBH2-R2 GCTATTGGACATGCCGTCGATG
EG1-UF1 AGCCTCATGTTCTTCTCCCAGAC
EG1-R GTCTGCTCAGGCATTATCTTCACTGC
EG1-DR1 CTGACGGGATCTTTTGCCTGCA
EG2-UF1 GTCTTATTGGCGCTGCATGCT
EG2-R CTGAGCTGATCTATGAGTCATAAGCTTC
EG2-DR1 GTATCAGATGTGAACTGCGCTG
实施例14:里氏木霉mus53基因修复载体pMDT05-mus53(KI)构建
以里氏木霉基因组DNA为模板，使用引物mus53-up-F和mus53-up-R扩增包含其5’端同源臂序列和重复序列(DR)在内的片段(2209bp)。以质粒pMDT05-mus53KO为模板，使用引物mus53-pyr4-F和mus53-pyr4-R扩增pyr4表达框。将上述2个PCR扩增片段按照omega公司凝胶回收试剂盒方法进行回收，回收后的片段按照等摩尔比混合作为PCR扩增模板，使用引物mus53-up-F和mus53-pyr4-R为上下游引物SOE-PCR扩增约4.0kb的融合片段。使用引物pMDT-SpeI-R和pMDT-XbaI-F扩增线性化载体pMDT-05，扩增产物用DpnI消化3h。将上述2片段并按照omega公司凝胶回收试剂盒方法进行回收，回收后的目的片段按照
Figure PCTCN2018107053-appb-000018
II一步法克隆试剂盒的方法组装，转化大肠杆菌TOP10感受态细胞，将验证和测序正确的载体命名为pMDT05-mus53-pyr4。
以里氏木霉基因组DNA为模板，使用mus53-down-F和mus53-down-R扩增包含其3’端同源臂和mus53修复区的片段(4343bp)。使用限制性内切酶EcoRI酶切载体pMDT05-mus53-pyr43h。将上述PCR扩增片段和酶切线性化的pMDT05-mus53-pyr4(EcoRI)载体按照omega公司凝胶回收试剂盒方法进行回收，回收后的目的片段按照
Figure PCTCN2018107053-appb-000019
II一步法克隆试剂盒的方法组装，转化大肠杆菌TOP10感受态细胞，将验证和测序正确的载体命名为pMDT05-mus53(KI)，构建图谱参见图12。
实施例15:4拷贝菌株LYH-D4(pyr4 -，mus53 -)中mus53和pyr4基因修复
在里氏木霉LYH-D4(pyr4 -，mus53 -)菌株中对里氏木霉mus53基因进行修复，根据实施例4中所述的方法和步骤，将mus53基因修复载体pMDT05-mus53(KI)通过农杆菌介导转化的方法转化里氏木霉LYH-D4(pyr4 -，mus53 -)株，挑取27个转化体点板到固体MM培养基(300μg/mL头孢霉素)平板上复筛，28℃培养3d，挑取其中15个将生长良好的转化体菌丝点板至PDA平板，28℃生孢培养7d。
通过引物MUS-F/TrpC-CX-F和MUS-YZ-F2/MUS-R做PCR筛选15个转化体，以确定是否在mus53基因座位点通过其5’端同源臂和3’端同源臂发生同源重组。通过引物RB-YZ-F和引物RB-YZ-R做PCR扩增筛选转化体，以确定是否在mus53基因座位点外发生随机整合。按照实施例11中方法删除pyr4基因表达框，并使用引物mus3-YZ-F和MUS-YZ-R2进行验证是否删除pyr4基因表达框，将mus53基因修复的阳性菌株命名为LYH-D4(pyr4 -)。验证引物序列(部分见表5)：
MUS-YZ-F2：GTGCTGGGAGACGATGTGATG
mus3-YZ-F：CAGCAGCGACGCGATTCCTTC
MUS-YZ-R2：CTGCTTCAGAATGATGCGGATG
在mus53基因修复后，按照实施例10中的方法和步骤，通过pyr4基因修复载体pMDT05-pyr4KI将LYH-D4(pyr4 -)中的pyr4基因进行修复，最终获得的阳性菌株命名为LYH-D4。
实施例16:定点整合4拷贝菌株LYH-D4摇瓶发酵优化
由于里氏木霉LYH-D4株的4个主要的纤维素酶基因被敲除，不能利用微晶纤维素作为诱导物和碳源，所以在实施例6中用于随机整合转化菌株优化的培养基不适合于里氏木霉LYH-D4株。
本实施例通过一系列培养基组分单因素实验，PB实验及响应面曲线实验优化培养基配方，提高里氏木霉LYH-D4株单位体积发酵活力，优化结果表明，使用优化前的发酵培养基(组成：乳糖30g/L，玉米浆12g/L，(NH 4) 2SO 40.5g/L，MgSO 4·7H 2O 1.56g/L、CaCl 20.5g/L，KH 2PO 46g/L，麸皮粉2g/L，Mandels微量元素(1000X)1ml，MnCl 25mM，pH 4.0)，发酵168h发酵液上清中草酸脱羧酶活力在6800IU/L左右。使用优化的培养基，摇瓶发酵表达168h后上清发酵液中草酸脱羧酶活力可到达26500IU/L。优选的培养基组成为：葡萄糖3-6g/L，乳糖30-40g/L，玉米浆粉7-10g/L，(NH 4) 2SO 40.5-1g/L，MgSO 4·7H 2O 1.56g/L、CaCl 20.5g/L，KH 2PO 42-4g/L，尿素0-1g/L，麸皮粉10-20g/L，Mandels微量元素(1000X)1ml，MnCl 20.5-5mM，pH 3.5-4.0。
实施例17:定点整合4拷贝菌株LYH-D4发酵罐发酵
1、种子液的制备
将重组里氏木霉菌LYH-D4的菌丝接种于多个PDA固体斜面培养基中，28℃培养7d，待分生孢子长 成绿色后，用无菌水冲洗收集孢子悬液，并调整孢子浓度到1.0*10 8个/ml左右，以1％接种量接种于500mL MM液体培养基中，28℃避光震荡(170转/分)培养24-36h，以其作为7L发酵罐发酵的种子液。
2、里氏木霉菌株LYH-D4在7L发酵罐中发酵
里氏木霉的整个发酵过程分为以下两个阶段：第一阶段为菌丝体生长阶段(0～72h)：在7L发酵罐(上海保兴生物设备工程有限公司)中加入4.5L基础发酵培养基(葡萄糖20g/L，玉米浆粉7g/L，KH 2PO 44g/L，尿素1g/L，硫酸铵2g/L，MgSO 4·7H 2O 0.5g/L，CaCl 21g/L，MnCl 21mM，Mandels微量元素(1000X)1ml/L,pH 4.0)，按10％的比例接种制备好的里氏木霉种子液，28℃通气搅拌培养72小时左右，溶氧量维持在30％以上，搅拌转速根据溶氧量进行调整，一般控制在250-500转/分钟，pH维持在3.5-4.0左右。在菌丝体生长阶段，随着里氏木霉菌体的生长，一般在24-28h左右葡糖糖基本消耗完，此时以12ml/h的速率补加250g/L乳糖溶液。培养至72h左右菌体的干重达到15-18g/L。第二阶段为诱导表达阶段(72～168h)：在发酵开始的第72h，通过蠕动泵流加250g/L的乳糖溶液，使其工作浓度始终不超过2g/l，溶氧量始终大于20％，28℃通气搅拌培养至接种后168小时左右，pH维持在4.0左右。每隔24小时取样检测发酵液上清草酸脱羧酶活力，发酵160小时左右发酵液上清活力可达到271756U/L(图13)。取136h和160h发酵液上清稀释10倍进行SDS-PAGE检测，可明显看到分子量约为60KDa的目的蛋白条带(图14)。将160h发酵液样品稀释200倍和500倍进行Western Blot检测分析(图15)。
实施例18:重组草酸脱羧的回收提取
发酵液在常温下经5000rpm离心15分钟，留取上清。发酵液上清经过孔径100nm的无机陶瓷膜(三达膜环境技术股份有限公司)过滤，收集透过液，澄清后的透过液边搅拌边加入浓度为10％的单宁酸，使单宁酸终浓度在1％左右，室温下静置1h，然后在常温下8000rpm离心15分钟，收集草酸脱羧酶与单宁酸的复合物沉淀。此复合物沉淀物用1/2体积透过液的无菌水充分重悬清洗，8000rpm离心15分钟，留取复合物沉淀，如此重复1次。向此复合物沉淀中加入0.4倍澄清液体积的聚乙二醇溶液(聚乙二醇的用量为澄清液体积的0.3-0.5％)，常温下不断搅拌4h，利用聚乙二醇同单宁酸之间有更强的结合力而使草酸脱羧酶从单宁酸蛋白复合物中解析出来。然后室温下8000rpm离心15分钟去除单宁酸聚乙二醇聚合物，留取上清，该上清即为浓缩2.5倍的浓缩酶液。最后向浓缩酶液中加入糖用活性炭2％脱色，得到浅黄色的草酸脱羧酶酶液，酶回收率可达到90-95％。脱色后的草酸脱羧酶酶液用截留分子量为10KDa的超滤膜进行浓缩10-30倍，浓缩后经喷雾干燥得到草酸脱羧酶酶粉。
实施例19:重组草酸脱羧酶性质及对比分析
将通过里氏木霉丝状真菌宿主细胞表达的重组草酸脱羧酶与通过天然宿主茶树菇诱导表达的草酸脱羧酶，在pH值1.5-7.0条件下测定相对酶活力，结果如图16所示，在不同pH值条件下，重组草酸脱羧酶与天然茶树菇诱导表达草酸脱羧酶具有相似的相对酶活力保持。重组草酸脱羧酶在pH值1.5-7.0的条件下保持其全部或部分酶活力，且在pH值1.5-2.5的条件下保持不低于其在最适pH条件下酶活力的10％，在pH值2.5-4.5的条件下保持不低于其在最适pH条件下酶活力的50％，在pH值4.5-7.0的条件下保持不低于其在最适pH条件下酶活力的25％，最适pH值为2.5-3.5。
将通过里氏木霉丝状真菌宿主细胞表达的重组草酸脱羧酶，天然宿主茶树菇诱导表达的草酸脱羧酶以及原核表达的草酸脱羧酶进行SDS-PAGE分析，结果如图17所示，里氏木霉表达的重组草酸脱羧酶与茶树菇表达的草酸脱羧酶由于糖基化修饰形式和程度的不同，在表观分子量上有差异，天然宿主茶树菇表达的草酸脱羧酶的分子量70kDa左右，而里氏木霉表达的重组草酸脱羧酶的分子量在60kDa左右，但两者都比无糖基化修复的原核表达的草酸脱羧酶的分子量大，原核表达的无糖基化修饰的草酸脱羧酶分子量在50kDa左右。将里氏木霉表达的重组草酸脱羧酶用MALDI-TOF-MS进行分子量分析，如图18所示，其真实分子量为57.1kDa。
分别将上述三种表达系统表达的草酸脱羧酶用经过TPCK处理的胰蛋白酶酶解消化后，做MALDI-TOF-MS分析，其峰图如图19,20和21所示，这三种表达系统表达的草酸脱羧酶由于在糖基化修饰的形式和程度不同，其经胰蛋白酶酶解消化后的质谱峰图具有差异性，这种差异性具有宿主细胞特异性。
本发明所述SEQ ID NO.10-16序列在里氏木霉中进行单拷贝重组表达与SEQ ID NO.9能得到相似的实验结果。
以上所述实施例仅是为充分说明本发明而所举的较佳实施例，本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换，均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。

Claims (23)

  1. 一种重组草酸脱羧酶，其特征在于：通过丝状真菌宿主细胞重组表达，致使重组草酸脱羧酶的糖基化修饰形式和程度不同于原始宿主细胞表达的草酸脱羧酶，所述重组草酸脱羧酶具有丝状真菌宿主细胞特有的糖基化修饰形式和程度。
  2. 根据权利要求1所述的重组草酸脱羧酶，其特征在于，所述重组草酸脱羧酶在pH值1.5-7.0的条件下保持其全部或部分酶活力，且在pH值1.5-2.5的条件下保持不低于其在最适pH条件下酶活力的10％，在pH值2.5-4.5的条件下保持不低于其在最适pH条件下酶活力的50％，在pH值4.5-7.0的条件下保持不低于其在最适pH条件下酶活力的25％。
  3. 根据权利要求1所述的重组草酸脱羧酶，其特征在于，所述重组草酸脱羧酶的最适pH值为2.5-3.5。
  4. 根据权利要求1所述的重组草酸脱羧酶，其特征在于，所述重组草酸脱羧酶编码基因来源于真核生物，所述真核生物为茶树菇(Agrocybe aegirit)、杨树菇(Agrocybe Cylindracea)、金针菇(Flammulina velutipes)、云芝(Coriolus versicolor)，褐腐菌(Postia placenta)、琉球曲霉(Aspergillus luchuensis)、双孢蘑菇(Agaricus bisporus)或金福菇(Tricholoma Lobayensc Heim)等真菌。
  5. 根据权利要求1-4所述的重组草酸脱羧酶，其特征在于，所述重组草酸脱羧酶的氨基酸序列与SEQID NO.1或SEQ ID NO.5的第20～470位氨基酸序列、或SEQ ID NO.2的第25～472位氨基酸序列、或SEQ ID NO.3的第20～455位氨基酸序列、或SEQ ID NO.4的第21～447位氨基酸序列、或SEQ ID NO.6的第21～455的氨基酸序列、或SEQ ID NO.7的第25～440位氨基酸序列、SEQ ID NO.8的第24～472的氨基酸序列具有至少60％同一性，优选至少65％同一性、至少70％同一性、至少75％同一性、至少80％同一性、至少85％同一性、至少90％同一性、或至少95％同一性。
  6. 根据权利要求1-5所述的重组草酸脱羧酶，其特征在于，所述重组草酸脱羧酶的氨基酸序列由SEQID NO.1或SEQ ID NO.5的第20～470位氨基酸序列、或SEQ ID NO.2的第25～472位氨基酸序列、或SEQ ID NO.3的第20～455位氨基酸序列、或SEQ ID NO.4的第21～447位氨基酸序列、或SEQ ID NO.6的第21～455的氨基酸序列、或SEQ ID NO.7的第25～440位氨基酸序列、SEQ ID NO.8的第24～472的氨基酸序列构成。
  7. 一种重组丝状真菌宿主细胞，其特征在于，所述重组丝状真菌宿主细胞的染色体DNA中包含编码权利要求1-6中任一项所述的草酸脱羧酶基因序列。
  8. 根据权利要求7所述的重组丝状真菌宿主细胞，其特征在于，所述丝状真菌为曲霉属(Aspergillus)、革盖菌属(Coriolus)、毛霉属(Mucor)、白腐菌属(Phlebia)、枝顶孢属(Acremonium)、隐球菌属(Cryptococcus)、镰孢霉属(Fusarium)、腐质霉属(Humicola)、毁丝霉属(Myceliophthora)、短梗霉属(Aureobasidium)、栓菌属(Trametes)、侧耳属(Pleurotus)、脉孢菌属(Neurospora)、青霉属(Penicillium)、拟青霉属(Paecilomyces)、平革菌属(Phanerochaete)、烟管菌属(Bjerkandera)、拟蜡菌属(Ceriporiopsis)、梭孢壳属(Thielavia)、金孢子菌属(Chrysosporium)、裂褶菌属(Schizophyllum)、鬼伞属(Coprinus)、稻瘟菌属(Magnaporthe)、新美鞭菌属(Neocallimastix)、弯颈霉属(Tolypocladium)踝节菌属(Talaromyces)、嗜热子囊菌属(Thermoascus)或木霉属(Trichoderma)等真菌。
  9. 根据权利要求7所述的重组丝状真菌宿主细胞，其特征在于，所述丝状真菌为曲霉属的黑曲霉(A.niger)、构巢曲霉(A.nidulans)、米曲霉(A.oryzae)或泡盛曲霉(A.awamori)菌株。
  10. 根据权利要求7所述的重组丝状真菌宿主细胞，其特征在于，所述丝状真菌为木霉属的哈茨木霉(Trichoderma harzianum)、康宁木霉(T.koningii)、里氏木霉(T.reesei)、长梗木霉(T.longibrachiatum)或绿色木霉(T.viride)菌株。
  11. 根据权利要求7所述的重组丝状真菌宿主细胞，其特征在于，所述丝状真菌为里氏木霉。
  12. 根据权利要求7-11任一所述的重组丝状真菌宿主细胞，其特征在于，所述草酸脱羧酶编码基因的核苷酸序列中至少10％的碱基根据丝状真菌宿主细胞密码子偏爱性进行了密码子优化。
  13. 根据权利要求12所述的重组丝状真菌宿主细胞，其特征在于，所述核苷酸序列选自SEQ ID NO.9、SEQ ID NO.10、SEQ ID NO.11、SEQ ID NO.12、SEQ ID NO.13、SEQ ID NO.14、SEQ ID NO.15和SEQ ID NO.16的核酸序列；或与SEQ ID NO.9-16中任一具有至少50％同一性，优选至少60％同一性、至少70％同一性、至少80％同一性或至少90％同一性的序列。
  14. 一种构建权利要求7-12任一所述的重组丝状真菌宿主细胞的方法，其特征在于，所述重组丝状真菌宿主细胞包含一个或多个拷贝的整合在其基因组中的草酸脱羧酶表达盒，所述草酸脱羧酶表达盒包括启动子、信号肽编码序列、草酸脱羧酶编码基因和终止子，所述方法包括如下步骤：
    S1：构建至少一个整合型表达载体，所述整合型表达载体包括选择标记基因表达盒和草酸脱羧酶表达盒。
    S2:整合型表达载体在转化丝状真菌宿主细胞后，通过筛选获得含有一个拷贝或多个拷贝草酸脱羧酶表达盒的重组丝状真菌宿主细胞。
  15. 根据权利要求14所述的方法，其特征在于，其中步骤S2中所述的丝状真菌宿主细胞为人工构建的营养缺陷型细胞，所述整合型表达载体在整合进所述丝状真菌宿主细胞基因组中时能修复该类型营养缺陷。
  16. 根据权利要求14所述的方法，其特征在于，在转化丝状真菌宿主细胞后，所述整合型表达载体通过非同源重组而随机整合进丝状真菌宿主细胞基因组中。
  17. 根据权利要求14所述的方法，其特征在于，所述的整合型表达载体包含与丝状真菌宿主细胞基因组中特异性基因座一定长度核苷酸序列同源的5’端同源臂和3’端同源臂，从而在转化丝状真菌宿主细胞后所述整合型表达载体能通过同源重组的方式整合进基因组特定的位点；优选整合进编码胞外蛋白的基因中；更优选整合进编码胞外蛋白酶类或编码胞外糖苷水解酶类的基因中，最优选地整合进CBH1、CBH2、EG1或EG2基因中。
  18. 一种培养基，适用于权利要求16所述方法制备的宿主细胞的培养，其特征在于，组成为：葡萄糖3-8g/L，微晶纤维素10-25g/L，玉米浆粉5-15g/L，(NH 4) 2SO 40.5-5g/L，MgSO 4·7H 2O 1.56g/L、CaCl 20.5g/L，KH 2PO 42-8g/L，尿素0-1g/L，麸皮粉0.2-2g/L，Mandels微量元素(1000X)1ml，MnCl 20.5-5mM，pH 3.0-4.5。
  19. 一种培养基，适用于权利要求17方法制备的宿主细胞的培养，其特征在于，组成为：葡萄糖3-6g/L，乳糖30-40g/L，玉米浆粉7-10g/L，(NH 4) 2SO 40.5-1g/L，MgSO 4·7H 2O 1.56g/L、CaCl 20.5g/L，KH 2PO 42-4g/L，尿素0-1g/L，麸皮粉10-20g/L，Mandels微量元素(1000X)1ml，MnCl 20.5-5mM，pH 3.5-4.0。
  20. 一种生产重组草酸脱羧酶的方法，其特征在于，构建含有启动子、信号肽编码序列、草酸脱羧酶编码基因和终止子的草酸脱羧酶表达盒，通过表达载体转入丝状真菌宿主细胞，使宿主细胞基因组中整合一个或多个草酸脱羧酶表达盒，培养宿主细胞表达草酸脱羧酶，最后从宿主细胞培养基质中分离纯化表达产物。
  21. 权利要求1-6任一所述的重组草酸脱羧酶或权利要求7-13所述的重组丝状真菌宿主细胞培养后分泌表达的草酸脱羧酶在制备药物、食品中的应用。
  22. 根据权利要求21所述的应用，所述药物为预防和/或治疗泌尿系结石的药物。
  23. 一种药物组合物，用于预防或治疗尿草酸过多的疾病，其特征在于：其包含有如权利要求20方法制备的草酸脱羧酶。
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