CN117821277A - Recombinant saccharomyces cerevisiae for producing Fusarium acid through fermentation, method and application - Google Patents

Abstract

The invention belongs to the technical field of bioengineering, and discloses a recombinant saccharomyces cerevisiae for producing fusarium acid by fermentation, wherein the recombinant saccharomyces cerevisiae is prepared by taking intracellular L-aspartic acid of saccharomyces cerevisiae as a starting substrate, and encoding homoserine-O-acyltransferase FUB5, O-acetyl-L-homoserine sulfhydrylase FUB7, FMN-dependent hydroxy acid oxidase FUB9 and FA transporter FUB11 through expressing aspartokinase FUB 3. The invention provides a recombinant saccharomyces cerevisiae for producing fusarium acid, which is a novel way for synthesizing fusarium acid in saccharomyces cerevisiae.

Description

Recombinant saccharomyces cerevisiae for producing Fusarium acid through fermentation, method and application

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to recombinant saccharomyces cerevisiae for producing fusarium acid by fermentation, a method and application thereof.

Background

Fusarium Acid (FA) is a picolinic acid derivative, also known as wilting acid, originally isolated from the fungus Fusarium. At present, the traditional Chinese medicine composition has positive effects in treating diseases such as depression, parkinsonism, attention deficit hyperactivity disorder and the like. Fusarium acid is an inhibitor of dopamine beta-hydroxylase (the enzyme that converts dopamine to norepinephrine) and also has the effect of inhibiting cell proliferation and DNA synthesis. Fusarium acid and analogues thereof have also been reported to have quorum sensing inhibition. Therefore, the FA has important application prospect in the fields of plant protection and medical health.

FA is a secondary metabolite of many fusarium fungi. At present, the synthesis of Fusarium acid mainly comprises the following methods:

1) Biosynthesis method: the method utilizes microorganisms such as Fusarium to synthesize Fusarium acid. The method has the advantages of high yield, easily available raw materials and the like, but the existing method lacks efficient microorganism strains and generally has the problems of low synthesis level, complex process, instability and the like.

2) Total synthesis: this method is to synthesize Fusarium acid from scratch. The method has the defects of low yield, high cost and the like, but can obtain the fusarium acid with high purity and definite structure.

3) Semi-synthesis method: the method is to synthesize Fusarium acid by chemical reaction by using natural products or compounds as starting materials. The method has the advantages of high yield, simple process and the like, but has the defects of limited source of the starting materials, uncertain structure and the like.

Biosynthesis is the main method for the current industrial production of Fusarium acid. Although the synthetic pathways of Fusarium acid have been resolved, no method has been created to construct engineered strains that can ferment efficiently to produce Fusarium acid by synthetic biology means.

Saccharomyces cerevisiae (Saccharomyces cerevisiae) is a eukaryotic single-cell organism, has strong metabolic and synthetic capabilities, and is an ideal cell factory for synthesizing compounds. The advantages of Saccharomyces cerevisiae mainly include the following aspects:

1) The metabolic pathway is perfect: saccharomyces cerevisiae has an intact metabolic pathway and can utilize various carbon sources and nitrogen sources, which provides a rich raw material for the production of synthetic compounds.

2) The synthesis capability is strong: saccharomyces cerevisiae is capable of synthesizing a variety of compounds including amino acids, carbohydrates, lipids, alcohols, vitamins, and the like.

3) The biological safety is high: saccharomyces cerevisiae is a safe microorganism and is not pathogenic, which is beneficial for industrial production. Saccharomyces cerevisiae has been widely used in industrial production as a cell factory for synthesizing compounds. For example, saccharomyces cerevisiae is used to produce compounds such as glutamic acid, gluconic acid, lactic acid, citric acid, and the like.

However, no metabolic engineering studies on Fusarium acid using Saccharomyces cerevisiae as a chassis cell have been found.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides a recombinant saccharomyces cerevisiae for producing fusarium acid by fermentation, a method and application thereof.

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

a recombinant saccharomyces cerevisiae for producing fusarium acid by fermentation is prepared from saccharomyces cerevisiae intracellular L-aspartic acid serving as a starting substrate, through expressing aspartokinase FUB3, encoding homoserine-O-acyltransferase FUB5, O-acetyl-L-homoserine sulfhydrylase FUB7, FMN dependent hydroxy acid oxidase FUB9 and FA transporter FUB11, and is obtained.

Further, the Saccharomyces cerevisiae host was Saccharomyces cerevisiae BY4741 (MATA, his 3. DELTA.1, leu2. DELTA.0, met15. DELTA.0, ura 3. DELTA.0).

Further, the gene encoding the pathway is derived from bakanae disease germ and comprises an aspartokinase encoding gene ffFUB3 with a nucleotide sequence shown as SEQ ID NO.1, a homoserine-O-acyltransferase encoding gene ffFUB5 with a nucleotide sequence shown as SEQ ID NO.2, an O-acetyl-L-homoserine sulfhydrylase encoding gene ffFUB7 with a nucleotide sequence shown as SEQ ID NO.3, an FMN-dependent hydroxy acid oxidase encoding gene ffFUB9 with a nucleotide sequence shown as SEQ ID NO.4 and an FA transporter encoding gene ffFUB11 with a nucleotide sequence shown as SEQ ID NO. 5.

A method of constructing a recombinant saccharomyces cerevisiae as described above comprising the steps of:

construction of P represented by the nucleotide sequence SEQ ID NO.6, respectively _PYK1 And P with nucleotide sequence shown as SEQ ID NO.8 _HXT7 The mediated aspartokinase ffFUB3 and encoding homoserine-O-acyltransferase encoding gene ffFUB5, and URA3 auxotroph screening marker gene expression cassette with nucleotide sequence shown as SEQ ID NO.12 are integrated and inserted into GAL10 gene locus through GAL10-up sequence with upstream homology arm nucleotide sequence shown as SEQ ID NO.10 and GAL10 homology arm GAL10-down sequence with nucleotide sequence shown as SEQ ID NO. 11; construction of a P represented by the nucleotide sequence SEQ ID NO.21 _TRP1 An mediated O-acetyl-L-homoserine sulfhydrylase ffFUB7 expression cassette and an LEU2 auxotroph screening marker gene expression cassette with a nucleotide sequence shown as SEQ ID NO.20 are integrated and inserted into the RND18-1 gene locus through an upstream homology arm RND18-1-up of an RND18-1 gene with a nucleotide sequence shown as SEQ ID NO.22 and a downstream homology arm RND18-1-down of an RND18-1 gene with a nucleotide sequence shown as SEQ ID NO. 23; construction of P represented by the nucleotide sequence SEQ ID NO.13, respectively _TPI1 And P with nucleotide sequence shown as SEQ ID NO.15 _ENO1 The mediated FMN-dependent hydroxy acid oxidase encoding gene ffFUB9 and FA transporter encoding gene ffFUB11, and kanMX resistance screening marker gene expression cassette with the nucleotide sequence shown as SEQ ID NO.19 are inserted into the TRP1 gene locus through the upstream homology arm TRP1-up of the TRP1 gene with the nucleotide sequence shown as SEQ ID NO.17 and the upstream homology arm TRP1-down integration of the TRP1 gene with the nucleotide sequence shown as SEQ ID NO. 18.

A method for producing fusaric acid by fermentation using recombinant saccharomyces cerevisiae as described above, comprising the steps of:

inoculating the recombinant saccharomyces cerevisiae strain into YPD liquid culture medium, and performing shake culture at 28 ℃ and 220rpm for 18-20 hours to obtain seed liquid; seed solution was inoculated in a Fusarium acid fermentation medium at an inoculum size of 1%, cultured in a shaker at 28℃to 30℃and 220rpm for 4 days, and a fermentation broth containing Fusarium acid was obtained.

Further, the YPD liquid medium comprises the following components: glucose 20g/L, yeast extract 10g/L, peptone 20g/L, and sterilizing at 115℃for 20min.

Further, the fusarium acid fermentation medium is an inorganic salt medium taking sucrose or glucose as a carbon source, and comprises the following formula: inorganic salt culture medium with sucrose or glucose as carbon source: glucose 25g/L, (NH) ₄ ) ₂ SO ₄ 15 g/L，KH ₂ PO ₄ 8g/L，MgSO ₄ 3 g/L，CaCl ₂ 0.1 g/L，ZnSO ₄ ·4H ₂ 0.8g/L of O, and sterilizing for 20min at 115 ℃; after cooling, 1%Trace Element Solution,1.2%Multivitamin Solution was added.

Wherein Trace Element Solution comprises the following components: EDTA 15g/L, mnCl ₂ ·4H ₂ O 0.5g/L，CuSO ₄ 0.5 g/L，CoCl ₂ 0.5 g/L，Na ₂ MoO ₄ 0.5 g/L，CaCl ₂ 3.7 g/L，FeSO ₄ ·7H ₂ O5.1 g/L, dissolved in water, and sterilized at 115℃for 20min.

Multivitamin Solution is composed of: 0.05g/L of biotin, 4-aminobenzoic acid 0.2.2 g/L of vitamin PP (polypropylene) 1.0g/L, 11.0 g/L of vitamin B, 61.0 g/L of vitamin B, 51.0 g/L of vitamin B and 825 g/L of vitamin B, dissolved in water and filtered through a 0.22 mu m filter membrane for use.

Use of a recombinant saccharomyces cerevisiae as described above for the production of fusaric acid.

Further, the fusaric acid can be used in medicine or in plant protection.

The invention has the advantages and positive effects that:

1. the invention provides a recombinant saccharomyces cerevisiae for producing fusarium acid, which is a novel way for synthesizing fusarium acid in saccharomyces cerevisiae.

2. The invention discloses a recombinant saccharomyces cerevisiae for producing fusarium acid by fermentation and a construction method thereof, wherein the construction strategy comprises the steps of introducing aspartokinase ffFUB3 and an encoding homoserine-O-acyltransferase ffFUB5 expression cassette, an O-acetyl-L-homoserine sulfhydrylase FUB7 expression cassette and an FMN-dependent hydroxy acid oxidase ffFUB9 and FA transporter ffFUB11 expression cassette into saccharomyces cerevisiae GAL10, RDN-18-1 and TRP1 sites by a homologous recombination method, and finding that the recombinant saccharomyces cerevisiae FA-C can synthesize the fusarium acid by shake flask fermentation.

3. The starting material for synthesizing the fusaric acid by the saccharomyces cerevisiae is the aspartic acid, the material can be converted from oxaloacetic acid in the tricarboxylic acid cycle, and the conversion from the aspartic acid to the fusaric acid only has 4 steps of reactions, so that the anabolism regulation network is simpler.

4. The saccharomyces cerevisiae is used as a host cell, the culture is simple, the fermentation period is short, and a foundation is laid for economically, efficiently and environmentally synthesizing the Fusarium acid.

Drawings

FIG. 1 is a diagram of a synthetic pathway for the synthesis of Fusarium acid constructed in Saccharomyces cerevisiae in accordance with the present invention;

FIG. 2 is a map of a gene expression cassette constructed in the present invention for the synthesis of Fusarium acid;

FIG. 3 is a diagram showing the HPLC qualitative analysis and detection of FA standard, saccharomyces cerevisiae strain and recombinant Saccharomyces cerevisiae fermentation broth in the present invention.

FIG. 4HPLC analysis shows a standard curve for Fusarium acid detection.

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 recombinant saccharomyces cerevisiae for producing fusarium acid by fermentation is prepared from saccharomyces cerevisiae intracellular L-aspartic acid serving as a starting substrate, through expressing aspartokinase FUB3, encoding homoserine-O-acyltransferase FUB5, O-acetyl-L-homoserine sulfhydrylase FUB7, FMN dependent hydroxy acid oxidase FUB9 and FA transporter FUB11, and is obtained.

Preferably, the Saccharomyces cerevisiae host is Saccharomyces cerevisiae BY4741 (MATA, his 3.DELTA.1, leu2.DELTA.0, met 15.DELTA.0, ura 3.DELTA.0).

Preferably, the gene encoding the pathway is derived from bakanae disease of rice, and comprises an aspartokinase encoding gene ffFUB3 with a nucleotide sequence shown as SEQ ID NO.1, a homoserine-O-acyltransferase encoding gene ffFUB5 with a nucleotide sequence shown as SEQ ID NO.2, an O-acetyl-L-homoserine sulfhydrylase encoding gene ffFUB7 with a nucleotide sequence shown as SEQ ID NO.3, an FMN-dependent hydroxy acid oxidase encoding gene ffFUB9 with a nucleotide sequence shown as SEQ ID NO.4, and an FA transporter encoding gene ffFUB11 with a nucleotide sequence shown as SEQ ID NO. 5.

A method of constructing a recombinant saccharomyces cerevisiae as described above comprising the steps of:

construction of P represented by the nucleotide sequence SEQ ID NO.6, respectively _PYK1 And P with nucleotide sequence shown as SEQ ID NO.8 _HXT7 The mediated aspartokinase ffFUB3 and encoding homoserine-O-acyltransferase encoding gene ffFUB5, and URA3 auxotroph screening marker gene expression cassette with nucleotide sequence shown as SEQ ID NO.12 are integrated and inserted into GAL10 gene locus through GAL10-up sequence with upstream homology arm nucleotide sequence shown as SEQ ID NO.10 and GAL10 homology arm GAL10-down sequence with nucleotide sequence shown as SEQ ID NO. 11; construction of a P represented by the nucleotide sequence SEQ ID NO.21 _TRP1 An mediated O-acetyl-L-homoserine sulfhydrylase ffFUB7 expression cassette and an LEU2 auxotroph screening marker gene expression cassette with a nucleotide sequence shown as SEQ ID NO.20 are integrated and inserted into the RND18-1 gene locus through an upstream homology arm RND18-1-up of an RND18-1 gene with a nucleotide sequence shown as SEQ ID NO.22 and a downstream homology arm RND18-1-down of an RND18-1 gene with a nucleotide sequence shown as SEQ ID NO. 23; construction of P represented by the nucleotide sequence SEQ ID NO.13, respectively _TPI1 And P with nucleotide sequence shown as SEQ ID NO.15 _ENO1 The mediated FMN-dependent hydroxy acid oxidase encoding gene ffFUB9 and FA transporter encoding gene ffFUB11, and kanMX resistance screening marker gene expression cassette with the nucleotide sequence shown as SEQ ID NO.19 are inserted into the TRP1 gene locus through the upstream homology arm TRP1-up of the TRP1 gene with the nucleotide sequence shown as SEQ ID NO.17 and the upstream homology arm TRP1-down integration of the TRP1 gene with the nucleotide sequence shown as SEQ ID NO. 18.

A method for producing fusaric acid by fermentation using recombinant saccharomyces cerevisiae as described above, comprising the steps of:

inoculating the recombinant saccharomyces cerevisiae strain into YPD liquid culture medium, and performing shake culture at 28 ℃ and 220rpm for 18-20 hours to obtain seed liquid; seed solution was inoculated in a Fusarium acid fermentation medium at an inoculum size of 1%, cultured in a shaker at 28℃to 30℃and 220rpm for 4 days, and a fermentation broth containing Fusarium acid was obtained.

Preferably, the YPD liquid medium comprises the following components: glucose 20g/L, yeast extract 10g/L, peptone 20g/L, and sterilizing at 115℃for 20min.

Preferably, the fusarium acid fermentation medium is an inorganic salt medium taking sucrose or glucose as a carbon source, and the formula of the fusarium acid fermentation medium is as follows: inorganic salt culture medium with sucrose or glucose as carbon source: glucose 25g/L, (NH) ₄ ) ₂ SO ₄ 15 g/L，KH ₂ PO ₄ 8g/L，MgSO ₄ 3 g/L，CaCl ₂ 0.1 g/L，ZnSO ₄ ·4H ₂ 0.8g/L of O, and sterilizing for 20min at 115 ℃; after cooling, 1%Trace Element Solution,1.2%Multivitamin Solution was added.

Wherein Trace isThe Element Solution composition is: EDTA 15g/L, mnCl ₂ ·4H ₂ O 0.5g/L，CuSO ₄ 0.5 g/L，CoCl ₂ 0.5 g/L，Na ₂ MoO ₄ 0.5 g/L，CaCl ₂ 3.7 g/L，FeSO ₄ ·7H ₂ O5.1 g/L, dissolved in water, and sterilized at 115℃for 20min.

Multivitamin Solution is composed of: 0.05g/L of biotin, 4-aminobenzoic acid 0.2.2 g/L of vitamin PP (polypropylene) 1.0g/L, 11.0 g/L of vitamin B, 61.0 g/L of vitamin B, 51.0 g/L of vitamin B and 825 g/L of vitamin B, dissolved in water and filtered through a 0.22 mu m filter membrane for use.

Use of a recombinant saccharomyces cerevisiae as described above for the production of fusaric acid.

Preferably, the Fusarium acid is capable of use in medicine or plant protection.

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

saccharomyces cerevisiae (Saccharomyces cerevisiae BY 474) is disclosed to enable one skilled in the art to better understand the invention, but is not limiting.

FA analysis by high performance liquid chromatography: the sample was taken with a high performance liquid chromatograph (Agilent 1260Infinity II Multicolumn Thermostat (MCT)) (G7116A) using a WondaSil C18 Superb liquid chromatography column (4.6 mm. Times.250 mm,5 μm) with a sample loading of 10. Mu.L, column temperature of 50 ℃, mobile phase of methanol: 0.43% phosphoric acid (68:32,% v/v), flow rate of 1mL/min, detection wavelength: 271nm. Setting an FA standard curve, and calculating the content of Fusarium acid in the sample according to the standard curve. Fusarium acid standard was purchased from MCE China (HY-128483).

Example 1: construction method of recombinant saccharomyces cerevisiae FA-A containing encoding aspartokinase ffFUB3 and encoding homoserine-O-acyltransferase ffFUB5

The aspartokinase ffFUB3 and the encoding homoserine-O-acyltransferase ffFUB5 used in the invention are derived from the bakanae disease germ of rice through codon optimization and are synthesized by a chemical synthesis method. Promoter P _PYK1 (SEQ ID NO. 6) and terminator T _PYK1 (SEQ ID NO. 7), promoter P _HXT7 (SEQ ID NO. 8) and terminator T _HXT7 (SEQ ID NO. 9), and the homology arm sequences GAL10-up (SEQ ID NO. 10) and GAL10-down (SEQ ID NO. 11) are derived from Saccharomyces cerevisiae BY474. The selectable marker URA3 expression cassette was derived from plasmid pFA6aURA3 (Addegen: # 61924).

The synthesized aspartokinase ffFUB3 is used as a template, FUB3-F/FUB3-R is used as a primer, and a standard PCR amplification system and a standard PCR amplification program are adopted to obtain the ffFUB3 coding gene. The Saccharomyces cerevisiae BY474 genome is used as a template, primers GAL10-up-F/GAL10-up-R, GAL10-down-F/GAL10-down-R, ppyk1-F/Ppyk1-R, tpyk1-F/Tpyk1-R, phxt7-F/Phxt7-R and thox 7-F/thox 7-R are respectively designed to amplify the upstream and downstream homology arms GAL10-up and GAL10-down of the GAL1 gene and the promoter P BY adopting a standard PCR amplification system and a standard program _PYK1 And terminator T _PYK1 Promoter P _hxt7 And terminator T _HXT7 . The PCR system (50. Mu.L) and amplification conditions used in the present invention were as follows:

an amplification program was set on the PCR instrument. The amplification conditions were:

p was obtained by an overlap PCR strategy _PYK1 ::ffFUB3::T _PYK1 -P _HXT7 ::ffFUB5::T _HXT7 Expression cassette DNA fragments. The screening marker gene URA3 expression cassette (SEQ ID No. 12) is obtained by amplification using pFA6aURA3 (Addegen: # 61924) as a template and a URA3-F/URA3-Rn primer pair by using a standard PCR amplification system and procedure. GAL10-up, P obtained _PYK1 ::ffFUB3::T _PYK1 -P _HXT7 ::ffFUB5::T _HXT7 Screening marker gene URA3 expression cassette sequence and GAL10-down fragment byAn overlap PCR strategy to obtain a nucleic acid sequence containing an insertable GAL10 site and containing P _PYK1 ::ffFUB3::T _PYK1 -P _HXT7 ::ffFUB5::T _HXT7 And a selectable marker gene and a DNA fragment of a selectable marker gene URA3 expression cassette. The fragment is transformed into a saccharomyces cerevisiae BY4741 strain, and a recombinant saccharomyces cerevisiae strain FA-A containing medium chain dehydrogenase/reductase ffFUB6 and encoding NRPS-like carboxylic acid reductase ffFUB8 can be obtained through auxotroph plate screening.

The overlap PCR system was (to construct P _PYK1 ::ffFUB3::T _PYK1 Expression box as an example):

taking P containing homology arm _PYK1 、ffFUB3、T _PYK1 1 μl of each fragment; ppyk1-F/Tpyk1-R each 1. Mu.L; dNTP Mix (2.5 mM each), 2. Mu.L; 2X Phanta Max Buffer, 10. Mu.L; ddH ₂ O was fixed to a volume of 20. Mu.L. An overlap amplification procedure was set on the PCR instrument. The amplification conditions were:

related primer sequence information: 5'-3' direction

Example 2: construction method of recombinant saccharomyces cerevisiae FA-B containing encoding O-acetyl-L-homoserine sulfhydrylase ffFUB7

The O-acetyl-L-homoserine sulfhydrylase ffFUB7 used in the invention is derived from bakanae disease of rice through codon optimization and is synthesized by a chemical synthesis method. Promoter P _TRP1 (SEQ ID NO. 15) was derived from plasmid p404TEF1 (Addegen: # 15972.). The selectable marker gene LEU2 was derived from plasmid pMV-LEU2 (Addegen: # 65334). The homology arms RDN18-1-up and RDN18-1-down are derived from Saccharomyces cerevisiaeBY474 genome.

The chemically synthesized O-acetyl-L-homoserine sulfhydrylase ffFUB7 gene site template, FUB7-F/FUB7-R as primer, the encoding DNA fragment of ffFUB7 obtained by amplification, and P _TRP1 -F/P _TRP1 R is a primer pair, P404TEF1 plasmid is used as a template, and standard PCR amplification system and procedure are adopted to amplify and obtain P _TRP1 A DNA fragment. Then P was obtained using the overlap procedure in example 1 _TRP1 The ffFUB7 expression cassette. Screening marker gene LEU2 expression cassette is obtained by amplification with pMV-LEU2 (Addegen: # 65334) as template and LEU2-F/LEU2-R primer pair using standard PCR amplification system and procedure. Primers RDN18-1-DOWN-R/RDN18-1-DOWN-F and RDN18-1-UP-F/RDN18-1-UP-R are designed BY taking a Saccharomyces cerevisiae BY474 genome as a template, and homologous arms RDN18-1-UP (SEQ ID NO. 16) and RDN18-1-DOWN (SEQ ID NO. 17) are amplified respectively BY adopting a standard PCR amplification system and a standard PCR amplification program. The PCR system (50. Mu.L) and amplification conditions used in the present invention are described in example 1. Obtained RDN18-1-up, P _TRP1 ffFUB7 expression cassette, P _TEF1 ::ffFUB4::T _CYC1 Expression cassette, selection marker gene LEU2 expression cassette, RDN18-1-Down sequence, P-containing insertionable RND18-1 locus was obtained by the overlap strategy as in example 1 _TEF1 ::ffFUB4::T _CYC1 Expression cassette and P _TRP1 The ffFUB7 expression cassette and the auxotroph screening marker gene LEU2 expression cassette. The fragment is transformed into a saccharomyces cerevisiae FA-A strain, and the recombinant saccharomyces cerevisiae FA-B strain containing O-acetyl-L-homoserine sulfhydrylase ffFUB7 recombinant saccharomyces cerevisiae is obtained through auxotroph plate screening.

Related primer sequence information: 5'-3' direction

Example 3: construction method of synthesizable Fusarium acid recombinant Saccharomyces cerevisiae FA-C containing encoding FMN-dependent hydroxy acid oxidase ffFUB9 and encoding FA transporter ffFUB11

P containing homology arms upstream and downstream of TRP1 gene was also constructed according to the method of example 1 _TPI1 ::ffFUB9::T _TPI1 -P _ENO1 ::ffFUB11::T _ENO1 -P _TEF ::kanMX::T _TEF Expression cassette DNA fragments. The construction strategy is as follows:

the invention synthesizes the hydroxy acid oxidase ffFUB9 and the encoding FA transporter ffFUB11 by a chemical synthesis method, and both genes are derived from the rice bakanae disease germ which is optimized by codons. Promoter P _TPI1 (SEQ ID NO. 21) and terminator T _TPI1 (SEQ ID NO. 14), promoter P _ENO1 (SEQ ID NO. 15) and terminator T _ENO1 (SEQ ID NO. 16), and the homology arm sequences TRP1-up (SEQ ID NO. 17) and TRP1-down (SEQ ID NO. 18) are derived from Saccharomyces cerevisiae BY474. The gentamicin resistance gene kanMX expression cassette was derived from pFA6a-kanMX6 (addagen: # 39296).

The synthesized aspartokinase encoding gene ffFUB9 is used as a template, FUB9-F/FUB9-R is used as a primer, and a standard PCR amplification system and a standard PCR amplification program are adopted to obtain the ffFUB9 encoding gene. The synthesized FA transporter encoding gene ffFUB11 is used as a template, FUB11-F/FUB11-R is used as a primer, and a standard PCR amplification system and a standard PCR amplification program are adopted to obtain the ffFUB11 encoding gene. The Saccharomyces cerevisiae BY474 genome is used as a template, primers TRP1-up-F/TRP1-up-R, TRP1-down-F/TRP1-down-R, ptpi-F/Ptpi 1-R, ttpi1-F/Ttpi1-R, peno1-F/Peno1-R and Teno1-F/Teno1-R are respectively designed, and a standard PCR amplification system and a standard program are adopted to obtain homology arms TRP1-up and TRP1-down and a promoter P _TPI1 And terminator T _TPI1 Promoter P _ENO1 And terminator T _ENO1 . The kanMX resistance gene expression cassette (SEQ ID No. 30) was obtained using a standard PCR amplification system and procedure with plasmid pFA6a-kanMX6 (addagen: # 39296) as template and kanMX-F/kanMX-R as template. See example 1 for PCR systems (50. Mu.L) and amplification conditions used in the present invention. By the overlap strategy as in example 1, P containing the upstream and downstream homology arms of TRP1 gene was obtained _TPI1 ::ffFUB9::T _TPI1 -P _ENO1 ::ffFUB11::T _ENO1 -P _TEF ::kanMX::T _TEF Expression cassette DNA fragments. The fragment is transformed into saccharomyces cerevisiae FA-B strain, and the recombinant saccharomyces cerevisiae FA-C strain containing FMN dependent hydroxy acid oxidase ffFUB9 and FA transporter ffFUB11 can be obtained through screening of gentamicin resistance plate and auxotroph plate.

Related primer sequence information: 5'-3' direction:

thus, the Saccharomyces cerevisiae BY4741 strain is subjected to iterative assembly in examples 1-3, so that the recombinant Saccharomyces cerevisiae strain capable of fermenting and synthesizing Fusarium acid can be obtained.

The synthetic route of Fusarium acid constructed in the present invention is shown in FIG. 1.

The individual gene expression cassettes and the nutritional screening marker expression cassettes and combinations thereof for constructing the fusarium acid synthesis pathway of the present invention are shown in fig. 2.

Example 4: HPLC detection of fermentation of recombinant Saccharomyces cerevisiae and fermentation products

The FA-C Saccharomyces cerevisiae strain of example 3 was inoculated into an appropriate amount of YPD liquid medium and shake-cultured at 28℃and 220rpm for 18-20 hours to obtain a seed solution. Seed solution was inoculated in 50mL of Fusarium acid fermentation medium at an inoculum size of 1%, and cultured in a shaker at 28℃and 220rpm for 4 days to obtain a fermentation broth containing Fusarium acid. The fermentation broth was transferred to a 50mL centrifuge tube at 12000rpm for 5min and the supernatant was collected. After proper dilution, the sample was filtered through a 0.22 μm filter to be detected.

The fusarium acid fermentation medium is an inorganic salt medium taking sucrose or glucose as a carbon source, and comprises the following formula: inorganic salt culture medium with sucrose or glucose as carbon source: glucose 25g/L, (NH) ₄ ) ₂ SO ₄ 15 g/L，KH ₂ PO ₄ 8 g/L，MgSO ₄ 3g/L，CaCl ₂ 0.1 g/L，ZnSO ₄ ·4H ₂ 0.8g/L of O, and sterilizing for 20min at 115 ℃; after cooling, 1%Trace Element Solution,1.2%Multivitamin Solution was added.

Wherein Trace Element Solution comprises the following components: EDTA 15g/L, mnCl ₂ ·4H ₂ O 0.5g/L，CuSO ₄ 0.5 g/L，CoCl ₂ 0.5 g/L，Na ₂ MoO ₄ 0.5 g/L，CaCl ₂ 3.7 g/L，FeSO ₄ ·7H ₂ O5.1 g/L, dissolved in water, and sterilized at 115℃for 20min.

Multivitamin Solution is composed of: 0.05g/L of biotin, 4-aminobenzoic acid 0.2.2 g/L of vitamin PP (polypropylene) 1.0g/L, 11.0 g/L of vitamin B, 61.0 g/L of vitamin B, 51.0 g/L of vitamin B and 825 g/L of vitamin B, dissolved in water and filtered through a 0.22 mu m filter membrane for use.

The sample was taken with a high performance liquid chromatograph (Agilent 1260Infinity II Multicolumn Thermostat (MCT)) (G7116A) using a WondaSil C18 Superb liquid chromatography column (4.6 mm. Times.250 mm,5 μm) with a sample loading of 10. Mu.L, column temperature of 50 ℃, mobile phase of methanol: 0.43% phosphoric acid (68:32,% v/v), flow rate of 1mL/min, detection wavelength: 271nm. Setting an FA standard curve, and calculating the content of Fusarium acid in the sample according to the standard curve. Fusarium acid standard was purchased from MCE China (HY-128483).

The fermentation result is shown in FIG. 3, the fermentation broth of recombinant strain S.cerevisiae FA-C has a corresponding characteristic peak at the retention time of 4.126min of the FA standard, while the fermentation broth of starting strain S.cerevisiae BY4741 has no characteristic peak of Fusarium acid. This suggests that a novel anabolic pathway for Fusarium acid was successfully constructed in Saccharomyces cerevisiae.

In the invention, HPLC is utilized to quantitatively analyze the Fusarium acid in the fermentation broth.

Firstly, setting an FA standard curve, and calculating the content of the fusaric acid in the sample according to the standard curve.

The FA standard curve was made as follows:

accurately weighing 50mg of Fusarium acid, adding 500 μl of methanol, and dissolving thoroughly to obtain a concentrateFusarium acid mother liquor with the concentration of 100mg/mL is sucked, then proper amount of mother liquor is absorbed, and is subjected to gradient dilution with deionized water to form standard solutions with the concentration of 20, 50, 100, 200, 500 and 1000 mug/mL, and the standard solutions are filtered by a 0.22 mu m filter membrane and then analyzed and quantified by HPLC. Fusarium acid standard curves were constructed with response values (mAU) of different concentration Fusarium acid standard solutions as ordinate and standard solution concentrations as abscissa (FIG. 4). Standard curve formula y=28.11×x+0.8509 (R ² = 0.9958) shows that there is a good linear relationship between the concentration of sickle acid (in the range of 20-1000 μg/mL) and the response value of the liquid chromatograph.

The FA content in the fermentation broth of S.cerevisiae FA-C was 1.9g/L by HPLC quantitative analysis.

There is no strategy reported to synthesize Fusarium acid by constructing a microbial cell factory. The invention provides a new idea for realizing the stable green biological production of the fusarium acid.

The sequence table is related nucleotide sequence information:

(1) SEQ ID NO.1 shows the coding sequence of the coding gene ffFUB3 of the aspartokinase derived from the bakanae disease of rice after codon optimization.

(2) SEQ ID NO.2 shows the coding sequence of homoserine-O-acyltransferase ffFUB5 gene from bakanae disease germ of rice with optimized codons.

(3) SEQ ID NO.3 shows the coding sequence of the O-acetyl-L-homoserine sulfhydrylase ffFUB7 gene derived from the bakanae disease germ of paddy rice with optimized codons.

(4) SEQ ID NO.4 is a codon optimized coding sequence of the FMN dependent hydroxy acid oxidase ffFUB9 gene derived from Miao ethnomea oryzae.

(5) SEQ ID No.5 is a codon optimized coding sequence of Fusarium acid transporter ffFUB11 derived from bakanae disease germ.

(6) SEQ ID NO.6 is pyruvate kinase gene promoter P _PYK1 Sequence.

(7) SEQ ID NO.7 is pyruvate kinase Gene T _PYK1 Sequence.

(8) SEQ ID NO.8 is a high affinity glucose transporter gene promoter P _HXT7 Sequence(s)

(9) SEQ ID NO.9 is a high affinity glucose transporter gene terminator T _HXT7 Sequence(s)

(10) SEQ ID NO.10 shows the upstream homology arm GAL10-up sequence of UDP glucose-4-cyclohexanol dehydrogenase gene

(11) SEQ ID NO.11 shows the GAL10-down sequence of the downstream homology arm of the UDP glucose-4-cyclohexanol dehydrogenase gene

(12) SEQ ID NO.12 shows the sequence of the guanine nucleotide 5' -phosphate decarboxylase URA3 expression cassette

(13) SEQ ID NO.13 shows a phosphoglycerate isomerase gene promoter P _TPI1 Sequence(s)

(14) SEQ ID NO.14 is a phosphoglycerate isomerase gene terminator T _TPI1 Sequence(s)

(15) SEQ ID NO.15 is phosphopyruvate hydratase promoter P _ENO1 Sequence(s)

(16) SEQ ID NO.16 is phosphopyruvate hydratase terminator T _ENO1 Sequence(s)

(17) SEQ ID NO.17 shows the upstream homology arm TRP1-up sequence of phosphoribosyl anthrone acid isomerase gene

(18) SEQ ID NO.18 shows the downstream homology arm TRP1-Down sequence of phosphoribosyl anthrone acid isomerase gene

(19) SEQ ID NO.19 is an aminoglycoside phosphotransferase kanMX expression cassette sequence

(20) SEQ ID NO.20 is a sequence of a nutritional screening marker gene 3-isopropyl malonate dehydrogenase LEU2 expression cassette

(21) SEQ ID NO.21 shows phosphoribosyl anthrone acid isomerase gene promoter P _TRP1 Sequence(s)

(22) SEQ ID NO.22 shows the upstream homology arm RDN18-1-up sequence of the small subunit 18S ribosomal RNA gene

(23) SEQ ID NO.23 shows the coding sequence of the upstream homology arm RDN18-1-down sequence 1SEQ ID NO.1ffFUB3 of the small subunit 18S ribosomal RNA gene:

ATGAGATCTAGAAGAGATAATTCTTGGGTTGCTCAAAAGTTTGGTGGTACCTCTATCGGT

AAGTTTCCTGATAAAGTGGCTGAAATCGTGAAATCAGCAAGATTGGGTGGCGATAGACC

TGCAGTAATCTGCAGCGCTAGATCATCAGGTAAAAAAGTTTTTGGTACAACTTCAAGATT

ATTACAAGTTTATAGGACTTTGAGAGGTATCGTTGCTATTACTCAGGATCCCGATATGCAA

GAATTGTTGTTCGATAGATTGAGAAGTATTATTAGAGATATTAGAGACGATCAAGTTGCTA

CAGTTCAGATGTACATCTTAAGACAAGATATTAGAGATGATACAATTAGACAAATTACTG

CTGATTGTCAAGAATTGTTGGATTATACATCAGCAGCTAAAAGATTTAATTTGGATATTAA

CGGTAAGGCTAAAGACAAGATGGTATCTTTTGGTGAAAAATTGTCTTGCAGATTGATGGT

TGCTATGTTGAGAGATAGAGATATTCCTGCTGAATATGTTGACTTGAGCGATATAGTCCCC

TCTAATAATTTAGATCAGTTAAGACCAGATTTCTTCCACGAAGCCGCTGCAGTGTTCGGT

AAAAGAGTGGAAGCATGTAACGGTAGAGTACCAGTTATTACGGGCTTTTTCGGCGCTGT

TCCCGGTTCTTTGATTGACTCTGGTATTGGTAGAGGTTACAGTGACCTTTGTGCTGTTTTG

GTTGCTATTGGTTTGCATGCTGAAAGAGTGCAAATTTGGAAAGAAGTTGACGGTATTTTT

ACAGCTGATCCAAGAGAAGTGCCAGATGCTAGATGTTTGCCAAGTATTACCCCTTCTGA

AGCTGCTGAATTAACTTTTTACGGTTCCGAAGTTATTCACCATTTAGCTTTATCCTTGGCT

ATCCAAGCTAAACCTCCAGTTTCTATTTTTGTCAAGAACGTTCAAAAGCCTTGGGGACA

GGGTACAGTCGTTGTTCCAACTGATGGTGATGATACCTCATCATGGCCTATAGATTATTTG

GATCCATCAGACTCCGATTCAACATCTTCTACCGCCTTACCCAAAATGCCAACTGCTGTT

ACGATAAAACGTGATATTACAATATTTAATATTTTGTCTAATAAACAAAGTATGTCCCATG

GTTTCTTTGTTAAAGTTTTTACTATCTTAGCAGAACATGACATTTCTGTGGATTTAATAAG

TACGTCAGAAGTTCATGTTTCCATGGCAATCAACAGTTCCAATATGGATCCAAGTCAAAT

TAAAAACGTCCAATGTAGATTGGCTGAGGAAGGTGAAGTTAACGTATTGCCAGACATGG

CTATTTTGTCTTTGGTTGGTGCAGAGTTGAAAAATATGACCGGCATTGCCGGCAAAATGT

TTGCAATTTTAGGTGAACAAGATGTTAATATTGAAATGATATCTCAAGGTGCTTCTGAAAT

TAATATTTCATGTGTCATACCAGATAAAGATGCAACCAGAGCATTGAATATGTTACATGAT

GAATTGTTCACTAAAAATGCTATTTAA

2SEQ ID NO.2ffFUB5 coding sequence:

ATGACCACAACTACTACTGCTCCAGCCTTGCCAACACCAATTCATGACGGTTTAGGTAAT

GGTACCACGTATGAAAGATCGATACCAAGACCAGTCAATCCTTTTTCAAATAGAGTTCCA

GGTAGAGAGATTATAACAGTTCCAAATTTTACCTTGGAAAGTGGTGTTGAGATGAGAAA

TGTCCCAGTTGCATATATGTCTTGGGGTAAGCTATCTCCTAAAGCTAATAATGTCATGATTA

TTTGTCATGCCTTATCTGGTTCTGCAGATGTTTCTGATTGGTGGGGTCCACTGTTAGGTCC

AGGTAAAGCCTTCGATACTGATAAATTTTTTGTCATTTGTATGAACTCTTTAGGTTCTCCA

TATGGCACGGCCTCACCAGTCACAGCTAAGAATGGTGATTATTCCGAAGGCTGGTATGG

GGCCGATTTTCCAGCCACAACTATTAGAGACGATGTTAGATTACATAAATTGGTGTTGGAT

AGATTGGGTGTGAGAAAAGTTGCTGCAGTTATCGGTGGTTCTATGGGCGGTATGCATGTT

CTTGAATGGGCTTTTTTCGGTAAGGATTACGTTAGATGTATTGTGCCCGCTGCTACTTCAT

CACATCAATCAGCTTGGGCTATTGGTTGGGGAGAAGCTCAACGTCATGCTATTAGATCAG

ATGTTAAATATAAAAACGGTAGGTATGGTTTTGATGATCCACCTATCTTGGGTTTGGAAGC

CGCCAGAATGACAGCTTTGTTAACCTATAGATCCAGAGATAGTTTAGAGAGAAGATTTGG

TAGAGATACCGGTAATAAAAAAAAAGCTAAGAATAAGGGTTCAGAAACATTGCCATCAA

ACTCTACTCCAATTCATTCACAAGGCGGAGCCGATGAGACACCTGTTGCATTTGATAGAG

CTGATTCTAACTTTGCTGCTCAATCATACTTGAGATATCAAGCTAAGAAATTCTCTGATAG

ATTTGACTCAAACTGTTATATCGCATTAACAAACAAATTAGATACTCATGATCTTGCTAGA

GGTAGGACGAGAACTATCACAGAAGCTCTGAGTTTGATTGAACAGCCAACATTGGTTTT

GGGTATTAGATCAGACGGTTTGTACACGTTGGCAGAACAAGAACAAATCGCGAGAACA

GTTCCAAACGCAAAGTTGAGGGAGATCGTCTCTGATGATGGTCATGATGCTTTCTTGATT

GAATGGAGTCAACTGAATTGGTTATTAGTTGGTTTTTTGCATGAAAGTTTACCAGATATTA

TGCAAAGAGCAGCATTGTAA

3SEQ ID NO.3ffFUB7 coding sequence:

ATGGCTGAGCAAGTTTTTCAAAATTTTGAAACTTTACAATTACACGCTGGTTACACCCCA

GATCCACATACTAGATCCACAGCTGTGCCAATTTATGCCACATCATCTTACACATTCAATG

ACTCTGCTCATGGCGCTAGATTGTTCGGTTTGAAAGAATTGGGTAACATTTACTCAAGGT

TAATGAACCCAACCGTTGATGTATTTGAAAAGAGGATTGCAGCATTGGAAGGTGGTATTG

CAGCTGCAGCTACAAGCTCTGGACAAGCCGCTCAATTTTTGACCATCGCTACTTTGGCTA

AAGCTGGTGATAATATTGTCGCTTCTTCTCATTTGTACGGTGGTACTTATAACCAGTTGAA

TGTTTTATTACCTAGATTCGGTATTAAAACAAAGTTTGTTAGGTCAGGTAAATTAGAAGAT

TATGCTGCTGCAATTGATGACCAAACTAGAGCTATTTATGTAGAGTCTATGTCCAATCCAG

ATTACGTTGTTCCAGATTTCGAAGGTATTGCTAAGATTGCTCATGAACATGGTATTCCGTT

GGTTGTTGACAATACCTTGGGTGCTGGTGGTTATTACATTAGACCCATAGAACATGGTGC

CGATATCGTTGTTCATTCTGCAACTAAGTGGATTGGTGGTCATGGTACTACTATTGGAGGT

GTGATAGTCGATTCAGGTAGATTCAACTGGAATAAACATTCAGACAGATTTCCAGAAATG

GTTGAACCTTCACCAAGTTACCACGGTTTGAAGTACTGGGAAGCTTTTGGTCCAGCTAC

ATTCATTACAAGAATCAGAGTTGAAATGTTAAGAGATATAGGTGCTTGCTTGAGTCCATT

CTCAGCTCAACAGTTATTGTTGGGAATTGAAACTTTGGGTTTAAGAGCCGAGAGACACG

CTCAAAACACTGAAAAGTTATCTAAGTATTTTGAATCTAGTCCAAATGTTTCCTGGGTCT

TGTGGCCTGGTTCTGAATCTCATCCTACTTATTCACAAGCTAAGAAGTATTTGACTAGAG

GTTTTGGTGCCATGTTATCAATCGGGGTTAAAGGTGATGCCTCAGCTGGTAGTAAAGTTG

TAGATGGTTTAAAGCTTGTTTCTAACTTAGCAAATGTTGGTGATGCTAAATCCTTAGCTAT

TCATCCTTGGTCTACTACACACGAACAATTATCAGAAGATGAAAGATTAGCATCCGGTGT

TACAGAAGATATGATCAGAATTTCTGTGGGTATAGAACATGTGGATGACATTATTGCTGAT

TTTGAACAATCCTTCCAAAAAGCCTATGGTTCTTAA

4SEQ ID NO.4ffFUB9 coding sequence:

ATGTCTGATGCCACTTCTTCCAAGCCACAAATTTTTTCTATTCAAGATTTGAAACAAGCT

GCTAGTGATAAGATGTCTCAAATGTATAGAGATTATTACAATGGTGGTGCAATGGATAATA

TTACATTAGCCAATAATGAAGCAGCTTTCGATAGATATTTGTTGAGGCCAAGAGTATTAA

GAAATGTTTCTAATATTGATATGACTACCACATTATGGGGCACAAAGGCTGCATTACCTTT

GGGTGTTTCTCCATCAGCAATGCATAGATTGGCTCATGCTGATGGTGAAGTTGGCACTTC

AAAGGCATGCGCAGCCAGGCATGTTCCAATGATTTTGAGTGCCCTGTCAAATGATACTTT

GGAAGATGTTTCAGGACAATCATCTGATGGTTCTACCCCATATGCTATTCAAGTTTCTCCA

TTTAAGAATAGACAAATCACAACTAATTTGTTGAATAGAGCCAAGGCTGCTGGCTACAA

AGCTGTAGTTTTAACTGTTGACGCTCCTATGTTTGGAAGAAGATTGGATGATTTGAGAAA

CGGATTCTCTTCGGGTGGCTTAGGTGGTGGTATACCCGATTTATCTTTTGATACTTCCGCA

ACCTGGGAAGAAAAGATAGCTTGGATGAAATCACAAACGGACCTAGAAATCTGGGTTA

AAGGTGTTACATCTCCATTAGATGCTCAAATAGCCATTGAACAAGGTGTCGATGGAATTA

TAATTTCCAACCATGGTGGTAGACAATTGGATACAACTCCTGCAACCATTGATATTTTGCG

TGAAATTGCACCTATTGCCAAAGGAAAGACCAGGATTGCCATTGATGGTGGCTTTAGAA

GAGGTTCTGACATTTTCAAAGCTGTCGCTTTAGGCGCCGATTTCGTTTTTGTTGGTAGAA

TTGCTATCTGGGGTTTAGCTTACGATGGTTCTAATGGTGTTGGGCTAGCTTTGGACCTTTT

AATAAATGAATTTAAATTGTGTATGGGTTTGGCCGGTTGTTCCAAAATTTCTGATATTTCT

CCTGCCCATTTGTCTATTTTGAATGCTAGAGGGGTTTTGGAGTCAGTCTATTAA

5SEQ ID NO.5ffFUB11 coding sequence:

ATGGCTATTGATCCTCAACCATCTAGCCCATCTTTAAGTTCTGAAACTATCGCTAACGATA

CAATTGGTAACGATAATAACGTAAACGAGCCATCAGTAGAACCAAAGACCCAGGAACAT

CAACATACCGTTCCACCTAGATTGTCAAGAATTTACTCTCAGGCTCATCATATTTCACAAT

CTTTTATTGATCAGAATTATCCTGGTGAAGGTACTACTCAAGTGCCATACAGAATTAACTT

TTTGCCAGATGATTCACAAAATGCACAATTGCCACCACCAATGGAAGAAGTTGGTATTTG

TGGTATAAAACAAGTCATTAGAGCATTTGGTATTTCGCAAGAAGTTGCAACATTAGGTAT

TAGTTTATATGTTTTGGGATTTACATTTGGTCCATTAATTTGGGCTCCTTTGTCAGAACTTT

ACGGTAGAAAAAAAGTTTTTTTTTTCACATTCATGGTTGCAACTGCCTTCAGTGCTGGTG

CTGCTGGTGCAGGTTCTATTGCTTCTTTATTGGTTTTGAGATTTTTGACAGGGTCCATCGG

TTCTGCACCTTTGAGTAACGCTCCAGCTTTAATCGCCGATATGTTTGATAAGTCCGAAAG

AGGTTTGGCAATGTGTATGTTCTCTGGTGCTCCATTTTTGGGCCCAGCCATCGGTCCAAT

CGCTGGTGGTTTCTTAGGTGAAACTGCAGGTTGGAGATGGCTTCACGGATTAATGGCAG

CTTTTACTGGTGTTACTTGGATTGCTTGTACTGTTTTTATCCCTGAAACCTATGCTCCGTAT

ATTTTGAGAAAAAGAGCACAACATATGAGCAAGTTGACTGGGAAAGTCTACATTAGCAC

ATTAGATGCTGATAAGCCTCCATCCTCTGCTGCTCACCAATTAAAAAATGCCTTGACTAG

ACCATGGTTACTATTGTTCAAAGAACCAATCGTGTTCATCACTAGTATCTATATAAGTATTA

TATACGGTACTATGTATATGTGTTTTGCTGCCTTTCCAATAGTTTTTCAACAAGGAAGAGG

TTGGTCTCAAGGTATTGGTGGTTTAGCCTTCACTGGCATTGTTATTGGTGTTATTTTGAGC

ATCATCAGCTTTGCATTTGAGGATAAAAGATATGCGAGAGCTGCGCAAAGAAGAGGTGC

TCCAATGGAACCAGAAGATAGATTACCACCAGCTATCATGGGTAGTTTGTTAATACCAAT

AGGTTTGTTTTGGTTCGCCTGGACAACCTTCGCATCCATTCACTGGATAGTGCCTATTATT

GGTACCGTTTTTTTCGCATGGGGATTAGTTTTAGTTTTCATGGCATTGCTAAATTATTTAAT

AGATTCATATGTTATATTTGCTGCTAGTATTATGGCCGCAAACTCCGCTCTTAGAAGTTTAT

TTGGTGCTGCCTTTCCTTTGTTTACTAGGCAGATGTATGATGGTTTAGGTGTTCAATGGGC

ATCTTCCATACCAGCTTTCTTGGCACTAGCATGTGTACCATTTCCTTTTTTATTTTATAAAT

ATGGTAGACAAATTAGAATGAAATGTGAATACGCTGCTGAAGCGGCTAATGTATTGCAAA

AAATGAGATCTTTGCATGTTACAGTTACAGAAGACGATGCTATGAACGAAGCTGAAGAA

ATGTGGAGAGCTAGAACTCATAACTCTCATACCTCTGCCACTCACTCTCATGGTCACAGG

AGATCTTTATCCTGTACTAGATCCGTCTAA

6SEQ ID NO.6 promoter P _PYK1 Sequence:

GTGATTTCCTTTCCTTCCCATATGATGCTAGGTACCTTTAGTGTCTTCCTAAAAAAAAAAA

AAGGCTCGCCATCAAAACGATATTCGTTGGCTTTTTTTTCTGAATTATAAATACTCTTTGG

TAACTTTTCATTTCCAAGAACCTCTTTTTTCCAGTTATATCATGGTCCCCTTTCAAAGTTAT

TCTCTACTCTTTTTCATATTCATTCTTTTTCATCCTTTGGTTTTTTATTCTTAACTTGTTTATT

ATTCTCTCTTGTTTCTATTTACAAGACACCAATCAAAACAAATAAAACATCATCACA 7SEQ ID NO.7 terminator T _PYK1 Sequence:

AAAAAGAATCATGATTGAATGAAGATATTATTTTTTTGAATTATATTTTTTAAATTTTATATA

AAGACATGGTTTTTCTTTTCAACTCAAATAAAGATTTATAAGTTACTTAAATAACATACAT

TTTATAAGGTATT

8SEQ ID NO.8 promoter P _HXT7 Sequence:

ATAATTTTCAGAGGCAACAAGGAAAAATTAGATGGCAAAAAGTCGTCTTTCAAGGAAA

AATCCCCACCATCTTTCGAGATCCCCTGTAACTTATTGGCAACTGAAAGAATGAAAAGG

AGGAAAATACAAAATATACTAGAACTGAAAAAAAAAAAGTATAAATAGAGACGATATAT

GCCAATACTTCACAATGTTCGAATCTATTCTTCATTTGCAGCTATTGTAAAATAATAAAAC

ATCAAGAACAAACAAGCTCAACTTGTCTTTTCTAAGAACAAAGAATAAACACAAAAAC

AAAAAGTTTTTTTAATTTTAATCAAAAA

9SEQ ID NO.9 terminator T _HXT7 Sequence:

TTTGCGAACACTTTTATTAATTCATGATCACGCTCTAATTTGTGCATTTGAAATGTACTCTA

ATTCTAATTTTATATTTTTAATGATATCTTGAAAAGTAAATACGTTTTTAATATATACAAAAT

AATACAGTTTAATTTTCAAGTTTTTGATCATTTGTTCTCAGAAAGTTGAGTGGGACGGAG

ACAAAGAAACTTTAAAGAGAAATGCAAAGTGGGAAGAAGTCA

10seq ID No.10 homology arm GAL10-up sequence:

ATCATCGCTTCGCTGATTAATTACCCCAGAAATAAGGCTAAAAAACTAATCGCATTATCAT

CCTATGGTTGTTAATTTGATTCGTTAATTTGAAGGTTTGTGGGGCCAGGTTACTGCCAATT

TTTCCTCTTCATAACCATAAAAGCTAGTATTGTAGAATCTTTATTGTTCGGAGCAGTGCGG

CGCGAGGCACATCTGCGTTTCAGGAACGCGACCGGTGAAGACGAGGACGCACGGAGG

AGAGTCTTCCGTCGGAGGGCTGTCGCCCGCTCGGCGGCTTCTAATCCGTACTTCAATATA

GCAATGAGCAGTTAAGCGTATTACTGAAAGTTCCAAAGAGAAGGTTTTTTTAGGCTAAG

ATAATGGGGCTCTTTACATTTCCACAACATATAAGTAAGATTAGATATGGATATGTATATGG

TGGTAATGCCATGTAATATGATTATTAAACTTCTTTGCGTCCATCCAAAAAAAAAGTAAG

AATTTTTGAAAATTCAATATAA

11SEQ ID NO.11 homology arm GAL10-Down sequence:

TTTGCCAGCTTACTATCCTTCTTGAAAATATGCACTCTATATCTTTTAGTTCTTAATTGCAA

CACATAGATTTGCTGTATAACGAATTTTATGCTATTTTTTAAATTTGGAGTTCAGTGATAAA

AGTGTCACAGCGAATTTCCTCACATGTAGGGACCGAATTGTTTACAAGTTCTCTGTACCA

CCATGGAGACATCAAAAATTGAAAATCTATGGAAAGATATGGACGGTAGCAACAAGAAT

ATAGCACGAGCCGCGGAGTTCATTTCGTTACTTTTGATATCACTCACAACTATTGCGAAG

CGCTTCAGTGAAAAAATCATAAGGAAAAGTTGTAAATATTATTGGTAGTATTCGTTTGGT

AAAGTAGAGGGGGTAATTTTTCCCCTTTATTTTGTTCATACATTCTTAAATTGCTTTGCCT

CTCCTTTTGGAAAGCTATACTTCGGAGCACTGTTGAGCGAAGGCTCATTAGATATATTTTC

TGTCATTTTCCTTAACCCAA

12SEQ ID NO.12URA3 expression cassette sequence:

TTCAATTCATCTTTTTTTTTTTTGTTCTTTTTTTTGATTCCGGTTTCTTTGAAATTTTTTTGA

TTCGGTAATCTCCGAGCAGAAGGAAGAACGAAGGAAGGAGCACAGACTTAGATTGGTA

TATATACGCATATGTGGTGTTGAAGAAACATGAAATTGCCCAGTATTCTTAACCCAACTGC

ACAGAACAAAAACCTGCAGGAAACGAAGATAAATCATGTCGAAAGCTACATATAAGGA

ACGTGCTGCTACTCATCCTAGTCCTGTTGCTGCCAAGCTATTTAATATCATGCACGAAAA

GCAAACAAACTTGTGTGCTTCATTGGATGTTCGTACCACCAAGGAATTACTGGAGTTAGT

TGAAGCATTAGGTCCCAAAATTTGTTTACTAAAAACACATGTGGATATCTTGACTGATTTT

TCCATGGAGGGCACAGTTAAGCCGCTAAAGGCATTATCCGCCAAGTACAATTTTTTACTC

TTCGAAGACAGAAAATTTGCTGACATTGGTAATACAGTCAAATTGCAGTACTCTGCGGG

TGTATACAGAATAGCAGAATGGGCAGACATTACGAATGCACACGGTGTGGTGGGCCCAG

GTATTGTTAGCGGTTTGAAGCAGGCGGCGGAAGAAGTAACAAAGGAACCTAGAGGCCT

TTTGATGTTAGCAGAATTGTCATGCAAGGGCTCCCTAGCTACTGGAGAATATACTAAGGG

TACTGTTGACATTGCGAAGAGCGACAAAGATTTTGTTATCGGCTTTATTGCTCAAAGAGA

CATGGGTGGAAGAGATGAAGGTTACGATTGGTTGATTATGACACCCGGTGTGGGTTTAG

ATGACAAGGGAGACGCATTGGGTCAACAGTATAGAACCGTGGATGATGTGGTCTCTACA

GGATCTGACATTATTATTGTTGGAAGAGGACTATTTGCAAAGGGAAGGGATGCTAAGGTA

GAGGGTGAACGTTACAGAAAAGCAGGCTGGGAAGCATATTTGAGAAGATGCGGCCAGC

AAAACTAA

13SEQ ID NO.13 promoter P _TPI1 Sequence:

TTCTGGCATCCAGTTTTTAATCTTCAGTGGCATGTGAGATTCTCCGAAATTAATTAAAGCA

ATCACACAATTCTCTCGGATACCACCTCGGTTGAAACTGACAGGTGGTTTGTTACGCATG

CTAATGCAAAGGAGCCTATATACCTTTGGCTCGGCTGCTGTAACAGGGAATATAAAGGGC

AGCATAATTTAGGAGTTTAGTGAACTTGCAACATTTACTATTTTCCCTTCTTACGTAAATAT

TTTTCTTTTTAATTCTAAATCAATCTTTTTCAATTTTTTGTTTGTATTCTTTTCTTGCTTAAA

TCTATAACTACAAAAAACACATACATAAACTAAAA

14SEQ ID NO.14 terminator T _TPI1 Sequence:

GATTAATATAATTATATAAAAATATTATCTTCTTTTCTTTATATCTAGTGTTATGTAAAATAAA

TTGATGACTACGGAAAGCTTTTTTATATTGTTTCTTTTTCATTCTGAGCCACTTAAATTTCG

TGAATGTTCTTGTAAGGGACGGTAGATTTACAAGTGATACAACAAAAAGCAAGGCGCTT

TTTCTAATAAAAAGAAGAAAAGCATTTAACAATTGAACACCTCTATATCAACGAAGAATA

TTACTTTGTCTCTAAATCCTTGTAAAATGTGTACGATCTCTATATGGGTTACTCATAAGTGT

ACCGAAGACTGCAT

15SEQ ID NO.15 promoter P _ENO1 Sequence:

GCCTCTCCCCGGAAACTGTGGCCTTTTCTGGCACACATGATCTCCACGATTTCAACATAT

AAATAGCTTTTGATAATGGCAATATTAATCAAATTTATTTTACTTCTTTCTTGTAACATCTC

TCTTGTAATCCCTTATTCCTTCTAGCTATTTTTCATAAAAAACCAAGCAACTGCTTATCAA

CACACAAACACTAAATCAAA

16SEQ ID NO.16 terminator T _ENO1 Sequence(s)：

AGCTTTTGATTAAGCCTTCTAGTCCAAAAAACACGTTTTTTTGTCATTTATTTCATTTTCTT

AGAATAGTTTAGTTTATTCATTTTATAGTCACGAATGTTTTATGATTCTATATAGGGTTGCA

AACAAGCATTTTTCATTTTATGTTAAAACAATTTCAGGTTTACCTTTTATTCTGCTTGTG 17SEQ ID NO.17 homology arm TRP1-up sequence:

TGGACGCGCCGCTCACCCGCACGGCAGAGACCAATCAGTAAAAATCAACGGTTAACGA

CATTACTATATATATAATATAGGAAGCATTTAATAGAACAGCATCGTAATATATGTGTACTTT

GCAGTTATGACGCCAGATGGCAGTAGTGGAAGATATTCTTTATTGAAAAATAGCTTGTCA

CCTTACGTACAATCTTGATCCGGAGCTTTTCTTTTTTTGCCGATTAAGAATTCGGTCGAAA

AAAGAAAAGGAGAGGGCCAAGAGGGAGGGCATTGGTGACTATTGAGCACGTGAGTATA

CGTGATTAAGCACACAAAGGCAGCTTGGAGTATGTCTGTTATTAATTTCACAGGTAGTTC

TGGTCCATTGGTGAAAGTTTGCGGCTTGCAGAGCACAGAGGCCGCAGAATGTGCTCTAG

ATTCCGATGCTGACTTGCTGGGTATTATATGTGTGCCCAATAGAAAGAGAACAATTGACC

CGGTTATTGCAAGGAAAATTTCAAG

18SEQ ID NO.18 homology arm TRP1-Down sequence:

TCGTCTTTTCGCTGTAAAAACTTTATCACACTTATCTCAAATACACTTATTAACCGCTTTT

ACTATTATCTTCTACGCTGACAGTAATATCAAACAGTGACACATATTAAACACAGTGGTTT

CTTTGCATAAACACCATCAGCCTCAAGTCGTCAAGTAAAGATTTCGTGTTCATGCAGATA

GATAACAATCTATATGTTGATAATTAGCGTTGCCTCATCAATGCGAGATCCGTTTAACCGG

ACCCTAGTGCACTTACCCCACGTTCGGTCCACTGTGTGCCGAACATGCTCCTTCACTATT

TTAACATGTGGAATTCTTGAAAGAATGAAATCGCCATGCCAAGCCATCACACGGTCTTTT

ATGCAATTGATTGACCGCCTGCAACACATAGGCAGTAAAATTTTTACTGAAACGTATATA

ATCATCATAAGCGACAAGTGAGGCAACACCTTTGTTACCACATTGACAACCCCAGGTATT

CATACTTCCTATTAGCG

19SEQ ID NO.19kanMX expression cassette sequences

GACATGGAGGCCCAGAATACCCTCCTTGACAGTCTTGACGTGCGCAGCTCAGGGGCATG

ATGTGACTGTCGCCCGTACATTTAGCCCATACATCCCCATGTATAATCATTTGCATCCATAC

ATTTTGATGGCCGCACGGCGCGAAGCAAAAATTACGGCTCCTCGCTGCAGACCTGCGAG

CAGGGAAACGCTCCCCTCACAGACGCGTTGAATTGTCCCCACGCCGCGCCCCTGTAGAG

AAATATAAAAGGTTAGGATTTGCCACTGAGGTTCTTCTTTCATATACTTCCTTTTAAAATC

TTGCTAGGATACAGTTCTCACATCACATCCGAACATAAACAACCATGGGTAAGGAAAAG

ACTCACGTTTCGAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAAT

GGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGATTGTATGGGAAGCCCG

ATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGAT

GAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTT

ATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGCAAAACAGCATTC

CAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTC

CTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTC

GTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATG

ACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAGCTTTTGCCA

TTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACG

AGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAG

GATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTT

TTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGA

TGAGTTTTTCTAATCAGTACTGACAATAAAAAGATTCTTGTTTTCAAGAACTTGTCATTTG

TATAGTTTTTTTATATTGTAGTTGTTCTATTTTAATCAAATGTTAGCGTGATTTATATTTTTTT

TCGCCTCGACATCATCTGCCCAGATGCGAAGTTAAGTGCGCAGAAAGTAATATCATGCGT

CAATCGTATGTGAATGCTGGTCGCTATACTG

20SEQ ID NO.20LEU2 expression cassette sequences

AACTGTGGGAATACTCAGGTATCGTAAGATGCAAGAGTTCGAATCTCTTAGCAACCATTA

TTTTTTTCCTCAACATAACGAGAACACACAGGGGCGCTATCGCACAGAATCAAATTCGAT

GACTGGAAATTTTTTGTTAATTTCAGAGGTCGCCTGACGCATATACCTTTTTCAACTGAA

AAATTGGGAGAAAAAGGAAAGGTGAGAGCGCCGGAACCGGCTTTTCATATAGAATAGA

GAAGCGTTCATGACTAAATGCTTGCATCACAATACTTGAAGTTGACAATATTATTTAAGG

ACCTATTGTTTTTTCCAATAGGTGGTTAGCAATCGTCTTACTTTCTAACTTTTCTTACCTTT

TACATTTCAGCAATATATATATATATATTTCAAGGATATACCATTCTAATGTCTGCCCCTAAG

AAGATCGTCGTTTTGCCAGGTGACCACGTTGGTCAAGAAATCACAGCCGAAGCCATTAA

GGTTCTTAAAGCTATTTCTGATGTTCGTTCCAATGTCAAGTTCGATTTCGAAAATCATTTA

ATTGGTGGTGCTGCTATCGATGCTACAGGTGTCCCACTTCCAGATGAGGCGCTGGAAGC

CTCCAAGAAGGTTGATGCCGTTTTGTTAGGTGCTGTGGGTGGTCCTAAATGGGGTACCG

GTAGTGTTAGACCTGAACAAGGTTTACTAAAAATCCGTAAAGAACTTCAATTGTACGCC

AACTTAAGACCATGTAACTTTGCATCCGACTCTCTTTTAGACTTATCTCCAATCAAGCCA

CAATTTGCTAAAGGTACTGACTTCGTTGTTGTCAGAGAATTAGTGGGAGGTATTTACTTT

GGTAAGAGAAAGGAAGACGATGGTGATGGTGTCGCTTGGGATAGTGAACAATACACCG

TTCCAGAAGTGCAAAGAATCACAAGAATGGCCGCTTTCATGGCCCTACAACATGAGCCA

CCATTGCCTATTTGGTCCTTGGATAAAGCTAATGTTTTGGCCTCTTCAAGATTATGGAGAA

AAACTGTGGAGGAAACCATCAAGAACGAATTCCCTACATTGAAGGTTCAACATCAATTG

ATTGATTCTGCCGCCATGATCCTAGTTAAGAACCCAACCCACCTAAATGGTATTATAATCA

CCAGCAACATGTTTGGTGATATCATCTCCGATGAAGCCTCCGTTATCCCAGGTTCCTTGG

GTTTGTTGCCATCTGCGTCCTTGGCCTCTTTGCCAGACAAGAACACCGCATTTGGTTTGT

ACGAACCATGCCACGGTTCTGCTCCAGATTTGCCAAAGAATAAGGTCAACCCTATCGCC

ACTATCTTGTCTGCTGCAATGATGTTGAAATTGTCATTGAACTTGCCTGAAGAAGGTAAG

GCCATTGAAGATGCAGTTAAAAAGGTTTTGGATGCAGGTATCAGAACTGGTGATTTAGG

TGGTTCCAACAGTACCACCGAAGTCGGTGATGCTGTCGCCGAAGAAGTTAAGAAAATCC

TTGCTTAA

21SEQ ID NO.21 promoter P _TRP1 Sequence:

AACGACATTACTATATATATAATATAGGAAGCATTTAATAGACAGCATCGTAATATATGTGT

ACTTTGCAGTTATGACGCCAGATGGCAGTAGTGGAAGATATTCTTTATTGAAAAATAGCT

TGTCACCTTACGTACAATCTTGATCCGGAGCTTTTCTTTTTTTGCCGATTAAGAATTAATT

CGGTCGAAAAAAGAAAAGGAGAGGGCCAAGAGGGAGGGCATTGGTGACTATTGAGCA

CGTGAGTATACGTGATTAAGCACACAAAGGCAGCTTGGAGT

22SEQ ID NO.22 homology arm RDN18-1-up sequence:

TATCTGGTTGATCCTGCCAGTAGTCATATGCTTGTCTCAAAGATTAAGCCATGCATGTCTA

AGTATAAGCAATTTATACAGTGAAACTGCGAATGGCTCATTAAATCAGTTATCGTTTATTT

GATAGTTCCTTTACTACATGGTATAACTGTGGTAATTCTAGAGCTAATACATGCTTAAAAT

CTCGACCCTTTGGAAGAGATGTATTTATTAGATAAAAAATCAATGTCTTCGGACTCTTTGA

TGATTCATAATAACTTTTCGAATCGCATGGCCTTGTGCTGGCGATGGTTCATTCAAATTTC

TGCCCTATCAACTTTCGATGGTAGGATAGTGGCCTACCATGGTTTCAACGGGTAACGGGG

AATAAGGGTTCGATTCCGGAGAGGGAGCCTGAGAAACGGCTACCACATCCAAGGAAGG

CAGCAGGCGCGCAAATTACCCAATCCTAATTCAGGGAGGTAGTGACAATAAATAACGAT

ACAGGGCCCATTCGGGTCTTGTAATTGGAATG

23SEQ ID NO.23 homology arm RDN18-1-Down sequence:

TTAGTTGGTGGAGTGATTTGTCTGCTTAATTGCGATAACGAACGAGACCTTAACCTACTA

AATAGTGGTGCTAGCATTTGCTGGTTATCCACTTCTTAGAGGGACTATCGGTTTCAAGCC

GATGGAAGTTTGAGGCAATAACAGGTCTGTGATGCCCTTAGACGTTCTGGGCCGCACGC

GCGCTACACTGACGGAGCCAGCGAGTCTAACCTTGGCCGAGAGGTCTTGGTAATCTTGT

GAAACTCCGTCGTGCTGGGGATAGAGCATTGTAATTATTGCTCTTCAACGAGGAATTCCT

AGTAAGCGCAAGTCATCAGCTTGCGTTGATTACGTCCCTGCCCTTTGTACACACCGCCCG

TCGCTAGTACCGATTGAATGGCTTAGTGAGGCCTCAGGATCTGCTTAGAGAAGGGGGCA

ACTCCATCTCAGAGCGGAGAATTTGGACAAACTTGGTCATTTAGAGGAACTAAAAGTCG

TAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTA

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 recombinant saccharomyces cerevisiae for the fermentative production of fusarium acid, characterized in that: the recombinant saccharomyces cerevisiae is prepared by taking intracellular L-aspartic acid of saccharomyces cerevisiae as a starting substrate, expressing aspartokinase FUB3, encoding homoserine-O-acyltransferase FUB5, O-acetyl-L-homoserine sulfhydrylase FUB7, FMN dependent hydroxy acid oxidase FUB9 and FA transporter FUB11.

2. The recombinant saccharomyces cerevisiae according to claim 1, wherein: the Saccharomyces cerevisiae host was Saccharomyces cerevisiae BY4741 (MATA, his 3.DELTA.1, leu2.DELTA.0, met 15.DELTA.0, ura 3.DELTA.0).

3. The recombinant saccharomyces cerevisiae according to claim 1 or 2, wherein: the gene for coding the pathway is derived from bakanae disease germ and comprises an aspartokinase coding gene ffFUB3 with a nucleotide sequence shown as SEQ ID NO.1, a homoserine-O-acyltransferase coding gene ffFUB5 with a nucleotide sequence shown as SEQ ID NO.2, an O-acetyl-L-homoserine sulfhydrylase coding gene ffFUB7 with a nucleotide sequence shown as SEQ ID NO.3, an FMN dependent hydroxy acid oxidase coding gene ffFUB9 with a nucleotide sequence shown as SEQ ID NO.4 and an FA transporter coding gene ffFUB11 with a nucleotide sequence shown as SEQ ID NO. 5.

4. A method of constructing a recombinant saccharomyces cerevisiae according to any of claims 1 to 3, wherein: the method comprises the following steps:

construction of P represented by the nucleotide sequence SEQ ID NO.6, respectively _PYK1 And P with nucleotide sequence shown as SEQ ID NO.8 _HXT7 The mediated aspartokinase ffFUB3 and encoding homoserine-O-acyltransferase encoding gene ffFUB5, and URA3 auxotroph screening marker gene expression cassette with nucleotide sequence shown as SEQ ID NO.12 are integrated and inserted into GAL10 gene locus through GAL10-up sequence with upstream homology arm nucleotide sequence shown as SEQ ID NO.10 and GAL10 homology arm GAL10-down sequence with nucleotide sequence shown as SEQ ID NO. 11; construction of a P represented by the nucleotide sequence SEQ ID NO.21 _TRP1 An mediated O-acetyl-L-homoserine sulfhydrylase ffFUB7 expression cassette and an LEU2 auxotroph screening marker gene expression cassette with a nucleotide sequence shown as SEQ ID NO.20 are integrated and inserted into the RND18-1 gene locus through an upstream homology arm RND18-1-up of an RND18-1 gene with a nucleotide sequence shown as SEQ ID NO.22 and a downstream homology arm RND18-1-down of an RND18-1 gene with a nucleotide sequence shown as SEQ ID NO. 23; construction of P represented by the nucleotide sequence SEQ ID NO.13, respectively _TPI1 And P with nucleotide sequence shown as SEQ ID NO.15 _ENO1 Mediated FMN dependent hydroxy acid oxidase compilationsThe gene ffFUB9 and FA transporter encoding gene ffFUB11, and kanMX resistance screening marker gene expression cassette with nucleotide sequence shown as SEQ ID NO.19 are inserted into the TRP1 gene locus through the integration of the upstream homology arm TRP1-up of the TRP1 gene with nucleotide sequence shown as SEQ ID NO.17 and the upstream homology arm TRP1-down of the TRP1 gene with nucleotide sequence shown as SEQ ID NO. 18.

5. A method for producing fusaric acid by fermentation using the recombinant saccharomyces cerevisiae according to any of claims 1 to 3, characterized in that: the method comprises the following steps:

inoculating the recombinant saccharomyces cerevisiae strain into YPD liquid culture medium, and performing shake culture at 28 ℃ and 220rpm for 18-20 hours to obtain seed liquid; seed solution was inoculated in a Fusarium acid fermentation medium at an inoculum size of 1%, cultured in a shaker at 28℃to 30℃and 220rpm for 4 days, and a fermentation broth containing Fusarium acid was obtained.

6. The method according to claim 5, wherein: the YPD liquid culture medium comprises the following components: glucose 20g/L, yeast extract 10g/L, peptone 20g/L, and sterilizing at 115℃for 20min.

7. The method according to claim 5 or 6, characterized in that: the fusarium acid fermentation medium is an inorganic salt medium taking sucrose or glucose as a carbon source, and comprises the following formula: inorganic salt culture medium with sucrose or glucose as carbon source: glucose 25g/L, (NH) ₄ ) ₂ SO ₄ 15 g/L，KH ₂ PO ₄ 8 g/L，MgSO ₄ 3 g/L，CaCl ₂ 0.1 g/L，ZnSO ₄ ·4H ₂ 0.8g/L of O, and sterilizing for 20min at 115 ℃; after cooling, 1%Trace Element Solution,1.2%Multivitamin Solution was added;

wherein Trace Element Solution comprises the following components: EDTA 15g/L, mnCl ₂ ·4H ₂ O 0.5g/L，CuSO ₄ 0.5 g/L，CoCl ₂ 0.5 g/L，Na ₂ MoO ₄ 0.5 g/L，CaCl ₂ 3.7 g/L，FeSO ₄ ·7H ₂ O5.1 g/L, after dissolution with water,sterilizing at 115 ℃ for 20min;

multivitamin Solution is composed of: 0.05g/L of biotin, 4-aminobenzoic acid 0.2.2 g/L of vitamin PP (polypropylene) 1.0g/L, 11.0 g/L of vitamin B, 61.0 g/L of vitamin B, 51.0 g/L of vitamin B and 825 g/L of vitamin B, dissolved in water and filtered through a 0.22 mu m filter membrane for use.

8. Use of a recombinant saccharomyces cerevisiae according to any of claims 1 to 3 for the production of fusarium acid.

9. The use according to claim 8, characterized in that: the Fusarium acid can be used in medicine or plant protection.