CN111484942A

CN111484942A - Method for producing adipic acid by using saccharomyces cerevisiae

Info

Publication number: CN111484942A
Application number: CN201911212824.4A
Authority: CN
Inventors: 邓禹; 张熙; 赵运英; 毛银; 李国辉; 周胜虎
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2019-12-02
Filing date: 2019-12-02
Publication date: 2020-08-04

Abstract

The invention discloses a method for producing adipic acid BY using saccharomyces cerevisiae, belonging to the field of biological engineering.A wild strain of saccharomyces cerevisiae BY4741 or a strain of saccharomyces cerevisiae BY4741 with L SC1 gene knocked out is used as a host, a β -ketothiolase gene, a 3-hydroxyacyl-coenzyme A dehydrogenase gene, a 3-hydroxyadipoyl dehydrogenase gene, a 5-carboxyl-2-pentenoyl coenzyme A reductase gene and adipoyl coenzyme A in a reverse degradation way of T.fusca adipic acid are expressed in a modular over-expression manner, and the yield of the adipic acid can reach 0.033 to 0.113 g/L.

Description

Method for producing adipic acid by using saccharomyces cerevisiae

Technical Field

The invention relates to a method for producing adipic acid by using saccharomyces cerevisiae, belonging to the field of bioengineering.

Background

Adipic acid (also known as Adipic acid) is an important organic dibasic acid and is widely used in chemical production, organic synthesis industry, medicine, lubricant manufacturing and the like.

The main mode of production of adipic acid is chemical synthesis at present, but the yield of the product of the method is not high. In addition, benzene is mainly used as a raw material in the chemical synthesis process of adipic acid, and the benzene is synthesized by a chemical method, so that the toxicity of the raw material and an intermediate product is strong, and a large amount of N is generated in the process₂And greenhouse gases such as O and the like cause serious and non-sustainable environmental pollution.

To solve the above problems, people focus on the way to biosynthesize adipic acid, and do a lot of basic work. The main methods reported to date for the biosynthesis of adipic acid are the biocatalytic method and the total biosynthetic method. The method for fully biologically synthesizing the adipic acid by taking the glucose as the substrate has the outstanding advantages of simple process flow, low total input cost, recycling and the like, and is favored by researchers. At present, the main biological catalysis method reported is mainly to use escherichia coli as a host to catalyze and synthesize adipic acid precursor cis, cis-muconic acid, and then to catalyze and synthesize adipic acid by using a metal catalyst. In addition, Escherichia coli totally biosynthesizes adipic acid by using glucose as a substrate and using acetyl CoA and succinyl CoA as substrates, and a higher yield is also achieved. But the adipic acid synthesized by the escherichia coli in a full-biological way has some defects, for example, the escherichia coli belongs to prokaryotes, has poor stress resistance, is not acid-resistant, has poor genetic stability of strains, and is not easy to carry out large-scale industrial fermentation production; in addition, Escherichia coli belongs to non-food safe strains, and the biosynthesis products of Escherichia coli are not easy to apply to the fields of food manufacturing and the like.

Based on the above problems, more and more researchers have chosen Saccharomyces cerevisiae as a host for the biosynthesis of adipic acid. The saccharomyces cerevisiae is the simplest eukaryotic organism, has clear genetic background, easy gene operation, strong genetic stability, vigorous vitality, strong acid resistance and stress resistance, can produce various types of organic acids, has a plurality of endogenous metabolic pathways supporting the synthesis of the organic acids, and is one of the most common strains for large-scale industrial fermentation production. This has made Saccharomyces cerevisiae attractive to researchers in adipic acid biosynthesis. Among them, some researchers have utilized the saccharomyces cerevisiae fatty acid oxidation method to biologically synthesize adipic acid completely, and have obtained more ideal yield. Currently, researchers have reported that adipic acid is synthesized de novo by using saccharomyces cerevisiae as a host and glucose as a substrate, but the yield is low, probably because no suitable metabolic pathway is found for the de novo synthesis of adipic acid by using glucose as a substrate in the saccharomyces cerevisiae host.

The six enzymes involved in the pathway are β -ketothiolase (Tfu _0875), 3-hydroxyacyl-CoA dehydrogenase (Tfu _2399), 3-hydroxyadipyl-CoA dehydrogenase (Tfu _0068), 5-carboxy-2-pentenyl-CoA reductase (Tfu _1648) and succinyl CoA synthetase (Tfu _2576, Tfu _2577) respectively, however, due to the lack of tools for genetically engineering the strain, the content of adipic acid is increased, compared with the current adipic acid biosynthetic pathway, the yield of the adipic acid reverse degradation pathway is higher, the yield of the adipic acid reverse degradation pathway can be increased by extremely high means of glucose engineering, the energy consumption for fermentation is lower than that of the current strain, the production cost of glucose is lower than that of glucose, the production cost is high, the production cost is lower than that of glucose by fermentation, the current strain is 355, the fermentation cost is lower than that of glucose fermentation, the production cost is high, the production cost is lower than that of glucose, the current strain is required for fermentation, the production of glucose, the production cost is high, the energy consumption is lower than that of glucose, the current strain is required for fermentation, the production cost is high, the production cost of glucose is lower than that of glucose, the current strain is high, the production cost of glucose is high, the strain is high, the production cost is high is required for fermentation process is high, the production cost is high is required for production of glucose is high, the production of glucose is required for the strain is high is required for fermentation process is high, the strain is required for production of glucose, the strain.

Theoretically, by combining the advantages of the Tfu adipic acid reverse degradation pathway and the advantages of a Saccharomyces cerevisiae host, the method not only can obtain higher yield and yield of adipic acid, but also can overcome various problems in large-scale industrial production, such as strain stress resistance, acid resistance, genetic stability and the like, and is a good strategy.

Disclosure of Invention

The invention firstly provides a recombinant saccharomyces cerevisiae for producing adipic acid, which over-expresses genes from Thermobifida fusca β -ketothiolase, 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipoyl dehydrogenase, 5-carboxy-2-pentenyl-CoA reductase and adipoyl-CoA synthetase.

In one embodiment of the invention, the overexpression is a modularized overexpression, and specifically comprises β -ketothiolase and 3-hydroxyacyl-CoA dehydrogenase genes co-expressed by using the same vector, 3-hydroxyadipyl dehydrogenase genes and 5-carboxy-2-pentenyl-CoA reductase genes co-expressed by using the same vector, and adipyl-CoA synthetase genes expressed by using one vector.

In one embodiment of the invention, the recombinant saccharomyces cerevisiae takes saccharomyces cerevisiae BY4741 or saccharomyces cerevisiae BY4741 with L SC1 gene knocked out as a host.

In one embodiment of the invention, the modular overexpression is to express β -ketothiolase gene and 3-hydroxyacyl-CoA dehydrogenase gene by using pRS423 as an expression vector, express 3-hydroxyadipoyl dehydrogenase gene and 5-carboxy-2-pentenyl-CoA reductase gene by using pHAC181 as an expression vector, and express adipoyl-CoA synthetase gene by using Y42 as an expression vector.

In one embodiment of the invention, the Accession number of the β -ketothiolase gene (Ttu _0875) is MN550906, the nucleotide sequence is shown as SEQ ID NO.1, the Accession number of the 3-hydroxyacyl-coenzyme A dehydrogenase gene (Ttu _2399) is MN550907, the nucleotide sequence is shown as SEQ ID NO.2, the Accession number of the 3-hydroxyadipyl dehydrogenase gene (Ttu _0068) is MN550908, the nucleotide sequence is shown as SEQ ID NO.3, the Accession number of the 5-carboxy-2-pentenyl-coenzyme A reductase gene (Ttu _1648) is MN550909, the nucleotide sequence is shown as SEQ ID NO.4, and the Accession number of the adipyl-coenzyme A synthetases (Ttu _2576, Ttu _2577) is MN550910 and MN550911, and the nucleotide sequences are shown as SEQ ID NO.5 and SEQ ID NO. 6.

The second purpose of the invention is to provide a method for constructing the recombinant saccharomyces cerevisiae, which comprises the following steps:

(1) connecting genes Tfu _0875 and Tfu _2399 by taking the plasmid pRS423 as a skeleton vector to obtain a recombinant plasmid pRS423-Tfu _0875-Tfu _ 2399;

(2) plasmid pHAC181 is used as a skeleton vector to connect genes Ttu _0068 and Ttu _1648, so as to obtain recombinant plasmid pHAC 181-Ttu _ 0068-Ttu _ 1648;

(3) connecting genes Tfu _2576 and Tfu _2577 by taking a plasmid Y42 as a skeleton vector to obtain a recombinant plasmid Y42-Tfu _2576-Tfu _ 2577;

(4) pRS 423-Ttu _ 0875-Ttu _2399, pHAC 181-Ttu _ 0068-Ttu _1648 and Y42-Ttu _ 2576-Ttu _2577 are respectively transferred into wild type BY4741 or a saccharomyces cerevisiae BY4741 with L SC1 gene knocked out to obtain recombinant saccharomyces cerevisiae P123 and P123 delta L SC 1.

In one embodiment of the invention, the step (1) uses gDNA of Saccharomyces cerevisiae BY4741 as a template, uses primers CN9847-1-AF/R, CN9847-1-BF/R, CN9847-1-DF/R, CN9847-1-EF/R, CN9847-1-GF/R to respectively amplify gene segments Tcyc1-1, Ttef1-1, Ptef1-1, Pgpd1-1 and Ttdh2-1, and homologous recombination and connection are carried out on the gene segments Tcyc1-1, Ttu 36ef 1-1, Ttu _0875 and Ttu _2399 shown in SEQ ID NO.1/2 obtained BY gene synthesis according to the sequence of a plasmid map, the full length of each segment is connected to a T vector, and the intermediate construction of pUCmpmT-Ttu _ 0875-Ttu _2399 is obtained; intermediate construct pUCMT-Tfu _0875-Tfu _2399 was digested with plasmid pRS423 using XhoI and SacI, followed by T₄The DNA ligase is connected to obtain a recombinant plasmid pRS 423-Ttu _ 0875-Ttu _ 2399.

In one embodiment of the present invention, the step (2) uses gDNA of Saccharomyces cerevisiae BY4741 as a template, and uses primers CN9847-2-AF/R, CN9847-2-BF/R, CN9847-2-DF/R, CN9847-2-EF/R, CN9847-2-GF/R to amplify gene segments Ttdh2-2, Tadh1-2, Padh1-2, Ppgk1-2, Tpgk1-2 and genes thereof respectivelyHomologous recombination and connection are carried out on the Tfu _0068 and Tfu _1648 fragments of which the synthesized nucleotide sequences are shown as SEQ ID NO.3/4 according to the sequence of a plasmid map, the full length of each fragment is connected to a T vector, and the intermediate constructed pUCMT-Tfu _0068-Tfu _1648 is obtained; the intermediate construct pUCMT-Tfu _0068-Tfu _1648 was digested simultaneously with plasmid pHAC181 using NdeI and EcoRI, followed by T₄The DNA ligase is connected to obtain a recombinant plasmid pHAC 181-Ttu _ 0068-Ttu _ 1648.

In one embodiment of the invention, the step (3) uses the obtained gDNA of Saccharomyces cerevisiae BY4741 as a template, and uses primers CN9847-3-AF/R, CN9847-3-BF/R, CN9847-3-DF/R, CN9847-3-EF/R, CN9847-3-GF/R to respectively amplify gene segments Tpgk1-3, Ttpi1-3, Ptpi1-3, Ptdh3-3 and Tfab1-3, and homologous recombination and connection are carried out on the Tfu _2576 and the Tfu _2577 segments which have nucleotide sequences obtained BY gene synthesis (Suzhou news) such as shown in SEQ ID NO.5/6, and all the segments are connected to a T vector in full length to obtain intermediate pUCMT-Tfu _2576-Tfu _ 2577; the intermediate construct pUCMT-Tfu _2576-Tfu _2577 was digested doubly with plasmid Y42 using BamHI and EcoRI, followed by T₄The DNA ligase is connected to obtain a recombinant plasmid Y42-Tfu _2576-Tfu _ 2577.

The third purpose of the invention is to provide a method for producing adipic acid by fermentation, wherein the method uses recombinant saccharomyces cerevisiae P123 and P123 delta L SC1 for fermentation.

In one embodiment of the invention, the method cultures the recombinant Saccharomyces cerevisiae at 30 ℃ to OD₆₀₀Transferring to YPD medium at an inoculum size of 10% when the inoculum size is 1.0-1.2, and fermenting at 30 deg.C for 96 hr.

In one embodiment of the invention, the fermentation medium is SD (-Ura-His-L eu) medium.

The invention also claims the application of the recombinant saccharomyces cerevisiae for producing the adipic acid in the preparation of products containing the adipic acid.

The invention has the beneficial effects that: the efficient adipic acid biosynthesis pathway, namely the Tfu adipic acid reverse degradation pathway is successfully introduced into the saccharomyces cerevisiae recombinant bacteria, and the de novo synthesis of adipic acid in a saccharomyces cerevisiae host is realized. Compared with the synthesis of adipic acid by a chemical method and the synthesis of adipic acid by a full biological method, the recovery of the product is more convenient and simpler, and the pollution degree to the environment is greatly reduced; compared with other adipic acid synthesis hosts, the saccharomyces cerevisiae provides a foundation for industrial fermentation production of adipic acid by virtue of excellent stress resistance, acid resistance and genetic stability; meanwhile, the saccharomyces cerevisiae is used as a food safety (GRAS) strain, and can realize the direct application of biosynthesis of adipic acid in the food field.

The recombinant saccharomyces cerevisiae constructed by the invention can produce adipic acid by taking glucose as a unique carbon source, and the fermentation yield of the P123 △L SC1 strain reaches 33 mg/L by introducing a Tfu adipic acid reverse degradation way and controlling the fermentation conditions on the level of a shake flask, so that the crossing of the synthesis of the adipic acid in a saccharomyces cerevisiae host from scratch is realized, and the yield of the adipic acid in the saccharomyces cerevisiae host P123 is improved to 0.113 g/L by fermentation optimization.

Drawings

FIG. 1 is a schematic representation of the Tfu adipate reverse degradation pathway;

FIG. 2 is a colony pcr verification map of BY4741 knock-out L SC1 gene, 1, 2, 5, 6 and 7: knockout of L SC1 successful strain, 3 and 4: no knockout successful strain, 8: blank control and M: 5000bp Marker;

FIG. 3 is a plasmid map of pRS 423-Ttu _ 0875-Ttu _ 2399;

FIG. 4 is a map of the pHAC 181-Ttu _ 0068-Ttu _1648 plasmid;

FIG. 5 is a plasmid map of Y42-Tfu _2576-Tfu _ 2577;

FIG. 6 is a verification map of recombinant plasmid pcr. 1: pRS 423-Ttu _ 0875-Ttu _ 2399; 2: pHAC181-Tfu _0068-Tfu _ 1648; 3: Y42-Tfu _2576-Tfu _ 2577; m: 5000bp Marker;

FIG. 7 is a verification map of recombinant Saccharomyces cerevisiae P123 colony pcr. M: 5000bp Marker; 1: gene fragment Tfu _ 0875; 2: gene fragment Tfu _ 2399; 3: gene fragment Tfu _ 0068; 4: gene fragment Tfu _ 1648; 5: gene fragment Tfu _ 2576; 6: gene fragment Tfu _ 2577;

FIG. 8 is a BY4741 wild type, BY 4741. DELTA. L SC1, P123 and P123. DELTA. L SC1 adipic acid production profiles in YPD medium.

Detailed Description

TABLE 1 primer sequence Listing in relation to the following examples

β -ketothiolase gene (Ttu _0875) has the nucleotide sequence shown in SEQ ID NO.1, 3-hydroxyacyl-coenzyme A dehydrogenase gene (Ttu _2399) has the nucleotide sequence shown in SEQ ID NO.2, 3-hydroxyadipyl dehydrogenase gene (Ttu _0068) has the nucleotide sequence shown in SEQ ID NO.3, 5-carboxy-2-pentenyl-coenzyme A reductase gene (Ttu _1648) has the nucleotide sequence shown in SEQ ID NO.4, and adipoyl-coenzyme A synthetase genes (Ttu _2576, Ttu _2577) have the nucleotide sequences shown in SEQ ID NO.5 and SEQ ID NO.6, respectively.

Example 1 knock-out of the succinate- -CoA ligand (GDP-forming) subBunit alpha (L SC1) Gene in BY4741

Obtaining a recombinant saccharomyces cerevisiae L SC1 gene knockout frame, namely amplifying a DNA fragment with the size of 1700bp BY using delta L SC1-pUG6-F and delta L SC1-pUG6-R as primers and a pUG6 vector as a template through PCR reaction, wherein the fragment is homologous sequences with 50bp at the head and the tail of an L SC1 gene ORF frame on a BY4741 genome respectively.

Transformation of recombinant Saccharomyces cerevisiae Gene knockout cassette plate-activated Saccharomyces cerevisiae BY4741 single colony was inoculated into 100m L conical flask containing 10m L YPD liquid medium at 250 r.min^-1Shaking overnight at 30 deg.C as fresh seed solution, transferring 1% into 250m L conical flask containing 50m L YPD medium, and culturing under the same condition for 5-8 hr to OD₆₀₀Stopping culturing to about 0.8-1.0: (2) collecting bacterial liquid with a 50m L centrifuge tube, 6000 r.min^-1Centrifuging at 4 deg.C for 10min, and collecting bacteria(3) pouring out supernatant, washing thalli with precooled 25m L sterile water, discarding supernatant and collecting thalli after low-temperature centrifugation, (4) repeating the operation for 3 times, (4) discarding supernatant and collecting thalli, adding 1m L sterile cell suspension (5% glycerol, 10% dimethyl sulfoxide, v/v) to resuspend thalli, subpackaging according to 100 mu L as competence for transformation, storing in a refrigerator at-80 ℃ for subsequent experiments, (5) incubating competent cells at 30 ℃ for 2min, 8000 r.min^-1Centrifuging for 2min, removing supernatant, preparing a transformation system which comprises 260 mu L50% PEG 4000, 36 mu L1M lithium acetate, 2 mu G DNA fragment to be transformed, supplementing 10 mu L ssDNA with sterile water to 360 mu L, resuspending for 1min, (6) heat shock at 42 ℃ for 30min, centrifuging at 6000rpm for 5min, removing supernatant, resuspending thallus with sterile water, centrifuging at 6000rpm for 5min, discarding supernatant, and directly coating thallus on YPD (G418 resistance) plate.

And obtaining a BY4741 delta L SC1 strain, namely picking fresh single colonies growing on the plate into 20 mu L sterile water, uniformly mixing, taking 1 mu L as a PCR reaction template, using a knockout verification primer to carry out PCR reaction, preparing a reaction system according to a table 2, carrying out agarose gel electrophoresis on a PCR product, selecting a single colony with the band size of 3190bp, namely a correct knockout strain (the contrast is 2572bp), and naming the strain as BY4741 delta L SC 1.

TABLE 2 colony PCR reaction System

Weighing 0.3g of agarose, dissolving in 25m L1 × TAE Buffer solution, heating for 30-60s to completely dissolve the agarose, adding 5 mu L EB, shaking uniformly, pouring into a horizontal gel frame inserted with a comb to avoid generating bubbles, standing until the agarose is solidified, slightly pulling out the comb, putting the gel into an electrophoresis tank, pouring the electrophoresis Buffer solution to enable the gel to be submerged in a gel block, adding a proper amount of 10 ×L applying Buffer according to the amount of the agarose, vertically dropping into a sample loading hole, inserting an electrode, running for 30min at 100V, taking out the gel block, and putting into a gel imaging instrument for taking a picture.

Example 2: construction of recombinant plasmid and obtaining of recombinant saccharomyces cerevisiae

The sequences of Tfu _0875, Tfu _2399, Tfu _0068, Tfu _1648, Tfu _2576, Tfu _2577 have been published in NCBI.

Carrying out PCR amplification BY using the obtained gDNA of the Saccharomyces cerevisiae BY4741 as a template and using a primer (table 1) with a homologous arm to obtain gene fragments Tcyc1-1, Tref 1-1, Ptef1-1, Pgpd1-1 and Ttdh2-1, wherein Tcyc1-1 is amplified BY using a primer CN 9847-1-AF/R; ttef1-1 was amplified with primer CN 9847-1-BF/R; amplifying Ptef1-1 by using a primer CN 9847-1-DF/R; pgpd1-1 was amplified with the primer CN 9847-1-EF/R; ttdh2-1 is amplified by adopting a primer CN 9847-1-GF/R; and (3) carrying out homologous recombination and connection on the amplified Tcyc1-1, Tref 1-1, Ptef1-1, Pgpd1-1 and Ttdh2-1 and synthesized Ttu _0875 and Ttu _2399 fragments with sequences shown in SEQ ID NO.1 and SEQ ID NO.2, and connecting all the fragments to a T vector in the sequence shown in the figure 3 to obtain the intermediate constructed pUCt-Ttu _ 0875-Ttu _ 2399. Intermediate construct pUCMT-Tfu _0875-Tfu _2399 was digested with plasmid pRS423 using XhoI and SacI, followed by T₄The DNA ligase is connected to obtain a recombinant plasmid pRS 423-Ttu _ 0875-Ttu _ 2399.

Performing PCR amplification BY using the obtained gDNA of the Saccharomyces cerevisiae BY4741 as a template and using a primer (table 1) with a homologous arm to obtain gene segments Ttdh2-2, Tadh1-2, Padh1-2, Ppgk1-2 and Tpgk1-2, wherein the Ttdh2-2 is amplified BY using a primer CN 9847-2-AF/R; tadh1-2 was amplified with primer CN 9847-2-BF/R; the primer CN9847-2-DF/R is adopted to amplify the Padh 1-2; adopting a primer CN9847-2-EF/R to amplify Ppgk 1-2; amplifying Tpgk1-2 by using a primer CN 9847-2-GF/R; and performing homologous recombination and connection on the amplified Ttdh2-2, Tadh1-2, Padh1-2, Ppgk1-2 and Tpgk1-2 and gene-synthesized Tfu _0068 and Tfu _1648 fragments shown in SEQ ID NO.3 and 4, and connecting all the fragments to a T vector in the sequence shown in the figure 4 to obtain an intermediate construct pUCMT-Tfu _0068-Tfu _ 1648. The intermediate construct pUCMT-Tfu _0068-Tfu _1648 was digested simultaneously with plasmid pHAC181 using NdeI and EcoRI, followed by T₄The DNA ligase is connected to obtain a recombinant plasmid pHAC 181-Ttu _ 0068-Ttu _1648

Carrying out PCR amplification BY using the obtained gDNA of the Saccharomyces cerevisiae BY4741 as a template and using a primer (table 1) with a homologous arm to obtain gene fragments Tpgk1-3, Ttpi1-3, Ptpi1-3, Ptdh3-3 and Tfab1-3, wherein the Tpgk1-3 is amplified BY using a primer CN 9847-3-AF/R; amplification with primer CN9847-3-BF/RTtpi 1-3; amplifying Ptpi1-3 by using a primer CN 9847-3-DF/R; amplifying Ptdh3-3 by using a primer CN 9847-3-EF/R; adopting a primer CN9847-3-GF/R to amplify Tfab 1-3; the amplified Tpgk1-3, Ttpi1-3, Ptpi1-3, Ptdh3-3 and Tfab1-3 are subjected to homologous recombination and connection with Tfu _2576 and Tfu _2577 fragments of which the sequences synthesized by the genes are shown in SEQ ID NO.5 and 6, and the fragments are connected to a T vector in full length according to the sequence shown in the figure 5 to obtain an intermediate construct pUCMT-Tfu _2576-Tfu _ 2577. The intermediate construct pUCMT-Tfu _2576-Tfu _2577 was digested doubly with plasmid Y42 using BamHI and EcoRI, followed by T₄The DNA ligase is connected to obtain a recombinant plasmid Y42-Tfu _2576-Tfu _ 2577.

pRS 423-Ttu _ 0875-Ttu _2399, pHAC 181-Ttu _ 0068-Ttu _1648 and Y42-Ttu _ 2576-Ttu _2577 are respectively transferred into wild Saccharomyces cerevisiae BY4741 and a L SC1 gene-knocked Saccharomyces cerevisiae BY4741 to obtain recombinant Saccharomyces cerevisiae P123 and P123 delta L SC 1.

Example 3: recombinant saccharomyces cerevisiae shake flask fermentation

YPD culture medium comprising yeast extract 10 g/L, peptone 20 g/L, and glucose 20 g/L, wherein 2.0% agar is added into the solid YPD culture medium, the pH value is 5.4-5.6, the medium is sterilized by high pressure steam at 121 ℃ for 30 min;

SD medium: glucose 20g, YNB Medium (No. (NH))₄)₂SO₄1.7g),(NH₄)₂SO₄5g, adding 1000m L deionized water, the pH value of which is 5.5, sterilizing for 15min by high pressure steam at 115 ℃, and adding proper sterilized amino acid, pyrimidine and agar powder according to a selective marker before inoculation.

L B Medium (g/L) 10 tryptone +5 Yeast powder +10 NaCl.

The fermentation condition is that the recombinant Saccharomyces cerevisiae is cultured at 30 ℃ to OD with SD (-Ura-His-L eu) culture medium as fermentation culture medium₆₀₀1.0-1.2 times, washing with sterile water for 2 times, inoculating to YPD medium at an inoculum size of 10%, fermenting for 96h, loading liquid at 50m L/250 m L, rotating at 250rpm, and temperature of 30 deg.C.

BY4741 wild type strain, L SC1 gene knock-out strain BY4741 delta L SC1 as control strain, and culturing under the same conditions, wherein the yield of adipic acid is 0, the highest yield of BY4741 delta L SC1 adipic acid is 0, the highest yield of P123 adipic acid is 0.113 g/L, and the highest yield of P123 delta L SC1 adipic acid is 0.033 g/L.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

<110> university of south of the Yangtze river

<120> method for producing adipic acid by using saccharomyces cerevisiae

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ctgaatgacg cagctaggat gataggcgat gggtatgcga ccgccgacga tgtggataca 1020

gccatgcgtc tgggttgcgg ttaccccaag ggcttagccg ccatgctgga cgagcgtgga 1080

gtcaagaacg tgactgagac tttggcagag ctggcagcgg caggactttt tacggatgat 1140

acagcaccgt tgcttacaat gttagcgaag caaggaaaag acactctgag aagctga 1197

<210>3

<211>771

<212>DNA

<213>Saccharomyces cerevisiae

<400>3

atgggtgagt tcattagatt tgagtctgac gggcccgtga gacatatagt gttaaatgca 60

ccccaaagac ttaatgcact tgatcgtcct atgttagctg aattagctga ggccgttcgt 120

gccgtggccg cggatgaaga ggcccgtgca ttggttgtga gcggcgctgg tagagccttc 180

tgcgctgggg ccgatgtcac ctcattgttt ggcgatccaa cccgtccccc ggcagtaatc 240

cgtgatgaac taaaacaagt atatgcgtct tttctgtcta tcgctgatct gacgattccc 300

accattgccg ccgtaggggg tattgctgtg ggagcgggag taaacatagc gatggcctgt 360

gacatggtcg ttgctgggcc aaaagccaaa ttcgctatca cttttgccga aatggggtta 420

catcctgggg gtgggtgttc ctggttcctt acaaggagga tggggggtca cagggcactg 480

gcgaccctac ttgatgctga gaggattgat gcggaagaag cattcagggc cggattagtt 540

acaaggctag tggaagatcc cgtggctgaa gcgctggcaa tggcacacag ctatgctgag 600

agagatcctg gccttgttcg tgatatgaaa agggcagtca gaatggcaga aaccgccgac 660

ctagctactg tgctagaatt tgaaagctgg gcacaagcaa gtagcgtcaa tagcccaaga 720

tttcaggagt tcctggcaga gtttgcagcg aggaaaaaca aaaaagaatg a 771

<210>4

<211>1158

<212>DNA

<213>Saccharomyces cerevisiae

<400>4

atgtctgatt tcgatttata tagaccgacc gaagagcatg aagcattaag ggaggccatt 60

aggtccgtgg cagaggataa aatagctcct cacgctgcag atgtggatga gcagagcagg 120

ttcccacaag aagcgtacga ggctctgcgt gcaagtgact tccacgctcc tcatgtggct 180

gaagagtacg gcggtgtcgg cgctgatgcg ctggcgactt gtattgtaat tgaagaaatc 240

gccagagtat gtgcgagctc ctccttaatc ccagcagtga acaaattggg tagtatgcct 300

ttgatactgt ctgggagtga cgaagtaaaa cagcgttact tgcccgaatt ggccagcgga 360

gaggctatgt ttagttatgg gctatctgaa agggaagctg gtagtgacac tgcctccatg 420

agaactcgtg cggtgagaga cggggatgac tggatactga atggccaaaa gtcctggata 480

accaatgctg gaatttccaa gtactatacc gttatggctg taactgaccc agacggaccc 540

aggggtagga atattagcgc atttgtagta cacatagatg atcccggttt tagtttcgga 600

gaacctgaga gaaagctagg aataaagggg tctccaactc gtgagctaat cttcgataat 660

gttaggattc cgggggatag attggtgggt aaggtcggtg aaggtttaag gactgctcta 720

aggactttgg accatacgag ggtaactatt ggagctcaag ccgttgggat tgcgcagggc 780

gcattagatt acgcacttgg ttatgtaaag gagaggaagc agttcggtaa ggcaattgct 840

gacttccagg ggatccaatt catgctggct gatatggcca tgaaattaga ggctgcacgt 900

cagatggtgt atgtcgccgc agcgaaatct gaacgtgatg acgccgactt atccttttac 960

ggcgcggcag caaagtgctt tgcgtccgat gtcgcaatgg aaataaccac cgacgccgtt 1020

cagttgttag gcgggtatgg atatactaga gactatccag tagaacgtat gatgcgtgat 1080

gccaaaataa cgcagatcta cgaaggtacg aatcagatcc aacgtgtagt catggcaagg 1140

cagctgctga aaaaatga 1158

<210>5

<211>879

<212>DNA

<213>Saccharomyces cerevisiae

<400>5

atggctatct tcttaaccaa agactccaaa gtccttgtgc agggcatgac tgggagtgag 60

ggaacaaagc ataccagaag aatgttggca gccgggacaa acatagttgg cggggttaac 120

ccgcgtaagg cgggtcaagt tgtggacttc gatggtacac aggtgcctgt attcggatca 180

gtcgctgagg gtatgaaagc tactggtgcc gatgtcaccg ttatctttgt accgccgaaa 240

tttgcaaaag atgctgtaat agaggcgatt gatgcggaga ttggtctagc tgtagttatc 300

actgagggca ttccggttca tgataccgca accttctggg ctcatgcttg cagtaagggt 360

aacaaaacac gtattattgg gcccaactgt cctgggctta ttactccagg ccaatcaaat 420

gcaggaataa tacccgccga tataacgaaa ccgggccgta ttggtcttgt aagtaagtcc 480

ggtacgctaa cttatcagat gatgtacgag ctaagggata tcgggtttag cacttgtgta 540

gggattggcg gggacccgat tatagggaca acccacattg acgcgctagc ggcttttgag 600

gcagaccccg ataccgatgt tatcgttatg atcggcgaaa ttggcgggga tgccgaggaa 660

agggccgccg aatatataaa aaagcatgtt accaagccgg tagtcggtta catagctggt 720

ttcacagcac ctgagggtaa gactatgggg catgctggcg cgattgtatc tggtagctct 780

ggaacagccg ctgcgaagaa ggaggcgtta gaagccgtcg gagtaaaagt tggtaagact 840

ccgtctgagg ctgcgaaatt ggtgaggagt ttgttctaa 879

<210>6

<211>1233

<212>DNA

<213>Saccharomyces cerevisiae

<400>6

atgaacgact tgaggagaaa ccccgcttct gagtcccaag gcagaactct ggtggatttg 60

ttcgagtatc aggctaaggc gctatttgcg gaatatggcg tgcctgtccc acaagggaag 120

gtggcctcaa cgccagaaga agtgcgtgct atcgcagagg agtttgcggc agcggggaag 180

ccaagggtcg ttgtgaaggc gcaggttaag actggcggtc gtggaaaagc aggcggcgtg 240

aaggtggcgg atggtcccga tgacgccgtc gctaaagcaa aacaaatatt aggtatggat 300

ataaaaggcc atactgttca tcgtgtactt gtagaggaag cgagcgatat agctgaagaa 360

tattacttta gttttctgtt agacagggcg aacagaagtt tcctttcaat ttgctcagcg 420

gaaggcggaa tggagattga ggaagtcgct gccacaaacc cggacgcagt cgcgaaggtt 480

ccgatctcac cccttaaagg tgcccctgcc gatgtggcgg cagacatcgt agctcagggt 540

aaattgcctg aagctgccgc acagggggct gttgatgtta ttacgaaatt atggaaagta 600

tttgttgaga aggacgcgac ccttgtggaa gtgaatccac taattctgac aaaggatggt 660

cgtgttgtgg ctttagacgg caaggtcaca ctagacgata atgcggagtt taggcaggat 720

ttggagtctc ttgcatctgc tgccgaaggt gatcctctag aagtaaaagc taaggagaaa 780

ggtctgaatt atgtaaagtt ggacggagaa gtgggtataa tcgggaatgg tgctggctta 840

gtaatgagta cattagacgt cgtggcctac gcgggagagc aacatggagg agtaaaacct 900

gcgaactttc tggatatcgg tggcggtgca tctgcggaag ttatggctaa tgggttggag 960

ataattctgt ccgatcctgc agtgaagagt gtgtttgtta atgtctttgg cggcattaca 1020

gcgtgcgacg cagtggcgaa tggcattgtt caagcactag agctgcttga gagtaggggg 1080

gaggatgtga caaaacccct ggttgtaaga ttagacggga acaacgcgga actaggcagg 1140

tcaatactga acgagaggaa ccatcctgcc gtacgtcaag ttgacacgat ggacggggca 1200

gctgctctgg ccgcggaact agcggcgaaa taa 1233

Claims

1. A recombinant saccharomyces cerevisiae for producing adipic acid is characterized in that genes of β -ketothiolase, 3-hydroxyacyl-coenzyme A dehydrogenase, 3-hydroxyadipyl dehydrogenase, 5-carboxy-2-pentenyl-coenzyme A reductase and adipyl-coenzyme A synthetase are expressed in a module, wherein the expression in the module is specifically that β -ketothiolase and 3-hydroxyacyl-coenzyme A dehydrogenase genes are co-expressed by adopting the same vector, 3-hydroxyadipyl dehydrogenase genes and 5-carboxy-2-pentenyl-coenzyme A reductase genes are co-expressed by adopting the same vector, and adipyl-coenzyme A synthetase genes are co-expressed by adopting the same vector.

2. The recombinant saccharomyces cerevisiae of claim 1, wherein the modular overexpression is realized by expressing β -ketothiolase gene and 3-hydroxyacyl-coa dehydrogenase gene with pRS423 as an expression vector, expressing 3-hydroxyadipyl dehydrogenase gene and 5-carboxy-2-pentenyl-coa reductase gene with pHAC181 as an expression vector, and expressing adipyl-coa synthase gene with Y42 as an expression vector.

3. The recombinant Saccharomyces cerevisiae according to claim 1 or 2, wherein Saccharomyces cerevisiae BY4741 or Saccharomyces cerevisiae BY4741 with L SC1 gene deleted is used as the host.

4. A method for constructing the recombinant saccharomyces cerevisiae of any one of claims 1 to 3, which is characterized by comprising the following steps:

(1) connecting β -ketothiolase gene Ttu _0875 and 3-hydroxyacyl-coenzyme A dehydrogenase gene Ttu _2399 by taking plasmid pRS423 as a skeleton vector to obtain a recombinant plasmid pRS 423-Ttu _ 0875-Ttu _ 2399;

(2) plasmid pHAC181 is used as a skeleton vector to connect gene 3-hydroxyadipyl dehydrogenase gene Tfu _0068 and 5-carboxyl-2-pentenyl coenzyme A reductase gene Tfu _1648, so as to obtain recombinant plasmid pHAC181-Tfu _0068-Tfu _ 1648;

(3) connecting adipoyl-CoA synthetase genes Tfu _2576 and Tfu _2577 by taking a plasmid Y42 as a skeleton vector to obtain a recombinant plasmid Y42-Tfu _2576-Tfu _ 2577;

(4) and (3) transferring the pRS 423-Ttu _ 0875-Ttu _2399, pHAC 181-Ttu _ 0068-Ttu _1648 and Y42-Ttu _ 2576-Ttu _2577 constructed in the steps (1) to (3) into a saccharomyces cerevisiae cell.

5. The method according to claim 4, wherein the Saccharomyces cerevisiae of step (4) is Saccharomyces cerevisiae BY4741 or Saccharomyces cerevisiae BY4741 with L SC1 gene knocked out.

6. A method for producing adipic acid by fermentation, which is characterized in that the recombinant Saccharomyces cerevisiae of any one of claims 1-3 is inoculated into a fermentation medium and fermented at 28-32 ℃ for at least 48 h.

7. The method of claim 6, wherein the recombinant Saccharomyces cerevisiae is cultured at 28-30 ℃ to OD₆₀₀When the amount is 1.0-1.2%, transferring the strain to a fermentation medium by 8-12% by volume, and fermenting for 48-96 h at 28-30 ℃.

8. The method of claim 6 or 7, wherein the fermentation medium is glucose as a carbon source.

9. The method of claim 6 or 7, wherein the fermentation medium is SD (-Ura-His-L eu) medium.

10. The use of the recombinant Saccharomyces cerevisiae of any of claims 1-3 in the preparation of adipic acid or its derivatives in the fields of chemical production, organic synthesis industry, medicine, and lubricant manufacture.