CN112852859B

CN112852859B - Method for improving synthesis capacity of filamentous fungi organic acid

Info

Publication number: CN112852859B
Application number: CN202010973401.0A
Authority: CN
Inventors: 田朝光; 李金根; 陈炳琛; 赵祯; 顾淑莹; 刘倩; 孙涛; 孙文良
Original assignee: Tianjin Institute of Industrial Biotechnology of CAS
Current assignee: Tianjin Institute of Industrial Biotechnology of CAS
Priority date: 2020-09-16
Filing date: 2020-09-16
Publication date: 2022-07-12
Anticipated expiration: 2040-09-16
Also published as: CN112852859A

Abstract

The invention provides a method for improving the organic acid synthesis capacity of filamentous fungus recombinant bacteria, and an obtained recombinant bacteria and a method for producing organic acid. The recombinant strain of the invention is introduced with the organic acid synthesis positive regulation gene and/or expresses the organic acid synthesis negative regulation gene in a down-regulation way, and compared with the original strain, the recombinant strain has obviously improved organic acid production capacity. Experiments prove that monosaccharide, polysaccharide, glycan or mixed sugar can be effectively utilized and CO is fixed simultaneously through introducing one or more positive regulatory genes and/or genetic modification engineering strains for down-regulating the negative regulatory genes₂To synthesize organic acid efficiently.

Description

Method for improving synthesis capacity of filamentous fungi organic acid

Technical Field

The present invention belongs to the field of gene engineering and biotechnology. In particular, the invention relates to a method for improving the synthesis capacity of filamentous fungi organic acid, and an obtained recombinant bacterium and a method for producing the organic acid.

Background

Lignocellulose is an abundant renewable resource in nature, and has the advantages of low cost, wide distribution, easy acquisition, large storage capacity and the like. The annual lignocellulose content produced by photosynthesis worldwide is as high as 1550 million tons, and only 2% is utilized. The cellulose raw materials in China are also quite rich, and the agricultural waste biomass resources generated each year are up to about 7 hundred million tons. The utilization of lignocellulose is limited by the excessive production cost of lignocellulose hydrolase (cellulase, hemicellulase, etc.), and has become a core problem restricting the development of the whole biorefinery industry. In nature, various microorganisms have the capacity of degrading and rapidly utilizing cellulose, and particularly filamentous fungi such as ascomycetes and basidiomycetes can secrete various lignocellulose hydrolase, so that the cellulose hydrolase has a complete cellulase system compared with other microorganisms. Through metabolic pathway design and modification, a chassis strain capable of directly utilizing biomass as a raw material to ferment and produce a large amount of organic acid is constructed, the biomass smelting cost can be effectively reduced, and the method has a very wide application prospect.

In addition, CO₂Is the main gas for generating the greenhouse effect and simultaneously CO₂And is one of the most abundant carbon resources on the earth. By using the synthetic biology enabling technology, the microbial metabolism is improved, and the fixation of CO by a biological method is realized₂The synthesis of a large amount of chemicals has important significance for solving the environmental problems and energy crisis. Currently, 6 naturally-immobilized COs are found in the biological world₂Pathways in which the Calvin cycle (CBB cycle) is the major CO of the biosphere₂Fixed pathway, estimated as CO fixed by the Calvin cycle every year₂Up to 5X 10¹⁴Kilogram. Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase, RubisCO) is a key enzyme for carbon assimilation in the CBB cycle. RuBisCO is rich in nature and can catalyze CO₂And ribulose diphosphate (RuBP) to two molecules of 3-phosphoglycerate. Type II RuBisCO consists of 2-8 identical large subunits, whichHeterologous expression involves fewer genes and is of greater concern. Medium II-type RuBisCO derived from photoautotrophic bacteria (such as Rhodospirillum rubrum, Thiobacillus densificans) has been functionally expressed in Escherichia coli and Saccharomyces cerevisiae for recovery of CO released by carbon metabolism₂To improve the yield of the target product. Construction of CBB cycle in heterotrophic traditional fermentation strains for utilization of atmospheric CO₂The synthesis of bulk chemicals and biofuels is of interest.

Myceliophthora thermophila is a high-temperature filamentous fungus for degrading natural cellulose, is an excellent producer of high-temperature resistant cellulase and has the capability of secreting a large number of biomass degrading enzymes naturally. The optimal growth temperature of myceliophthora thermophila is 45-48 ℃, the optimal enzyme activity temperature of the myceliophthora thermophila is very close to 50 ℃ of the optimal enzyme activity temperature of cellulase, the myceliophthora thermophila has very good cellulose degradation capability, and various saccharides such as cellobiose, glucose, xylose and the like generated by degradation can be fully utilized, so that biomass can be used as a raw material for fermentation. Meanwhile, myceliophthora thermophila can be used for producing a large number of chemicals such as malic acid, fumaric acid and the like through fermentation after certain metabolic engineering transformation, and is proved to be used for large-scale industrial fermentation.

The main constituent units of biomass include xylose, arabinose, glucose, and the like. In the industrial fermentation process, the rapid utilization of three monosaccharides by the fermentation strain is the key for realizing the high-efficiency conversion of biomass. In cells, pentose sugars (xylose and arabinose) are substrates for ribose 5-phosphate kinase (PRK) in the CBB cycle via ribose 5-phosphate, an intermediate in the metabolism of pentose phosphate pathway, so in biomass utilization, pentose sugars can act as a CBB pathway to fix CO₂The driving substrate of (1). However, it has not been reported to introduce a CBB pathway in filamentous fungi to increase biomass carbon source utilization, enabling biomass and CO utilization₂Is a carbon source, thereby enhancing the synthesis of organic acid.

Disclosure of Invention

As a result of extensive and intensive studies, the present inventors have found that a positive regulator gene for organic acid synthesis or/and a negative regulator gene for organic acid synthesis can be introduced into filamentous fungi by genetic engineeringCompared with the original strain, the recombinant strain has obviously improved production capacity of the dibasic organic acid. Proved by verification, the strain can obviously improve the substrate consumption rate and CO of the organic acid engineering strain₂Immobilization efficiency and production efficiency of organic acids thereof.

To achieve the purpose, the invention adopts the following technical scheme.

The invention provides a method for improving the organic acid synthesis capacity of a filamentous fungus recombinant strain, which introduces an organic acid synthesis positive regulation gene or/and a lower regulation organic acid synthesis negative regulation gene into the filamentous fungus by a genetic engineering method, wherein the production capacity of binary organic acid of the recombinant strain is remarkably improved compared with that of the original strain; wherein, the organic acid comprises malic acid, succinic acid, fumaric acid and oxaloacetic acid, and malic acid and/or succinic acid are preferred.

In specific embodiments, the organic acid synthesis positive regulator gene is selected from one or more of 5-phosphoribosyl kinase,1, 5-bisphosphate ribocarboxylase/oxygenase, sugar transporter; the down regulation organic acid synthesis negative regulation gene is selected from one or more of lactate dehydrogenase, pyruvate decarboxylase or pyruvate carboxykinase. Preferably, the simultaneous introduction of one or more exogenous organic acid synthesis positive regulatory genes and the down-regulation of the expression of one or more of said negative regulatory genes.

In particular, the filamentous fungal cell is selected from the group consisting of Neurospora (Neurospora), Aspergillus (Aspergillus), Trichoderma (Trichoderma), Penicillium (Penicillium), Myceliophthora (Myceliophthora), Torulaspora (Sporotrichum), Fusarium (Fusarium), Rhizopus (Rhizopus), Mucor (Mucor), and Paecilomyces (Paecilomyces), more preferably, the Myceliophthora thermophila (Myceliophthora thermophila), Myceliophthora heterolytica (Myceliophthora).

Wherein, compared with the original strain, the recombinant strain has the advantage that the organic acid production capacity is enhanced or improved by at least 10 percent; preferably at least 10-50%; more preferably, at least 50% to 500%.

In one embodiment, the amino acid sequence of the 5-phosphoribosyl kinase is shown in SEQ ID No.3, the amino acid sequence of the 1,5-bisphosphate ribocarboxylase/oxygenase is shown in SEQ ID No.1, and the amino acid sequence of the sugar transporter is shown in SEQ ID No. 56; the amino acid sequence of the lactate dehydrogenase is shown as SEQ ID NO.15, the amino acid sequence of the pyruvate decarboxylase is shown as SEQ ID NO.13, and the amino acid sequence of the pyruvate carboxykinase is shown as SEQ ID NO. 17.

In a specific embodiment, the method for introducing the organic acid synthesis positive regulatory gene is realized by introducing an expression vector containing the positive regulatory gene; the down regulation of the negative regulation gene of the synthesis of organic acid is realized by gene knockout or gene editing or an inhibitor, wherein the inhibitor is selected from the group consisting of the antibody, inhibitory mRNA, antisense RNA, microRNA, miRNA, siRNA, shRNA or an activity inhibitor; preferably, downregulating the negative regulatory gene expression level refers to being achieved by a CRISPR/Cas 9-based genome editing method.

The invention also provides the recombinant bacterium prepared by the method.

The invention further provides a method for producing organic acid by using the recombinant bacterium, which is characterized in that monosaccharide, glycan and/or plant biomass are used as substrates for fermentation production of organic acid by using the recombinant bacterium.

In a specific implementation scope, the monosaccharide is selected from glucose, xylose, arabinose or a combination thereof; the polysaccharide, the peritectic cellulose, the hemicellulose or the combination thereof; the plant biomass is selected from crop straws, forestry wastes, energy plants or partial or complete decomposition products thereof; wherein the crop straws comprise corn straws, wheat straws, rice straws, sorghum straws, soybean straws, cotton straws, bagasse and corn cobs; the forestry waste comprises branches and leaves and sawdust; the energy plant comprises sweet sorghum, switchgrass, miscanthus, reed, or combinations thereof.

Preferably, the recombinant bacterium is myceliophthora thermophila, more preferably myceliophthora thermophila, myceliophthora isocarboxamide; the organic acid refers to malic acid and/or succinic acid.

The recombinant strain of the invention is introduced with organic acid synthesis positive regulation gene, and/or down-regulatedThe organic acid synthesis negative regulation gene is expressed, and compared with the original strain, the recombinant strain has obviously improved organic acid production capacity. The inventor proves through experiments that monosaccharide, polysaccharide, glycan or mixed sugar can be effectively utilized and CO is fixed simultaneously through introducing one or more positive regulatory genes and/or genetic modification engineering strains for down regulating the negative regulatory genes₂To efficiently synthesize an organic acid.

Drawings

FIG. 1 phenotypic analysis of myceliophthora thermophila strain CP-1. (A) Measuring the enzyme activity of RuBisCO in the recombinant strain CP-1; (B) analyzing the yield of the organic acid; (C) strain CP-1 was biomass analyzed under xylose and arabinose conditions.

FIG. 2 carbon fixation efficiency assay of myceliophthora thermophila strain CP-51.

FIG. 3 analysis of fermentation behavior of myceliophthora thermophila strain CP-1. (A) (C) and (D) show the malic acid production of strain CP-51 under xylose, arabinose and glucose conditions, respectively; (B) biomass analysis of strain CP-1 under xylose conditions

FIG. 4 analysis of the efficiency of substrate transport by myceliophthora thermophila strain Gal-1.

FIG. 5 the efficiency of substrate utilization by myceliophthora thermophila strain Gal-1 under monosaccharide or mixed sugar conditions.

FIG. 6 organic acid production by myceliophthora thermophila strain Gal-1 using a mixed sugar consisting of glucose, xylose and arabinose as a carbon source.

FIG. 7 malic acid production by myceliophthora thermophila strain Gal-1 under conditions of polysaccharides (xylan, crystalline cellulose and corncob residue).

FIG. 8 succinic acid production by myceliophthora thermophila strain Gal-1 under conditions of polysaccharides (xylan, crystalline cellulose and corncob residue).

Detailed Description

To further illustrate the technical means and effects thereof, the technical solutions of the present invention are further described below with reference to the preferred embodiments of the present invention, and it should be understood that these embodiments are only used for illustrating the present invention and are not used to limit the scope of the present invention.

The methods used in the following examples are conventional methods, unless otherwise specified, such as molecular cloning, described in Sambrook et al: the conditions described in the Laboratory Manual (New York: Cold spring harbor Laboratory Press, 1989),

the examples do not specify particular techniques or conditions, and are to be construed in accordance with the description of the art in the literature or with the specification of the product. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer. The percentage concentrations are mass percentage concentrations unless otherwise specified. Both primers and nucleic acid sequencing were performed by GENEWIZ, national genistein, Inc. Wherein, "MYCTH _ … …" is the gene locus number of myceliophthora thermophila.

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. It is to be understood that the described embodiments are exemplary only and are not limiting upon the scope of the invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and substitutions are intended to be within the scope of the invention.

Example 1 construction of a recombinant Strain of myceliophthora thermophila having a significantly enhanced ability to organic acids

In this example, Myceliophthora thermophila malic acid fermentation strain JG207 obtained in the previous stage is mainly used as a starting strain (Li J, et al. direct production of chemistry from lignocellulose using Myceliophthora thermophila. metabolic engineering 2019.DOI:10.1016/J. ymben), and key enzymes 1,5-bisphosphate ribocarboxylase/oxygenase (Ribulose-1,5-bisphosphate (RubP) carboxylase-oxydagenase, RubiCO) and 5-phosphoriboskinase (phosphoribukinase, PRK) in CBB cycle (Calvin-Benson-Basshmyte) are introduced to enhance the pentose utilization ability of the malic acid fermentation strain and fix CO utilization at the same time₂To improve the product yield.

Construction of RuBisCO and PRK expression vectors

Expression vectors for the construction of prk and cbbm (rubisco) respectively were constructed using plasmid pAN52-bar (Gu SY, Li JG, Chen BC, Sun T, Liu Q, Xiao DG, tie cg. metabolic engineering of the thernophilic membrane genes fungus Myceliophthora thermophila to product fumaric acid. biotechnology for biofuels.2018,11:323.) as backbone, and the constitutive strong promoters selected were Ppdc, the promoter of the Myceliophthora thermophila pyruvate deacidification enzyme gene pdc (Mycth _112121), and the promoter pda, the glyceraldehyde-3-phosphate dehydrogenase encoding gene gpdA (Mycth _2311855), respectively.

The amino acid sequence of the RuBisCO is shown as SEQ ID No. 1; the coding nucleotide sequence (cbbM) of the RuBisCO is shown in SEQ ID No. 2; the PRK amino acid sequence is shown as SEQ ID No. 3; the PRK coding nucleotide sequence (cbbM) is shown as SEQ ID No. 4; the nucleotide sequence of the promoter Ppdc is shown as SEQ ID No. 5; the nucleotide sequence of the promoter pgpdA is shown as SEQ ID No. 6.

After PCR amplification, the required DNA fragments are obtained, the primers are shown in Table 1, and the multiple PCR fragments are quickly assembled on a skeleton plasmid pAN52-bar which is subjected to double enzyme digestion by restriction enzymes Bgl II and BamH I by adopting a Gibson Assembly technology system, so that prk and cbbM recombinant expression vectors pAN52-prk and pAN52-cbbM are constructed,

the PCR reaction system is as follows: 5 XPhusion HF buffer 10. mu.L, 10mM dNTPs 1. mu.L, 10mM primer-F and primer-R each 2.0. mu.L, template DNA 1. mu.L, Phusion DNA polymerase 0.5. mu.L, water 33.5. mu.L. The PCR reaction conditions are as follows: firstly, the temperature is 98 ℃ for 30 s; then the temperature is 98 ℃ for 10s, the temperature is 65 ℃ for 30s, the temperature is 72 ℃ for 1.5min, and 35 cycles are carried out; finally, the temperature is 72 ℃ for 10min, and the temperature is 4 ℃ for 10min.

Recombinant expression of key genes prk and cbbm of CBB in myceliophthora thermophila

10 mu g of the recombinant expression vectors pAN52-prk and pAN52-cbbM linearized by restriction endonuclease Hind III were simultaneously transformed and introduced into protoplast cells of myceliophthora thermophila strain JG207, and transformants were selected by adding glufosinate-p (PPT) to the plates, as follows:

A. culture of myceliophthora thermophila strains

Myceliophthora thermophila malic acid strain JG207 is cultured on MM medium at 45 ℃ for 10 days for later use.

MM medium: 50 XVogel's salt 20mL, sucrose 20g, agar 15g, constant volume to 1L, autoclaving.

The used reagent formula is as follows:

50 XVogel's salt (1L): trisodium citrate (1/2H)₂O)150g, anhydrous KH₂PO₄250g, anhydrous NH₄NO₃100g，MgSO₄·7H₂O 10g，CaCl₂·2H₂O5 g, trace element salt solution 5mL, biotin (0.1mg/mL)2.5mL, and the volume is up to 1L.

Solution of trace element salts: c₆H₈O·7H₂ O.5g/L，ZnSO₄·7H₂O 0.5g/L，Fe(NH₄)₂(SO₄)·6H₂O 0.1g/L，CuSO₄·5H₂O 0.025g/L，MnSO₄·H₂O 0.005g/L，H₃BO₃ 0.005g/L，NaMoO₄·2H₂O 0.005g/L

B. Transformation of myceliophthora thermophila protoplasts

a. Mycelium preparation: collecting mature myceliophthora spores with 0.05% Tween-80 sterile water, filtering to remove mycelia via lens-wiping paper, spreading on MM plate paved with glassine paper, and culturing at 45 deg.C for 16 h.

b. Preparing protoplasts: placing the cellophane with hyphae in 30mL of lysis solution (formula: 0.15g lyase, adding 30mL of solution A in sterile operation, filtering for sterilization, solution A: 1.0361g potassium dihydrogen phosphate, 21.864g sorbitol, dissolving in 90mL deionized water, adjusting pH to 5.6 with potassium hydroxide, quantifying to 100mL, sterilizing at high temperature), lysing for 2h at 30 ℃, and gently shaking every 20 min. Then filtering by cellophane, centrifuging at 2000rpm for 10min at 4 ℃, discarding the supernatant, adding 4mL of solution B (0.735g of calcium chloride, 18.22g of sorbitol, 1mL of Tris-HCl 1M, pH7.5, dissolving in 90mL of deionized water, adjusting pH to 7.6 by hydrochloric acid, quantifying to 100mL, sterilizing at high temperature), and centrifuging at 2000rpm for 10min at 4 ℃; the supernatant was discarded and a volume of solution B was added at 200. mu.L/plasmid.

c. Protoplast transformation: pre-cooled 15mL centrifuge tubes were sequentially added 50. mu.L of pre-cooled PEG (12.5g PEG 6000, 0.368g calcium chloride, 500. mu.L Tris HCl 1M pH7.5) and the transformed DNA fragments were added to 200. mu.L protoplasts. After 20min on ice, 2mL of precooled PEG was added, 5min at room temperature, 4mL of solution B was added and mixed gently. 3mL of the above solution was added to 12mL of the thawed MM medium containing the corresponding antibiotic, plated on a plate, incubated at 35 ℃ and 3 days later, individual mycelia were picked and grown on the corresponding resistant plates.

C. Myceliophthora thermophila transformant verification

a. Genome extraction:

extracting genome DNA from the transformant selected from the above-mentioned transformation by phenol chloroform method, which comprises the following steps:

1) to a 2.0mL sterile DNA extraction tube, 200mg of zirconium beads and 1mL of lysis solution (lysis buffer, formulation: 0.2M Tris-HCl (pH7.5), 0.5M NaCl, 10mM EDTA, 1% SDS (w/v)), myceliophthora thermophila mycelia growing in the plate were picked up in a DNA extraction tube;

2) placing all DNA extraction tubes on a grinding aid, oscillating at the maximum rotation speed for 30s, and repeating twice;

3) carrying out water bath at 65 ℃ for 30 minutes, and taking out the mixture every few minutes during the water bath process to carry out vortex oscillation;

4) taking out after the water bath is finished, and adding 80 mu L of 1M Tris & HCl with the pH value of 7.5 into each tube for neutralization;

5) add 400 μ L of phenol: chloroform (1:1), 5 minutes at 13000 rpm;

6) take 300. mu.L of supernatant into a new 1.5mL EP tube, add 600. mu.L of 95% ethanol (DNA grade);

7) after one hour incubation on ice followed by centrifugation at 13000rpm at 4 ℃ white DNA was visible to precipitate to the bottom of the EP tube;

8) washing with 400. mu.L of 75% alcohol (DNA grade), centrifuging at 13000rpm at 4 ℃, and gently taking out the supernatant;

9) putting the EP tube into a vacuum concentrator, and drying alcohol in vacuum;

10) add 50. mu.L of ddH₂And O, dissolving the DNA, measuring the DNA concentration by using the NanoDrop, and storing the extracted DNA in a refrigerator at the temperature of-20 ℃ after the concentration is measured so as to prepare for the next PCR verification.

B, verifying myceliophthora thermophila transformants by PCR:

the extracted genomic DNA was used as a template, and the transformants were subjected to gene PCR verification using primers Ppdc _ prk-F/OEprk-R and OEcbbm-F/R, respectively (Table 1).

The PCR reaction system is as follows: 5 XPisuion GC buffer 4. mu.L, 10mM dNTPs 0.2. mu.L, primers each 1. mu.L, genome 1. mu.L, DMSO 0.6. mu.L, Phusion DNA polymerase 0.1. mu.L, water 12.1. mu.L. The PCR reaction conditions are as follows: firstly, the temperature is 98 ℃ for 30 s; then the temperature is 98 ℃ for 10s, 62 ℃ for 30s and 72 ℃ for 1.5min for 30 cycles; finally, the temperature is 72 ℃ for 10min, and the temperature is 4 ℃ for 10min.

And (3) carrying out 1% agarose gel electrophoresis (120V voltage, 30 minutes) on the PCR amplification product, and observing a gene amplification band under a gel imaging system, wherein the result shows that both prk and cbbM are successfully introduced into the genome of the myceliophthora thermophila malic acid fermentation strain JG207, so as to obtain the recombinant strain CP-1.

TABLE 1 primers used for vector construction in this example

Example 2 phenotypic analysis of the recombinant Strain of myceliophthora thermophila CP-1

1. Enzyme activity determination of RuBisCO in recombinant strain CP-1

After the hyphae of the recombinant strain CP-1 are cultured, the hyphae are frozen by liquid nitrogen and ground, then 100mM Tris (pH 7.4) solution is added, the mixture is centrifuged at 4 ℃, and the protein concentration and the RuBisCO activity in the supernatant are measured.

Protein concentration in the supernatant was determined using the berle Bradford protein rapid test kit.

The method for measuring the RuBisCO activity comprises the following steps: the reaction mixture (100mM Tris, (pH 7.4),10mM MgCl2,20mM NaHCO3,10mM KCl,1mM DTT,2mM oxaloacetate,5mM growth phosphate,10U 3-phosphoglycerate kinase,10U growth phosphate dehydrogenase,10U growth phosphate, 0.2mM NADH) was allowed to stand at 30 ℃ for 15min, and thereafter RubP (final concentration of 0.5mM) was added to start the reaction, and the change in absorbance at 340nm was measured for 5min at the start.

The result shows (figure 1A) that the activity of the recombinant strain RuBisCO reaches 22.3U/mg protein, and the corresponding enzyme activity is detected in the control strain JG207, which indicates that the recombinant strain RuBisCO can be correctly expressed in myceliophthora thermophila.

2. Analysis of yield of recombinant Strain CP-1 organic acid

Inoculating the obtained recombinant strain CP-1 and a control strain myceliophthora thermophila JG207 strain in 50mL of malic acid fermentation medium (150 mg/L potassium dihydrogen phosphate, 150mg/L dipotassium hydrogen phosphate, 100mg/L magnesium sulfate, 100mg/L calcium chloride, 1mL/L biotin, 1mL/L trace element liquid and 80g/L calcium carbonate), wherein carbon sources of the recombinant strain CP-1 and the control strain are respectively 75g/L xylose and 75g/L arabinose, and the inoculation amount is 2.5 to 10⁵The culture medium is 50 mL/mL in volume, and is cultured at 45 ℃ and the rotating speed of a shaking table is 150 rpm. After 8 days of fermentation, 1mL of sample was taken to determine the yield of organic acid in the fermentation broth.

The sample processing method comprises the following steps: 1mL of the fermentation broth was taken in a 15mL centrifuge tube and 1mL of 1M H was added₂SO₄Then, the mixture was left at 80 ℃ for 30min, and sufficiently shaken every 0min. Then 2mL of double distilled water is added into a centrifuge tube, after sufficient shaking, 1mL of liquid is taken out and placed into a 1.5mL centrifuge tube, centrifugation is carried out at 12000rpm for 10min, and supernatant is taken out to determine the content of C4-dicarboxylic acid.

C4-dicarboxylic acid content determination: measuring the contents of malic acid and succinic acid in the treated sample by high performance liquid chromatography, wherein the detector is ultraviolet detector, 5mM H₂SO₄As a mobile phase, the flow rate was 0.5 mL/min.

As shown in FIG. 1B, the malic acid and succinic acid yields of the recombinant strain CP-1 were significantly improved after the CBB pathway was introduced. Under the condition of xylose, the malic acid yield of the recombinant strain CP-1 strain reaches 41.4g/L, which is 38% higher than the malic acid yield of 30g/L of the strain JG207 fermented for 8 days; meanwhile, under the condition of arabinose, the malic acid yield of the recombinant strain CP-1 strain reaches 60.2g/L, which is improved by 15.1% compared with 52.3g/L of malic acid yield of the strain JG207 fermented for 8 days.

Under the condition of xylose and arabinose, the succinic acid content in the fermentation liquor of the CP-1 strain is respectively improved by 15 percent and 7 percent.

3. Recombinant strain CP-1 bioassay

Measuring the biomass in the fermentation liquor on the 4 th day of the fermentation of the recombinant strain CP-1, wherein the method comprises the following steps: taking 2mL of fermentation liquor into a weighed 15mL centrifuge tube (M1), adding 2mL of dilute hydrochloric acid (prepared by concentrated hydrochloric acid and water in a volume ratio of 1: 5), uniformly mixing, centrifuging until the supernatant is clear, discarding the supernatant, repeating for 2-3 times, washing with 2mL of water for 3 times, placing the centrifuge tube into an oven at 80 ℃ until the weight is constant, weighing M2, and weighing the dry weight of hyphae as M2-M1.

As a result, as shown in FIG. 1C, the biomass of the recombinant strain CP-1 was increased 1.38-fold and 1.18-fold under xylose and arabinose conditions, respectively.

Example 3 further engineering of organic acid fermentation strains by means of Metabolic engineering

In this example, a genome editing technology (Liu Q, Gao RR, Li JG, Lin LC, Zhao JQ, Sun WL, tie cg. development of a genome-editing CRISPR/Cas9 system in a thermal engineering genetic engineering biofunctional. biofuels.2017,10:1.) based on CRISPR/Cas9 was used to insert CBB pathway gene prk, rubisco (cbbm) and screening gene neo into the sites of lactate dehydrogenase ldh, pyruvate decarboxylase gene pdc and pyruvate carboxykinase pck in strain JG207 gene, respectively, so as to knock out metabolic branches and improve the organic acid synthesis performance of the strain while overexpressing the target gene. Among them, Cas9 protein expression plasmid was also constructed as described in the above article.

The PDC amino acid sequence is shown as SEQ ID No. 13; the PDC encoding nucleotide sequence is shown as SEQ ID No. 14; the LDH amino acid sequence is shown as SEQ ID No. 15; the LDH encoding nucleotide sequence is shown as SEQ ID No. 16; the PCK amino acid sequence is shown as SEQ ID No. 17; the PCK coding nucleotide sequence is shown as SEQ ID No. 18.

Construction of sgRNA transcription cassette vector

Target sites for the genes of interest pdc (Mycth _112121), ldh (Mycth _110317) and pck (Mycth _2315623) were designed by the software sgRNAcas9 tool, respectively. A fusion PCR method is adopted to connect a sequence U6p promoter, a protospacer and the sgRNA together, and a gene overlap extension (SOE) method is adopted to construct a sgRNA expression cassette vector.

The PCR reaction system is as follows:

max Buffer 25. mu.L, 10mM dNTPs 1. mu.L, upstream/downstream primers 1.5. mu.L each, template 1. mu.L,

max Super-Fidelity DNA Polymerase 1. mu.l, water 19. mu.l.

The PCR reaction conditions are as follows: firstly, 30s at 95 ℃; then, the temperature is 15s at 98 ℃, 15s at 58 ℃ and 45s at 72 ℃ for 32 cycles; finally, 5min at 72 ℃ and 10min at 4 ℃.

The sgRNA expression plasmids U6p-pdc-sgRNA, U6p-ldh-sgRNA and U6p-pck-sgRNA, whose sequences are shown in SEQ ID No.19, SEQ ID No.20 and SEQ ID No.21, respectively, were formed by amplification by SOE-PCR.

2. Donor DNA vector construction

In the present invention, donor DNA fragments, donor-ldh-prk, donor-pdc-cbbM, and donor-pck-neo, whose nucleic acid sequences are shown in SEQ ID No.22, SEQ ID No.23, and SEQ ID No.24, respectively, are finally constructed by ligating a homologous fragment of about 1000bp upstream/downstream of a target gene, a target gene (cbbM and prk), or a gene expression cassette PtrpC-neo of a geneticin (G418), to a plasmid PPk2BarGFP linearized with restriction enzymes XbaI and EcoRV, by the method of Gibson Assembly.

The sequences of PCR primers required for constructing the donor DNA fragments are shown in Table 1,

the PCR reaction system and the PCR reaction conditions are the same as those in the construction of the sgRNA expression cassette vector.

3. Transformation of myceliophthora thermophila protoplasts

Following the procedure of example 1, the Cas9 protein expression plasmids p0380-Ptef1-Cas9, sgRNA transcription cassettes (U6p-pdc-sgRNA, U6p-ldh-sgRNA, and U6p-pck-sgRNA), donor DNAs (donor-pdc-prk, donor-ldh-cbbM, and donor-pck-neo) were mixed in equal proportions and co-transformed into myceliophthora thermophila strain JG207 protoplast cells, Cas9 cleaved at a target site by target sequence pairing with the DNA strand of the target gene on the host cell genome under the mediation of the gRNA, followed by homologous recombination of the donor DNA fragment with sequences flanking the target site to achieve the purpose of targeted insertion of the target gene in the genome, and transformants were selected by adding a medium plate of geneticin G418 to the plate.

4. Myceliophthora thermophila transformant verification

The genome extraction method was identical to that described above, and then transformants were verified by PCR.

The genomic DNA extracted above was used as a template, and the transformants were subjected to gene PCR using the primers pdc-out-F/R, ldh-out-F/R and pck-out-F/R, respectively. PCR reagents were purchased from Biotech, Inc. of Nanjing Novozam.

The PCR reaction system is as follows: 2 μ L of 10 XTaq Buffer, 0.2 μ L of 10mM dNTP Mix, 0.4 μ L of each of the upstream and downstream primers, 1 μ L of DNA template, 0.2 μ L of Taq DNA Polymerase, and 15.8 μ L of water.

The PCR reaction conditions were: firstly, 94 ℃ for 5 min; then 30 cycles of 94 ℃ for 30s, 55 ℃ for 30s and 72 ℃ for 1.2 min; finally, the temperature is 72 ℃ for 7min, and the temperature is 4 ℃ for 10min.

The PCR amplification product was subjected to 1% agarose gel electrophoresis (110V for 30 min) to show an obvious gene amplification band under a gel imaging system, and the results are shown in FIG. 5, which indicates that homologous recombination occurs between the donor DNA fragment and the sequences on both sides of the target site, thereby obtaining a recombinant strain CP-51.

TABLE 2 primers used for vector construction in this example

Example 4 biological phenotypic analysis of the recombinant Strain of myceliophthora thermophila CP-51 in organic acid Synthesis

1. Analysis of carbon fixation efficiency of recombinant strain CP-51

For the carbon-fixing efficiency of the strain CP-51, CP-51 and a control strain myceliophthora thermophila JG207 are inoculated in 50mL of malic acid fermentation medium (150 mg/L potassium dihydrogen phosphate, 150mg/L dipotassium hydrogen phosphate, 100mg/L magnesium sulfate, 100mg/L calcium chloride, 1mL/L biotin and 1mL/L trace element liquid), the carbon sources are respectively 75g/L of xylose, and the neutralizing agent is 80g/L of Ca¹³CO₃The inoculum size was 2.5 x 10⁵The culture medium is 50 mL/mL in volume, and is cultured at 45 ℃ and the rotating speed of a shaking table is 150 rpm. The end of fermentation is determined by LC-MS/MS to contain¹³Malic acid ratio of C. As shown in FIG. 2, the fermentation broth of strain CP-51 contained 1 strain¹³The proportion of malic acid containing 2C atoms in the total malic acid is 50.9%¹³The proportion of malic acid of C atom is 8.8%, which are higher than that of the control strain JG207 (23.1% and 7.2%, respectively). 17.1% of carbon atoms in the recombinant strain CP-51 synthetic malic acid are fixed with external CO₂Is obviously higher than the original strain JG207 (9.4%).

2. Synthetic analysis of recombinant strain CP-51 organic acid

The obtained recombinant strain CP-51 and a control strain myceliophthora thermophila JG207 strain are inoculated in 50mL of malic acid fermentation medium (150 mg/L potassium dihydrogen phosphate, 150mg/L dipotassium hydrogen phosphate, 100mg/L magnesium sulfate, 100mg/L calcium chloride, 1mL/L biotin, 1mL/L trace element liquid and 80g/L calcium carbonate), carbon sources of the recombinant strain CP-51 and the control strain are respectively 75g/L xylose, 75g/L arabinose and 75g/L glucose, and the inoculation amounts are 2.5 x 10⁵The culture medium is 50 mL/mL in volume, and is cultured at 45 ℃ and the rotating speed of a shaking table is 150 rpm. After 8 days of fermentation, 1mL of sample was taken to determine the yield of organic acid in the fermentation broth.

The sample treatment and detection methods were the same as described in example 2.

As shown in FIG. 3, at the end of fermentation, the malic acid yields of the recombinant strain CP-51 under the conditions of xylose, arabinose and glucose reached 46.7g/L, 64.5g/L and 74.3g/L, respectively, which were significantly higher than those of the original strain JG207 and the constructed strain CP-1 in example 1. Meanwhile, the succinic acid content in the recombinant strain CP-51 fermentation liquor is also obviously improved.

3. Analysis of cell dry weight in the Synthesis of recombinant Strain CP-51 organic acid

Measuring biomass in fermentation liquor at the 4 th day of fermentation of the recombinant strain CP-51 under the xylose condition, wherein the method comprises the following steps: taking 2mL of fermentation liquor into a weighed 15mL centrifuge tube (M1), adding 2mL of dilute hydrochloric acid (prepared by concentrated hydrochloric acid and water in a volume ratio of 1: 5), uniformly mixing, centrifuging until the supernatant is clear, discarding the supernatant, repeating for 2-3 times, washing with 2mL of water for 3 times, placing the centrifuge tube into an oven at 80 ℃ until the weight is constant, weighing M2, and weighing the dry weight of hyphae as M2-M1. As shown in FIG. 3B, the biomass of the recombinant strain CP-51 was increased 1.54-fold under xylose conditions.

Example 5 construction of organic acid fermentation Strain with significantly enhanced substrate transport

In this example, the transporter Gal-2M was introduced into the recombinant strain CP-51 constructed in example 3 by genetic means, as follows:

gla2M overexpression vector construction

An expression vector of gal-2M is constructed by using plasmid pAN52-bar (Gu SY, Li JG, Chen BC, Sun T, Liu Q, Xiao DG, Tien CG. Metabolic engineering of the therophilic membrane culture funus Myceliophthora thermophila to product genetic acid. Biotechnology for biofuels.2018,11:323.) as a skeleton construction gene, and the selected constitutive strong promoter is the Peif of a Myceliophthora thermophila pyruvate deacidification transcription initiation factor eif (Mycth _ 2297659). The transporter Gal2M was constructed by mutating the amino acid sequence from the Saccharomyces cerevisiae transporter Ga1-2 (N376F).

The Gal-2M amino acid sequence is shown as SEQ ID No. 56; the Gal-2M coding nucleotide sequence (cbbM) is shown in SEQ ID No. 57; the nucleotide sequence of the promoter Peif is shown as SEQ ID No. 58.

A point mutation (N376F) was introduced into the gal-1() coding nucleotide by means of SOE-PCR, and thereby the gal2M nucleotide was obtained. And then obtaining a required DNA fragment after PCR amplification, wherein primers are used for quickly assembling the plurality of PCR fragments on a skeleton plasmid pAN52-bar which is subjected to double enzyme digestion by restriction enzymes Bgl II and BamH I by adopting a Gibson Assembly technology system as shown in Table 3, so that a gla2M recombinant expression vector pAN52-gla2M is constructed. The PCR reaction system is as follows: 5 XPisuion HF buffer 10u L, 10mM dNTPs 1 u L, 10mM primer-F and primer-R each 2.0 u L, template DNA 1 u L, Phusion DNA polymerase 0.5 u L, water 33.5 u L. The PCR reaction conditions are as follows: firstly, the temperature is 98 ℃ for 30 s; then the temperature is 98 ℃ for 10s, the temperature is 65 ℃ for 30s, the temperature is 72 ℃ for 1.5min, and 35 cycles are carried out; finally, the temperature is 72 ℃ for 10min, and the temperature is 4 ℃ for 10min.

Recombinant expression of gla-2M in myceliophthora thermophila

Mu.g of the recombinant expression vector pAN52-gal2M linearized with restriction enzyme Hind III was introduced into the cells of the protoplast of the myceliophthora thermophila strain CP-51, and transformants were selected by adding glufosinate-P (PPT) to the plates. After the genomic DNA of the transformants was extracted, the transformants were subjected to gene PCR verification using primers Gal1-F and Gal 2-R. The PCR reaction system is as follows: 2 μ L of 10 XTaq Buffer, 0.2 μ L of 10mM dNTP Mix, 0.4 μ L of each of the upstream and downstream primers, 1 μ L of DNA template, 0.2 μ L of Taq DNA Polymerase, and 15.8 μ L of water. The PCR reaction conditions are as follows: firstly, 94 ℃ for 5 min; then 30 cycles of 94 ℃ for 30s, 55 ℃ for 30s and 72 ℃ for 1.2 min; finally, the temperature is 72 ℃ for 7min, and the temperature is 4 ℃ for 10min.

1% agarose gel electrophoresis (110V voltage, 30 minutes) is carried out on the PCR amplification product, an obvious gene amplification band is shown under a gel imaging system, and the result is shown in figure 5, which shows that homologous recombination occurs between the donor DNA fragment and sequences on two sides of the target site, and then the recombinant strain Gal-1 is obtained.

In this example, myceliophthora thermophila protoplast preparation and transformation, and transformant genome extraction were performed as described in example 1.

TABLE 2 primers used for vector construction in this example

Example 6 biological phenotypic analysis of recombinant Strain Gal-1 in organic acid Synthesis

1. Recombinant strain Gal-1 substrate transport efficiency analysis

Culturing the recombinant strain Gal-1 and a control strain CP-51 thereof in 100mL glucose culture medium for 18h, collecting hyphae, washing with 1 XVogel's salt solution (formula shown in example 1) for 3 times, and then respectively transferring to 0.5% glucose culture medium, 0.5% xylose culture medium or 0.5% arabinose culture medium for culturing for 4 h; 10mL of the culture solution was centrifuged, washed with 1 XVogel's salt solution, and resuspended in 1mL of sterile water to which cycloheximide (100. mu.g/mL) was added, to which 100. mu.L of glucose (10mM), 100. mu.L of xylose (10mM), or 100. mu.L of arabinose (10mM), respectively, was added; after 20min of reaction, the dry weight of the mycelia was removed by centrifugation, and the residual sugar concentration in the supernatant was determined.

The experimental result shows (figure 4) that the transport rate of the recombinant strain Gal-1 to glucose is similar to that of the control strain CP-51, while the transport rate of the recombinant strain Gal-1 to pentose (xylose and arabinose) is obviously and rapidly increased, which indicates that the overexpression of the sugar transporter Gal-2M in myceliophthora thermophila can obviously improve the transport efficiency of the recombinant strain to a substrate.

2. Analysis of utilization efficiency of recombinant strain Gal-1 on mixed substrates

Respectively inoculating the recombinant strain Gal-1 and a control strain CP-51 thereof into a1 XVogel's salt solution culture medium, wherein the carbon source is composed of two or three monosaccharides (20g/L xylose, 20g/L arabinose and 40g/L glucose), and the inoculation amount is 2.5 x 10⁵The cells/mL, the volume of the culture medium is 100 mL/bottle, the culture is carried out at 45 ℃, and the rotation speed of a shaking table is 150 rpm. Sampling at intervals of a certain period of time to determine the residual sugar content in the fermentation liquor.

The results show that overexpression of Gal-2M significantly increases the rate of substrate efficiency of recombinant strain Gal-1 in monosaccharides and mixtures thereof. The consumption rate of xylose and arabinose by strain Gal-1 was significantly higher than that of the control strain CP-51. Meanwhile, in the mixture of two or three monosaccharides, the substrate utilization efficiency of the strain Gal-1 is obviously better than that of the control strain CP-51.

3. Synthesis of organic acid by using recombinant strain Gal-1 to convert biomass monosaccharide mixture

The obtained recombinant strain Gal-1 and the control strain CP-51 were inoculated into 50mL of a malic acid fermentation medium (150 mg/L potassium dihydrogenphosphate, 150mg/L dipotassium hydrogenphosphate, 100mg/L magnesium sulfate, 100mg/L calcium chloride, 1mL/L biotin, 1mL/L trace element liquid, 80g/L calcium carbonate) with a carbon source of a monosaccharide mixture containing 20g/L xylose, 20g/L arabinose, and 40g/L glucose,the inoculation amount is 2.5 x 10⁵The culture medium is 50 mL/mL in volume, and is cultured at 45 ℃ and the rotating speed of a shaking table is 150 rpm. A1 mL sample was taken every 2 days to determine the residual sugar content and the organic acid content in the fermentation broth.

As shown in FIG. 6, at the end of fermentation, the rate of utilization of the mixture of xylose, arabinose and glucose by the recombinant strain Gal-1 was significantly faster than that of the control strain CP-51, and the content of organic acids in the fermentation broth was also significantly increased. At the end of the fermentation, the concentration of malic acid in the fermentation liquor of the strain Gal-1 reaches 84.6g/L, which is 22% higher than that of the control strain CP-51(69.5 g/L).

Example 7 application of organic acid high-producing Strain Gal-1 in conversion of Biomass Synthesis Chemicals

The obtained recombinant strain Gal-1 and the original strain JG207 were inoculated in 50mL of malic acid fermentation medium (150 mg/L potassium dihydrogen phosphate, 150mg/L dipotassium hydrogen phosphate, 100mg/L magnesium sulfate, 100mg/L calcium chloride, 1mL/L biotin, 1mL/L trace element solution, 80g/L calcium carbonate), and the carbon sources were polysaccharides (75g/L crystalline cellulose, 75g/L xylan and 75g/L corn cob residue) with the inoculum size of 2.5 × 10⁵Culturing at 45 ℃ at the rotating speed of a shaking table of 150rpm in a culture medium volume of 50 mL/mL per bottle, and taking out 1mL of sample after fermenting for 8 days to determine the residual sugar content and the organic acid content in the fermentation liquid.

The results show (figure 7) that under the conditions of xylan, crystalline cellulose and corncob residue, the malic acid content of the recombinant strain Gal-1 reaches 56.8g/L, 69.9g/L and 44.4g/L respectively, and is increased by 17%, 7% and 23.2% respectively compared with the strain JG 207. Meanwhile, the succinic acid content in the fermentation liquor of the strain Gal-1 respectively reaches 12.6g/L, 11.4g/L and 3.7 g/L.

Sequence listing

<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences

<120> a method for improving the organic acid synthesis ability of filamentous fungi

<130>

<160> 64

<170> PatentIn version 3.5

<210> 1

<211> 466

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<213> Myceliophthora thermophila

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Met Asp Gln Ser Ser Arg Tyr Val Asn Leu Ala Leu Lys Glu Glu Asp

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Leu Ile Ala Gly Gly Glu His Val Leu Cys Ala Tyr Ile Met Lys Pro

20 25 30

Lys Ala Gly Tyr Gly Tyr Val Ala Thr Ala Ala His Phe Ala Ala Glu

35 40 45

Ser Ser Thr Gly Thr Asn Val Glu Val Cys Thr Thr Asp Asp Phe Thr

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Arg Gly Val Asp Ala Leu Val Tyr Glu Val Asp Glu Ala Arg Glu Leu

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Thr Lys Ile Ala Tyr Pro Val Ala Leu Phe Asp Arg Asn Ile Thr Asp

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Gly Lys Ala Met Ile Ala Ser Phe Leu Thr Leu Thr Met Gly Asn Asn

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Gln Gly Met Gly Asp Val Glu Tyr Ala Lys Met His Asp Phe Tyr Val

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Pro Glu Ala Tyr Arg Ala Leu Phe Asp Gly Pro Ser Val Asn Ile Ser

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Ala Leu Trp Lys Val Leu Gly Arg Pro Glu Val Asp Gly Gly Leu Val

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Val Gly Thr Ile Ile Lys Pro Lys Leu Gly Leu Arg Pro Lys Pro Phe

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Ala Glu Ala Cys His Ala Phe Trp Leu Gly Gly Asp Phe Ile Lys Asn

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Asp Glu Pro Gln Gly Asn Gln Pro Phe Ala Pro Leu Arg Asp Thr Ile

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Ala Leu Val Ala Asp Ala Met Arg Arg Ala Gln Asp Glu Thr Gly Glu

210 215 220

Ala Lys Leu Phe Ser Ala Asn Ile Thr Ala Asp Asp Pro Phe Glu Ile

225 230 235 240

Ile Ala Arg Gly Glu Tyr Val Leu Glu Thr Phe Gly Glu Asn Ala Ser

245 250 255

His Val Ala Leu Leu Val Asp Gly Tyr Val Ala Gly Ala Ala Ala Ile

260 265 270

Thr Thr Ala Arg Arg Arg Phe Pro Asp Asn Phe Leu His Tyr His Arg

275 280 285

Ala Gly His Gly Ala Val Thr Ser Pro Gln Ser Lys Arg Gly Tyr Thr

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Ala Phe Val His Cys Lys Met Ala Arg Leu Gln Gly Ala Ser Gly Ile

305 310 315 320

His Thr Gly Thr Met Gly Phe Gly Lys Met Glu Gly Glu Ser Ser Asp

325 330 335

Arg Ala Ile Ala Tyr Met Leu Thr Gln Asp Glu Ala Gln Gly Pro Phe

340 345 350

Tyr Arg Gln Ser Trp Gly Gly Met Lys Ala Cys Thr Pro Ile Ile Ser

355 360 365

Gly Gly Met Asn Ala Leu Arg Met Pro Gly Phe Phe Glu Asn Leu Gly

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Asn Ala Asn Val Ile Leu Thr Ala Gly Gly Gly Ala Phe Gly His Ile

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Asp Gly Pro Val Ala Gly Ala Arg Ser Leu Arg Gln Ala Trp Gln Ala

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Trp Arg Asp Gly Val Pro Val Leu Asp Tyr Ala Arg Glu His Lys Glu

420 425 430

Leu Ala Arg Ala Phe Glu Ser Phe Pro Gly Asp Ala Asp Gln Ile Tyr

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Pro Gly Trp Arg Lys Ala Leu Gly Val Glu Asp Thr Arg Ser Ala Leu

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Pro Ala

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atggaccagt cgagccggta cgtcaacctc gccctgaagg aggaggattt aattgccggg 60

ggcgaacacg tgctctgcgc gtacatcatg aagccgaagg ccggctatgg ctatgtcgcc 120

acggccgccc attttgccgc cgagtcgtcg acgggcacca atgtcgaggt gtgcaccacg 180

gacgacttta cccgcggcgt ggatgccctc gtgtacgagg tcgacgaggc gcgggagctc 240

acgaagatcg cctacccggt cgccctgttc gaccggaaca tcaccgacgg caaggcgatg 300

atcgcgtcgt tcctcacgct gactatgggt aacaaccaag gcatgggcga cgtcgagtac 360

gcgaagatgc acgacttcta cgtcccggag gcctatcggg ccctgtttga cggccccagc 420

gtcaacatca gcgccctctg gaaggtcctg gggcgcccgg aggtcgatgg cggcctcgtg 480

gtcggcacca ttatcaagcc gaagctgggc ctgcgcccga agccctttgc ggaggcctgt 540

catgccttct ggctgggcgg cgatttcatt aagaacgacg agccccaggg caaccaaccg 600

tttgcccccc tccgcgacac cattgccctc gtcgcggacg ccatgcgccg ggcccaggac 660

gaaaccggcg aggccaagct gttcagcgcc aacatcaccg cggatgaccc gttcgagatc 720

atcgcgcggg gcgagtacgt cttagaaacc ttcggcgaga acgcgagcca cgtcgccctc 780

ctggtcgacg gctacgtcgc cggcgccgcc gccattacga ccgcccgccg gcggtttccc 840

gacaacttcc tccattacca ccgcgccggc cacggcgccg tgaccagccc ccagtcgaag 900

cgcggctaca ccgccttcgt gcactgcaaa atggcccgcc tccagggcgc gagcggcatc 960

cacacgggga ctatgggttt cggcaagatg gagggggaga gcagcgatcg cgccatcgcc 1020

tacatgctca cgcaggacga ggcccagggg ccgttttacc gccagtcgtg ggggggcatg 1080

aaggcctgca cccccattat cagcggcggg atgaatgccc tgcgcatgcc ggggtttttt 1140

gagaacctcg gcaacgccaa cgtcattctc accgccggcg gcggcgcctt tggccatatt 1200

gacggccccg tggcgggcgc gcgctcgctc cgccaggcct ggcaggcgtg gcgcgatggc 1260

gtcccggtcc tcgactatgc ccgggaacac aaggagctcg cccgcgcctt tgagagcttc 1320

ccgggcgacg cggaccagat ttaccccggc tggcgcaagg ccctgggcgt cgaggacacc 1380

cgctcggcgc tgccggcctg a 1401

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<213> Myceliophthora thermophila

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Met Ala Val Cys Thr Val Tyr Thr Ile Pro Thr Thr Thr His Leu Gly

1 5 10 15

Ser Ser Phe Asn Gln Asn Asn Lys Gln Val Phe Phe Asn Tyr Lys Arg

20 25 30

Ser Ser Ser Ser Asn Asn Thr Leu Phe Thr Thr Arg Pro Ser Tyr Val

35 40 45

Ile Thr Cys Ser Gln Gln Gln Thr Ile Val Ile Gly Leu Ala Ala Asp

50 55 60

Ser Gly Cys Gly Lys Ser Thr Phe Met Arg Arg Leu Thr Ser Val Phe

65 70 75 80

Gly Gly Ala Ala Glu Pro Pro Lys Gly Gly Asn Pro Asp Ser Asn Thr

85 90 95

Leu Ile Ser Asp Thr Thr Thr Val Ile Cys Leu Asp Asp Phe His Ser

100 105 110

Leu Asp Arg Asn Gly Arg Lys Val Glu Lys Val Thr Ala Leu Asp Pro

115 120 125

Lys Ala Asn Asp Phe Asp Leu Met Tyr Glu Gln Val Lys Ala Leu Lys

130 135 140

Glu Gly Lys Ala Val Asp Lys Pro Ile Tyr Asn His Val Ser Gly Leu

145 150 155 160

Leu Asp Pro Pro Glu Leu Ile Gln Pro Pro Lys Ile Leu Val Ile Glu

165 170 175

Gly Leu His Pro Met Tyr Asp Ala Arg Val Arg Glu Leu Leu Asp Phe

180 185 190

Ser Ile Tyr Leu Asp Ile Ser Asn Glu Val Lys Phe Ala Trp Lys Ile

195 200 205

Gln Arg Asp Met Lys Glu Arg Gly His Ser Leu Glu Ser Ile Lys Ala

210 215 220

Ser Ile Glu Ser Arg Lys Pro Asp Phe Asp Ala Tyr Ile Asp Pro Gln

225 230 235 240

Lys Gln His Ala Asp Val Val Ile Glu Val Leu Pro Thr Glu Leu Ile

245 250 255

Pro Asp Asp Asp Glu Gly Lys Val Leu Arg Val Arg Met Ile Gln Lys

260 265 270

Glu Gly Val Lys Phe Phe Asn Pro Val Tyr Leu Phe Asp Glu Gly Ser

275 280 285

Thr Ile Ser Trp Ile Pro Cys Gly Arg Lys Leu Thr Cys Ser Tyr Pro

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Gly Ile Lys Phe Ser Tyr Gly Pro Asp Thr Phe Tyr Gly Asn Glu Val

305 310 315 320

Thr Val Val Glu Met Asp Gly Met Phe Asp Arg Leu Asp Glu Leu Ile

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Tyr Val Glu Ser His Leu Ser Asn Leu Ser Thr Lys Phe Tyr Gly Glu

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Val Thr Gln Gln Met Leu Lys His Gln Asn Phe Pro Gly Ser Asn Asn

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Gly Thr Gly Phe Phe Gln Thr Ile Ile Gly Leu Lys Ile Arg Asp Leu

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Phe Glu Gln Leu Val Ala Ser Arg Ser Thr Ala Thr Ala Thr Ala Ala

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<213> Myceliophthora thermophila

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atggcggtct gcaccgtgta caccatcccc acgacgaccc acctgggcag cagcttcaac 60

caaaacaaca aacaggtctt cttcaactac aagcgcagca gcagcagcaa caacacgctg 120

　　ttcaccacgc ggccgagcta tgtcatcacc tgctcgcagc agcagaccat cgtcattggc 180

　　ctcgccgcgg actcgggctg tggcaaaagc acgttcatgc gccgcctcac cagcgtcttt 240

　　ggcggcgcgg ccgagccgcc caagggcggc aacccggact cgaacacgct catctcggac 300

　　accaccacgg tgatctgcct cgacgacttt cactcgctcg accggaatgg ccgcaaggtc 360

　　gagaaggtca ccgccctcga ccccaaggcg aacgacttcg acctgatgta cgagcaggtg 420

　　aaggccctga aggaaggcaa ggcggtcgac aagcccatct acaaccatgt ctcgggcctc 480

　　ctcgatccgc ccgaactcat ccagccgccg aagatcctgg tcatcgaagg gctccatccg 540

　　atgtacgacg cccgcgtgcg cgagctcctg gacttctcga tctatctcga catctcgaac 600

　　gaggtcaaat tcgcctggaa gatccagcgc gacatgaagg agcgcggcca ctcgttagag 660

　　tctattaagg cgtcgatcga gtcgcggaag cccgactttg acgcctacat cgacccgcag 720

　　aagcagcatg cggacgtcgt catcgaagtc ctgcccaccg agctcatccc ggacgatgac 780

　　gagggcaagg tcctccgcgt ccggatgatc cagaaggagg gcgtcaagtt cttcaacccc 840

　　gtctacctct tcgacgaggg cagcacgatc tcgtggattc cttgtggtcg taaactcacg 900

　　tgctcgtacc cgggcattaa gttcagctac gggcccgaca cgttctacgg gaacgaggtc 960

　　acggtcgtgg agatggacgg catgttcgac cggctcgacg agctgatcta tgtcgagtcg 1020

　　cacctgtcga acctcagcac gaagttttac ggcgaggtga cccagcaaat gctgaagcac 1080

　　cagaacttcc ccggcagcaa caacgggacg ggcttctttc aaaccatcat cggcctcaag 1140

　　atccgggacc tcttcgagca gctcgtcgcc tcgcggagca ccgccaccgc caccgccgcc 1200

　　aaggcctga 1209

　　<210> 5

　　<211> 701

　　<212> DNA

　　<213> Myceliophthora thermophila

　　<400> 5

　　cattcgatat gcaacccgat ctcaagcaga gaaaggtgtt agggccgacg aaaagaccag 60

　　tcttgcgagc caacctgcgc tggacctatg gtactgcgtg tgtgtgggcg gataccgagt 120

　　gtactccgta aggagggttg gtctcatgcc tcttggcggg agccgcccga taactagtat 180

　　aactagttgt aactccgtat ccggttacgg aaacggaaag gcccgctcgg ctgttctccg 240

　　gcggctcccc gatcgctgat cagagcatgg aacagatgtc aattacatca ctcccgcgta 300

　　aacgaaccat agttatcgaa ccacagttat cgaaccacag agccagccca tgggaacgtc 360

　　tgaacagctc ggaggatgca accgatattg caatgcaaaa cgtcacccat gctacaatta 420

　　attccctgca caactacttg taagccgcga ggcctagaac acagttgcag aacctgggta 480

　　tcgtgcctgt ggtctgatgc agatatgtgt caccactcaa gaccccgcca acacgccgct 540

　　tcgaggccct gaacagtaca aagggcgctt caaattcgta caagcccccc cgaggccgtt 600

　　ttcaagtctt tgtatgacca tctattttcc gattgacgtc cctcacggat tctctttcgt 660

　　tgctgacctc cttgtgacca caaacatcgc caacaacaga c 701

　　<210> 6

　　<211> 1464

　　<212> DNA

　　<213> Myceliophthora thermophila

　　<400> 6

　　tgacggtgct tttcacctct cgatgcccga aatcgggtct aagctgagtt tgatcaaata 60

　　tgtgactcca acatcgcccc cttcggcaaa ccccgtcgac acgtgtgtca tccttccatt 120

　　gcaagcgatc actcgcaggg cgtgacgatg aacgagattt ttgcccggac cgattcgcgg 180

　　atatagcggc agccgaccag ccctaccaca ctgatggccg tgtccctagt gtatgctccc 240

　　agaaccgcaa gcatacactg ggcaatgctt ggtatgcagt tgaggcagct ttatgtttcc 300

　　atacccttcc acttcggctc ggggactcgg cggggtcgcg gaagtttgac ggcagccgtc 360

　　gggccttagg ccgagattac cgtggttgtg gcccagtttt agccgttccc gtccgtttcc 420

　　taccggacca tgattttcgt gaaccattgc aatcccgaag cgcatttccg acgttaagga 480

　　gttacctccg ctgcccacaa ttcatgatcg tggccggctc aaggcagcgt ggcggggcat 540

　　ccgtgtcaag ctcccaggag gaggtgcgcg atttcaaatc cgggccaaaa caggccaaga 600

　　ctggctggcc aaaaaaagga gcgtagacgg cccgggacat cggacgtcag ctcgcagcca 660

　　cccaaaaccg gtccgatcta ctcgcttact gtggtagttc aggtactttt gagtagtaaa 720

　　aacgctacgg cagggccggg gggttccccg gtgacggagg tgcctctgcg gtggcgaaca 780

　　tcccacgcac tatcgagcta cggtgacacc tcgtgtcctg ttggtcttgc aatgctgggg 840

　　cggcaggaaa tgcgtcgcgc tcctcccggc caagacctaa aacagacagc gccgcaaagt 900

　　cgctcactag caccgcgaaa cgaagatgcc ccacctcaac gcaatctgtg atgcaagcaa 960

　　ttgggaaggc tcaccccacc tcagcgaggg gctcaaccat ttttattatc agctcatgcc 1020

　　accacaacat gactgttttc tttccttgct catcccacat ttgacaaaaa tcgtcgatta 1080

　　atctctttcc atacaggccg tccgcgctct gataaccaca taaaagtctc ttcagtcaac 1140

　　agctcaaagc tccctcatcc ctccaggtaa gcagccaaag agctccccca cggaccccgc 1200

　　actgcctcat cccgcctgta tcggacctgc gcgacccagc agagaatccc aaacctttgc 1260

　　tgcttgctgc ccggttccgg actgagctgc aacccaagcc tttaaaaagc tattcccttc 1320

　　tcccacggtg tcaactctgt cctatccctc cgacatccgt tgagctcaac aactccccga 1380

　　accttttacc ccgcgccgag ctacccctcc atcaaaccac cctgacagct cgctcactca 1440

　　cctccccaca tcacagaaat caaa 1464

　　<210> 7

　　<211> 54

　　<212> DNA

<213> artificially synthesized sequence

　　<400> 7

　　tttgcagttg gctgacttga agtaatctct gcacattcga tatgcaaccc gatc 54

　　<210> 8

　　<211> 55

　　<212> DNA

<213> artificially synthesized sequence

　　<400> 8

　　tcgtggggat ggtgtacacg gtgcagaccg ccatgtctgt tgttggcgat gtttg 55

　　<210> 9

<211> 52

<212> DNA

<213> artificially synthesized sequence

<400> 9

cttgtgacca caaacatcgc caacaacaga catggcggtc tgcaccgtgt ac 52

<210> 10

<211> 52

<212> DNA

<213> artificially synthesized sequence

<400> 10

gtttgatgat ttcagtaacg ttaagtggat ctcaggcctt ggcggcggtg gc 52

<210> 11

<211> 53

<212> DNA

<213> artificially synthesized sequence

<400> 11

tcactcacct ccccacatca cagaaatcaa aaatggacca gtcgagccgg tac 53

<210> 12

<211> 54

<212> DNA

<213> artificially synthesized sequence

<400> 12

ctgtttgatg atttcagtaa cgttaagtgg atctcaggcc ggcagcgccg agcg 54

<210> 13

<211> 574

<212> PRT

<213> Myceliophthora thermophila

<400> 13

Met Thr Asp Ile Arg Glu Gln Gly Leu Lys Lys Pro Val Asn Val Ala

1 5 10 15

Glu Tyr Leu Phe Arg Arg Leu His Glu Ile Gly Ile Arg Ser Val His

20 25 30

Gly Leu Pro Gly Asp Phe Asn Leu Val Ala Leu Asp Tyr Ile Pro Lys

35 40 45

Ala Gly Leu Lys Trp Val Gly Ser Val Asn Glu Leu Asn Ala Ala Tyr

50 55 60

Ala Ala Asp Gly Tyr Ala Arg Thr Lys Gly Ile Ser Ala Ile Phe Thr

65 70 75 80

Thr Phe Gly Val Gly Glu Leu Ser Ala Ile Asn Gly Ile Ala Gly Ala

85 90 95

Phe Ser Glu His Val Pro Val Val His Ile Val Gly Cys Pro Ser Thr

100 105 110

Ile Ser Gln Arg Asn Gly Met Leu Leu His His Thr Leu Gly Asn Gly

115 120 125

Asp Phe Asn Val Phe Ala Asn Met Ser Ser Gln Ile Ser Cys Asp Val

130 135 140

Ala Arg Leu Asn Lys Arg Ala Glu Ile Ala Asp Gln Ile Asp His Ala

145 150 155 160

Leu Arg Glu Cys Trp Ile Arg Ser Arg Pro Val Tyr Ile Met Leu Pro

165 170 175

Thr Asp Met Val Glu Arg Lys Val Glu Gly Ala Arg Leu Asp Thr Pro

180 185 190

Ile Asp Leu Thr Glu Pro Ala Asn Gln Ser Glu Arg Glu Asp Tyr Val

195 200 205

Val Asp Val Val Leu Arg Tyr Leu His Ala Ala Lys Gln Pro Val Ile

210 215 220

Leu Val Asp Ala Cys Ala Ile Arg His Arg Val Leu Lys Glu Val His

225 230 235 240

Asp Leu Val Glu Lys Thr Gln Leu Pro Val Phe Val Thr Pro Met Gly

245 250 255

Lys Gly Ala Ile Asn Glu Asp His Pro Asn Tyr Gly Gly Val Tyr Ala

260 265 270

Gly Thr Gly Ser Gln Pro Ala Val Ala Glu Arg Val Glu Thr Ala Asp

275 280 285

Leu Val Leu Ser Ile Gly Ala Leu Lys Ser Asp Phe Asn Thr Ala Gly

290 295 300

Phe Ser Tyr Arg Thr Ser Gln Leu Asn Thr Ile Asp Phe His Ser Asp

305 310 315 320

His Cys Thr Val Arg Tyr Ser Glu Tyr Pro Gly Val Ala Met Arg Gly

325 330 335

Val Leu Arg Lys Val Val Glu Arg Val Asp Leu Ser Lys Leu Ser Arg

340 345 350

Pro Pro Ser Pro Glu Val Val Asn Glu Val Thr Lys Asn Arg Asp Ser

355 360 365

Ser Gln Thr Ile Thr Gln Ala Phe Phe Trp Pro Arg Ile Gly Glu Tyr

370 375 380

Leu Lys Glu Asn Asp Ile Val Val Thr Glu Thr Gly Thr Ser Asn Phe

385 390 395 400

Gly Ile Trp Glu Thr Lys Tyr Pro Arg Gly Val Thr Gly Ile Thr Gln

405 410 415

Ile Leu Trp Gly Ser Ile Gly Trp Ser Val Gly Ala Ala Gln Gly Ala

420 425 430

Ala Leu Ala Ala Lys Asp Met Gly Val Asp Arg Arg Thr Ile Leu Phe

435 440 445

Val Gly Asp Gly Ser Phe Gln Leu Thr Ala Gln Glu Val Ser Thr Met

450 455 460

Ile Arg His Asp Leu Arg Ile Thr Ile Phe Leu Ile Phe Asn Gly Gly

465 470 475 480

Phe Thr Ile Glu Arg Phe Ile His Gly Met Glu Ala Glu Tyr Asn Asp

485 490 495

Ile Thr Arg Trp Asn Tyr Ile Asp Val Pro Thr Ala Phe Gly Gly Ser

500 505 510

Glu Lys Gln Val Arg Lys Phe Val Val Lys Thr Lys Asp Glu Leu Glu

515 520 525

Glu Leu Leu Thr Asp Thr Asp Phe Asn Glu Ala Arg Gly Leu Gln Phe

530 535 540

Val Glu Leu Trp Met Arg Lys Asp Asp Ala Pro Arg Ala Leu Lys Ile

545 550 555 560

Thr Ala Glu Ile Ala Ala Arg Asn Asn Ala Ser Met Ser Glu

565 570

<210> 14

<211> 1725

<212> DNA

<213> Myceliophthora thermophila

<400> 14

atgacagaca tcagagagca gggccttaag aaacccgtca acgtcgccga gtatctgttc 60

aggagactcc acgaaatcgg catccgctcc gttcatggcc tcccaggtga cttcaacctg 120

gttgcattgg actacattcc caaagctggc ctcaaatggg tgggcagcgt gaatgagctg 180

aatgcagcat acgctgccga cgggtatgcc cgcacaaagg gcatctcagc tatttttact 240

acctttggag tgggagagtt atctgcaatc aacggtatcg ccggcgcctt ctccgaacac 300

gttcccgtag tacacattgt tggctgcccg tcgacgattt cacaacggaa tggcatgctg 360

cttcaccaca cgcttggcaa tggagacttt aacgttttcg ccaacatgag ctctcagatc 420

tcttgcgatg tggctcgtct caataaacgg gccgagatag ccgaccagat cgaccatgcc 480

ctgcgcgagt gctggatccg cagtcggccc gtatacatca tgcttccgac agacatggtc 540

gagaggaagg ttgagggcgc ccgtctggac acgcccattg acctcacaga acccgccaac 600

cagtcagagc gggaggacta tgtcgttgac gtcgtcctga ggtatctcca cgcggctaag 660

caacctgtaa ttttggtcga tgcctgcgcc attcgccata gggtgctcaa ggaggttcat 720

gacttggtgg agaaaaccca gctgccggta ttcgttaccc caatgggcaa gggcgccatc 780

aacgaggacc atcccaacta tggcggagta tatgcgggaa ctggttcgca gcccgcggtg 840

gctgagcgtg ttgagacagc ggacctggtg ctttcgatcg gcgccctcaa gagcgacttc 900

aacacggcag gcttttcgta ccggacgtcc caactcaaca cgatcgactt ccactcggac 960

cattgcacgg tgcggtattc cgagtacccc ggcgtggcca tgcgcggcgt gctccgcaag 1020

gtggtggaac gggttgatct tagcaaactg tctaggcctc catcgcccga ggtggtcaac 1080

gaggtgacta agaacaggga tagttcccaa accatcacac aggcgttctt ctggccacgc 1140

attggggaat atctgaagga gaacgacatc gtggtgacag aaacgggcac gtccaacttc 1200

ggcatttggg aaaccaagta cccacggggt gtgactggga tcacacaaat tctttggggc 1260

agcattggtt ggtcggtggg cgccgcccaa ggtgccgcgc tagctgcaaa ggacatgggc 1320

gtcgaccgcc gaaccatctt gtttgtcggc gacggctcgt tccagctcac ggcccaagag 1380

gtgtcaacca tgatccgaca tgatttgagg atcaccatat tcctgatttt caacggcggc 1440

ttcaccatcg agcgctttat ccatggcatg gaggccgagt acaatgatat cacccgttgg 1500

aactacattg acgtgccaac agcattcggc ggttcggaaa aacaggttcg caagtttgtc 1560

gtcaagacca aggatgagct cgaggagcta ttgacggaca cagacttcaa tgaggctaga 1620

gggttacagt ttgttgagct gtggatgcga aaggatgacg caccgcgggc cctgaagatc 1680

actgctgaga ttgccgctag aaacaacgca agtatgagtg agtaa 1725

<210> 15

<211> 319

<212> PRT

<213> Myceliophthora thermophila

<400> 15

Met Pro Gln Pro Asn Ala Ala Asp Val Lys Ser Ile Lys Ile Val Ile

1 5 10 15

Val Gly Ala Gly Ser Val Gly Val Thr Thr Ala Tyr Ala Leu Leu Leu

20 25 30

Asp Arg Leu Ala Ala Asp Ile Val Leu Ile Asp Ile Asp Lys Asn Arg

35 40 45

Ala Met Gly Glu Val Met Asp Leu Ser His Ala Ala His Phe Ala Gln

50 55 60

Ala Arg Val Arg Val Gly Asp Tyr Glu Asp Cys Ala His Ala Ala Ala

65 70 75 80

Val Ile Ile Thr Ala Gly Val Asn Gln Lys Pro Gly Gln Thr Arg Leu

85 90 95

Asp Leu Val Lys Thr Asn Tyr Ala Leu Phe Arg Asp Val Val Pro Arg

100 105 110

Ile Ala Arg His Ala Pro Asp Thr Ile Leu Val Val Ala Thr Asn Pro

115 120 125

Val Asp Val Leu Thr His Ala Ala His His Leu Ser Gly Phe Pro Leu

130 135 140

Glu Arg Val Ile Gly Ser Gly Thr Ala Met Asp Thr Thr Arg Phe Arg

145 150 155 160

His Glu Leu Gly Lys His Phe Gly Val Asn Pro Arg Asn Val His Ala

165 170 175

Met Ile Val Gly Glu His Gly Asp Ser Gln Leu Pro Val Trp Ser Leu

180 185 190

Ala Thr Ile Cys Gly Met Arg Leu His Asp Tyr Cys Arg Ala Ala Arg

195 200 205

Met Glu His Asp Glu Ala Ala Leu Glu Ala Cys Ala Lys Arg Thr Arg

210 215 220

Glu Ala Ala Tyr Glu Ile Ile Arg Arg Lys Gly Lys Thr Asn Tyr Gly

225 230 235 240

Val Ala Ser Val Leu Val Ser Ile Leu Gln Pro Ile Val Thr Asp Ser

245 250 255

Asp Ala Ile Met Thr Val Ser Arg Val Gly Thr Tyr Ala Gly Ile Gln

260 265 270

Asp Val Ala Leu Ser Met Pro Cys Lys Leu Asn Arg His Gly Ala Tyr

275 280 285

Gln Asp Val Pro Leu Leu Leu Ser Glu Leu Glu Glu Ala Glu Leu Arg

290 295 300

Glu Ser Ala Gln Ser Ile Lys Glu Val Leu Met Ser Leu Glu Lys

305 310 315

　　<210> 16

　　<211> 960

　　<212> DNA

　　<213> Myceliophthora thermophila

　　<400> 16

　　atgcctcagc caaacgcagc agatgtcaag tcaatcaaga tcgttatcgt cggtgccggc 60

　　tcggtaggcg tgacgactgc gtacgcgctg ctccttgata ggttagcggc cgacattgtg 120

　　ctcatcgaca tcgacaagaa ccgggcgatg ggtgaggtga tggacctgag ccacgcggcg 180

　　cactttgcac aggcgcgcgt gcgcgtcggc gactacgagg actgcgccca cgcggcggcg 240

　　gtcatcatca cggcgggcgt caaccaaaag cccggccaga cgcgcctcga cctcgtcaag 300

　　accaactacg cgctattccg ggacgtagtg ccccgcatcg cacgccacgc gcccgacacc 360

　　atcctcgtcg tggccaccaa cccggtcgac gtgctcacgc acgcggccca ccacctctcc 420

　　ggcttcccac tcgagagggt gatcgggtcc ggcacggcca tggacaccac caggttccgg 480

　　cacgagctgg gcaagcactt tggggtcaac ccgcggaacg tgcacgccat gatcgtgggc 540

　　gagcacggcg acagccagct gcccgtgtgg tcgctcgcca ccatctgcgg gatgcggctg 600

　　cacgactact gcagggcggc ccgcatggag cacgacgagg ccgcactcga ggcctgcgcc 660

　　aagcggacca gggaggccgc ctacgagatc atccggcgca agggcaagac caactacggc 720

　　gtcgcctcgg tgctcgtcag catcctgcag cccatcgtca ccgacagcga cgccatcatg 780

　　acggtctcga gggtcggcac gtacgccgga atccaggacg tggccctcag catgccctgc 840

　　aagctgaacc ggcatggcgc gtaccaggac gtgcccctac tcctcagcga gttggaggag 900

　　gccgaattga gagagtcggc ccaaagcatt aaggaggtcc tcatgtcgtt agagaaataa 960

　　<210> 17

　　<211> 591

　　<212> PRT

　　<213> Myceliophthora thermophila

　　<400> 17

Met Asn Gly Pro Val Ala Arg Thr Asp Ser Pro Tyr Thr Asp Pro Ser

1 5 10 15

Val Lys Gly Pro Arg Thr Lys Leu Leu Ser Asn Arg Ser Pro Ala Val

20 25 30

Ala Ala Ala Ala Asn Met Val Asn Thr Val Asn Lys Thr Gly Leu His

35 40 45

Pro Gly Gly Ile Val Pro His Pro Lys Thr Glu Leu Glu Glu Glu Leu

50 55 60

His Glu Lys Ala His Ile Asp Tyr Thr Arg Val Ala Ile Ile Pro Asn

65 70 75 80

Pro Ser Val Ala Ala Leu Tyr Glu Asp Ala Leu Val Tyr Glu Thr Gly

85 90 95

Thr Ala Ile Thr Ser Ser Gly Ala Leu Thr Ala Tyr Ser Gly Lys Lys

100 105 110

Thr Gly Arg Ser Pro Gln Asp Lys Arg Ile Val Lys Glu Pro Thr Ser

115 120 125

Glu Asn Asp Ile Trp Trp Gly Pro Val Asn Lys Pro Met Asp Pro Glu

130 135 140

Ile Trp Lys Ile Asn Arg Glu Arg Ala Val Asp Tyr Leu Asn Thr Arg

145 150 155 160

Ser Arg Ile Tyr Val Val Asp Gly Tyr Ala Gly Trp Asp Glu Lys Tyr

165 170 175

Arg Ile Lys Val Arg Val Val Cys Ala Arg Ala Tyr His Ala Leu Phe

180 185 190

Met Arg Asn Met Leu Ile Arg Pro Ser Arg Glu Glu Leu Glu His Phe

195 200 205

His Pro Asp Tyr Thr Ile Tyr Asn Ala Gly Arg Phe Pro Ala Asn Arg

210 215 220

Tyr Thr His Gly Met Thr Ser Ala Thr Ser Val Ala Ile Asn Phe Ala

225 230 235 240

Glu Lys Glu Met Val Ile Leu Gly Thr Glu Tyr Ala Gly Glu Met Lys

245 250 255

Lys Gly Ile Phe Thr Val Met Phe Tyr Glu Gly Pro Ile Lys His Asn

260 265 270

Ile Leu Thr Leu His Ser Ser Ala Asn Glu Gly Lys Asp Gly Asp Val

275 280 285

Thr Leu Phe Phe Gly Leu Ser Gly Thr Gly Lys Thr Thr Leu Ser Ala

290 295 300

Asp Pro Asn Arg Arg Leu Ile Gly Asp Asp Glu His Cys Trp Ser Asp

305 310 315 320

Arg Gly Ile Phe Asn Ile Glu Gly Gly Cys Tyr Ala Lys Thr Ile Gly

325 330 335

Leu Ser Ala Glu Lys Glu Pro Asp Ile Tyr Asn Ala Ile Arg Phe Gly

340 345 350

Ser Val Leu Glu Asn Val Val Phe Asp Gln Glu Thr Arg Glu Val Asp

355 360 365

Tyr Asp Asp Ser Thr Leu Thr Glu Asn Thr Arg Cys Ala Tyr Pro Ile

370 375 380

Glu Tyr Ile Ser Asn Ala Lys Ile Pro Cys Leu Ser Asn Asn His Pro

385 390 395 400

Ser Asn Ile Ile Leu Leu Thr Cys Asp Ala Arg Gly Val Leu Pro Pro

405 410 415

Ile Ser Lys Leu Asn Ser Ala Gln Thr Met Phe His Phe Ile Ser Gly

420 425 430

Tyr Thr Ser Lys Met Ala Gly Thr Glu Asp Gly Val Leu Glu Pro Gln

435 440 445

Ala Thr Phe Ser Ser Cys Phe Ala Gln Pro Phe Leu Ala Leu His Pro

450 455 460

Met Arg Tyr Ala Lys Met Leu Ala Glu Lys Ile Glu Lys His Asn Ala

465 470 475 480

Asn Ala Trp Leu Leu Asn Thr Gly Trp Val Gly Ala Gly Phe Ala Gln

485 490 495

Gly Gly Lys Arg Cys Pro Leu Lys Tyr Thr Arg Ala Ile Leu Asp Ala

500 505 510

Ile His Ser Gly Glu Leu Ala Lys Val Glu Tyr Glu Asn Tyr Glu Val

515 520 525

Phe Asn Leu Gln Val Pro Lys Ser Cys Pro Asn Val Pro Ser Glu Leu

530 535 540

Leu Asn Pro Lys Lys Ala Trp Thr Ala Gly Ala Asp Ser Phe Asn Ala

545 550 555 560

Glu Val Arg Lys Leu Gly Ala Leu Phe Leu Glu Asn Phe Lys Lys Tyr

565 570 575

Glu Ser Glu Ala Thr Glu Asp Val Ile Lys Ala Gly Pro Val Leu

580 585 590

　　<210> 18

　　<211> 1776

　　<212> DNA

　　<213> Myceliophthora thermophila

　　<400> 18

　　atgaacggcc ccgtcgccag aaccgactct ccctacactg atccctccgt caaaggtcct 60

　　cgtaccaagc ttcttagtaa cagatctccc gccgtcgccg ccgccgccaa catggtcaac 120

　　accgtcaaca agacaggcct ccatcctggt ggcattgtgc cccacccaaa gactgagcta 180

　　gaggaggaac tccacgagaa agctcacatc gactacacca gagtcgcaat catcccaaac 240

　　ccttcagtcg ccgcgctcta cgaagatgca ctcgtatacg agacaggcac tgccatcacc 300

　　tcatccggtg ccttgactgc ttactccggc aagaagaccg gccggtcacc gcaggacaag 360

　　cgcattgtca aggagcccac atccgaaaac gacatctggt ggggacccgt caacaagcct 420

　　atggacccag agatctggaa gatcaaccgt gagcgcgccg tcgattatct caacacccgg 480

　　agtcgcatct acgtcgttga tggatatgcc ggctgggatg agaagtaccg catcaaggtg 540

　　cgcgtcgtct gcgcgcgcgc ctaccacgcc ctcttcatgc gcaacatgct catccgcccg 600

　　tcacgggagg agctcgagca tttccacccc gattacacaa tctacaatgc cgggcgtttc 660

　　cccgccaacc gctacaccca cggcatgacc tcagccacgt cggttgctat caacttcgcc 720

　　gagaaggaga tggtcatctt gggtaccgag tacgccggcg agatgaagaa gggcatcttc 780

　　accgtcatgt tctacgaggg tcccatcaag cacaacatct tgactctgca ctcgtccgcc 840

　　aacgagggca aggacggcga cgtcaccctc ttcttcggtc tgtccggcac tggcaagacc 900

　　accctgtcgg ccgaccccaa ccggcgcctg atcggcgacg acgagcactg ctggagcgat 960

　　cgcggcatct tcaacatcga aggcggctgc tacgccaaga cgatcggcct ctcggccgag 1020

　　aaggagcccg atatctacaa cgccatccgc ttcggctccg ttctggagaa cgtcgtcttt 1080

　　gaccaggaga cgcgcgaggt tgactacgac gactcgaccc tgaccgagaa cacgcgctgc 1140

　　gcctatccga tcgagtacat cagcaacgcc aagatcccct gcctgtccaa caaccacccg 1200

　　tccaacatca tcctgctcac ctgcgacgct aggggcgtcc tcccccccat ctctaagctc 1260

　　aacagcgctc agaccatgtt ccacttcatc agcggctaca cctccaagat ggccggtacc 1320

　　gaggacggcg tgctcgagcc gcaggcgacc ttctcgtctt gcttcgcgca gcccttcctc 1380

　　gcgctgcacc cgatgcgcta cgccaagatg ctggccgaga agattgagaa gcacaacgcc 1440

　　aacgcctggc tgctcaacac cggctgggtc ggcgccggct tcgcccaggg cggcaagcgc 1500

　　tgcccgctca agtacacgcg tgccatcctc gacgccatcc acagcggcga gctcgccaag 1560

　　gtggagtacg agaactacga ggtgttcaac ctccaggtgc ccaagtcgtg ccctaatgtc 1620

　　ccgtccgagc tcctcaaccc caagaaggcc tggaccgccg gtgccgacag cttcaatgcc 1680

　　gaggtcagga agcttggcgc tctcttcttg gagaacttca agaagtatga gagcgaggcg 1740

　　accgaggatg tcatcaaggc agggccggtt ctttaa 1776

　　<210> 19

　　<211> 565

　　<212> DNA

　　<213> Myceliophthora thermophila

　　<400> 19

　　aggatcggtg gagtgaagtt cggaatcgag gttcggcgat gggtcgtaag catggcgact 60

　　tcgaacttac ttgcactggc aagcgttgcc agaacggcga gaaaaagaag ggtaagcgat 120

　　attcgcgtca tgatggactg ttccttttgg aacagtagtt gttgtgggaa gactatgtca 180

　　cacttgccca cctgcaaggc cagggtcgtg gtcgaacgag accagcctcg gcgctgctgg 240

　　gagctcaaga tgggcacgtt tgattcgtta gacgtcaaca aggctggagt tcctagtgac 300

　　agccaaaggc acagccacat taagtggcgc tttatctgtc cactaaggtt caattgtggc 360

　　tttgagccgc gcagtgtgca gtcgtgcatt ggccacctag ctagcagtat ttaagatcct 420

　　cttctctccc gagatcttcc tcctcttctt ttctttcttt cctcgacggg tatgcccgca 480

　　caaagtttta gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa 540

　　aagtggcacc gagtcggtgc ttttt 565

　　<210> 20

　　<211> 565

　　<212> DNA

　　<213> Myceliophthora thermophila

　　<400> 20

　　aggatcggtg gagtgaagtt cggaatcgag gttcggcgat gggtcgtaag catggcgact 60

　　tcgaacttac ttgcactggc aagcgttgcc agaacggcga gaaaaagaag ggtaagcgat 120

　　attcgcgtca tgatggactg ttccttttgg aacagtagtt gttgtgggaa gactatgtca 180

　　cacttgccca cctgcaaggc cagggtcgtg gtcgaacgag accagcctcg gcgctgctgg 240

　　gagctcaaga tgggcacgtt tgattcgtta gacgtcaaca aggctggagt tcctagtgac 300

　　agccaaaggc acagccacat taagtggcgc tttatctgtc cactaaggtt caattgtggc 360

　　tttgagccgc gcagtgtgca gtcgtgcatt ggccacctag ctagcagtat ttaagatcct 420

　　cttctctccc gagatcttcc tcctcttctt ttctttcttt cctcgccaaa cgcagcagat 480

　　gtcagtttta gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa 540

　　aagtggcacc gagtcggtgc ttttt 565

　　<210> 21

　　<211> 565

　　<212> DNA

　　<213> Myceliophthora thermophila

　　<400> 21

　　aggatcggtg gagtgaagtt cggaatcgag gttcggcgat gggtcgtaag catggcgact 60

　　tcgaacttac ttgcactggc aagcgttgcc agaacggcga gaaaaagaag ggtaagcgat 120

　　attcgcgtca tgatggactg ttccttttgg aacagtagtt gttgtgggaa gactatgtca 180

　　cacttgccca cctgcaaggc cagggtcgtg gtcgaacgag accagcctcg gcgctgctgg 240

　　gagctcaaga tgggcacgtt tgattcgtta gacgtcaaca aggctggagt tcctagtgac 300

　　agccaaaggc acagccacat taagtggcgc tttatctgtc cactaaggtt caattgtggc 360

　　tttgagccgc gcagtgtgca gtcgtgcatt ggccacctag ctagcagtat ttaagatcct 420

　　cttctctccc gagatcttcc tcctcttctt ttctttcttt cctcgatgca ctcgtatacg 480

　　agacgtttta gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa 540

　　aagtggcacc gagtcggtgc ttttt 565

　　<210> 22

　　<211> 3071

　　<212> DNA

　　<213> Myceliophthora thermophila

　　<400> 22

　　gtgacaaata ccagagtcta gtgtaaaatt agacaaaaaa gttattttga gatcggagca 60

　　aaatagttcg acccagccag gattcgaacc tggaaccttt accagccatg tttaccggaa 120

　　agtaacgcgc taccattgcg ccactaggcc cctttttgtt tcgtcggtca attatttaat 180

　　ataaatacta aggaagagtg aggttctgag acatctggtg ccccttatag catatgatgc 240

　　ggtcgtagcc ttccgaatgt gggagtagca attcaccgat gtaaacggag gaaggtgggc 300

　　tgcggcgtac aggctatata aagtgctctg ttccacagaa cccgggcttc gcttccagaa 360

　　tgtgatattg aaacaagtcg gacaaatgtg tggtattgta tataggcttg catgtgatct 420

　　tgaccgagtt tatgtggaaa atggcatttg aaatgagggg tattcggaat ctagggcgtt 480

　　tgctactgcg attagatgac gtcaggtgtc tgttctcgcg aaatacatat aatctcctcg 540

　　gttagatttg ttcaggagaa agatgatggt gccaaactct acagtttagg cttctctacc 600

　　gaggaaagag aatgagaggt cggaccacca gcgtgctaac aggacgagac ttgagccaat 660

　　ttgtcaatag acaagatagt ctcgtgtgcc aagccaacat catcatccga tctcagctga 720

　　gcccctcctt ctgaaaagag tataattaca gcagcttcag acgttgaggc atcactgttg 780

　　ctgccgcacg tcagtcaacc acttaatccg ctctgtgtga ccggtgacgg attcgatcat 840

　　tctacgatcc gctccgcatc gccggttaaa gggggtgacg agttatttat tacctttacg 900

　　gattcaagat ggcggtctgc accgtgtaca ccatccccac gacgacccac ctgggcagca 960

　　gcttcaacca aaacaacaaa caggtcttct tcaactacaa gcgcagcagc agcagcaaca 1020

　　acacgctgtt caccacgcgg ccgagctatg tcatcacctg ctcgcagcag cagaccatcg 1080

　　tcattggcct cgccgcggac tcgggctgtg gcaaaagcac gttcatgcgc cgcctcacca 1140

　　gcgtctttgg cggcgcggcc gagccgccca agggcggcaa cccggactcg aacacgctca 1200

　　tctcggacac caccacggtg atctgcctcg acgactttca ctcgctcgac cggaatggcc 1260

　　gcaaggtcga gaaggtcacc gccctcgacc ccaaggcgaa cgacttcgac ctgatgtacg 1320

　　agcaggtgaa ggccctgaag gaaggcaagg cggtcgacaa gcccatctac aaccatgtct 1380

　　cgggcctcct cgatccgccc gaactcatcc agccgccgaa gatcctggtc atcgaagggc 1440

　　tccatccgat gtacgacgcc cgcgtgcgcg agctcctgga cttctcgatc tatctcgaca 1500

　　tctcgaacga ggtcaaattc gcctggaaga tccagcgcga catgaaggag cgcggccact 1560

　　cgttagagtc tattaaggcg tcgatcgagt cgcggaagcc cgactttgac gcctacatcg 1620

　　acccgcagaa gcagcatgcg gacgtcgtca tcgaagtcct gcccaccgag ctcatcccgg 1680

　　acgatgacga gggcaaggtc ctccgcgtcc ggatgatcca gaaggagggc gtcaagttct 1740

　　tcaaccccgt ctacctcttc gacgagggca gcacgatctc gtggattcct tgtggtcgta 1800

　　aactcacgtg ctcgtacccg ggcattaagt tcagctacgg gcccgacacg ttctacggga 1860

　　acgaggtcac ggtcgtggag atggacggca tgttcgaccg gctcgacgag ctgatctatg 1920

　　tcgagtcgca cctgtcgaac ctcagcacga agttttacgg cgaggtgacc cagcaaatgc 1980

　　tgaagcacca gaacttcccc ggcagcaaca acgggacggg cttctttcaa accatcatcg 2040

　　gcctcaagat ccgggacctc ttcgagcagc tcgtcgcctc gcggagcacc gccaccgcca 2100

　　ccgccgccaa ggcctgagtc accgacagcg acgccatcat gacggtctcg agggtcggca 2160

　　cgtacgccgg aatccaggac gtggccctca gcatgccctg caagctgaac cggcatggcg 2220

　　cgtaccagga cgtgccccta ctcctcagcg agttggagga ggccgaattg agagagtcgg 2280

　　cccaaagcat taaggaggtc ctcatgtcgt tagagaaata actctgttaa ctctgttcat 2340

　　tttgttcttt aattattcaa tccttggcat tgttaattta actatcgatt taagaaaatt 2400

　　gggcgctgtg atcttgtgaa gctatcacaa cgtgcctata caccggatca ggtaattagt 2460

　　aggagaaagg tcaggaaact ctgccaagtc tgcagttcaa atttcctggt tcccacattg 2520

　　ttgccctggt acatgaacag ccttacattt cggagaatcc agatcctgta cacacaatac 2580

　　tcaccgggcc cctttcttgc tgccagaaac ccataagtac gagataatcg atctggcatt 2640

　　gacatttctt cccatggatg ctcgtccaat tcgcggccat gatacaggcg tacacctact 2700

　　ctcggcttgt gaaatataaa tgccatatag tacatggcaa ccaagtcctc aagacaggat 2760

　　ggacaggccg gaagaggctg cggacggttc tgaaagacga cgagctctgc ccgcgatgtg 2820

　　agcgacggct cgaagagcaa atgtgtcgca cccaggactg gctacaagac caccggaagg 2880

　　acttcaagac ggaagtgatt cacatttacc gaaatgacga agagccaatg acaagccccg 2940

　　agtggccttt gttcaccgag tccgagagct ccaacgaatc cggagaggat cggaaacaaa 3000

　　cctcggcctc gaacaaggag ggagacccat ttctggggac caaccttcca actgggtctg 3060

　　gagcgggcga c 3071

　　<210> 23

　　<211> 3111

　　<212> DNA

　　<213> Myceliophthora thermophila

　　<400> 23

　　ggagggttgg tctcatgcct cttggcggga gccgcccgat aactagtata actagttgta 60

　　actccgtatc cggttacgga aacggaaagg cccgctcggc tgttctccgg cggctccccg 120

　　atcgctgatc agagcatgga acagatgtca attacatcac tcccgcgtaa acgaaccata 180

　　gttatcgaac cacagttatc gaaccacaga gccagcccat gggaacgtct gaacagctcg 240

　　gaggatgcaa ccgatattgc aatgcaaaac gtcacccatg ctacaattaa ttccctgcac 300

　　aactacttgt aagccgcgag gcctagaaca cagttgcaga acctgggtat cgtgcctgtg 360

　　gtctgatgca gatatgtgtc accactcaag accccgccaa cacgccgctt cgaggccctg 420

　　aacagtacaa agggcgcttc aaattcgtac aagccccccc gaggccgttt tcaagtcttt 480

　　gtatgaccat ctattttccg attgacgtcc ctcacggatt ctctttcgtt gctgacctcc 540

　　ttgtgaccac aaacatcgcc aacaaatgga ccagtcgagc cggtacgtca acctcgccct 600

　　gaaggaggag gatttaattg ccgggggcga acacgtgctc tgcgcgtaca tcatgaagcc 660

　　gaaggccggc tatggctatg tcgccacggc cgcccatttt gccgccgagt cgtcgacggg 720

　　caccaatgtc gaggtgtgca ccacggacga ctttacccgc ggcgtggatg ccctcgtgta 780

　　cgaggtcgac gaggcgcggg agctcacgaa gatcgcctac ccggtcgccc tgttcgaccg 840

　　gaacatcacc gacggcaagg cgatgatcgc gtcgttcctc acgctgacta tgggtaacaa 900

　　ccaaggcatg ggcgacgtcg agtacgcgaa gatgcacgac ttctacgtcc cggaggccta 960

　　tcgggccctg tttgacggcc ccagcgtcaa catcagcgcc ctctggaagg tcctggggcg 1020

　　cccggaggtc gatggcggcc tcgtggtcgg caccattatc aagccgaagc tgggcctgcg 1080

　　cccgaagccc tttgcggagg cctgtcatgc cttctggctg ggcggcgatt tcattaagaa 1140

　　cgacgagccc cagggcaacc aaccgtttgc ccccctccgc gacaccattg ccctcgtcgc 1200

　　ggacgccatg cgccgggccc aggacgaaac cggcgaggcc aagctgttca gcgccaacat 1260

　　caccgcggat gacccgttcg agatcatcgc gcggggcgag tacgtcttag aaaccttcgg 1320

　　cgagaacgcg agccacgtcg ccctcctggt cgacggctac gtcgccggcg ccgccgccat 1380

　　tacgaccgcc cgccggcggt ttcccgacaa cttcctccat taccaccgcg ccggccacgg 1440

　　cgccgtgacc agcccccagt cgaagcgcgg ctacaccgcc ttcgtgcact gcaaaatggc 1500

　　ccgcctccag ggcgcgagcg gcatccacac ggggactatg ggtttcggca agatggaggg 1560

　　ggagagcagc gatcgcgcca tcgcctacat gctcacgcag gacgaggccc aggggccgtt 1620

　　ttaccgccag tcgtgggggg gcatgaaggc ctgcaccccc attatcagcg gcgggatgaa 1680

　　tgccctgcgc atgccggggt tttttgagaa cctcggcaac gccaacgtca ttctcaccgc 1740

　　cggcggcggc gcctttggcc atattgacgg ccccgtggcg ggcgcgcgct cgctccgcca 1800

　　ggcctggcag gcgtggcgcg atggcgtccc ggtcctcgac tatgcccggg aacacaagga 1860

　　gctcgcccgc gcctttgaga gcttcccggg cgacgcggac cagatttacc ccggctggcg 1920

　　caaggccctg ggcgtcgagg acacccgctc ggcgctgccg gcctgatgat agtaccggac 1980

　　gtcccaactc aacacgatcg acttccactc ggaccattgc acggtgcggt attccgagta 2040

　　ccccggcgtg gccatgcgcg gcgtgctccg caaggtggtg gaacgggttg atcttagcaa 2100

　　actgtctagg cctccatcgc ccgaggtggt caacgaggtg actaagaaca gggatagttc 2160

　　ccaaaccatc acacaggcgt tcttctggcc acgcattggg gaatatctga aggagaacga 2220

　　catcgtggtg acagaaacgg gcacgtccaa cttcggcatt tgggaaacca agtacccacg 2280

　　gggtgtgact gggatcacac aaattctttg gggcagcatt ggttggtcgg tgggcgccgc 2340

　　ccaaggtgcc gcgctagctg caaaggacat gggcgtcgac cgccgaacca tcttgtttgt 2400

　　cggcgacggc tcgttccagc tcacggccca agaggtgtca accatgatcc gacatgattt 2460

　　gaggatcacc atgtgagttt tcccctagtc ttctcgcaaa cacgatgctc acaaccccta 2520

　　gattcctgat tttcaacggc ggcttcacca tcgagcgctt tatccatggc atggaggccg 2580

　　agtacaatga tatcacccgt tggaactaca ttgacgtgcc aacagcattc ggcggttcgg 2640

　　aaaaacaggt tcgcaagttt gtcgtcaaga ccaaggatga gctcgaggag ctattgacgg 2700

　　acacagactt caatgaggct agagggttac agtttgttga gctgtggatg cgaaaggatg 2760

　　acgcaccgcg ggccctgaag atcactgctg agattgccgc tagaaacaac gcaagtatga 2820

　　gtgagtaaga tggtggcttg gtggctgaag gaggagaaat ggcggagcgg acagtccggt 2880

　　gtcggctgcg gtgatgagaa atctctttct taattgagcc atagtgaaat aagggactga 2940

　　gctgccttgc tgtgtttggc cattctcaac ttcagtgaca catttccctc cctccctacc 3000

　　accttgtctc gttttctctt gggattgcta tgtattttct gaacaaaggg gcgcaggaaa 3060

　　aggaggtggt aacacgtgac tcgttctcgg agtccatctg cagcggctct t 3111

　　<210> 24

　　<211> 3219

　　<212> DNA

　　<213> Myceliophthora thermophila

　　<400> 24

　　gctcgagttt tgtcgcattc gacgcacacc ccaagatcaa ttccatccag agactgtgtg 60

　　attgcattat cgccgccgcc tagccagccc gtaaccgacc tatgagactg gggcgtcgcc 120

　　gtagtccttc ccgaacggtg acaggggtgc cgtcaacgag ctccggaaac atttcgtgcc 180

　　ccagccgccc aatcaccgcc cgcccgactc gtcatccacc ggtatccggg ctccgttccg 240

　　gcctggcgca cgttcagttc cccgtggaat gctgtcgtca ctcgtcattg gacgtgactt 300

　　cgctcacggc cccacggctt tatatactct cgctcctcga cctcgggtgc agcaggacgg 360

　　tctaaacggt ctatcttctg ctatcagcac tttgacatcc cgaagctttt tatcccctgt 420

　　catccccagc aggaagtgga tttgccgtta gcccacattg ccgaactgcg aagccacccg 480

　　aactcgagct cagcagaaag cacttaagaa ccatgaacgg ccccgtcgcc agaaccgact 540

　　ctccctacac tgatccctcc gtcaaaggtc ctcgtaccaa gcttcttagt aacagatctc 600

　　ccgccgtcgc cgccgccgcc aacagtgagt cgtcccggtg gccaagtatg cttccaatcc 660

　　ttcgtcgtcc atcgctgaca ctggggcttc acctcaacag tggtcaacac cgtcaacaag 720

　　acaggcctcc atcctggtgg cattgtgtta gtgccctcac gacccttcgt tcttgataca 780

　　tgttctaact ggaagtgatg caggccccac ccaaagactg agctaggtat ggactacgaa 840

　　agcatcaccg agacgtttcg acgtcgatgg cggctctcgg aattgccctt ctaacacgtg 900

　　acgcagagga ggaactccac gagaaagctc acatcgacta caccagagtc gcaatcgtga 960

　　gtcccttcga ctatgccctc gttctcttgg ttcaaaccac tgacggtatc tttcaccaga 1020

　　tcccaaaccc ttcagtcgcc gcgctctact gacgacgtta actgatattg aaggagcatt 1080

　　ttttgggctt ggctggagct agtggaggtc aacaatgaat gcctattttg gtttagtcgt 1140

　　ccaggcggtg agcacaaaat ttgtgtcgtt tgacaagatg gttcatttag gcaactggtc 1200

　　agatcagccc cacttgtagc agtagcggcg gcgctcgaag tgtgactctt attagcagac 1260

　　aggaacgagg acattattat catctgctgc ttggtgcacg ataacttggt gcgtttgtca 1320

　　agcaaggtaa gtggacgacc cggtcatacc ttcttaagtt cgcccttcct ccctttattt 1380

　　cagattcaat ctgacttacc tattctaccc aagcatccaa atgattgaac aagatggatt 1440

　　gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca 1500

　　gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct 1560

　　ttttgtcaag accgacctgt ccggtgccct gaatgaactg caagacgagg cagcgcggct 1620

　　atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc 1680

　　gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct 1740

　　tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga 1800

　　tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg 1860

　　gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc 1920

　　agccgaactg ttcgccaggc tcaaggcgag catgcccgac ggcgaggatc tcgtcgtgac 1980

　　ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat 2040

　　cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga 2100

　　tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc 2160

　　cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagtcag 2220

　　gtcgactata tgccgtcgct ctcaattgcg gccaaaatct ggcccaattg ccaatggacg 2280

　　tcatttgtcc ttggcgttgc gcacatcacg cgagcgatcg gatagccgga tgctgcgata 2340

　　gccggctgcg acgtgcggcg acatcatcgt ttgagtcgtg cgttttgccg cggcctgccg 2400

　　gatcccccac ctttgtcgct gtcgtcatac cctgcatccc ctcgtcccaa ttgggtctcc 2460

　　acttccccca atagccgatc gtctcgttcc gcccccgcaa aatgcccacc ctaccaatca 2520

　　catgtcattc tcgatcgtgg aaccagtttc gctaaccaca aatggtctcg cgtatagatc 2580

　　tggaagatca accgtgagcg cgccgtcgat tatctcaaca cccggagtcg catctacgtc 2640

　　gttgatggat atgccggctg ggatgagaag taccgcatca aggtgcgcgt cgtctgcgcg 2700

　　cgcgcctacc acgccctctt catgcgcaac atgctcatcc gcccgtcacg ggaggagctc 2760

　　gagcatttcc accccgatta cacaatctac aatgccgggc gtttccccgc caaccgctac 2820

　　acccacggca tgacctcagc cacgtcggtt gctatcaact tcgccgagaa ggagatggtc 2880

　　atcttgggta ccgagtacgc cggcgagatg aagaagggca tcttcaccgt catgttctac 2940

　　gagggtccca tcaagcacaa catcttgact ctgcactcgt ccgccaacga gggcaaggac 3000

　　ggcgacgtca ccctcttctt cggtctgtcc ggcactggca agaccaccct gtcggccgac 3060

　　cccaaccggc gcctgatcgg cgacgacgag cactgctgga gcgatcgcgg catcttcaac 3120

　　atcgaaggcg gctgctacgc caagacgatc ggcctctcgg ccgagaagga gcccgatatc 3180

　　tacaacgcca tccgcttcgg ctccgttctg gagaacgtc 3219

　　<210> 25

　　<211> 25

　　<212> DNA

<213> artificially synthesized sequence

　　<400> 25

　　aggatcggtg gagtgaagtt cggaa 25

　　<210> 26

　　<211> 53

　　<212> DNA

<213> artificially synthesized sequence

　　<400> 26

　　ctagctctaa aacgtctcgt atacgagtgc atcgaggaaa gaaagaaaag aag 53

　　<210> 27

　　<211> 58

　　<212> DNA

<213> artificially synthesized sequence

　　<400> 27

　　tatttctagc tctaaaactg acatctgctg cgtttggcga ggaaagaaag aaaagaag 58

　　<210> 28

　　<211> 49

　　<212> DNA

<213> artificially synthesized sequence

　　<400> 28

　　ctctaaaact ttgtgcgggc atacccgtcg aggaaagaaa gaaaagaag 49

　　<210> 29

　　<211> 52

　　<212> DNA

<213> artificially synthesized sequence

　　<400> 29

　　tctttctttc ctcgatgcac tcgtatacga gacgttttag agctagaaat ag 52

　　<210> 30

　　<211> 56

　　<212> DNA

<213> artificially synthesized sequence

　　<400> 30

　　ttttctttct ttcctcgcca aacgcagcag atgtcagttt tagagctaga aatagc 56

　　<210> 31

　　<211> 25

　　<212> DNA

<213> artificially synthesized sequence

　　<400> 31

　　aaaaagcacc gactcggtgc cactt 25

　　<210> 32

　　<211> 63

　　<212> DNA

<213> artificially synthesized sequence

　　<400> 32

　　gtggagatgt ggagtgggcg cttacacagt acacgaggac ttgctcgagt tttgtcgcat 60

　　tcg 63

　　<210> 33

　　<211> 56

　　<212> DNA

<213> artificially synthesized sequence

　　<400> 33

　　caaaaaatgc tccttcaata tcagttaacg tcgtcagtag agcgcggcga ctgaag 56

　　<210> 34

　　<211> 23

　　<212> DNA

<213> artificially synthesized sequence

　　<400> 34

　　cgacgttaac tgatattgaa gga 23

　　<210> 35

　　<211> 20

　　<212> DNA

<213> artificially synthesized sequence

　　<400> 35

　　tcagaagaac tcgtcaagaa 20

　　<210> 36

　　<211> 62

　　<212> DNA

<213> artificially synthesized sequence

　　<400> 36

　　gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gtcaggtcga ctatatgccg 60

　　tc 62

　　<210> 37

　　<211> 59

　　<212> DNA

<213> artificially synthesized sequence

　　<400> 37

　　caagtcatgt gattgtaatc gaccgacgga attgaggatg acgttctcca gaacggagc 59

　　<210> 38

　　<211> 56

　　<212> DNA

<213> artificially synthesized sequence

　　<400> 38

　　tgtggagtgg gcgcttacac agtacacgag gacttggtaa accgaaagct ggggaa 56

　　<210> 39

　　<211> 21

　　<212> DNA

<213> artificially synthesized sequence

　　<400> 39

　　gtctgttgtt ggcgatgttt g 21

　　<210> 40

　　<211> 55

　　<212> DNA

<213> artificially synthesized sequence

　　<400> 40

　　ctccttgtga ccacaaacat cgccaacaac agacatggac cagtcgagcc ggtac 55

　　<210> 41

　　<211> 57

　　<212> DNA

<213> artificially synthesized sequence

　　<400> 41

　　agtcgatcgt gttgagttgg gacgtccggt actatcatca ggccggcagc gccgagc 57

　　<210> 42

　　<211> 22

　　<212> DNA

<213> artificially synthesized sequence

　　<400> 42

　　gtaccggacg tcccaactca ac 22

　　<210> 43

　　<211> 56

　　<212> DNA

<213> artificially synthesized sequence

　　<400> 43

　　catgtgattg taatcgaccg acggaattga ggataagagc cgctgcagat ggactc 56

　　<210> 44

　　<211> 59

　　<212> DNA

<213> artificially synthesized sequence

　　<400> 44

　　agatgtggag tgggcgctta cacagtacac gaggacttgt gacaaatacc agagtctag 59

　　<210> 45

　　<211> 23

　　<212> DNA

<213> artificially synthesized sequence

　　<400> 45

　　cttgaatccg taaaggtaat aaa 23

　　<210> 46

　　<211> 51

　　<212> DNA

<213> artificially synthesized sequence

　　<400> 46

　　cgagttattt attaccttta cggattcaag atggcggtct gcaccgtgta c 51

　　<210> 47

　　<211> 42

　　<212> DNA

<213> artificially synthesized sequence

　　<400> 47

　　gatggcgtcg ctgtcggtga ctcaggcctt ggcggcggtg gc 42

　　<210> 48

　　<211> 21

　　<212> DNA

<213> artificially synthesized sequence

　　<400> 48

　　gtcaccgaca gcgacgccat c 21

　　<210> 49

　　<211> 58

　　<212> DNA

<213> artificially synthesized sequence

　　<400> 49

　　aagtcatgtg attgtaatcg accgacggaa ttgaggatgt cgcccgctcc agacccag 58

　　<210> 50

　　<211> 21

　　<212> DNA

<213> artificially synthesized sequence

　　<400> 50

　　acacgtgacg cagaggagga a 21

　　<210> 51

　　<211> 24

　　<212> DNA

<213> artificially synthesized sequence

　　<400> 51

　　gtactcggta cccaagatga ccat 24

　　<210> 52

　　<211> 23

　　<212> DNA

<213> artificially synthesized sequence

　　<400> 52

　　gtgaccacaa acatcgccaa caa 23

　　<210> 53

　　<211> 20

　　<212> DNA

<213> artificially synthesized sequence

　　<400> 53

　　atcgtgttga gttgggacgt 20

　　<210> 54

　　<211> 21

　　<212> DNA

<213> artificially synthesized sequence

　　<400> 54

　　gtgacggatt cgatcattct a 21

　　<210> 55

　　<211> 21

　　<212> DNA

<213> artificially synthesized sequence

　　<400> 55

　　gccgaccctc gagaccgtca t 21

　　<210> 56

　　<211> 574

　　<212> PRT

　　<213> Myceliophthora thermophila

　　<400> 56

Met Ala Val Glu Glu Asn Asn Met Pro Val Val Ser Gln Gln Pro Gln

1 5 10 15

Ala Gly Glu Asp Val Ile Ser Ser Leu Ser Lys Asp Ser His Leu Ser

20 25 30

Ala Gln Ser Gln Lys Tyr Ser Asn Asp Glu Leu Lys Ala Gly Glu Ser

35 40 45

Gly Ser Glu Gly Ser Gln Ser Val Pro Ile Glu Ile Pro Lys Lys Pro

50 55 60

Met Ser Glu Tyr Val Thr Val Ser Leu Leu Cys Leu Cys Val Ala Phe

65 70 75 80

Gly Gly Phe Met Phe Gly Trp Asp Thr Gly Thr Ile Ser Gly Phe Val

85 90 95

Val Gln Thr Asp Phe Leu Arg Arg Phe Gly Met Lys His Lys Asp Gly

100 105 110

Thr His Tyr Leu Ser Asn Val Arg Thr Gly Leu Ile Val Ala Ile Phe

115 120 125

Asn Ile Gly Cys Ala Phe Gly Gly Ile Ile Leu Ser Lys Gly Gly Asp

130 135 140

Met Tyr Gly Arg Lys Lys Gly Leu Ser Ile Val Val Ser Val Tyr Ile

145 150 155 160

Val Gly Ile Ile Ile Gln Ile Ala Ser Ile Asn Lys Trp Tyr Gln Tyr

165 170 175

Phe Ile Gly Arg Ile Ile Ser Gly Leu Gly Val Gly Gly Ile Ala Val

180 185 190

Leu Cys Pro Met Leu Ile Ser Glu Ile Ala Pro Lys His Leu Arg Gly

195 200 205

Thr Leu Val Ser Cys Tyr Gln Leu Met Ile Thr Ala Gly Ile Phe Leu

210 215 220

Gly Tyr Cys Thr Asn Tyr Gly Thr Lys Ser Tyr Ser Asn Ser Val Gln

225 230 235 240

Trp Arg Val Pro Leu Gly Leu Cys Phe Ala Trp Ser Leu Phe Met Ile

245 250 255

Gly Ala Leu Thr Leu Val Pro Glu Ser Pro Arg Tyr Leu Cys Glu Val

260 265 270

Asn Lys Val Glu Asp Ala Lys Arg Ser Ile Ala Lys Ser Asn Lys Val

275 280 285

Ser Pro Glu Asp Pro Ala Val Gln Ala Glu Leu Asp Leu Ile Met Ala

290 295 300

Gly Ile Glu Ala Glu Lys Leu Ala Gly Asn Ala Ser Trp Gly Glu Leu

305 310 315 320

Phe Ser Thr Lys Thr Lys Val Phe Gln Arg Leu Leu Met Gly Val Phe

325 330 335

Val Gln Met Phe Gln Gln Leu Thr Gly Asn Asn Tyr Phe Phe Tyr Tyr

340 345 350

Gly Thr Val Ile Phe Lys Ser Val Gly Leu Asp Asp Ser Phe Glu Thr

355 360 365

Ser Ile Val Ile Gly Val Val Phe Phe Ala Ser Thr Phe Phe Ser Leu

370 375 380

Trp Thr Val Glu Asn Leu Gly His Arg Lys Cys Leu Leu Leu Gly Ala

385 390 395 400

Ala Thr Met Met Ala Cys Met Val Ile Tyr Ala Ser Val Gly Val Thr

405 410 415

Arg Leu Tyr Pro His Gly Lys Ser Gln Pro Ser Ser Lys Gly Ala Gly

420 425 430

Asn Cys Met Ile Val Phe Thr Cys Phe Tyr Ile Phe Cys Tyr Ala Thr

435 440 445

Thr Trp Ala Pro Val Ala Trp Val Ile Thr Ala Glu Ser Phe Pro Leu

450 455 460

Arg Val Lys Ser Lys Cys Met Ala Leu Ala Ser Ala Ser Asn Trp Val

465 470 475 480

Trp Gly Phe Leu Ile Ala Phe Phe Thr Pro Phe Ile Thr Ser Ala Ile

485 490 495

Asn Phe Tyr Tyr Gly Tyr Val Phe Met Gly Cys Leu Val Ala Met Phe

500 505 510

Phe Tyr Val Phe Phe Phe Val Pro Glu Thr Lys Gly Leu Ser Leu Glu

515 520 525

Glu Ile Gln Glu Leu Trp Glu Glu Gly Val Leu Pro Trp Lys Ser Glu

530 535 540

Gly Trp Ile Pro Ser Ser Arg Arg Gly Asn Asn Tyr Asp Leu Glu Asp

545 550 555 560

Leu Gln His Asp Asp Lys Pro Trp Tyr Lys Ala Met Leu Glu

565 570

　　<210> 57

　　<211> 1725

　　<212> DNA

　　<213> Myceliophthora thermophila

　　<400> 57

　　atggcagttg aggagaacaa tatgcctgtt gtttcacagc aaccccaagc tggtgaagac 60

　　gtgatctctt cactcagtaa agattcccat ttaagcgcac aatctcaaaa gtattctaat 120

　　gatgaattga aagccggtga gtcagggtct gaaggctccc aaagtgttcc tatagagata 180

　　cccaagaagc ccatgtctga atatgttacc gtttccttgc tttgtttgtg tgttgccttc 240

　　ggcggcttca tgtttggctg ggataccggt actatttctg ggtttgttgt ccaaacagac 300

　　tttttgagaa ggtttggtat gaaacataag gatggtaccc actatttgtc aaacgtcaga 360

　　acaggtttaa tcgtcgccat tttcaatatt ggctgtgcct ttggtggtat tatactttcc 420

　　aaaggtggag atatgtatgg ccgtaaaaag ggtctttcga ttgtcgtctc ggtttatata 480

　　gttggtatta tcattcaaat tgcctctatc aacaagtggt accaatattt cattggtaga 540

　　atcatatctg gtttgggtgt cggcggcatc gccgtcttat gtcctatgtt gatctctgaa 600

　　attgctccaa agcacttgag aggcacacta gtttcttgtt atcagctgat gattactgca 660

　　ggtatctttt tgggctactg tactaattac ggtacaaaga gctattcgaa ctcagttcaa 720

　　tggagagttc cattagggct atgtttcgct tggtcattat ttatgattgg cgctttgacg 780

　　ttagttcctg aatccccacg ttatttatgt gaggtgaata aggtagaaga cgccaagcgt 840

　　tccattgcta agtctaacaa ggtgtcacca gaggatcctg ccgtccaggc agagttagat 900

　　ctgatcatgg ccggtataga agctgaaaaa ctggctggca atgcgtcctg gggggaatta 960

　　ttttccacca agaccaaagt atttcaacgt ttgttgatgg gtgtgtttgt tcaaatgttc 1020

　　caacaattaa ccggtaacaa ttattttttc tactacggta ccgttatttt caagtcagtt 1080

　　ggcctggatg attcctttga aacatccatt gtcattggtg tagtcttctt tgcctccact 1140

　　ttctttagtt tgtggactgt cgaaaacttg ggacatcgta aatgtttact tttgggcgct 1200

　　gccactatga tggcttgtat ggtcatctac gcctctgttg gtgttactag attatatcct 1260

　　cacggtaaaa gccagccatc ttctaaaggt gccggtaact gtatgattgt ctttacctgt 1320

　　ttttatattt tctgttatgc cacaacctgg gcgccagttg cctgggtcat cacagcagaa 1380

　　tcattcccac tgagagtcaa gtcgaaatgt atggcgttgg cctctgcttc caattgggta 1440

　　tgggggttct tgattgcatt tttcacccca ttcatcacat ctgccattaa cttctactac 1500

　　ggttatgtct tcatgggctg tttggttgcc atgttttttt atgtcttttt ctttgttcca 1560

　　gaaactaaag gcctatcgtt agaagaaatt caagaattat gggaagaagg tgttttacct 1620

　　tggaaatctg aaggctggat tccttcatcc agaagaggta ataattacga tttagaggat 1680

　　ttacaacatg acgacaaacc gtggtacaag gccatgctag aataa 1725

　　<210> 58

　　<211> 1346

　　<212> DNA

　　<213> Myceliophthora thermophila

　　<400> 58

　　cacagcagtt cgcacgctcc cattgggttc ctcatcacgc agtcgctctc cccgccaacc 60

　　agcgccaggt ccgggaacag cggcgcaaat gcgtatttga gggcgcctcg ctcgagcaac 120

　　ctgtgcctga ccttctcctc ctccttctgc accttgcatc tcgtcgcgtc cactcgcagg 180

　　caaccacaca tcctcctcct ctcccaaaac ccccccgctt tttctttccc ttgttggaat 240

　　tcgattgaaa aagaagacgg gtccgtctag agaccgcctt ctcacctttc tctcgacttc 300

　　tttctaggaa aagaagcaag agtcattctt cttgtccacc ttctggttca cggaaggtcg 360

　　aggagaagat tgcctctgcc cccaaagtcg ccaacctgga ctttgaagca cgtgttccgg 420

　　tccctttcag tgtcttcccg tcctcgtaca gggagtccga gaccgccacc caaacccact 480

　　cccacgaaga ggttgagatc aagctccccc agctcgccgg acgggaaggt caacactctt 540

　　cattccaagc ccaagcacat cttcctccca gcggagaggg tcgcttcaga gaagaagagg 600

　　tccgcatcac tcgtcaagag gaacatcacc gccgtcccgg catccgtgaa gagttcgttc 660

　　accgcgagga gcgtcaccgg taagtttagt ttttgttttg attcaccacc cattgtcttc 720

　　cccgcctttt tctttttctt cccttgctct cttgcccctg tctagtgtag ggcattgcca 780

　　aggccatctt cacacacaca cacccccccc cccaccctca gctggggggg ggggggtggc 840

　　ctgggttgac caagggacgg tgaagactac tactacttga gccactcaaa cccatgcatg 900

　　acacagggtt ttcctttttc ttttctcttt tcctttaact aaccaaccac tccaacatta 960

　　gccctcagtc aacctactcc gagtctcgca tcgagttcga tactgagcac cgcactcaca 1020

　　actccgtcat tgacgttgct gagagcgagt atcgtgcccg tgtccagccc aactaccgca 1080

　　aggaagcttc cgtagtcggt accaccgtcg acggatcccg cttcagccac agccgcaagg 1140

　　ccagcagcac cacctccacc cacaccgacg agtacaccgt cgatccccct agccaccgcc 1200

　　ccgtctacaa gaaggagtcg gttgaagtcg ccggtaccac tgttgacccc cctgctcctc 1260

　　gttcgaccta ccacgagcag gtgaacattg ttgaagagac cgttgacgct caccgttacg 1320

　　ctcctcaaca caacaacaac aacaag 1346

　　<210> 59

　　<211> 59

　　<212> DNA

<213> artificially synthesized sequence

　　<400> 59

　　ttggctgact tgaagtaatc tctgcagatc tttaattaac acagcagttc gcacgctcc 59

　　<210> 60

　　<211> 58

　　<212> DNA

<213> artificially synthesized sequence

　　<400> 60

　　gtgaaacaac aggcatattg ttctcctcaa ctgccatctt gttgttgttg ttgtgttg 58

　　<210> 61

　　<211> 60

　　<212> DNA

<213> artificially synthesized sequence

　　<400> 61

　　cgctcaccgt tacgctcctc aacacaacaa caacaacaag atggcagttg aggagaacaa 60

　　<210> 62

　　<211> 47

　　<212> DNA

<213> artificially synthesized sequence

　　<400> 62

　　caaactaaag aaagtggagg caaagaagac tacaccaatg acaatgg 47

　　<210> 63

　　<211> 46

　　<212> DNA

<213> artificially synthesized sequence

　　<400> 63

　　tccattgtca ttggtgtagt cttctttgcc tccactttct ttagtt 46

　　<210> 64

　　<211> 58

　　<212> DNA

<213> artificially synthesized sequence

　　<400> 64

　　tcaagctgtt tgatgatttc agtaacgtta agtggatctt attctagcat ggccttgt 58

Claims

1. A method for improving the organic acid synthesis capacity of a filamentous fungus recombinant strain is characterized in that 5-phosphoribosyl kinase and 1, 5-diphospho ribose carboxylation/oxygenase in an organic acid synthesis positive control gene are introduced into a filamentous fungus for producing dibasic organic acid by a genetic engineering method; the dibasic organic acid is selected from malic acid and/or succinic acid;

the filamentous fungus is selected from myceliophthora thermophila (Myceliophthorathermophila) Malic acid fermentation strain JG 207.

2. The method of claim 1, wherein a negative organic acid synthesis regulating gene selected from one or more of lactate dehydrogenase, pyruvate decarboxylase, or pyruvate carboxykinase is also downregulated.

3. The method of claim 1, wherein an exogenous organic acid is introduced simultaneously with the synthesis of the up-regulated gene sugar transporter.

4. The method according to any one of claims 1 to 3, wherein the recombinant strain has an enhanced or increased organic acid production capacity of at least 10% compared to its starting strain.

5. A method according to any one of claims 1 to 3, wherein the amino acid sequence of the 5-phosphoribosyl kinase is as shown in SEQ ID No.3, the amino acid sequence of the 1,5-bisphosphate ribocarboxylase/oxygenase is as shown in SEQ ID No.1, and the amino acid sequence of the sugar transporter is as shown in SEQ ID No. 56; the amino acid sequence of the lactate dehydrogenase is shown as SEQ ID NO.15, the amino acid sequence of the pyruvate decarboxylase is shown as SEQ ID NO.13, and the amino acid sequence of the pyruvate carboxykinase is shown as SEQ ID NO. 17.

6. The method according to any one of claims 1 to 3, wherein the introduction of the positive regulator gene for organic acid synthesis is carried out by introducing an expression vector containing the positive regulator gene.

7. The method of any one of claims 2 to 3, wherein said down-regulating an organic acid synthesis negative regulator gene is achieved by gene knock-out or gene editing or an inhibitor selected from said antibody, inhibitory mRNA, antisense RNA, microRNA, miRNA, siRNA, shRNA or activity inhibitor.

8. The method of claim 7, wherein down-regulating the organic acid synthesis negative regulatory gene expression level is achieved by a CRISPR/Cas 9-based genome editing method.

9. A recombinant bacterium produced by the method according to any one of claims 1 to 8.

10. The method for producing organic acid by using the recombinant bacterium of claim 9, wherein the recombinant bacterium is used for producing organic acid by fermentation using monosaccharide, glycan and/or plant biomass as a substrate.

11. The method of claim 10, wherein the monosaccharide is selected from the group consisting of glucose, xylose, arabinose, and combinations thereof; the glycan is selected from crystalline cellulose, hemicellulose or a combination thereof; the plant biomass is selected from crop straws, forestry wastes, energy plants or partial or whole decomposition products thereof; wherein the crop straw is selected from corn straw, wheat straw, rice straw, sorghum straw, soybean straw, cotton straw, bagasse and corn cob; the forestry waste is selected from branches and leaves and sawdust; the energy plant is selected from sweet sorghum, switchgrass, miscanthus, reed, or combinations thereof.