CN117701617A - Method for improving kojic acid yield and application - Google Patents

Abstract

The invention belongs to the technical field of bioengineering, and discloses a method for improving the yield of kojic acid, which is realized by utilizing aspergillus niger engineering bacteria of a kojic acid-producing strain, wherein the method simultaneously overexpresses a plurality of copy numbers of KojA proteins and VHb proteins in the aspergillus niger strain; kojic acid producing strain Aspergillus niger engineering bacteria which over express KojA protein and VHb protein can produce kojic acid in fermentation, and the yield of kojic acid reaches 40g/L to 100g/L. The kojA gene is over-expressed in the aspergillus niger and copied into the strain by a molecular cloning technology and a genetic engineering technology, and the exogenous gene vgb gene which can improve the growth of the strain, promote the synthesis of protein and improve the yield of metabolites can be over-expressed on the basis. Finally, the recombinant strain can produce kojic acid with high yield. Finally, the kojic acid yield of the recombinant strain can reach 40-100 g/L. The invention can be used for the industrial production of kojic acid.

Description

Method for improving kojic acid yield and application

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to a method for improving the yield of kojic acid and application thereof.

Background

Kojic acid is an important industrial raw material, and can be used as a preservative, an oxidant for fats and oils, and an antibacterial agent in foods and pesticides. The cosmetic industry can be used as skin brightening agent and ultraviolet protecting agent in soap, facial cream, etc., and has effects of whitening skin and resisting aging. In the production and preparation of chemicals, metal adsorbents are used as binders between metals and organic materials. The pharmaceutical aspect is antifungal medicine or component as effective bactericide. At present, kojic acid is mainly produced by fermenting Aspergillus oryzae, but the fermentation strain of Aspergillus oryzae adopted at present and the fermentation performance thereof are difficult to further improve and optimize, the yield is generally maintained at about 30g/L, and the yield of glucose serving as a substrate is not more than 0.3g/g. With the increasing demands of various industries for kojic acid, there is a more urgent need for cheaper, convenient and low-cost kojic acid production methods. The engineering strain for obtaining the high-yield kojic acid by adopting biological engineering, metabolic engineering and synthetic biological methods is a potential direction.

Given that the biosynthesis pathway of kojic acid is not resolved, there are few reports of achieving heterologous synthesis of kojic acid in other strains incapable of naturally synthesizing kojic acid.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides a method for improving the yield of kojic acid and application thereof.

The technical scheme adopted for solving the technical problems is as follows:

a method for increasing kojic acid production by using a kojic acid producing strain, the method over-expressing a KojA protein and a VHb protein in the kojic acid producing strain; the kojic acid producing engineering strain which over-expresses the KojA protein and the VHb protein can produce kojic acid in fermentation, and the kojic acid yield reaches 40g/L to 100g/L.

Further, the kojic acid producing strain is aspergillus nidulans and aspergillus niger.

Further, the method of overexpressing the KojA protein is that the copy number of the gene kojA encoding the KojA protein in the engineering strain is 2 to 5.

Further, the method for realizing the overexpression of the KojA protein and the overexpression of the kojA genes with the copy number of 2 to 5 is specifically as follows: the kojA gene overexpression plasmid pLH1081 is obtained by artificially synthesizing a kojA gene nucleotide sequence and connecting the kojA gene nucleotide sequence to a plasmid pLH 454; and transferring pLH1081 into a kojic acid-producing strain by using an agrobacterium-mediated method to obtain a kojA gene over-expression engineering bacterium, then after losing a hygromycin resistance screening marker hph in the engineering bacterium, transferring the engineering bacterium into a kojic acid-producing strain engineering bacterium in which a next round of agrobacterium-mediated pLH1081 is lost and the hygromycin resistance screening marker hph is obtained in the last round, and thus obtaining the kojic acid-producing strain kojic acid-producing engineering bacterium in which the KojA protein over-expression and the copy number of 2 to 5 kojA genes are over-expressed through 2 to 5 rounds of circulation.

Further, the nucleotide sequence of the kojA gene is SEQ NO.1, and the amino acid sequence of the KojA protein is SEQ NO.2.

Further, the method for over-expressing the VHb protein comprises the following steps: the nucleotide sequence of the vgb gene is artificially synthesized and connected to a plasmid pLH454 to obtain a vgb gene overexpression plasmid pLH1261; and transferring the pLH1261 into kojic acid producing strain engineering bacteria capable of producing kojic acid by using an agrobacterium-mediated method, so as to realize the over-expression of VHb protein in Aspergillus niger engineering bacteria capable of producing kojic acid.

Further, the nucleotide sequence of the vgb is SEQ NO.3, and the amino acid sequence of the VHb protein is SEQ NO.4.

Use of a method as described above for increasing the yield of kojic acid.

A method for fermentative production of kojic acid using the method as described above comprising the steps of:

obtaining kojic acid producing strain Aspergillus niger engineering bacteria which over express KojA protein and VHb proteinInoculating the obtained kojic acid-producing engineering bacteria into a potato dextrose agar culture medium for 5 days; then adding sterile water, and washing out spores by using a cotton swab; the number of spores in the spore liquid was counted by using a hemocytometer at 5X 10 ⁷ Inoculating the spores/mL into a fermentation culture medium, and culturing in a shaking table at 28 ℃ for 5-7 days at 100-300 rpm;

wherein, the formula of the fermentation medium is as follows: 0.1 to 3.5g/L of yeast extract, 0.1 to 2g/L of monopotassium phosphate, 0.1 to 1g/L of magnesium sulfate heptahydrate, 0.1 to 200g/L of glucose and 0.1 to 1.5mol/L of MES morpholinoethanesulfonic acid, and the pH value is regulated to 6.5 by using sodium hydroxide solid.

The invention has the advantages and positive effects that:

1. the invention constructs a series of new kojic acid production engineering strains by adopting a new method, the obtained aspergillus nidulans and aspergillus niger engineering bacteria can be applied to the kojic acid biological fermentation production, the obtained strains are fermented for 3-7 days under the shake flask condition, the kojic acid yield is 40-90g/L, and the glucose is used as a substrate, the yield is more than 0.7g/g, compared with the yield and the yield of the aspergillus oryzae strains commonly used in industry, the yield of kojic acid produced by the microbial fermentation method is greatly improved, and the invention provides new excellent strains and an optimization technology for improving the yield of kojic acid produced by the microbial fermentation method. The invention has obvious innovation and provides a new technology for the field of kojic acid biosynthesis.

2. The invention over-expresses the reported gene kojA gene for key kojic acid production in aspergillus flavus in aspergillus niger and copies the gene into a strain by a molecular cloning technology and a genetic engineering technology, and on the basis, the over-expression can improve the growth of the strain, promote the protein synthesis and improve the exogenous gene VHb gene of metabolite yield. Finally, the recombinant strain can produce kojic acid with high yield. Finally, the kojic acid yield of the recombinant strain can reach 40-100 g/L. The invention can be used for producing kojic acid from Aspergillus niger and Aspergillus nidulans in industry.

Drawings

FIG. 1 is a map of a starting plasmid pLH454 in the present invention;

FIG. 2 is a map of a kojA gene expression plasmid pLH1081 constructed in the present invention;

FIG. 3 is a map of the VHb gene expression plasmid pLH1261 constructed in the present invention;

FIG. 4 is a diagram showing the digestion verification of the kojA gene expression plasmid pLH1081 and the VHb gene expression plasmid pLH1261 according to the present invention; wherein M is DNA Marker,1, 2 are single restriction enzyme verification diagrams of kojA gene expression plasmid pLH1081 and VHb gene expression plasmid pLH1261; 3. 4 is a VHb gene expression plasmid pLH1261 double restriction enzyme verification graph (NcoI, speI, 7530bp/2887 bp); 5. 6 is a single restriction map (EcoRI, 8814bp/2464 bp) of the kojA gene expression plasmid pLH1081;

FIG. 5 shows the kojic acid yield after shaking flask fermentation for 5 days of fermentation broth supernatant after 2-5 copies of kojA gene and overexpression of vgb gene are introduced into the strain;

Detailed Description

The invention will now be further illustrated by reference to the following examples, which are intended to be illustrative, not limiting, and are not intended to limit the scope of the invention.

The various experimental operations involved in the specific embodiments are conventional in the art, and are not specifically noted herein, and may be implemented by those skilled in the art with reference to various general specifications, technical literature or related specifications, manuals, etc. before the filing date of the present invention.

A method for increasing kojic acid production by using a kojic acid producing strain, the method over-expressing a KojA protein and a VHb protein in the kojic acid producing strain; the kojic acid producing engineering strain which over-expresses the KojA protein and the VHb protein can produce kojic acid in fermentation, and the kojic acid yield reaches 40g/L to 100g/L.

Preferably, the kojic acid producing strain is Aspergillus nidulans and Aspergillus niger.

Preferably, the method for over-expressing the KojA protein is that the copy number of the gene kojA encoding the KojA protein in the engineering strain is 2 to 5.

Preferably, the method for realizing the overexpression of the KojA protein and the overexpression of the kojA genes with the copy number of 2 to 5 is specifically as follows: the kojA gene overexpression plasmid pLH1081 is obtained by artificially synthesizing a kojA gene nucleotide sequence and connecting the kojA gene nucleotide sequence to a plasmid pLH 454; and transferring pLH1081 into a kojic acid-producing strain by using an agrobacterium-mediated method to obtain a kojA gene over-expression engineering bacterium, then after losing a hygromycin resistance screening marker hph in the engineering bacterium, transferring the engineering bacterium into a kojic acid-producing strain engineering bacterium in which a next round of agrobacterium-mediated pLH1081 is lost and the hygromycin resistance screening marker hph is obtained in the last round, and thus obtaining the kojic acid-producing strain kojic acid-producing engineering bacterium in which the KojA protein over-expression and the copy number of 2 to 5 kojA genes are over-expressed through 2 to 5 rounds of circulation.

Preferably, the nucleotide sequence of the kojA gene is SEQ NO.1, and the amino acid sequence of the KojA protein is SEQ NO.2.

Preferably, the method for over-expressing the VHb protein comprises the following steps: the nucleotide sequence of the vgb gene is artificially synthesized and connected to a plasmid pLH454 to obtain a vgb gene overexpression plasmid pLH1261; and transferring the pLH1261 into kojic acid producing strain engineering bacteria capable of producing kojic acid by using an agrobacterium-mediated method, so as to realize the over-expression of VHb protein in Aspergillus niger engineering bacteria capable of producing kojic acid.

Preferably, the nucleotide sequence of the vgb is SEQ NO.3, and the amino acid sequence of the VHb protein is SEQ NO.4.

Use of a method as described above for increasing the yield of kojic acid.

A method for fermentative production of kojic acid using the method as described above comprising the steps of:

obtaining kojic acid producing strain Aspergillus niger engineering bacteria which over express KojA protein and VHb protein, inoculating the obtained kojic acid producing engineering bacteria into potato dextrose agar culture medium for 5 days; then adding sterile water, and washing out spores by using a cotton swab; the number of spores in the spore liquid was counted by using a hemocytometer at 5X 10 ⁷ Inoculating the spores/mL into a fermentation culture medium, and culturing in a shaking table at 28 ℃ for 5-7 days at 100-300 rpm;

wherein, the formula of the fermentation medium is as follows: 0.1 to 3.5g/L of yeast extract, 0.1 to 2g/L of monopotassium phosphate, 0.1 to 1g/L of magnesium sulfate heptahydrate, 0.1 to 200g/L of glucose and 0.1 to 1.5mol/L of MES morpholinoethanesulfonic acid, and the pH value is regulated to 6.5 by using sodium hydroxide solid.

Specifically, the preparation and detection of the correlation are as follows:

example 1: construction of kojA Gene overexpression plasmid pLH1081

The kojA gene used in the experiment is a gene fragment artificially synthesized after codon optimization. PCR amplification is carried out by adopting primers Primer3650 and Primer3651, ecoRI and SacI are subjected to double digestion and glue recovery, then the PCR amplification is carried out, the PCR amplification is connected with a vector pLH454 (shown in figure 1) obtained by treating with the same restriction enzyme by means of a One Step Clone Kit kit, the connected product is physically and chemically transferred into E.coli JM109 competent cells, the E.coli JM109 competent cells are evenly coated in LB solid medium containing 100 mug/mL kanamycin resistance, the culture is carried out after inversion overnight at 37 ℃, a monoclonal is selected, and the pLH1081 is obtained through colony PCR verification and plasmid double digestion verification, the map of the plasmid double digestion verification is shown in figure 2.

Example 2: construction of the vgb Gene overexpression plasmid pLH1261

The vgb gene used in the experiment is a gene fragment artificially synthesized after codon optimization. PCR amplification was performed using primers P454-vhb R and P454-vhb L, ecoRI and SacI were digested and gel recovered, and then ligated with the vector pLH454 (FIG. 1) obtained by the same restriction enzyme treatment by means of One Step Clone Kit kit, the ligation product was chemically transferred to E.coli JM109 competent cells, uniformly spread on LB solid medium containing 100. Mu.g/mL kanamycin resistance, cultured overnight at 37℃in an inverted state, and single clones were picked up and subjected to colony PCR and plasmid double digestion verification to obtain pLH1261, the map of which is shown in FIG. 3, and plasmid double digestion verification is shown in FIG. 4.

Table 1 primer sequences used in the examples

Example 3: construction of Gene expression Strain: taking construction of a kojA expression Strain as an example, construction of a kojA Gene-overexpressing Strain

Agrobacterium containing plasmid pLH1081 was co-cultured with spores of A.niger host strain S469 (see for details Engineering aPhosphoketolase Pathway to Supplement Cytosolic Acetyl-CoA in Aspergillus niger Enables aSignificant Increase in Citric Acid Production, J Fungi (Basel). 2023Apr 23;9 (5): 504.Doi:10.3390/jof 9050504) on IM plates, and the co-culture was then transferred to CM plates containing 200. Mu.M cefotaxime, 100. Mu.g/mL ampicillin, 100. Mu.g/mL streptomycin, 250. Mu.g/mL hygromycin B, and incubated at 28℃until a monoclonal was formed. Selecting a monoclonal and transferring the monoclonal to a potato dextrose agar culture medium plate containing hygromycin B (250 mug/mL) and a potato dextrose agar culture medium plate, extracting genome from a transformant, verifying to obtain a hygromycin-resistant expression strain containing a kojA gene, selecting one of correct clones to carry out resistance marker hygromycin induced recombination so as to obtain a single copy kojA gene overexpression strain S3145 without hygromycin resistance. The second copy of the kojA gene was overexpressed using S3145 as starting strain, thus until a copy number of 2-5 was reached.

The electrotransformation conditions of the agrobacterium-mediated method are as follows: capacitence 25uF, voltage 2.5kV,

Resistance:200Ω,Pulse:5msec。

the Agrobacterium strain used is an AGL-1 strain.

The preparation method of the IM culture medium comprises the following steps: 15g agar is added with water to a volume of 905.7mL, sterilized for 20min at 121 ℃, and added with sterile KBuffer 0.8mL, MN buffer 20mL, 1% CaCl2.2H2O 1mL, 0.01% FeSO410mL, IM Trace elements mL and 20% NH which are prepared in advance ₄ NO ₃ 2.5 mL, 50% glycerol 10mL, 1M MES 40mL, 20% glucose 5mL, kanamycin was added to a final concentration of 100. Mu.g/mL and acetosyringone was added to a final concentration of 200. Mu.M when the temperature was cooled to about 50 ℃.

The preparation method of the CM culture medium comprises the following steps: adding water into 20g agar to volume of 897mL, sterilizing at 121deg.C for 20min, adding 20mL sterile ASPN solution (KCl 26.1g/L, KH) ₂ PO ₄ 74.8 g/L,NaNO ₃ 297.5 g/L, pH adjusted to 5.5), 20mL of 50% glucose, 2mL of 1M MgSO ₄ 1, mL CM Trace elements (component per 100)The mL contains ZnSO ₄ ·7H ₂ O 2.1g,H ₃ BO ₃ 1.1g,MnCl ₂ ·4H ₂ O 0.5g,FeSO ₄ ·7H ₂ O 0.5g,CoCl ₂ ·6H2O 0.17g,CuSO ₄ ·5H ₂ O 0.16g,Na ₂ MoO ₄ ·2H ₂ O0.15 g, EDTA 5.1 g), 10mL 10% casein hydrolysate, 50mL 10% yeast extract, hygromycin was added to a final concentration of 250. Mu.g/mL, streptomycin to a final concentration of 100. Mu.g/mL, cefotaxime to a final concentration of 100. Mu.g/mL, ampicillin to a final concentration of 100. Mu.g/mL when the temperature was cooled to about 50 ℃.

The resistance marker induction recombination method comprises the following steps: spores of about 300 kojA gene-expressing clones were uniformly spread on MM medium containing 15mg/mL xylose, cultured at 28℃until single clones were grown, 200 single clones were randomly selected and transferred onto potato dextrose agar medium at 28℃for 24 hours, then clones were transferred onto potato dextrose agar medium containing hygromycin (250. Mu.g/mL) in one-to-one correspondence for 24 hours, and finally transformants were phenotypically selected for resistance marker-induced recombination, i.e., transformants which could not normally grow on potato dextrose agar medium containing hygromycin for successful induction of recombination.

The preparation method of the potato dextrose agar medium comprises the following steps: accurately weighing 500g of peeled potato, cutting into about 1cm ³ Adding distilled water, continuously stirring, boiling for 30min, filtering with double-layer gauze, collecting filtrate, adding 50g glucose, stirring to dissolve completely, metering distilled water to 2.5L, packaging in wide-mouth bottles, adding 1.5% agar, and sterilizing at 121deg.C under high pressure for 20min.

Example 4: the engineering strain simultaneously expressing the vgb and the multicopy kojA is constructed by over-expressing the vgb gene on the basis of the engineering strain expressing the kojA gene

Agrobacterium containing plasmid pLH1261 was co-cultured with the 2-5 copy kojA gene expressing Aspergillus niger spores obtained in the construction of example 3 on IM plates, and the co-culture was transferred to CM plates containing 200. Mu.M cefotaxime, 100. Mu.g/mL ampicillin, 100. Mu.g/mL streptomycin, 250. Mu.g/mL hygromycin B and cultured at 28℃until a monoclonal was formed. Selecting a monoclonal and transferring the monoclonal to a potato dextrose agar medium plate containing hygromycin B (250 mug/mL) and a potato dextrose agar medium plate, extracting genome from a transformant, verifying to obtain a hygromycin-resistant expression strain containing the vgb gene, selecting one of the correct clones to carry out resistance marker hygromycin induced recombination, thereby obtaining the Aspergillus niger engineering bacterium which does not have hygromycin resistance, over-expresses 2-5 copies of the kojA gene and over-expresses the vgb.

Example 5: application of engineering strain in fermentation to produce kojic acid

The method for producing kojic acid by fermenting the Aspergillus niger kojic acid production engineering bacteria in a shake flask, which is constructed by the invention, comprises the following specific steps:

fermentation medium: 0.1 to 3.5g/L of yeast extract, 0.1 to 2g/L of monopotassium phosphate, 0.1 to 1g/L of magnesium sulfate heptahydrate, 0.1 to 200g/L of glucose and 0.1 to 1.5mol/L of MES morpholinoethanesulfonic acid, and the pH value is regulated to 6.5 by using sodium hydroxide solid.

The fermentation method comprises the following steps: inoculating Aspergillus niger kojic acid production engineering bacteria into potato dextrose agar medium for culturing for 5 days. Then, a proper amount of sterile water was added and spores were washed off with a cotton swab. The number of spores in the spore liquid was counted by using a hemocytometer at 5X 10 ⁷ The individual spores/mL are inoculated in a fermentation culture medium and are cultured in a shaking table, wherein the culture condition is 28 ℃, the rotation speed is 100-300 rpm, and the time period is 5-7 days.

The preparation method of the detection sample comprises the following steps:

1mL of the bacterial liquid was collected in a shake flask fermented for 5 days, and the supernatant was collected by centrifugation at 12000rpm for 5 min. The supernatant was aspirated into a 1.5mL EP tube, diluted 10-fold with ultrapure water, and the sample was filtered using a 1mL syringe with a fluid 13mm0.22um filter.

The method for detecting the organic acid by the high performance liquid chromatography comprises the following steps: agilent high performance liquid chromatograph UV detector, aminexHPX 87H column (300 mM 7.8 mM), 5mM H ₂ SO ₄ Mobile phase, flow rate of 0.6mL/min, chromatographic column temperature of 65 ℃, detection wavelength of 210nm, 20 mu L sample injectionVolume.

The LB medium, potato dextrose agar medium, IM medium, CM medium, MM medium, and the like are the same as those described in publication Sun Jing and the like in the establishment of an Aspergillus niger genetic transformation system in which amdS is a selection marker (Sun Jing, et al, chinese agricultural science and technology, guide, 2020,22 (09): 179-187).

And finally, collecting fermentation products, preparing a detection sample, and measuring the content of kojic acid in the sample through high performance liquid chromatography. The results are shown in FIG. 5. As shown in FIG. 5, after 7 days of shake flask fermentation, the koji acid yield of the koji acid production engineering bacteria constructed by the invention gradually increases along with the increase of the copy number of the over-expressed koiA gene, and the koji acid final concentrations of the koiA gene over-expressed engineering bacteria with the single copy, double copy, triple copy, four copy and five copy numbers respectively reach 10g/L,25g/L,30g/L,33g/L and 36g/L. Wherein, the two Aspergillus niger engineering bacteria are obtained by further over-expressing the vgb genes on the basis of the three-copy number and five-copy number koiA gene over-expression engineering bacteria, and the final concentration of the kojic acid reaches 45g/L and 60g/L after 7 days of shake flask fermentation.

The invention provides a genetic engineering modification method for effectively improving the yield of kojic acid. The kojic acid yield of the kojic acid production strain Aspergillus oryzae commonly used in the industry at present is about 30g/L, and the kojic acid engineering strain constructed by the invention is greatly higher than the kojic acid production strain Aspergillus oryzae in terms of yield. The invention realizes the efficient synthesis of kojic acid in heterologous host Aspergillus niger, and the developed Aspergillus niger engineering strain and corresponding fermentation process are different from the existing Aspergillus oryzae kojic acid producing strain and related fermentation process, and has new technical characteristics, thus providing a new technical scheme for the efficient industrial production of kojic acid.

SEQ NO.1: nucleotide sequence 1281bp of KojA gene

CGTGTCGCGACACAGCTAAGGGTCGGCATCGTCGGTGGCGGATGGAACGGCTGCCATCTTGCTTTAGAGCTCAAGAAACAGGGCCACCGAGTTTCTTTGTTCGAGCAGAAGCCTGATATCTTTCAAGGCGTCTCAGGAAACTTTGGTATCCGCTTGCACAAGGGGCCACACTACCCTCGGTCTAAAGCAACTCGAGACAGCTGCCGTGAAGCCTTGGTCAAGTTCTGTGAGACGTATCCCGAACTAGTCGTCCACCATGAATCCGCGATATATGCACACGGCGAAGCGGACGCTCTGGGGAACCCATCGAAGGTCTCGGATGAGGCATTCAGGGATGTATGCTACGAATCTCCTGAGTGTACTGCGGTTGATCCGAAGGCGAATGGTTTCCAGGGCCTCATCAGCGCATACAATCTGGATGAACCCAGCGTCGCTATTGGCGACCGACTCCGGAATACATTCAAAGAGAAACTTGGTCGTGCAGGCATCTACGTGCACCTCAATGCAACAGTCGACCGTATCATCCACACCGAGGACACCAACCGCATTCAAACCGGGGACGGGCAGTACGTCTTTGACGTAGTGATCAACGCCACCGGTTATACCAGCCTTCTGCCACAAAATATTGCAGATGCTCTTCCGGTTGACATTGGCATCACCTATCAGACCTGTATTGCTCTCGTTTACGAAGACCAGCAGCCACAGGAAAAGCCTCTTTCCTTCATTGTCATGGATGGCTGGTTTCCCTGCGTGATGCCGGCGATCGACACAAACGAGCCCCTTCAGAAGAAGTATATCTTGACACACGGCAGTTACACCATCCTCGGATCATTCGACCGTCACGAGGAGGGTCAAGAGCTTCTGGATAGTCTGGACGAGGAAGCTATCGCTGCTCGGATCAAGCCCCACTGTGAGCGGGAGATTACCCGGTTCTGGCCCGGATTTCTCGATCGGTTCCAGTACCGCGGCTGGAAGGGCAGTGTCCTGGCCAAGTTAAAGACCACATCCGAATTCCGAAGCAGTTTGACGTTTGAAAAAGACGGCGTGATCCACATCTTCCCCGGCAAGGTGAGCAACGTTGTAACGGCTGCCGAGGAAGTGGTTCCTCTGATCAATGACATTGCGCGCCGACGGCACGGTGTGGTTCGGGAGTGGAATGGCGTACGCTTCACTGTGAGCAGCGCCTTCCACACCCACAGCAAGGAGATTGGCGATAAGCCAGGGCTGGGCGAGCACCACACTAGCAACTTGCAGACCTATGTTTCACTAGTGACTGCAAAC

SEQ NO.2: amino acid sequence 427aa of the kojA gene

RVATQLRVGIVGGGWNGCHLALELKKQGHRVSLFEQKPDIFQGVSGNFGIRLHKGPHYPRSKATRDSCREALVKFCETYPELVVHHESAIYAHGEADALGNPSKVSDEAFRDVCYESPECTAVDPKANGFQGLISAYNLDEPSVAIGDRLRNTFKEKLGRAGIYVHLNATVDRIIHTEDTNRIQTGDGQYVFDVVINATGYTSLLPQNIADALPVDIGITYQTCIALVYEDQQPQEKPLSFIVMDGWFPCVMPAIDTNEPLQKKYILTHGSYTILGSFDRHEEGQELLDSLDEEAIAARIKPHCEREITRFWPGFLDRFQYRGWKGSVLAKLKTTSEFRSSLTFEKDGVIHIFPGKVSNVVTAAEEVVPLINDIARRRHGVVREWNGVRFTVSSAFHTHSKEIGDKPGLGEHHTSNLQTYVSLVTANSEQ NO.3: nucleotide sequence 441bp of vgb gene

ATGCTGGATCAGCAGACCATCAACATCATCAAGGCCACCGTCCCCGTCCTGAAGGAGCACGGTGTCACTATTACCACCACCTTCTACAAGAACCTGTTCGCCAAGCACCCCGAGGTCCGCCCTTTGTTTGATATGGGCCGCCAGGAGTCCCTGGAGCAGCCTAAAGCTCTGGCTATGACCGTCCTGGCTGCTGCTCAAAATATCGAGAACCTGCCCGCTATTCTGCCCGCCGTCAAAAAGATCGCCGTCAAGCACTGCCAGGCCGGCGTTGCCGCTGCTCATTATCCTATTGTCGGTCAGGAGCTGCTGGGCGCCATTAAGGAAGTCCTGGGCGATGCCGCCACCGATGATATCCTGGATGCCTGGGGCAAGGCCTACGGCGTTATTGCCGATGTCTTTATCCAGGTCGAGGCCGATCTGTACGCCCAGGCCGTTGAATAA

SEQ NO.4: amino acid sequence 146aa of the VHb gene

MLDQQTINIIKATVPVLKEHGVTITTTFYKNLFAKHPEVRPLFDMGRQESLEQPKALAMTVLAAAQNIENLPAILPAVKKIAVKHCQAGVAAAHYPIVGQELLGAIKEVLGDAATDDILDAWGKAYGVIADVFIQVEADLYAQAVE

Although embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the disclosure of the embodiments.

Claims (9)

1. A method for increasing kojic acid yield, characterized by: the method utilizes a kojic acid producing strain to realize the improvement of kojic acid yield, and the method over-expresses KojA protein and VHb protein in the kojic acid producing strain; the kojic acid producing engineering strain which over-expresses the KojA protein and the VHb protein can produce kojic acid in fermentation, and the kojic acid yield reaches 40g/L to 100g/L.

2. The method according to claim 1, characterized in that: the kojic acid producing strain is Aspergillus niger.

3. The method according to claim 1, characterized in that: the method for over-expressing the KojA protein is that the copy number of the gene kojA encoding the KojA protein in the engineering strain is 2 to 5.

4. A method according to claim 3, characterized in that: the method for realizing the overexpression of the KojA protein and the overexpression of the kojA genes with the copy number of 2 to 5 comprises the following specific steps: the kojA gene overexpression plasmid pLH1081 is obtained by artificially synthesizing a kojA gene nucleotide sequence and connecting the kojA gene nucleotide sequence to a plasmid pLH 454; and transferring pLH1081 into a kojic acid-producing strain by using an agrobacterium-mediated method to obtain a kojA gene over-expression engineering bacterium, then after losing a hygromycin resistance screening marker hph in the engineering bacterium, transferring the engineering bacterium into a kojic acid-producing strain engineering bacterium in which a next round of agrobacterium-mediated pLH1081 is lost and the hygromycin resistance screening marker hph is obtained in the last round, and thus obtaining the kojic acid-producing strain Aspergillus niger engineering bacterium in which the KojA protein over-expression and the copy number of the kojA gene over-expression are 2 to 5 through 2 to 5 rounds of circulation.

5. The method according to claim 1, characterized in that: the nucleotide sequence of the kojA gene is SEQ NO.1, and the amino acid sequence of the KojA protein is SEQ NO.2.

6. The method according to any one of claims 1 to 5, wherein: the method for over-expressing the VHb protein comprises the following steps: the nucleotide sequence of the vgb gene is artificially synthesized and connected to a plasmid pLH454 to obtain a vgb gene overexpression plasmid pLH1261; and transferring the pLH1261 into Aspergillus niger engineering bacteria capable of producing kojic acid by using an agrobacterium-mediated method, so as to realize the over-expression of VHb protein in the Aspergillus niger engineering bacteria for producing kojic acid.

7. The method according to claim 6, wherein: the nucleotide sequence of the vgb is SEQ NO.3, and the amino acid sequence of the VHb protein is SEQ NO.4.

8. Use of the method according to any one of claims 1 to 7 for increasing the yield of kojic acid.

9. A process for the fermentative production of kojic acid using the process according to any one of claims 1 to 7, characterized in that: the method comprises the following steps:

obtaining kojic acid producing strain Aspergillus niger engineering bacteria which over express KojA protein and VHb protein, inoculating the obtained kojic acid producing engineering bacteria into potato dextrose agar culture medium for 5 days; then adding sterile water, and washing out spores by using a cotton swab; the number of spores in the spore liquid was counted by using a hemocytometer at 5X 10 ⁷ Inoculating the spores/mL into a fermentation culture medium, and culturing in a shaking table at 28 ℃ for 5-7 days at 100-300 rpm;

wherein, the formula of the fermentation medium is as follows: 0.1 to 3.5g/L of yeast extract, 0.1 to 2g/L of monopotassium phosphate, 0.1 to 1g/L of magnesium sulfate heptahydrate, 0.1 to 200g/L of glucose and 0.1 to 1.5mol/L of MES morpholinoethanesulfonic acid, and the pH value is regulated to 6.5 by using sodium hydroxide solid.