CN111484942A - Method for producing adipic acid by using saccharomyces cerevisiae - Google Patents

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

A kind of method utilizing Saccharomyces cerevisiae to produce adipic acid

technical field

The invention relates to a method for producing adipic acid by utilizing Saccharomyces cerevisiae, and belongs to the field of biological engineering.

Background technique

Adipic acid (adipate), also known as fatty acid, is an important organic dibasic acid, which is widely used in chemical production, organic synthesis industry, medicine, lubricant manufacturing and so on.

At present, the main production method of adipic acid is chemical synthesis, but the product yield of this method is not high. In addition, in the chemical synthesis process of adipic acid, benzene is mainly used as the raw material, and the raw materials and intermediate products are highly toxic, and a large amount of greenhouse gases such as N ₂ O is generated in the process, which causes serious and unsustainable environmental pollution.

In order to solve the above problems, people have focused their attention on the biosynthesis of adipic acid, and a lot of basic work has been done. The main methods of biosynthesis of adipic acid currently reported include biocatalysis and total biosynthesis. The total biosynthesis of adipic acid using glucose as a substrate has outstanding advantages such as simple process flow, low total input cost, and recyclability, so it is favored by researchers. The main biocatalytic methods reported at present are mainly to use Escherichia coli as the host to catalyze the synthesis of adipic acid precursor cis, cis-muconic acid, and then use metal catalysts to catalyze the synthesis of adipic acid. In addition, Escherichia coli also achieved high yields by using acetyl-CoA and succinyl-CoA as substrates to fully biosynthesize adipic acid from glucose. However, the total biosynthesis of adipic acid by Escherichia coli has some drawbacks. For example, Escherichia coli is a prokaryotic organism, with poor stress resistance, acid intolerant, and poor genetic stability of strains, so it is not easy to carry out large-scale industrial fermentation production; in addition, Escherichia coli belongs to non-food safety strains, and their biosynthetic products are not easily used in food manufacturing and other fields.

Based on the above problems, more and more researchers choose Saccharomyces cerevisiae as the host for the biosynthesis of adipic acid. Saccharomyces cerevisiae is the simplest eukaryotic organism with clear genetic background, easy genetic manipulation, strong genetic stability, strong vitality, strong acid resistance and stress resistance, and can produce various types of organic acids, And there are many endogenous metabolic pathways that support organic acid synthesis, and Saccharomyces cerevisiae is one of the most commonly used strains for large-scale industrial fermentation production. This has made Saccharomyces cerevisiae of great interest to researchers in adipic acid biosynthesis. Among them, some researchers used Saccharomyces cerevisiae fatty acid oxidation method to fully biosynthesize adipic acid, and obtained a relatively ideal yield. At present, researchers have reported the de novo synthesis of adipic acid using Saccharomyces cerevisiae as the host and glucose as the substrate, but the yield is low. The possible reason is that the de novo synthesis of adipic acid using glucose as the substrate under the host of Saccharomyces cerevisiae has not been found. appropriate metabolic pathways.

There are natural adipic acid synthesis or catabolism pathways in nature. Among them, the 3-oxoadipyl-CoA pathway of T. tanninosa can efficiently utilize adipic acid. At the same time, when the growth condition is good and the carbon When the source is sufficient, this pathway can also accumulate adipic acid, so this pathway is also called the T.fu adipic acid retrodegradation pathway. 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-pentenoyl-CoA reductase (Tfu_1648) and succinyl CoA synthase (Tfu_2576, Tfu_2577). However, boosting adipic acid was difficult due to a lack of tools to genetically engineer the strain. Compared with the currently reported adipic acid biosynthesis pathway, the Tfu adipic acid reverse degradation pathway is more efficient, and can produce adipic acid from carbon source substrates such as glucose with extremely high yields. At present, reconstitution of this pathway in E. coli can bring the production of adipic acid to 68 g/L, the highest value reported. However, in the actual production process of this strain, there are the following defects: 1. Poor acid resistance, the strain will crack when the pH is lower than 5; 2. The cost of feeding is high: high-cost carbon sources such as glucose must be used during production. Fermentation; 3. High energy consumption required for fermentation temperature: Since the fermentation temperature required by Escherichia coli is 37°C, the energy demand is high.

In theory, combining the advantages of the Tfu adipic acid reverse degradation pathway and the advantages of the Saccharomyces cerevisiae host, not only can higher adipic acid yield and yield be obtained, but also various problems encountered in large-scale industrial production can be overcome. Such as strain resistance, acid resistance, genetic stability, etc., is a good strategy.

SUMMARY OF THE INVENTION

The present invention first provides an adipic acid-producing recombinant Saccharomyces cerevisiae, which overexpresses β-ketothiolase, 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipoyl Genes for dehydrogenase, 5-carboxy-2-pentenoyl-CoA reductase, and adipoyl-CoA synthase.

In one embodiment of the present invention, the overexpression is submodule overexpression; specifically: β-ketothiolase and 3-hydroxyacyl-CoA dehydrogenase genes are co-expressed using the same vector; 3- The hydroxyadipoyl dehydrogenase gene and the 5-carboxy-2-pentenoyl-CoA reductase gene were co-expressed with the same vector; the adipoyl-CoA synthase gene was expressed with a single vector.

In one embodiment of the present invention, the recombinant Saccharomyces cerevisiae uses Saccharomyces cerevisiae BY4741 or Saccharomyces cerevisiae BY4741 with the LSC1 gene knocked out as a host.

In one embodiment of the present invention, the sub-module overexpression uses pRS423 as the expression vector to express β-ketothiolase gene and 3-hydroxyacyl-CoA dehydrogenase gene; pHAC181 is used as the expression vector to express 3 -Hydroxyadipoyl dehydrogenase gene and 5-carboxy-2-pentenoyl-CoA reductase gene; Y42 was used as the expression vector to express the adipoyl-CoA synthase gene.

In one embodiment of the present invention, the Accession number of the β-ketothiolase gene (Tfu_0875) is MN550906, and the nucleotide sequence is shown in SEQ ID NO. 1; The Accession number of the hydrogenase gene (Tfu_2399) is MN550907, and the nucleotide sequence is shown in SEQ ID NO.2; the Accession number of the 3-hydroxyadipoyl dehydrogenase gene (Tfu_0068) is MN550908, and the nucleotide sequence is MN550908. As shown in SEQ ID NO.3; the Accession number of the 5-carboxy-2-pentenoyl-CoA reductase gene (Tfu_1648) is MN550909, and the nucleotide sequence is shown in SEQ ID NO.4; The Accession numbers of the acyl-CoA synthases (Tfu_2576, Tfu_2577) are MN550910 and MN550911, respectively, and the nucleotide sequences are shown in SEQ ID NO.5 and SEQ ID NO.6.

The second object of the present invention is to provide a method for constructing the recombinant Saccharomyces cerevisiae, comprising the following steps:

(1) using plasmid pRS423 as backbone vector, connecting genes Tfu_0875 and Tfu_2399 to obtain recombinant plasmid pRS423-Tfu_0875-Tfu_2399;

(2) using plasmid pHAC181 as a backbone vector, connecting genes Tfu_0068 and Tfu_1648 to obtain recombinant plasmid pHAC181-Tfu_0068-Tfu_1648;

(3) using plasmid Y42 as a backbone vector, connecting genes Tfu_2576 and Tfu_2577 to obtain recombinant plasmid Y42-Tfu_2576-Tfu_2577;

(4) pRS423-Tfu_0875-Tfu_2399, pHAC181-Tfu_0068-Tfu_1648, Y42-Tfu_2576-Tfu_2577 were respectively transferred into wild-type BY4741 or Saccharomyces cerevisiae BY4741 with LSC1 gene knocked out to obtain recombinant Saccharomyces cerevisiae P123 and P123ΔLSC1.

In one embodiment of the present invention, step (1) takes the gDNA of Saccharomyces cerevisiae BY4741 as a template, and uses primers CN9847-1-AF/R, CN9847-1-BF/R, CN9847-1-DF/R, CN9847 -1-EF/R, CN9847-1-GF/R amplified gene fragments Tcyc1-1, Ttef1-1, Ptef1-1, Pgpd1-1, Ttdh2-1 respectively, and SEQ ID NO.1/ The Tfu_0875 and Tfu_2399 fragments shown in 2 are connected by homologous recombination according to the sequence of the plasmid map, and the full length of each fragment is connected to the T carrier to obtain the intermediate construction pUCmT-Tfu_0875-Tfu_2399; the intermediate construct pUCmT-Tfu_0875-Tfu_2399 and plasmid pRS423 use XhoI Double digestion with SacI, and then ligated with T ₄ DNA ligase to obtain the recombinant plasmid pRS423-Tfu_0875-Tfu_2399.

In one embodiment of the present invention, step (2) takes the 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 and CN9847-2-GF/R respectively amplify gene fragments Ttdh2-2, Tadh1-2, Padh1-2, Ppgk1-2, Tpgk1-2, and the nucleotide sequences synthesized with the genes are as shown in SEQID NO The Tfu_0068 and Tfu_1648 fragments shown in .3/4 were connected by homologous recombination according to the sequence of the plasmid map, and the full length of each fragment was connected to the T vector to obtain the intermediate construction pUCmT-Tfu_0068-Tfu_1648; pHAC181 was double digested with NdeI and EcoRI, and then ligated with T4 DNA ligase to obtain the recombinant plasmid _pHAC181 -Tfu_0068-Tfu_1648.

In one embodiment of the present invention, the gDNA of Saccharomyces cerevisiae BY4741 obtained in step (3) is used as a template, and primers CN9847-3-AF/R, CN9847-3-BF/R, CN9847-3-DF/R are used as a template. , CN9847-3-EF/R, CN9847-3-GF/R respectively amplified gene fragments Tpgk1-3, Ttpi1-3, Ptpi1-3, Ptdh3-3, Tfab1-3, and obtained with gene synthesis (Suzhou Hongxun) The nucleotide sequence of Tfu_2576 and Tfu_2577 fragments shown in SEQ ID NO.5/6 are connected by homologous recombination, and the full length of each fragment is connected to the T carrier to obtain the intermediate construct pUCmT-Tfu_2576-Tfu_2577; the intermediate construct pUCmT -Tfu_2576-Tfu_2577 and plasmid Y42 were double digested with BamHI and EcoRI, and then ligated with T4 DNA ligase to obtain recombinant plasmid Y42- _{Tfu_2576} -Tfu_2577.

The third object of the present invention is to provide a method for producing adipic acid by fermentation, which uses recombinant Saccharomyces cerevisiae P123 and P123ΔLSC1 for fermentation.

In one embodiment of the present invention, the method cultivates the recombinant Saccharomyces cerevisiae at 30°C until the OD ₆₀₀ is 1.0-1.2, transfers to YPD medium with 10% inoculum, and ferments at 30°C for 96 hours.

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

The present invention also claims the application of the adipic acid-producing recombinant Saccharomyces cerevisiae in preparing adipic acid-containing products.

The beneficial effects of the present invention are: the efficient adipic acid biosynthesis pathway, 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 the Saccharomyces cerevisiae host is realized. Compared with chemical synthesis of adipic acid, the whole biological synthesis of adipic acid is more convenient and simpler to recover the product, and greatly reduces the degree of environmental pollution; compared with other adipic acid synthesis hosts, Saccharomyces cerevisiae is Its excellent stress resistance, acid resistance and genetic stability provide the basis for the industrial fermentation production of adipic acid; at the same time, as a food safety (GRAS) strain, Saccharomyces cerevisiae can realize the direct application of biosynthesis of adipic acid in the food field .

The recombinant Saccharomyces cerevisiae constructed in the present invention can use glucose as the sole carbon source to produce adipic acid. At the shake flask level, through the introduction of the reverse degradation pathway of Tfu adipic acid and the control of fermentation conditions, the fermentation yield of P123△LSC1 strain reaches 100%. 33mg/L, realizing the leap of adipic acid synthesis in Saccharomyces cerevisiae host from scratch. And through fermentation optimization, the yield of adipic acid in Saccharomyces cerevisiae host P123 was increased to 0.113g/L. At the same time, during the adipic acid fermentation process, the host can tolerate pH values below 4 and has good genetic stability, which lays the foundation for the industrial fermentation of food-safe adipic acid with Saccharomyces cerevisiae as the host.

Description of drawings

Fig. 1 is a schematic diagram of the reverse degradation pathway of Tfu adipic acid;

Figure 2 is a PCR verification map of the colony that BY4741 knocked out the LSC1 gene. 1, 2, 5, 6, 7: strains successfully knocked out LSC1; 3, 4: strains without successful knockout; 8: blank control; M: 5000bp Marker;

Fig. 3 is pRS423-Tfu_0875-Tfu_2399 plasmid map;

Fig. 4 is pHAC181-Tfu_0068-Tfu_1648 plasmid map;

Fig. 5 is Y42-Tfu_2576-Tfu_2577 plasmid map;

Figure 6 is a PCR verification map of the recombinant plasmid. 1: pRS423-Tfu_0875-Tfu_2399; 2: pHAC181-Tfu_0068-Tfu_1648; 3: Y42-Tfu_2576-Tfu_2577; M: 5000bp Marker;

Figure 7 is the PCR verification map of the recombinant Saccharomyces cerevisiae P123 colony. 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;

Figure 8 is a graph of adipic acid production of BY4741 wild type, BY4741ΔLSC1, P123 and P123ΔLSC1 in YPD medium.

Detailed ways

Table 1 List of primer sequences involved in the following examples

The nucleotide sequence of β-ketothiolase gene (Tfu_0875) is shown in SEQ ID NO.1; the nucleotide sequence of 3-hydroxyacyl-CoA dehydrogenase gene (Tfu_2399) is shown in SEQ ID NO.2 The nucleotide sequence of 3-hydroxyadipoyl dehydrogenase gene (Tfu_0068) is shown in SEQ ID NO.3; the nucleotide sequence of 5-carboxy-2-pentenoyl-CoA reductase gene (Tfu_1648) The sequence is shown in SEQ ID NO.4; the nucleotide sequences of adipyl-CoA synthase genes (Tfu_2576, Tfu_2577) are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively.

Example 1: Knockout of succinate--CoA ligase (GDP-forming) subunit alpha (LSC1) gene in BY4741

Recombinant Saccharomyces cerevisiae LSC1 knockout box was obtained: using ΔLSC1-pUG6-F and ΔLSC1-pUG6-R as primers and pUG6 vector as template, a DNA fragment with a size of 1700 bp was amplified by PCR reaction. It is a homologous sequence with 50bp upstream and downstream of the ORF box of LSC1 gene in BY4741 genome. The PCR product was purified and reserved for future use.

Transformation of recombinant Saccharomyces cerevisiae gene knockout box: inoculate a single colony of Saccharomyces cerevisiae BY4741 activated on the plate into a 100 mL conical flask containing 10 mL of YPD liquid medium, and then rotate at 250 r·min ^-1 at a temperature of 30 ℃. Shaker overnight culture as fresh seed solution, transfer to 250mL Erlenmeyer flask with 50mL YPD medium at 1%, culture for 5-8h under the same conditions, and stop the culture when OD ₆₀₀ is about 0.8-1.0. (2) Collect the bacterial liquid with a 50 mL centrifuge tube, centrifuge at 6000 r·min ^-1 for 10 min at 4°C, and collect the bacterial cells. (3) Pour off the supernatant, wash the cells with 25 mL of pre-cooled sterile water, and then discard the supernatant to collect the cells after low-temperature centrifugation. Repeat the operation 3 times. (4) Discard the supernatant to collect the bacterial cells, add 1 mL of sterile cell suspension (5% glycerol, 10% dimethyl sulfoxide, v/v) to resuspend the bacterial cells, and divide them into 100 μL as the competent cells for transformation. Stored in a -80°C freezer for subsequent experiments. (5) Incubate the competent cells at 30°C for 2 min, centrifuge at 8000 r·min ^-1 for 2 min, remove the supernatant, and prepare a transformation system, including the following components: 260 μL 50% PEG 4000, 36 μL 1M lithium acetate, 2 μg DNA fragment to be transformed, 10 μL of ssDNA was supplemented to 360 μL with sterile water, and resuspended for 1 min. (6) Heat shock at 42°C for 30 min, centrifuge at 6000 rpm for 5 min, remove the supernatant and resuspend the bacteria with sterile water, centrifuge at 6000 rpm for 5 min, discard the supernatant, and directly coat the bacteria on a YPD (G418 resistant) plate.

Obtainment of BY4741ΔLSC1 strain: Pick a fresh single colony grown on the above plate into 20 μL of sterile water and mix well, take 1 μL as a PCR reaction template, and use knockout verification primers for PCR reaction. The reaction system is prepared according to Table 2. The PCR product was subjected to agarose gel electrophoresis, and a single colony with a band size of 3190 bp was selected, which was the correct knockout strain (the control was 2572 bp), and was named BY4741ΔLSC1.

Table 2 Colony PCR reaction system

Preparation for gel electrophoresis: Weigh 0.3g of agarose and dissolve it in 25mL of 1×TAE buffer, heat for 30-60s to completely dissolve, add 5μL of EB, shake well and pour into the horizontal gel box with the comb inserted, Avoid creating air bubbles and leave to solidify. Gently pull out the comb, put the gel into the electrophoresis tank, pour the electrophoresis buffer so that it does not cover the gel block, add an appropriate amount of 10×Loading Buffer according to the loading volume, point it vertically into the loading hole, and insert it well After the electrode, run at 100V for 30min and end, take out the gel block and put it into the gel imager to take pictures.

Example 2: Construction of recombinant plasmid and acquisition of recombinant Saccharomyces cerevisiae

The sequences of Tfu_0875, Tfu_2399, Tfu_0068, Tfu_1648, Tfu_2576, Tfu_2577 have been published in NCBI.

The obtained gDNA of Saccharomyces cerevisiae BY4741 was used as a template, and the primers with homology arms (Table 1) were used for PCR amplification to obtain gene fragments Tcyc1-1, Ttef1-1, Ptef1-1, Pgpd1-1, Ttdh2-1 Fragments, wherein Tcyc1-1 was amplified with primer CN9847-1-AF/R; Ttef1-1 was amplified with primer CN9847-1-BF/R; Ptef1-1 was amplified with primer CN9847-1-DF/R; The primer CN9847-1-EF/R was used to amplify Pgpd1-1; the primer CN9847-1-GF/R was used to amplify Ttdh2-1; the amplified Tcyc1-1, Ttef1-1, Ptef1-1, Pgpd1-1, Ttdh2-1 was connected by homologous recombination with the Tfu_0875 and Tfu_2399 fragments whose synthetic sequences are shown in SEQ ID NO.1 and 2, and each fragment was connected to the T vector in full length according to the sequence of Figure 3 to obtain the intermediate construction pUCmT-Tfu_0875-Tfu_2399. The intermediate construct pUCmT-Tfu_0875-Tfu_2399 and plasmid pRS423 were double digested with XhoI and SacI, and then ligated with T4 DNA ligase to obtain the recombinant plasmid _pRS423 -Tfu_0875-Tfu_2399.

The obtained gDNA of Saccharomyces cerevisiae BY4741 was used as a template, and the primers with homology arms (Table 1) were used for PCR amplification to obtain gene fragments Ttdh2-2, Tadh1-2, Padh1-2, Ppgk1-2, Tpgk1-2 , among which, primer CN9847-2-AF/R was used to amplify Ttdh2-2; primer CN9847-2-BF/R was used to amplify Tadh1-2; primer CN9847-2-DF/R was used to amplify Padh1-2; CN9847-2-EF/R was used to amplify Ppgk1-2; primer CN9847-2-GF/R was used to amplify Tpgk1-2; the amplified Ttdh2-2, Tadh1-2, Padh1-2, Ppgk1-2, Tpgk1 -2 was connected by homologous recombination with the Tfu_0068 and Tfu_1648 fragments shown in SEQ ID NO. 3 and 4 of gene synthesis, and each fragment was connected to the T vector in full length according to the sequence of Figure 4 to obtain the intermediate construct pUCmT-Tfu_0068-Tfu_1648. The intermediate construct pUCmT-Tfu_0068-Tfu_1648 and the plasmid pHAC181 were double digested with NdeI and EcoRI, and then ligated with T4 DNA ligase to obtain the recombinant plasmid _pHAC181 -Tfu_0068-Tfu_1648

The obtained gDNA of Saccharomyces cerevisiae BY4741 was used as a template, and the primers with homology arms (Table 1) were used for PCR amplification to obtain gene fragments Tpgk1-3, Ttpi1-3, Ptpi1-3, Ptdh3-3, Tfab1-3 , among which, primer CN9847-3-AF/R was used to amplify Tpgk1-3; primer CN9847-3-BF/R was used to amplify Ttpi1-3; primer CN9847-3-DF/R was used to amplify Ptpi1-3; CN9847-3-EF/R was used to amplify Ptdh3-3; primer CN9847-3-GF/R was used to amplify Tfab1-3; the amplified Tpgk1-3, Ttpi1-3, Ptpi1-3, Ptdh3-3, Tfab1 -3 and the Tfu_2576 and Tfu_2577 fragments whose sequences of gene synthesis are shown in SEQ ID NO.5 and 6 are connected by homologous recombination, and each fragment is connected to the T vector in full length according to the sequence of FIG. 5 to obtain the intermediate construct pUCmT-Tfu_2576- Tfu_2577. The intermediate construct pUCmT-Tfu_2576-Tfu_2577 and plasmid Y42 were double digested with BamHI and EcoRI, and then ligated with T4 DNA ligase to obtain recombinant plasmid Y42- _{Tfu_2576} -Tfu_2577.

pRS423-Tfu_0875-Tfu_2399, pHAC181-Tfu_0068-Tfu_1648, and Y42-Tfu_2576-Tfu_2577 were transferred into wild-type Saccharomyces cerevisiae BY4741 and Saccharomyces cerevisiae BY4741 with the LSC1 gene knocked out, respectively, to obtain recombinant Saccharomyces cerevisiae P123 and P123ΔLSC1.

Example 3: Recombinant Saccharomyces cerevisiae shake flask fermentation

YPD medium: yeast extract 10g/L, peptone 20g/L, glucose 20g/L, solid YPD medium with 2.0% agar, pH 5.4~5.6, 121℃, high pressure steam sterilization for 30min;

SD medium: glucose 20g, YNB medium (without (NH ₄ ) ₂ SO ₄ 1.7g), (NH ₄ ) ₂ SO ₄ 5g, add 1000mL deionized water pH=5.5, 115°C, autoclave for 15min . Before inoculation, add appropriate amount of sterilized amino acid, pyrimidine and agar powder according to the selectable marker.

LB medium (in g/L): 10 tryptone+5 yeast powder+10NaCl.

Fermentation conditions: Using SD (-Ura-His-Leu) medium as fermentation medium, the recombinant Saccharomyces cerevisiae was cultured at 30°C until the OD ₆₀₀ was 1.0-1.2, washed twice with sterile water, and the inoculum was 10%. Transfer to YPD medium and ferment for 96h, the filling volume is 50mL/250mL, the rotation speed is 250rpm, and the temperature is 30℃.

Taking BY4741 wild-type and LSC1 gene knockout strain BY4741ΔLSC1 as the control strain, cultured under the same conditions, the production of adipic acid was 0, the highest production of adipic acid of BY4741ΔLSC1 was 0, and the highest production of adipic acid of P123 was 0.113g/L, The highest yield of adipic acid in P123ΔLSC1 was 0.033 g/L.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone who is familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, The protection scope of the present invention should be defined by the claims.

SEQUENCE LISTING

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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 (10)

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.

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