CN106754435B

CN106754435B - Construction method and application of recombinant mold with increased citric acid yield

Info

Publication number: CN106754435B
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: 2020-04-21
Anticipated expiration: 2036-12-28
Also published as: CN106754435A

Abstract

The invention relates to a construction method of recombinant mold with improved citric acid yield, which comprises the following steps: constructing an HGT1 protein expression frame, wherein the HGT1 protein expression frame comprises a constitutive promoter, an HGT1 protein coding gene connected with the constitutive promoter and a terminator connected with the HGT1 protein coding gene; constructing a resistance gene expression frame, wherein the resistance gene expression frame comprises a constitutive promoter, an hph gene connected with the constitutive promoter and a terminator connected with the hph gene; and (3) transforming the HGT1 protein expression cassette and the resistance gene expression cassette into the mould to obtain the recombinant mould. The invention also provides an HGT1 protein expression cassette and a recombinant mould which are constructed by adopting the method. The invention also discloses application of the recombinant mold constructed by the method in fermentation production of citric acid. According to the invention, the high-affinity glucose transporter expression is started, so that the yield of citric acid in the mould is improved, and the fermentation time is greatly shortened.

Description

Construction method and application of recombinant mold with increased citric acid yield

Technical Field

The invention relates to the technical field of bioengineering, in particular to a construction method and application of recombinant mold with improved citric acid yield.

Background

Aspergillus niger is an important industrial production bacterium, and the current research on the metabolic modification of Aspergillus niger focuses on the modification of a central metabolic pathway and a respiratory chain, including single-gene expression and gene synergistic expression of key enzymes of a glycolysis pathway, a TCA cycle and an rTCA cycle, and overexpression and knockout of alternative oxidase, but the modification has little influence on the yield of citric acid, and only the enhancement of the rTCA cycle pathway in the method promotes the improvement of the yield of citric acid.

In the industrial production of citric acid, starch is used as a raw material, the starch is liquefied by amylase and then filtered to obtain clear liquid and mixed liquid, and the clear liquid and the mixed liquid are blended to obtain seeds and a fermentation culture medium, wherein about half of carbon sources in the culture medium are glucose, and the other half of carbon sources exist in the form of polyglucose. However, since industrial production bacteria synthesize a large amount of saccharifying enzymes at the initial stage of fermentation and decompose polyglucose into glucose, the carbon source is present substantially in the form of glucose in the medium at the early stage of fermentation, and thus the absorption of the carbon source by citric acid fermentation is actually referred to as the absorption of glucose.

Torres determines that 2 Km values of glucose absorption of Aspergillus niger are respectively 260 μ M and 3.67mM, which indicates that two sets of transport systems with high affinity and low affinity exist, and the metabolic flow required by citric acid fermentation is provided by the transport system with low affinity, but the transport system is only at glucose concentration>50g·L^-1It is effective. Since the glucose transport is the first step of citric acid fermentation, the transport system has direct influence on citric acid fermentation, so that the glucose transport system in the citric acid fermentation production process is adjusted, and the yield of citric acid can be enhanced.

Disclosure of Invention

In order to solve the technical problems, the invention aims to provide a construction method and application of recombinant mold with improved citric acid yield, wherein the absorption of glucose in the later fermentation stage is enhanced by starting the expression of high-affinity glucose transporter, so that the fermentation yield of citric acid in the mold is improved.

In order to achieve the purpose, the invention adopts the following technical scheme:

in one aspect, the present invention provides a method for constructing a recombinant mold with increased citric acid yield, comprising the steps of:

(1) constructing an HGT1 protein expression cassette, wherein the HGT1 protein expression cassette comprises a constitutive promoter, an HGT1 protein coding gene connected with the constitutive promoter and a terminator connected with the HGT1 protein coding gene;

(2) constructing a resistance gene expression frame, wherein the resistance gene expression frame comprises a constitutive promoter, an hph gene connected with the constitutive promoter and a terminator connected with the hph gene;

(3) and (3) transforming the HGT1 protein expression frame in the step (1) and the resistance gene expression frame obtained in the step (2) into a mould to obtain the recombinant mould.

Further, in step (1), the gene encoding HGT1 protein is derived from Aspergillus niger.

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

Further, the gene encoding HGT1 protein has a function of transporting glucose, and the encoded amino acid sequence thereof may have substitution, deletion or insertion of 1 or more amino acids.

Preferably, the amino acid sequence coded by the HGT1 protein coding gene is shown as SEQ ID NO. 2.

Further, in the step (1) and the step (2), the constitutive promoter is PgpdA promoter, gpdA promoter, Pmbf promoter or Pgla promoter.

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

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

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

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

Further, in step (2), a hygromycin resistance gene expression cassette is constructed to facilitate screening of positive clones. The resistance gene expression cassette was cloned using pAN7-1 as a template.

Further, in the step (3), the mold is Aspergillus niger, Aspergillus oryzae or Aspergillus nidulans.

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

Further, the construction method of the HGT1 protein expression cassette comprises the following steps:

(1) after the terminator sequence is cut by enzyme, the terminator sequence is connected to a pUC19 plasmid to obtain a pUC-trp vector;

(2) after the constitutive promoter sequence PgpdA is cut by enzyme, the promoter sequence PgpdA is connected to a pUC-trp vector to obtain a pUC-PgpdA-trp vector; the constitutive promoter sequence PgpdA may also be replaced by a gpdA promoter, a Pmbf promoter or a Pgla promoter;

(3) after double cleavage of the HGT1 sequence, the fragment was ligated into the pUC-PgpdA-trp vector to obtain the pUC-PgpdA-HGT1-trp expression cassette.

Further, the transformation method comprises the following steps:

(1) transferring the HGT1 protein expression frame and the resistance gene expression frame into a mould protoplast by a PEG mediated method;

(2) culturing on a hygromycin-resistant hypertonic soft agar PDA plate to obtain positive clones, transferring the positive clones cultured for 4-7 days to a hygromycin-containing plate containing 150-180mg/L of hygromycin for further culture to obtain the positive clones, and verifying that the positive clones are the recombinant molds.

Based on the research foundation that the moulds absorb glucose by expressing the high-affinity glucose transporter at the later fermentation stage, the invention realizes the expression of the high-affinity glucose transporter HGT1 in different moulds so as to enhance the absorption of glucose at the later fermentation stage.

In another aspect, the invention provides an HGT1 protein expression cassette comprising a constitutive promoter, an HGT1 protein encoding gene linked to the constitutive promoter, and a terminator linked to the HGT1 protein encoding gene.

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

Furthermore, the HGT1 protein coding gene is derived from Aspergillus niger, and the nucleotide sequence of the HGT1 protein coding gene is shown in SEQ ID NO. 1.

Further, the terminator is a trp terminator.

In yet another aspect, the present invention also provides a recombinant mold strain constructed by the above method.

The invention also provides application of the recombinant mold constructed by the method in fermentation production of citric acid.

Further, the conditions for producing citric acid by fermenting the recombinant mold are as follows: fermenting at 30-35 deg.C for 72-120h, preferably at 250 r/min.

By the scheme, the invention at least has the following advantages:

according to the invention, a constitutive promoter is utilized to start the HGT1 protein to express in the mould, and the glucose uptake at the later fermentation stage is enhanced, so that the yield of citric acid is increased; the method has universal applicability to Aspergillus niger strains for producing citric acid, can improve the yield of citric acid produced by fermenting Aspergillus niger by nearly 10 percent, improves the yield of citric acid by 14.7 percent by using Aspergillus niger H915-1 as a host, and shortens the fermentation period by 6 hours.

Drawings

FIG. 1 shows the results of the citric acid and glucose content test in Aspergillus niger strains;

FIG. 2 shows the results of the specific acid production rate test of citric acid in fermentation production.

Detailed Description

The following detailed description of embodiments of the present invention is provided in connection with the accompanying drawings and examples. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

EXAMPLE 1 extraction of Aspergillus niger RNA

Inoculating Aspergillus niger spores into a citric acid fermentation culture medium, culturing at 35 ℃ for 48h at 250r/min, collecting bacterial balls by using mirocloth filter cloth, washing for 3 times by using sterile ultrapure water, draining water, and quickly freezing in liquid nitrogen. The tissue was ground thoroughly by liquid nitrogen milling and Aspergillus niger total RNA was extracted using the QIAGEN RNeasy Plant Mini Kit. RNA was reverse transcribed into cDNA using the TAKARA PrimeScript RT reagent Kit with gDNA Eraser.

Example 2 extraction of genomic DNA from Aspergillus niger

Inoculating Aspergillus niger spores into ME liquid culture medium (3% malt extract, 0.5% tryptone), culturing at 35 deg.C for 48h at 250r/min, collecting the pellet with mirocloth filter cloth, washing with sterile ultrapure water for 3 times, draining, and quickly freezing in liquid nitrogen. The tissue was ground thoroughly by liquid nitrogen milling and the filamentous fungal genome was extracted by DNeasy Plant MiniKit from QIAGEN.

Example 3 construction of the HGT1 protein expression cassette and the hygromycin resistance expression cassette

The trp terminator is amplified by using primers trp-F (the sequence is shown as SEQ ID NO. 6) and trp-R (the sequence is shown as SEQ ID NO. 7) and taking pAN7-1 as a template, the upstream and the downstream of the sequence contain Pst I and HindIII enzyme cutting sites, the sequence is connected to pMD19 for sequencing, then the sequence is cut by the two restriction enzymes, and the sequence is connected to pUC19 cut by the same enzyme, so that pUC19-trp is obtained.

The PgpdA promoter was amplified from pAN7-1 using primers Pgpd-F (SEQ ID NO. 8) and Pgpd-R (SEQ ID NO. 9) containing Eco RI and Kpn I cleavage sites at both ends, and after cleavage, this sequence was ligated to pUC19-trp, which was cleaved similarly, to obtain pUC-PgpdA-trp.

The HGT1 gene was amplified from the A.niger cDNA obtained in example 1 using the primers HGT1-F (SEQ ID NO. 10) and HGT1-R (SEQ ID NO. 11), both ends of the gene containing Kpn I and Pst I cleavage sites, and ligated to pUC-PgpdA-trp digested with the same enzyme to form PgpdA-HGT1-trp expression cassette.

The hygromycin-resistant expression cassette was amplified from plasmid pAN7-1 using primers gpd-F (SEQ ID NO. 12) and Ttrp-R-2 (SEQ ID NO. 13).

In this example, the amplification conditions were as follows: pre-denaturation at 94 ℃ for 3min, followed by 30 cycles of 94 ℃ for 20s, 55 ℃ for 20s and 72 ℃ for 3min, and finally reaction at 72 ℃ for 10 min.

In this example, the enzyme digestion conditions were as follows: the enzyme was cleaved at 37 ℃ for 2 hours.

Example 4 preparation and transformation of Aspergillus niger protoplasts

According to 3X 10⁵Aspergillus niger spores were inoculated into PDA liquid medium at a concentration of 200r/min at 30 ℃ overnight. The pellet was collected with mirocloth filter cloth and washed with sterile water. Weighing a certain amount of lyase, dissolving the lyase by using an osmotic pressure stabilizer KMC, and filtering and sterilizing by using a sterile filter membrane.

Weighing a certain amount of bacteria balls, adding the bacteria balls into the enzymolysis liquid, carrying out shake culture at 37 ℃ and 100r/min for about 3h until hyphae are completely digested into protoplasts, centrifuging the protoplasts at 4 ℃ and 1000rpm for 10min, discarding the supernatant, adding precooled STC with the same volume, centrifuging the protoplasts at 4 ℃ and 1000rpm for 10min, discarding the supernatant, washing the protoplasts for 2 times, adding 100 mu L STC, and uniformly mixing.

mu.L of linearized nucleic acid fragment (HGT1 protein expression cassette and hygromycin resistance expression cassette) and 330. mu.L of PEG buffer were added to 100. mu.L of Aspergillus niger protoplasts, left on ice for 20min, 2mL of PEG was added, left at room temperature for 10min, 4mL of STC and 4mL of the upper medium preheated at 48 ℃ were added in this order, and plated on the lower medium containing 180mg/L of hygromycin. The plates were cultured in an inverted manner at 35 ℃ for 4-7 days until colonies appeared, and single colonies were picked for subculture. Each colony was subjected to 3 single spore subcultures.

Example 5 verification of the yield of A.niger transformants

Inoculating Aspergillus niger to PDA culture medium, culturing at 35 deg.C for 5-7 days, scraping spores, and culturing at a temperature of 10%⁶Inoculating the strain to seed culture medium at 37 deg.C and 250r/min for 24 hr, inoculating to fermentation culture medium at 1/10 deg.C, and fermenting at 35 deg.C and 250r/min for 72 hr. Centrifuging the fermentation liquor to remove thalli, diluting by 10 times, filtering by using a filter membrane, and detecting the content of citric acid by using an HPLC method, wherein the detection conditions of the HPLC method are as follows:

the instrument comprises the following steps: an Agilent 1200 high performance liquid chromatograph (provided with an ultraviolet visible detector, a differential detector and a workstation); chromatographic conditions are as follows: HPX87H chromatographic column (4.6X 250mM, 5 μm), mobile phase 5mM sulfuric acid solution, flow rate 0.6mL/min, sample amount 10 μ L, column temperature 30 deg.C, ultraviolet detection at 210nm wavelength.

FIG. 1 reflects the results of contents of citric acid and glucose in different strains, wherein H915-1 represents a wild Aspergillus niger strain, and HGT1 represents a recombinant strain of the present invention, and it can be seen from the figure that the yield of citric acid of the recombinant Aspergillus niger strain of the present invention is increased by about 14.6% compared with that of the wild strain, and the glucose content is lower in the later stage of fermentation, indicating that the HGT1 protein promotes glucose absorption, and the enhancement of glucose absorption can increase the yield of citric acid.

FIG. 2 shows the results of the citric acid ratio acid production rate test of the two strains, and it can be seen that the recombinant strain of the present invention has a higher citric acid production rate, because the overexpression of HGT1 protein enhances the glucose transport, and can increase the acid production rate, indicating that glucose absorption is the limiting step for increasing the citric acid production. Compared with the wild type aspergillus niger strain, the fermentation time of the recombinant aspergillus niger strain is shortened by 6 hours.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, it should be noted that, for those skilled in the art, many modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> university of south of the Yangtze river

<160>13

<170>PatentIn version 3.3

<210>1

<211>1644

<212>DNA

<213> HGT1 Gene

<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> amino acids encoded by HGT1 protein-encoding gene

<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 promoter

<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 terminator

<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 terminator

<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 method for constructing a recombinant mold with improved citric acid yield, comprising the following steps:

(1) constructing an HGT1 protein expression cassette, wherein the HGT1 protein expression cassette comprises a constitutive promoter, an HGT1 protein coding gene connected with the constitutive promoter and a terminator connected with the HGT1 protein coding gene; the nucleotide sequence of the HGT1 protein coding gene is shown in SEQ ID NO. 1;

(2) constructing a resistance gene expression cassette comprising a constitutive promoter, an hph gene linked to the constitutive promoter, and a terminator linked to the hph gene;

(3) and (3) transforming the HGT1 protein expression frame in the step (1) and the resistance gene expression frame obtained in the step (2) into a mould, wherein the mould is Aspergillus niger to obtain the recombinant mould.

2. The method of constructing a recombinant mold having an increased citric acid yield according to claim 1, wherein: in step (1) and step (2), the constitutive promoter is PgpdA promoter, gpdA promoter, Pmbf promoter or Pgla promoter.

3. The method of constructing a recombinant mold having an increased citric acid yield according to claim 1, wherein: in the step (1) and the step (2), the terminator is a trp terminator.

4. A recombinant mold constructed by the method of any one of claims 1-3.

5. Use of a recombinant mould constructed according to the method of any one of claims 1-3 for the fermentative production of citric acid.