CN106754435A

CN106754435A - The construction method of the restructuring mould of citric acid output increased and application

Info

Publication number: CN106754435A
Application number: CN201611236665.8A
Authority: CN
Inventors: 刘龙; 殷娴; 陈坚; 堵国成; 李江华
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2016-12-28
Filing date: 2016-12-28
Publication date: 2017-05-31
Anticipated expiration: 2036-12-28
Also published as: CN106754435B

Abstract

The present invention relates to a kind of construction method of the restructuring mould of citric acid output increased, comprise the following steps：HGT1 protein expression frames are built, HGT1 protein expressions inframe includes constitutive promoter and the constitutive promoter HGT1 protein coding genes for connecting and the terminator being connected with HGT1 protein coding genes；Tolerant gene expression frame is built, tolerant gene expression inframe includes constitutive promoter and the constitutive promoter hph genes for connecting and the terminator being connected with hph genes；HGT1 protein expressions frame and tolerant gene expression frame are transformed into mould, obtain recombinating mould.The HGT1 protein expressions frame built present invention simultaneously provides a kind of use above method and restructuring mould.The present invention discloses application of the restructuring mould in fermentation production of citric acid that a kind of use above method builds.The present invention is expressed by starting high-affinity GLUT, and then improves the yield of citric acid in mould, and fermentation time is greatly shortened.

Description

The construction method of the restructuring mould of citric acid output increased and application

Technical field

The present invention relates to technical field of bioengineering, more particularly to a kind of restructuring mould of citric acid output increased structure Method and application.

Background technology

Aspergillus niger is important industrial production bacterium, and carrying out Metabolically engineered research to aspergillus niger at present concentrates on central metabolites The transformation of path and respiratory chain, including glycolytic pathway, TCA circulation, the single-gene expression of the key enzyme of rTCA circulations and gene Coordinate expression, and alternative oxidase overexpression and knockout, but these influences to lemon acid yield of transformation are little, above-mentioned Only enhancing rTCA circulations approach promotes the raising of lemon acid yield in method.

In citric acid industry production, raw material is starch, and starch is filtered by after amylase liquefaction, is obtained clear liquid and is mixed Liquid, by clear liquid and mixed liquid blend and obtains seed and fermentation medium, and now the carbon source of about half is grape in culture medium Sugar, second half carbon source exists in the form of polyglucose.To be many and industrial production bacterium synthesizes a large amount of carbohydrase at the fermentation initial stage Polydextrose is decomposed into glucose, therefore, carbon source is basic in earlier fermentation culture medium exists in the form of glucose, so lemon Absorption of the lemon acid fermentation to carbon source actually refers to the absorption to glucose.

Torres determines aspergillus niger and absorbs glucose in the presence of 2 Km values, respectively 260 μM and 3.67mM, illustrates exist Two sets of movement systems of high-affinity and low-affinity, and the generation as needed for the movement system of low-affinity provides citric acid fermentation Xie Liu, but the movement system is only in concentration of glucose>50g·L^-1When work.Because the transhipment of glucose is citric acid fermentation First step, the movement system has in directly influence, therefore adjustment citric acid fermentation production process on citric acid fermentation Glucose transport systems, it is possible to strengthen the yield of citric acid.

The content of the invention

In order to solve the above technical problems, a kind of structure of the restructuring mould it is an object of the invention to provide citric acid output increased Construction method and application, by starting the expression of high-affinity GLUT, with the suction of strengthen glucose phase after fermentation Receive, and then improve the fermentation yield of citric acid in mould.

To achieve the above object, present invention employs following technical scheme：

On the one hand, it is the invention provides a kind of construction method for recombinating mould of citric acid output increased including following Step：

(1) HGT1 protein expression frames are built, HGT1 protein expressions frame connects comprising constitutive promoter and constitutive promoter The HGT1 protein coding genes for connecing and the terminator being connected with HGT1 protein coding genes；

(2) tolerant gene expression frame is built, tolerant gene expression frame connects comprising constitutive promoter and constitutive promoter The hph genes for connecing and the terminator being connected with hph genes；

(3) the tolerant gene expression frame that step (1) HGT1 protein expressions frame and step (2) are obtained is transformed into mould, Obtain recombinating mould.

Further, in step (1), HGT1 protein coding genes derive from aspergillus niger.

Further, in step (1), the nucleotide sequence of HGT1 protein coding genes is as shown in SEQ ID NO.1.

Further, HGT1 protein coding genes have the function of transhipment glucose, and the amino acid sequence of its coding can be sent out Give birth to substitution, missing or the insertion of one or more amino acid.

Preferably, the amino acid sequence of HGT1 protein coding genes coding is as shown in SEQ ID NO.2.

Further, in step (1) neutralization procedure (2), constitutive promoter be PgpdA promoters, gpdA promoters, Pmbf promoters or Pgla promoters.

Further, the sequence of PgpdA promoters is as shown in SEQ ID NO.3.

Further, in step (1) and step (2), terminator is trp terminators.

Further, the sequence of trp terminators is as shown in SEQ ID NO.4.

Further, in step (2), the sequence of hph genes is as shown in SEQ ID NO.5.

Further, in step (2), hygromycin gene expression cassette is built, is easy to the screening of positive clone molecule.It is anti- Property gene expression frame with pAN7-1 as template clone obtain.

Further, in step (3), mould is aspergillus niger, aspergillus oryzae or aspergillus nidulans.

Preferably, in step (3), mould be aspergillus niger Aspergillus niger H915-1, aspergillus oryzae 100-8 or Aspergillus nidulans FGSC A4.

Further, the construction method of HGT1 protein expressions frame, comprises the following steps：

(1) after by terminator sequence digestion, it is connected on pUC19 plasmids, obtains pUC-trp carriers；

(2) after by constitutive promoter sequence PgpdA digestions, it is connected on pUC-trp carriers, obtains pUC-PgpdA- Trp carriers；Constitutive promoter sequence PgpdA may be replaced by gpdA promoters, Pmbf promoters or Pgla promoters；

(3) after by HGT1 sequence double digestions, fragment is connected on pUC-PgpdA-trp carriers, obtains pUC-PgpdA- HGT1-trp expression cassettes.

Further, method for transformation is comprised the following steps：

(1) HGT1 protein expressions frame and tolerant gene expression frame are transferred in mould protoplast through PEG mediated methods；

(2) culture obtains positive colony on the hypertonic soft agar PDA plate of hygromycin resistance, then will be through culture 4-7 days Positive colony be transferred on the flat board containing 150-180mg/L hygromycin further culture and obtain positive colony, checking is correct As recombinate mould.

Due to mould, the phase absorbs glucose by expressing high-affinity GLUT after fermentation, based on this Research foundation, the present invention realizes expression of the high-affinity GLUT HGT1 in different moulds, to strengthen grape The absorption of sugared phase after fermentation.

On the other hand, opened the invention provides a kind of HGT1 protein expressions frame, including constitutive promoter and composing type The HGT1 protein coding genes of mover connection and the terminator being connected with HGT1 protein coding genes.

Further, constitutive promoter is PgpdA promoters, gpdA promoters, Pmbf promoters or Pgla promoters.

Further, HGT1 protein coding genes derive from aspergillus niger, and the nucleotide sequence of HGT1 protein coding genes is such as Shown in SEQ ID NO.1.

Further, terminator is trp terminators.

It yet still another aspect, present invention also offers a kind of restructuring trichoderma strain built by the above method.

Present invention also offers application of the restructuring mould constructed by the above method in fermentation production of citric acid.

Further, the condition of restructuring mold fermentation production citric acid of the invention is：Fermented at a temperature of 30-35 DEG C 72-120h, preferably rotating speed are 250r/min or so.

By such scheme, the present invention at least has advantages below：

The present invention starts HGT1 albumen and is expressed in mould using constitutive promoter, by strengthening fermentation later stage glucose Intake, so as to improve the yield of citric acid；The inventive method has general applicability to the Aspergillus niger strain for producing citric acid, can Fermentation of Aspergillus niger is set to produce citric acid output increased nearly 10%, by the use of Aspergillus niger H915-1 as host, lemon Lemon acid yield improves 14.7%, and fermentation period shortens 6h.

Brief description of the drawings

Fig. 1 is the content measuring result of citric acid and glucose in Aspergillus niger strain；

When Fig. 2 is fermenting and producing, citric acid than rate of producing acid test result.

Specific embodiment

With reference to the accompanying drawings and examples, specific embodiment of the invention is described in further detail.Hereinafter implement Example is not limited to the scope of the present invention for illustrating the present invention.

The extraction of the aspergillus niger RNA of embodiment 1

Aspergillus niger spore is inoculated into citric acid fermentation culture medium, 250r/min cultures 48h, uses at 35 DEG C Mirocloth filter clothes collect bacterium ball, then with aseptic milli-Q water 3 times, are filtered dry and rapid after moisture are freezed in liquid nitrogen.Use liquid nitrogen The method of grinding will be organized and fully ground, and aspergillus niger is extracted using QIAGEN companies RNeasy Plant Mini Kit kits Total serum IgE.With TAKARA companies PrimeScript RT reagent Kit with gDNA Eraser by RNA reverse transcriptions into cDNA。

The extraction of the aspergillus niger genomic DNA of embodiment 2

Aspergillus niger spore is inoculated into ME fluid nutrient mediums (3% malt extract, 0.5% tryptone), at 35 DEG C 250r/min cultivates 48h, and bacterium ball is collected with mirocloth filter clothes, then with aseptic milli-Q water 3 times, is filtered dry rapid after moisture Freezed in liquid nitrogen.To be organized with the method for liquid nitrogen grinding and fully ground, using QIAGEN companies DNeasy Plant Mini Kit extracts filamentous fungi genome.

The structure of the HGT1 protein expressions frame of embodiment 3 and hygromycin resistance expression cassette

Using primer trp-F (sequence is as shown in SEQ ID NO.6) and trp-R (sequence is as shown in SEQ ID NO.7) with PAN7-1 is template amplification trp terminators, and sequence upstream and downstream I containing Pst and Hin dIII restriction enzyme sites are connected on pMD19 and survey Sequence, then uses the two digestion with restriction enzyme, and the sequence is connected on the pUC19 of same digestion, obtains pUC19- trp。

Using primer Pgpd-F (sequence is as shown in SEQ ID NO.8) and Pgpd-R (sequence is as shown in SEQ ID NO.9) PgpdA promoters, sequence two RI containing Eco and Kpn I restriction enzyme sites are expanded from pAN7-1, after digestion, by sequence connection To the pUC19-trp of same digestion, pUC-PgpdA-trp is obtained.

Using primer HGT1-F (sequence is as shown in SEQ ID NO.10) and HGT1-R (sequence such as SEQ ID NO.11 institutes Show) HGT1 genes, gene two I containing Kpn and Pst I restriction enzyme sites are expanded in the aspergillus niger cDNA that is obtained from embodiment 1, It is connected on the pUC-PgpdA-trp of same digestion, forms PgpdA-HGT1-trp expression cassettes.

Hygromycin resistance expression cassette passes through primer gpd-F (sequence is as shown in SEQ ID NO.12) and Ttrp-R-2 (sequences As shown in SEQ ID NO.13) expanded from plasmid pAN7-1 and obtain.

In the present embodiment, amplification condition is as follows：94 DEG C of predegeneration 3min, then carry out 30 circulations, 94 DEG C of 20s, 55 DEG C 20s and 72 DEG C of 3min, last 72 DEG C of reactions 10min.

In the present embodiment, digestion condition is as follows：37 DEG C of digestions 2 hours.

The preparation and conversion of the aspergillus niger protoplast of embodiment 4

By 3 × 10⁵Aspergillus niger spore is seeded to PDA liquid medium by the concentration of/ml, the 200r/min cultures at 30 DEG C Overnight.Bacterium ball is collected with mirocloth filter clothes, and with sterile water wash bacterium ball.A certain amount of lyases is weighed, and uses osmotic pressure Stabilizer KMC dissolves, and uses sterilised membrane filter filtration sterilization.

Weigh a certain amount of bacterium ball, be added in enzymolysis liquid, 37 DEG C, 100r/min concussion and cultivates about 3h until mycelia disappear completely Turn to protoplast, 4 DEG C, 1000rpm centrifugation 10min abandon supernatant, add the STC of same volume precooling, 4 DEG C, 1000rpm from Heart 10min, abandons supernatant, washs 2 times, adds 100 μ L STC, mixes.

To adding 10 μ L linearized nucleic acids fragment (HGT1 protein expressions frame and hygromycin in 100 μ L aspergillus niger protoplasts Resistance expression's frame) and 330 μ L PEG buffer solutions, 20min is placed on ice, 2mL PEG are added, room temperature is placed 10min, is sequentially added 4mL STC and 4mL is plated on the lower floor's culture medium containing 180mg/L hygromycin in 48 DEG C of upper strata culture mediums of preheating.Flat board Culture 4-7 days is inverted at 35 DEG C, until there is bacterium colony, picking single bacterium colony subculture.Each bacterium colony carries out 3 monospore subcultures.

The aspergillus niger of embodiment 5 converts the checking of suboutput

Aspergillus niger is inoculated in PDA culture medium, raw spore culture 5-7 days, scrape spore, with 10 at 35 DEG C⁶The inoculation of/mL Amount is inoculated in seed culture medium, 37 DEG C, 250r/min culture 24h, with 1/10 inoculum concentration switching fermentation medium, 35 DEG C, 250r/ Min fermentations 72h.Zymotic fluid centrifugation removal thalline, dilutes 10 times, and citric acid content is detected with HPLC methods with after membrane filtration, HPLC method testing conditions are as follows：

Instrument：The high performance liquid chromatographs of Agilent 1200 (match somebody with somebody UV-vis detector, Composition distribution and work station)； Chromatographic condition：HPX87H chromatographic columns (4.6 × 250mm, 5 μm), mobile phase is 5mM sulfuric acid solutions, and flow is 0.6mL/min, is entered Sample amount is 10 μ L, and column temperature is 30 DEG C, the detection of 210nm length ultraviolets light.

Fig. 1 is reflected in different strains, and the content results of citric acid and glucose, wherein H915-1 represent wild black song Trichoderma strain, HGT1 represents recombinant bacterial strain of the invention, it can be seen that the citric acid of recombinant aspergillus niger bacterial strain of the invention Yield is improved, and its yield improves 14.6% or so, and fermentation later stage than wild-type strain, and the content of glucose is lower, Illustrate that HGT1 albumen promotes the absorption of glucose, and the enhancing of glucose absorption can increase lemon acid yield.

Fig. 2 is the citric acid of above two bacterial strain than rate of producing acid test result, it can be seen that recombinant bacterial strain of the invention Citric acid output speed it is higher, reason is that the overexpression of HGT1 albumen enhances the transhipment of glucose, can carry high acid Speed, illustrates that glucose absorption is to improve the Steps of lemon acid yield.Compared with wild type Aspergillus niger bacterial strain, the present invention Recombinant aspergillus niger bacterial strain fermentation time reduction 6h.

The above is only the preferred embodiment of the present invention, is not intended to limit the invention, it is noted that for this skill For the those of ordinary skill in art field, on the premise of the technology of the present invention principle is not departed from, can also make it is some improvement and Modification, these are improved and modification also should be regarded as protection scope of the present invention.

Sequence table

<110>Southern Yangtze University

<120>The construction method of the restructuring mould of citric acid output increased and application

<160> 13

<170> PatentIn version 3.3

<210> 1

<211> 1644

<212> DNA

<213>HGT1 genes

<400> 1

1 ATGTTGATTG GCAACATCTA CGTGATTGCG AGCGTCGCCG TCGTCGGCGG CGGCTTGTTT

61 GGCTTTGATA TTTCCTCCAT GTCCGCTCAG CTGAGCGAGA ATTCCTATCT ATGCTATTTC

121 AACCAGGGCC CTAAGGGTCC TCCCTTCACT GACGATGAGG ACTGTTCTGG TCCCACATCC

181 CTCAACCAGG GTGGCATCAC GGCAGCCATG GCAGCTGGAT CTTGGCTGGG TGCTTTGATT

241 TCAGGTCCGC TTTCCGATCG CATCGGACGT AAGACGTCCA TCATGGTCGG CTGTGTCGTT

301 TGGCTGATTG GTTCCACCAT TATGTGCGCT TCTCAGAACA TCGGTATGCT CGTTGTTGGG

361 CGGGTCATCA ACGGTCTCGC GGTGGGCATT GAGTCTGCTC AGGTCCCCGT CTACATCAGT

421 GAACTTTCGC CACCCTCCAA GCGCGGTCGG TTCGTAGGTA TGCAGCAATG GGCCATTACA

481 TGGGGTATCC TCATTATGTT CTATATCTCG TATGGCTGCT CCTTCATCGG GGGACAGAAG

541 TCCTACAACT ACAGCACTGC TTCTTGGCGA GTCCCCTGGG GCTTGCAGAT GCTGCCGGCT

601 GTTTTCCTTT TCCTTGGAAT GCTGGTTCTG CCCGAATCTC CTCGTTGGCT GGCGCGCAAG

661 GATCGTTGGG AGGACTGCCA TCGGGTCTTG GCTCTCGTCC ACGCCAAGGG GGATCTCAAC

721 CATCCTTTCG TTGCGCTAGA GTTGCAGGAC ATTCGGGATA TGTGTGAGCT TGAACGTCAG

781 TTCAAAGATG TCACTTACCT TGACCTGTTC AAACCGAGGA TGATCAACCG CACGTTGATC

841 GGTCTGTTCA TGCAGATCTG GTCTCAGCTG ACCGGCATGA ATGTGATGAT GTACTACATC

901 ACGTATCTCT TTTCTATGGC TGGATACACC GGCGATTCCA CCCTCCTTGC CTCCTCCATT

961 CAGTATATTA TCAATGTGTT TATGACCCTC CCGGCACTGA TCTGGATGGA CAAGTGGGGT

1021 CGTCGCATGC CATTGCTGGT TGGCGCAGCT CTGATGGCCA TCTTGATGTA TGCCAATGGT

1081 GCGATCATGG CGGTTCATGG TGTTGTGGTG CCCGGGGGCA TCAATGGGGT TGCAGCTGAG

1141 TCCATGCGTC TTCACGGCGC TCCTGCCAAA GGTTTGATTG CTTGCACATA CCTCTTCGTC

1201 GCGTCGTATG CGCCTACCTG GGGTCCTGTA TCCTGGACGT ACCCCCCTGA GCTGTATCCG

1261 CTGCGGCTGC GTGGTAAGGG AGTGGCTTTG TCGACCTCAG GTAACTGGGC GTTTAACACT

1321 GCATTGGGGC TGTTTACACC CACTGCATTT GAGAACATCC GCTGGAAGAC CTACATCATG

1381 TTTGGGGTGT TCAACACTGC CATGTTCTTG CACGTGCTGT TCCTGTTCCC GGAGACTGCA

1441 GGAAAGACGC TCGAAGAGAC CGAAGCCATG TTCGAGGATC CCAACGGCAT CAAGTACATT

1501 GGCACACCGG CCTGGAAGAC CAAGATGAAG ACCCGGCAGG TGGAGCAGCT GGAGCACGGT

1561 CAAGTGGATA TAGAGTCCAA GATTGAAACC CGCCACGCAG AGACCACTGA GACCGCTCCC

1621 AAATCCCAGG AGGCCACAGC ATAA

<210> 2

<211> 547

<212>Amino acid

<213>The amino acid of HGT1 protein coding genes coding

<400> 2

Met Leu Ile Gly Asn Ile Tyr Val Ile Ala Ser Val Ala Val Val

1 5 10 15

Gly Gly Gly Leu Phe Gly Phe Asp Ile Ser Ser Met Ser Ala Gln

20 25 30

Leu Ser Glu Asn Ser Tyr Leu Cys Tyr Phe Asn Gln Gly Pro Lys

35 40 45

Gly Pro Pro Phe Thr Asp Asp Glu Asp Cys Ser Gly Pro Thr Ser

50 55 60

Leu Asn Gln Gly Gly Ile Thr Ala Ala Met Ala Ala Gly Ser Trp

65 70 75

Leu Gly Ala Leu Ile Ser Gly Pro Leu Ser Asp Arg Ile Gly Arg

80 85 90

Lys Thr Ser Ile Met Val Gly Cys Val Val Trp Leu Ile Gly Ser

95 100 105

Thr Ile Met Cys Ala Ser Gln Asn Ile Gly Met Leu Val Val Gly

110 115 120

Arg Val Ile Asn Gly Leu Ala Val Gly Ile Glu Ser Ala Gln Val

125 130 135

Pro Val Tyr Ile Ser Glu Leu Ser Pro Pro Ser Lys Arg Gly Arg

140 145 150

Phe Val Gly Met Gln Gln Trp Ala Ile Thr Trp Gly Ile Leu Ile

155 160 165

Met Phe Tyr Ile Ser Tyr Gly Cys Ser Phe Ile Gly Gly Gln Lys

170 175 180

Ser Tyr Asn Tyr Ser Thr Ala Ser Trp Arg Val Pro Trp Gly Leu

185 190 195

Gln Met Leu Pro Ala Val Phe Leu Phe Leu Gly Met Leu Val Leu

200 205 210

Pro Glu Ser Pro Arg Trp Leu Ala Arg Lys Asp Arg Trp Glu Asp

215 220 225

Cys His Arg Val Leu Ala Leu Val His Ala Lys Gly Asp Leu Asn

230 235 240

His Pro Phe Val Ala Leu Glu Leu Gln Asp Ile Arg Asp Met Cys

245 250 255

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

260 265 270

Lys Pro Arg Met Ile Asn Arg Thr Leu Ile Gly Leu Phe Met Gln

275 280 285

Ile Trp Ser Gln Leu Thr Gly Met Asn Val Met Met Tyr Tyr Ile

290 295 300

Thr Tyr Leu Phe Ser Met Ala Gly Tyr Thr Gly Asp Ser Thr Leu

305 310 315

Leu Ala Ser Ser Ile Gln Tyr Ile Ile Asn Val Phe Met Thr Leu

320 325 330

Pro Ala Leu Ile Trp Met Asp Lys Trp Gly Arg Arg Met Pro Leu

335 340 345

Leu Val Gly Ala Ala Leu Met Ala Ile Leu Met Tyr Ala Asn Gly

350 355 360

Ala Ile Met Ala Val His Gly Val Val Val Pro Gly Gly Ile Asn

365 370 375

Gly Val Ala Ala Glu Ser Met Arg Leu His Gly Ala Pro Ala Lys

380 385 390

Gly Leu Ile Ala Cys Thr Tyr Leu Phe Val Ala Ser Tyr Ala Pro

395 400 405

Thr Trp Gly Pro Val Ser Trp Thr Tyr Pro Pro Glu Leu Tyr Pro

410 415 420

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

425 430 435

Trp Ala Phe Asn Thr Ala Leu Gly Leu Phe Thr Pro Thr Ala Phe

450 440 445 450

Glu Asn Ile Arg Trp Lys Thr Tyr Ile Met Phe Gly Val Phe Asn

455 460 465

Thr Ala Met Phe Leu His Val Leu Phe Leu Phe Pro Glu Thr Ala

470 475 480

Gly Lys Thr Leu Glu Glu Thr Glu Ala Met Phe Glu Asp Pro Asn

485 490 495

Gly Ile Lys Tyr Ile Gly Thr Pro Ala Trp Lys Thr Lys Met Lys

500 505 510

Thr Arg Gln Val Glu Gln Leu Glu His Gly Gln Val Asp Ile Glu

515 520 525

Ser Lys Ile Glu Thr Arg His Ala Glu Thr Thr Glu Thr Ala Pro

530 535 540

Lys Ser Gln Glu Ala Thr Ala

545

<210> 3

<211> 2310

<212> DNA

<213>PgpdA promoters

<400> 3

1 CAATTCCCTT GTATCTCTAC ACACAGGCTC AAATCAATAA GAAGAACGGT TCGTCTTTTT

61 CGTTTATATC TTGCATCGTC CCAAAGCTAT TGGCGGGATA TTCTGTTTGC AGTTGGCTGA

121 CTTGAAGTAA TCTCTGCAGA TCTTTCGACA CTGAAATACG TCGAGCCTGC TCCGCTTGGA

181 AGCGGCGAGG AGCCTCGTCC TGTCACAACT ACCAACATGG AGTACGATAA GGGCCAGTTC

241 CGCCAGCTCA TTAAGAGCCA GTTCATGGGC GTTGGCATGA TGGCCGTCAT GCATCTGTAC

301 TTCAAGTACA CCAACCCTCT TCTGATCCAG TCGATCATCC CGCTGAAGGG CGCTTTCGAA

361 TCGAATCTGG TTAAGATCCA CGTCTTCGGG AAGCCAGCGA CTGGTGACCT CCAGCGTCCC

421 TTTAAGGCTG CCAACAGCTT TCTCAGCCAG GGCCAGCCCA AGACCGACAA GGCCTCCCTC

481 CAGAACGCCG AGAAGAACTG GAGGGGTGGT GTCAAGGAGG AGTAAGCTCC TTATTGAAGT

541 CGGAGGACGG AGCGGTGTCA AGAGGATATT CTTCGCTCTG TATTATAGAT AAGATGATGA

601 GGAATTGGAG GTAGCATAGC TTCATTTGGA TTTGCTTTCC AGGCTGAGAC TCTAGCTTGG

661 AGCATAGAGG GTCCCTTTGG CTTTCAATAT TCTCAAGTAT CTCGAGTTTG AACTTATTCC

721 CGTGAACCTT TTATTCACCA ATGAGCATTG GAATGAACAT GAATCTGAGG ACTGCAATCG

781 CCATGAGGTT TTCGAAATAC ATCCGGATGT CGAAGGCTTG GGGCACCTGC GTTGGTTGAA

841 TTTAGAACGT GGCACTATTG ATCATCCGAT AGCTCTGCAA AGGGCGTTGC ACAATGCAAG

901 TCAAACGTTG CTAGCAGTTC CAGGTGGAAT GTTATGATGA GCATTGTATT AAATCAGGAG

961 ATATAGCATG ATCTCTAGTT AGCTCACCAC AAAAGTCAGA CGGCGTAACC AAAAGTCACA

1021 CAACACAAGC TGTAAGGATT TCGGCACGGC TACGGAAGAC GGAGAAGCCC ACCTTCAGTG

1081 GACTCGAGTA CCATTTAATT CTATTTGTGT TTGATCGAGA CCTAATACAG CCCCTACAAC

1141 GACCATCAAA GTCGTATAGC TACCAGTGAG GAAGTGGACT CAAATCGACT TCAGCAACAT

1201 CTCCTGGATA AACTTTAAGC CTAAACTATA CAGAATAAGA TGGTGGAGAG CTTATACCGA

1261 GCTCCCAAAT CTGTCCAGAT CATGGTTGAC CGGTGCCTGG ATCTTCCTAT AGAATCATCC

1321 TTATTCGTTG ACCTAGCTGA TTCTGGAGTG ACCCAGAGGG TCATGACTTG AGCCTAAAAT

1381 CCGCCGCCTC CACCATTTGT AGAAAAATGT GACGAACTCG TGAGCTCTGT ACAGTGACCG

1441 GTGACTCTTT CTGGCATGCG GAGAGACGGA CGGACGCAGA GAGAAGGGCT GAGTAATAAG

1501 CGCCACTGCG CCAGACAGCT CTGGCGGCTC TGAGGTGCAG TGGATGATTA TTAATCCGGG

1561 ACCGGCCGCC CCTCCGCCCC GAAGTGGAAA GGCTGGTGTG CCCCTCGTTG ACCAAGAATC

1621 TATTGCATCA TCGGAGAATA TGGAGCTTCA TCGAATCACC GGCAGTAAGC GAAGGAGAAT

1681 GTGAAGCCAG GGGTGTATAG CCGTCGGCGA AATAGCATGC CATTAACCTA GGTACAGAAG

1741 TCCAATTGCT TCCGATCTGG TAAAAGATTC ACGAGATAGT ACCTTCTCCG AAGTAGGTAG

1801 AGCGAGTACC CGGCGCGTAA GCTCCCTAAT TGGCCCATCC GGCATCTGTA GGGCGTCCAA

1861 ATATCGTGCC TCTCCTGCTT TGCCCGGTGT ATGAAACCGG AAAGGCCGCT CAGGAGCTGG

1921 CCAGCGGCGC AGACCGGGAA CACAAGCTGG CAGTCGACCC ATCCGGTGCT CTGCACTCGA

1981 CCTGCTGAGG TCCCTCAGTC CCTGGTAGGC AGCTTTGCCC CGTCTGTCCG CCCGGTGTGT

2041 CGGCGGGGTT GACAAGGTCG TTGCGTCAGT CCAACATTTG TTGCCATATT TTCCTGCTCT

2101 CCCCACCAGC TGCTCTTTTC TTTTCTCTTT CTTTTCCCAT CTTCAGTATA TTCATCTTCC

2161 CATCCAAGAA CCTTTATTTC CCCTAAGTAA GTACTTTGCT ACATCCATAC TCCATCCTTC

2221 CCATCCCTTA TTCCTTTGAA CCTTTCAGTT CGAGCTTTCC CACTTCATCG CAGCTTGACT

2281 AACAGCTACC CCGCTTGAGC AGACATCACC

<210> 4

<211> 771

<212> DNA

<213>Trp terminators

<400> 4

1 GATCCACTTA ACGTTACTGA AATCATCAAA CAGCTTGACG AATCTGGATA TAAGATCGTT

61 GGTGTCGATG TCAGCTCCGG AGTTGAGACA AATGGTGTTC AGGATCTCGA TAAGATACGT

121 TCATTTGTCC AAGCAGCAAA GAGTGCCTTC TAGTGATTTA ATAGCTCCAT GTCAACAAGA

181 ATAAAACGCG TTTCGGGTTT ACCTCTTCCA GATACAGCTC ATCTGCAATG CATTAATGCA

241 TTGGACCTCG CAACCCTAGT ACGCCCTTCA GGCTCCGGCG AAGCAGAAGA ATAGCTTAGC

301 AGAGTCTATT TTCATTTTCG GGAGACGAGA TCAAGCAGAT CAACGGTCGT CAAGAGACCT

361 ACGAGACTGA GGAATCCGCT CTTGGCTCCA CGCGACTATA TATTTGTCTC TAATTGTACT

421 TTGACATGCT CCTCTTCTTT ACTCTGATAG CTTGACTATG AAAATTCCGT CACCAGCCCC

481 TGGGTTCGCA AAGATAATTG CACTGTTTCT TCCTTGAACT CTCAAGCCTA CAGGACACAC

541 ATTCATCGTA GGTATAAACC TCGAAAATCA TTCCTACTAA GATGGGTATA CAATAGTAAC

601 CATGGTTGCC TAGTGAATGC TCCGTAACAC CCAATACGCC GGCCGAAACT TTTTTACAAC

661 TCTCCTATGA GTCGTTTACC CAGAATGCAC AGGTACACTT GTTTAGAGGT AATCCTTCTT

721 TCTAGAAGTC CTCGTGTACT GTGTAAGCGC CCACTCCACA TCTCCACTCG A

<210> 5

<211> 1020

<212> DNA

<213>Hph terminators

<400> 5

1 ATGCCTGAAC TCACCGCGAC GTCTGTCGAG AAGTTTCTGA TCGAAAAGTT CGACAGCGTC

61 TCCGACCTGA TGCAGCTCTC GGAGGGCGAA GAATCTCGTG CTTTCAGCTT CGATGTAGGA

121 GGGCGTGGAT ATGTCCTGCG GGTAAATAGC TGCGCCGATG GTTTCTACAA AGATCGTTAT

181 GTTTATCGGC ACTTTGCATC GGCCGCGCTC CCGATTCCGG AAGTGCTTGA CATTGGGGAA

241 TTCAGCGAGA GCCTGACCTA TTGCATCTCC CGCCGTGCAC AGGGTGTCAC GTTGCAAGAC

301 CTGCCTGAAA CCGAACTGCC CGCTGTTCTG CAGCCGGTCG CGGAGGCCAT GGATGCGATC

361 GCTGCGGCCG ATCTTAGCCA GACGAGCGGG TTCGGCCCAT TCGGACCGCA AGGAATCGGT

421 CAATACACTA CATGGCGTGA TTTCATATGC GCGATTGCTG ATCCCCATGT GTATCACTGG

481 CAAACTGTGA TGGACGACAC CGTCAGTGCG TCCGTCGCGC AGGCTCTCGA TGAGCTGATG

541 CTTTGGGCCG AGGACTGCCC CGAAGTCCGG CACCTCGTGC ACGCGGATTT CGGCTCCAAC

601 AATGTCCTGA CGGACAATGG CCGCATAACA GCGGTCATTG ACTGGAGCGA GGCGATGTTC

661 GGGGATTCCC AATACGAGGT CGCCAACATC TTCTTCTGGA GGCCGTGGTT GGCTTGTATG

721 GAGCAGCAGA CGCGCTACTT CGAGCGGAGG CATCCGGAGC TTGCAGGATC GCCGCGGCTC

781 CGGGCGTATA TGCTCCGCAT TGGTCTTGAC CAACTCTATC AGAGCTTGGT TGACGGCAAT

841 TTCGATGATG CAGCTTGGGC GCAGGGTCGA TGCGACGCAA TCGTCCGATC CGGAGCCGGG

901 ACTGTCGGGC GTACACAAAT CGCCCGCAGA AGCGCGGCCG TCTGGACCGA TGGCTGTGTA

961 GAAGTACTCG CCGATAGTGG AAACCGACGC CCCAGCACTC GTCCGAGGGC AAAGGAATAG

<210> 6

<211> 31

<212> DNA

<213>Primer trp-F

<400> 6

1 ctgcaggatc cacttaaacg ttactgaaat c

<210> 7

<211> 28

<212> DNA

<213>Primer trp-R

<400> 7

1 AAGCTTCTCG AGTGGAGATG TGGAGTGG

<210> 8

<211> 40

<212> DNA

<213>Primer Pgpd-F

<400> 8

1 GAATTCGCGG CCGCCAATTC CCTTGTATCT CTACACACAG

<210> 9

<211> 26

<212> DNA

<213>Primer Pgpd-R

<400> 9

1 GGTACCGGTG ATGTCTGCTC AAGCGG

<210> 10

<211> 45

<212> DNA

<213>Primer HGT1-F

<400> 10

1 CTTGAGCAGA CATCACCGGT ACCATGTTGA TTGGCAACAT CTACG

<210> 11

<211> 44

<212> DNA

<213>Primer HGT1-R

<400> 11

1 TAACGTTTAA GTGGATCGGA TCCTTATGCT GTGGCCTCCT GGGA

<210> 12

<211> 26

<212> DNA

<213>Primer gpd-F

<400> 12

1 CAATTCCCTT GTATCTCTAC ACACAG

<210> 13

<211> 22

<212> DNA

<213>Primer Ttrp-R-2

<400> 13

1 CTCGAGTGGA GATGTGGAGT GG

Claims

1. a kind of citric acid output increased restructuring mould construction method, it is characterised in that comprise the following steps：

(1) HGT1 protein expression frames are built, the HGT1 protein expressions frame starts comprising constitutive promoter and the composing type The HGT1 protein coding genes of son connection and the terminator being connected with the HGT1 protein coding genes；

(2) tolerant gene expression frame is built, the tolerant gene expression frame starts comprising constitutive promoter and the composing type The hph genes of son connection and the terminator being connected with the hph genes；

(3) the tolerant gene expression frame that step (1) the HGT1 protein expressions frame and step (2) are obtained is transformed into mould In, obtain the restructuring mould.

2. citric acid output increased according to claim 1 restructuring mould construction method, it is characterised in that：In step (1) in, the HGT1 protein coding genes derive from aspergillus niger.

3. citric acid output increased according to claim 2 restructuring mould construction method, it is characterised in that：In step (1) in, the nucleotide sequence of the HGT1 protein coding genes is as shown in SEQ ID NO.1.

4. citric acid output increased according to claim 1 restructuring mould construction method, it is characterised in that：In step (1) in neutralization procedure (2), the constitutive promoter is opened for PgpdA promoters, gpdA promoters, Pmbf promoters or Pgla Mover.

5. citric acid output increased according to claim 1 restructuring mould construction method, it is characterised in that：In step (1) and in step (2), the terminator is trp terminators.

6. citric acid output increased according to claim 1 restructuring mould construction method, it is characterised in that：In step (3) in, the mould is aspergillus niger, aspergillus oryzae or aspergillus nidulans.

7. a kind of HGT1 protein expressions frame, including constitutive promoter and the HGT1 albumen of constitutive promoter connection are compiled Code gene and the terminator being connected with the HGT1 protein coding genes.

8. HGT1 protein expressions frame according to claim 7, it is characterised in that：The constitutive promoter is opened for PgpdA Mover, gpdA promoters, Pmbf promoters or Pgla promoters.

9. HGT1 protein expressions frame according to claim 7, it is characterised in that：The HGT1 protein coding genes are derived from Aspergillus niger, the nucleotide sequence of the HGT1 protein coding genes is as shown in SEQ ID NO.1.

10. HGT1 protein expressions frame according to claim 7, it is characterised in that：The terminator is trp terminators.

A kind of 11. restructuring moulds built by any one of claim 1-6 method.

12. applications of the restructuring mould in fermentation production of citric acid built according to any one of claim 1-6 method.