Method for promoting recombinant yarrowia lipolytica to synthesize phloretin
阅读说明：本技术 一种促进重组解脂亚罗酵母菌合成根皮素的方法 (Method for promoting recombinant yarrowia lipolytica to synthesize phloretin ) 是由 孟永宏 杨雪言 郭玉蓉 邓红 李封辰 于 2021-02-04 设计创作，主要内容包括：本发明公开了一种促进重组解脂亚罗酵母菌合成根皮素的方法,所述方法以酪氨酸为原料,重组解脂亚罗酵母菌为宿主菌,经过宿主菌体内若干酶的催化反应获得根皮素。本发明利用解脂亚罗酵母菌细胞内乙酰辅酶A的高积累量,通过引入POX2启动子的有益突变体片段,驱动乙酰辅酶A转化为丙二酰辅酶A的关键基因ACC1和对羟基苯丙酰辅酶A合成关键基因4CL高效过表达,提高合成根皮素的两种重要前体物质丙二酰辅酶A和对羟基苯丙酰辅酶A积累量,以最终提升宿主菌内根皮素产量。本发明通过在微生物体内构建根皮素合成代谢通路,有效避免了大规模的提取、分离过程,环境友好、无污染,符合当今绿色化生产的新需求。(The invention discloses a method for promoting recombinant yarrowia lipolytica to synthesize phloretin, which takes tyrosine as a raw material and recombinant yarrowia lipolytica as host bacteria, and obtains phloretin through catalytic reaction of a plurality of enzymes in the host bacteria. According to the invention, the high accumulation amount of acetyl coenzyme A in the yarrowia lipolytica cell is utilized, and the beneficial mutant fragment of the POX2 promoter is introduced to drive the key gene ACC1 for converting acetyl coenzyme A into malonyl coenzyme A and the p-hydroxyphenylpropionyl coenzyme A to synthesize the key gene 4CL for high-efficiency overexpression, so that the accumulation amounts of malonyl coenzyme A and p-hydroxyphenylpropionyl coenzyme A which are two important precursor substances for synthesizing phloretin are increased, and the yield of the phloretin in the host bacterium is finally increased. The invention effectively avoids large-scale extraction and separation processes by constructing a phloretin anabolic pathway in a microorganism body, is environment-friendly and pollution-free, and meets the new requirements of current green production.)
1. A method for promoting recombinant yarrowia lipolytica to synthesize phloretin is characterized by comprising the following steps: the method takes tyrosine as a raw material, recombines yarrowia lipolytica as host bacteria, and obtains phloretin through catalytic reaction of a plurality of enzymes in the host bacteria;
the construction method of the recombinant yarrowia lipolytica yeast comprises the following steps:
(1) respectively connecting tyrosine ammonia lyase gene TAL, enoate reductase gene 2-ER, 4-coumaroyl-CoA ligase gene 4CL, chalcone synthase gene CHS and acetyl-CoA carboxylase gene ACC1 to a vector pJN44 to construct a plasmid pJN44-TAL containing a TAL expression cassette, a plasmid pJN44-2-ER containing a 2-ER expression cassette, a plasmid pJN44-4CL containing a 4CL expression cassette, a plasmid pJN44-CHS containing a CHS expression cassette and a plasmid pJN44-ACC1 containing an ACC1 expression cassette;
(2) carrying out enzyme digestion on expression cassettes containing target genes in the constructed plasmids pJN44-2-ER, pJN44-4CL, pJN44-CHS and pJN44-ACC1, and sequentially connecting the expression cassettes to a plasmid pJN44-TAL to obtain a recombinant plasmid pJN44-TAL/2-ER/4CL/CHS/ACC 1;
(3) extracting a yarrowia lipolytica genome, obtaining an upstream and downstream homology arm of an integration site GUT2 on the genome through PCR, and further constructing a vector pURA-GUT2L & R; carrying out enzyme digestion on an expression cassette containing a target gene in the recombinant plasmid pJN44-TAL/2-ER/4CL/CHS/ACC1 and connecting the expression cassette to a vector pURA-GUT2L & R to obtain a recombinant plasmid pURA-GUT2L & R-TAL/2-ER/4CL/CHS/ACC 1;
(4) using a POX2 promoter gene sequence as a template, obtaining an optimized POX2 promoter mutant through error-prone PCR reaction screening, connecting a POX2 promoter mutant fragment to a plasmid pURA-GUT2L & R-TAL/2-ER/4CL/CHS/ACC1, replacing the original promoters of ACC1 and 4CL expression cassettes respectively, and integrating the obtained plasmid to a lipolysis yarrowia yeast chromosome to obtain the recombinant lipolysis yarrowia yeast;
the POX2 promoter mutant has any one of SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No. 4.
2. The method for promoting the recombinant yarrowia lipolytica to synthesize phloretin according to claim 1, wherein: in the step (4), the error-prone PCR reaction system is as follows: 100 μ L, 8mmol/L MgCl2、0.6mmol/L MnCl250mmol/L KCl, 10mol/L Tris-HCl, Taq DNA polymerase of pH 8.3 at 25 ℃ and 6U, the concentrations of dATP, dGTP, dCTP and dTTP are 0.2, 1 and 1mmol/L respectively; the error-prone PCR reaction conditions were: pre-denaturation at 96 deg.C for 5min, denaturation at 96 deg.C for 2min, annealing at 58 deg.C for 2min, and extension at 72 deg.C for 1min, and performing 30 cycles.
3. The method for promoting the recombinant yarrowia lipolytica to synthesize phloretin according to claim 1, wherein: in step (4), the original promoters of ACC1 and 4CL expression cassettes on plasmid pURA-GUT2L & R-TAL/2-ER/4CL/CHS/ACC1 were double digested with restriction enzymes BamHI and HindIII, BamHI and HindIII cleavage sites were added to both ends of POX2 promoter mutant fragment, and POX2 promoter mutant fragment was ligated to plasmid pURA-GUT2L & R-TAL/2-ER/4 CL/CHS/1, respectively, replacing the original promoters of ACC1 and 4CL expression cassettes.
4. The method for promoting the recombinant yarrowia lipolytica to synthesize phloretin according to claim 1, wherein: in step (4), the POX2 promoter mutant with the sequence of SEQ ID No.1 is connected to the plasmid pURA-GUT2L & R-TAL/2-ER/4CL/CHS/ACC1 to replace the original promoter of the ACC1 expression cassette, the POX2 promoter mutant with the sequence of SEQ ID No.3 is connected to the plasmid pURA-GUT2L & R-TAL/2-ER/4CL/CHS/ACC1 to replace the original promoter of the 4CL expression cassette.
Technical Field
The invention belongs to the technical field of biosynthesis of phloretin, and particularly relates to a method for promoting recombinant yarrowia lipolytica to synthesize phloretin.
Background
Phloretin (Phloretin) is a novel natural skin whitening agent newly developed in foreign countries, and is mainly distributed in pericarp and root bark of juicy fruits such as apples and pears. The phloretin is pearl white crystal powder, can be dissolved in ethanol and acetone, is almost insoluble in water, has very strong moisturizing effect, can promote absorption and utilization of functional factors in a formula, exerts good effects, and can be applied to masks, skin cream, emulsion and essence. In addition, phloretin also has the function of improving memory and a plurality of important biological activities of cancer resistance, oxidation resistance, tumor resistance and the like, and has wide application prospect in the development of novel medicaments and natural health-care foods.
At present, the existing extraction method of phloretin mainly comprises acid hydrolysis, enzymolysis, direct extraction and the like. In actual production, naringin is usually used as a raw material, Raney nickel is used as a catalyst, naringin dihydrochalcone is synthesized by catalytic hydrogenation in a sodium hydroxide solution, and naringin dihydrochalcone is hydrolyzed by a hydrochloric acid solution to obtain phloretin. However, this method has a problem of chemical waste liquid discharge, which causes environmental pollution to some extent. The methods of enzymolysis, direct extraction and the like also have various problems of low extraction efficiency, serious pollution and the like. Therefore, the method for synthesizing phloretin by using microbes for heterologous organisms has the advantages of mild conditions and less pollution, and becomes an ideal synthetic method. The biosynthesis pathway of phloretin takes p-hydroxyphenylpropionyl coenzyme A and malonyl coenzyme A as precursor substances. Patent CN107805646A reports that phenylalanine is used as a raw material, and escherichia coli is used as a host bacterium to synthesize phloretin. Patent CN103571892A uses naringin or its aglycone as raw material, and uses colibacillus, saccharomyces cerevisiae or pichia pastoris as host bacteria; CN107586795A and CN109913508A both use p-hydroxyphenylpropionic acid as raw material, and respectively use saccharomyces cerevisiae and cyanobacteria cells as host bacteria. The patents are mostly limited to the construction of a phloretin synthesis path, only CN107586795A is used, and the accumulation amount of important precursor malonyl coenzyme A in the phloretin synthesis is increased by integrating key genes for strengthening the route from ethanol to malonyl coenzyme A in saccharomyces cerevisiae and introducing a route for converting malonic acid into malonyl coenzyme A, so that the yield of the phloretin is increased.
Disclosure of Invention
The invention aims to provide a method for microbial heterologous synthesis of phloretin by using yarrowia lipolytica as host bacteria, starting from two precursor substances of p-hydroxyphenylpropionyl coenzyme A and malonyl coenzyme A meeting the biosynthesis of the phloretin, and finally improving the yield of the phloretin in the host bacteria by improving the accumulation of the two precursor substances so as to solve the problems of low extraction efficiency, complicated separation and purification steps, serious chemical pollution and the like of the existing extraction method.
Aiming at the purposes, the technical scheme adopted by the invention is as follows: the phloretin is obtained by taking exogenous tyrosine as a raw material and recombinant yarrowia lipolytica as a host bacterium through catalytic reactions of a plurality of enzymes in the host bacterium, and the synthetic route is as follows:
determining key genes for phloretin biosynthesis: taking exogenous tyrosine as a substrate, generating 4-coumaric acid by tyrosine under the action of Tyrosine Ammonia Lyase (TAL), generating p-hydroxyphenylpropionic acid by the 4-coumaric acid under the action of enoate reductase (2-ER), further generating p-hydroxyphenylpropionyl-CoA under the action of 4-coumaroyl-CoA ligase (4CL), generating malonyl-CoA by the acetyl-CoA under the action of acetyl-CoA carboxylase (ACC1), and then generating 1 molecule of p-hydroxyphenylpropionyl-CoA and 3 molecules of malonyl-CoA under the catalytic action of chalcone synthase (CHS) to finally synthesize phloretin.
The construction method of the recombinant yarrowia lipolytica yeast comprises the following steps:
1. respectively connecting tyrosine ammonia lyase gene TAL, enoate reductase gene 2-ER, 4-coumaroyl-CoA ligase gene 4CL, chalcone synthase gene CHS and acetyl-CoA carboxylase gene ACC1 to a vector pJN44 to construct a plasmid pJN44-TAL containing a TAL expression cassette, a plasmid pJN44-2-ER containing a 2-ER expression cassette, a plasmid pJN44-4CL containing a 4CL expression cassette, a plasmid pJN44-CHS containing a CHS expression cassette and a plasmid pJN44-ACC1 containing an ACC1 expression cassette;
2. carrying out enzyme digestion on expression cassettes containing target genes in the constructed plasmids pJN44-2-ER, pJN44-4CL, pJN44-CHS and pJN44-ACC1, and sequentially connecting the expression cassettes to a plasmid pJN44-TAL to obtain a recombinant plasmid pJN44-TAL/2-ER/4CL/CHS/ACC 1;
3. extracting a yarrowia lipolytica genome, obtaining an upstream and downstream homology arm of an integration site GUT2 on the genome through PCR, and further constructing a vector pURA-GUT2L & R; the expression cassette containing the target gene in the recombinant plasmid pJN44-TAL/2-ER/4CL/CHS/ACC1 is digested and connected to the vector pURA-GUT2L & R to obtain the recombinant plasmid pURA-GUT2L & R-TAL/2-ER/4CL/CHS/ACC 1.
4. Using a POX2 promoter gene sequence as a template, obtaining an optimized POX2 promoter mutant through error-prone PCR reaction screening, connecting a POX2 promoter mutant fragment to a plasmid pURA-GUT2L & R-TAL/2-ER/4CL/CHS/ACC1, replacing the original promoters of ACC1 and 4CL expression cassettes respectively, and integrating the obtained plasmid to a lipolysis yarrowia yeast chromosome to obtain the recombinant lipolysis yarrowia yeast; wherein the POX2 promoter mutant has any one of SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No. 4.
In the step 4, the error-prone PCR reaction system is preferably: 100 μ L, 8mmol/L MgCl2、0.6mmol/L MnCl250mmol/L KCl, 10mol/L Tris-HCl, Taq DNA polymerase of pH 8.3 at 25 ℃ and 6U, the concentrations of dATP, dGTP, dCTP and dTTP are 0.2, 1 and 1mmol/L respectively; the error-prone PCR reaction conditions were: pre-denaturation at 96 deg.C for 5min, denaturation at 96 deg.C for 2min, annealing at 58 deg.C for 2min, and extension at 72 deg.C for 1min, and performing 30 cycles.
In step 4, the original promoters of ACC1 and 4CL expression cassettes of plasmid pURA-GUT2L & R-TAL/2-ER/4CL/CHS/ACC1 were double digested with restriction enzymes BamHI and HindIII, BamHI and HindIII sites were added to both ends of POX2 promoter mutant fragment, and POX2 promoter mutant fragment was ligated to plasmid pURA-GUT2L & R-TAL/2-ER/4 CL/CHS/1, respectively, replacing the original ACC promoters of ACC1 and 4CL expression cassettes.
In the above step 4, it is preferable that the POX2 promoter mutant with the sequence of SEQ ID No.1 is ligated to the plasmid pURA-GUT2L & R-TAL/2-ER/4CL/CHS/ACC1 to replace the original promoter of the ACC1 expression cassette, and the POX2 promoter mutant with the sequence of SEQ ID No.3 is ligated to the plasmid pURA-GUT2L & R-TAL/2-ER/4CL/CHS/ACC1 to replace the original promoter of the 4CL expression cassette.
The invention has the following beneficial effects:
1. the invention constructs a phloretin anabolic pathway in a microorganism body, produces phloretin by fermentation, effectively avoids large-scale extraction and separation processes, is environment-friendly and pollution-free, and meets the new requirements of current green production.
2. The invention takes yarrowia lipolytica engineering bacteria as host bacteria, and overexpresses a key gene ACC1 for converting acetyl coenzyme A into malonyl coenzyme A by utilizing the high accumulation of acetyl coenzyme A in cells so as to improve the content of malonyl coenzyme A in the host bacteria. Meanwhile, the over-expression of the p-hydroxyphenylpropionyl coenzyme A for synthesizing the key gene 4CL improves the accumulation of the p-hydroxyphenylpropionyl coenzyme A for synthesizing another important precursor substance of the phloretin, and finally improves the yield of the phloretin in host bacteria.
3. When two key genes of ACC1 and 4CL are overexpressed, beneficial mutant fragments of a POX2 promoter are respectively introduced to drive the genes to be efficiently overexpressed.
Detailed Description
The present invention will be described in further detail with reference to examples, but the scope of the present invention is not limited to these examples.
Example 1
1. Optimizing the base sequence of the target gene according to the amino acid sequence and codon preference of yarrowia lipolytica, and sending the optimized base sequence to commercial gene synthesisCompany synthesizes the target gene: tyrosine ammonia lyase Gene (TAL, Gene ID: 54319287), enoate reductase Gene (2-ER, Gene ID:44999865), 4-coumaroyl-CoA ligase Gene (4CL, Gene ID:110880015), chalcone synthase Gene (CHS, Gene ID:110908971), acetyl-CoA carboxylase Gene (ACC1, Gene ID: 2909424). After synthesis, codon optimized tyrosine ammonia lyase gene (TAL) and expression vector pJN44 (containing promoter P) were addedTEFAnd a terminator Txpr2) Respectively treating with restriction enzyme SmaI at 37 deg.C for 2h, purifying and recovering DNA fragments of target gene and vector; the two DNA fragments thus recovered were ligated by NEB DNA ligase (NEB, product No. M0367S) to obtain plasmid pJN44-TAL containing the TAL expression cassette. Similarly, plasmid pJN44-2-ER containing the 2-ER expression cassette, plasmid pJN44-4CL containing the 4CL expression cassette, plasmid pJN44-CHS containing the CHS expression cassette, and plasmid pJN44-ACC1 containing the ACC1 expression cassette were constructed.
2. Treating a plasmid pJN44-2-ER containing the 2-ER expression cassette and a plasmid pJN44-TAL containing the TAL expression cassette with restriction enzymes XbaI/SpeI and XbaI at 37 ℃ for 2h respectively, purifying and gluing to recover DNA fragments of the 2-ER expression cassette and the plasmid pJN 44-TAL; the two DNA fragments thus recovered were ligated by NEB DNA ligase (NEB, product No. M0367S) to obtain plasmid pJN 44-TAL/2-ER. Similarly, the 4CL expression cassette is digested and ligated to plasmid pJN44-TAL/2-ER to obtain plasmid pJN44-TAL/2-ER/4CL, the CHS expression cassette is digested and ligated to plasmid pJN44-TAL/2-ER/4CL to obtain plasmid pJN44-TAL/2-ER/4CL/CHS, the ACC1 expression cassette is digested and ligated to plasmid pJN44-TAL/2-ER/4CL/CHS to obtain recombinant plasmid pJN44-TAL/2-ER/4CL/CHS/ACC 1. According to the operation of a yeast transformation kit (Zymo Research corporation, USA), the recombinant plasmid pJN44-TAL/2-ER/4CL/CHS/ACC1 is transformed into yarrowia lipolytica for free expression, after a transformant grows out, a single clone is selected, the plasmid is extracted for verification, a strain which is verified to be correct is cultured, a tyrosine substrate is added into a cultured bacterial liquid, and the reaction is carried out for 120 hours at the temperature of 30 ℃ and the rpm/min. High Performance Liquid Chromatography (HPLC) is adopted for detection, and the result shows that phloretin is generated in the bacterial liquid, and the yield of the phloretin is 363.5 mg/L.
3. Extracting yarrowia lipolytica genome, obtaining the upstream and downstream homology arms of the integration site GUT2 on the genome through PCR, and further constructing a vector pURA-GUT2L & R. The plasmid pJN44-TAL/2ER/4CL/CHS/ACC1 and the vector pURA-GUT2L & R constructed in the step 2 are respectively treated with restriction endonucleases XbaI/SpeI and XbaI at 37 ℃ for 2h, purified and gel recovered to obtain a DNA fragment containing an expression cassette of five target genes (TAL, 2-ER, 4CL, CHS and ACC1) and the vector pURA-GUT2L & R, and the two recovered DNA fragments are subjected to ligation reaction under the action of NEB DNA ligase (NEB company, product number: M0367S) to obtain a recombinant plasmid pURA-GUT2L & R-TAL/2-CL/CHS/1.
4. Firstly, using a POX2 promoter gene sequence as a template, designing a primer POX2-EPf/POX2-EPr (POX2-EPf base sequence: cgcggatcctttcccttatacttttcccca; POX2-Epr base sequence: cccaagcttggcgtcgttgc), and obtaining a mutant POX2 promoter through error-prone PCR. The error-prone PCR reaction system is as follows: 100 μ L of MgCl containing 8mmol/L2、0.6mmol/L MnCl250mmol/L KCl, 10mol/L Tris-Cl, pH 8.3(25 ℃), 6U Taq DNA polymerase, dATP, dGTP, dCTP, dTTP concentrations of 0.2, 1 and 1mmol/L respectively. The error-prone PCR reaction conditions were: pre-denaturation at 96 deg.C for 5min, denaturation at 96 deg.C for 2min, annealing at 58 deg.C for 2min, and extension at 72 deg.C for 1min, and performing 30 cycles. The mutant promoters are subjected to enzyme digestion, then the mutant promoter mixed fragments are connected with plasmids pJN44 subjected to the same enzyme digestion to obtain a recombinant plasmid library, and the recombinant plasmids are converted into yarrowia lipolytica according to the operation of a yeast conversion kit (Zymo Research corporation, USA), so that the mutant POX2 promoter recombinant yarrowia lipolytica cell library is obtained. After transformation, coating the cells on a corresponding auxotroph culture medium, carrying out inverted culture at 30 ℃ for 3-4 days until single clones appear, identifying transformants by colony PCR, and selecting positive clones. And screening the constructed mutant by using a 96 deep-well plate through a high-throughput screening method, and detecting the fluorescence intensity of the yeast enhanced green fluorescent protein yEGFP of the recombinant bacteria by using a fluorescence microplate reader. Randomly selecting 200 mutants for screening, and detecting that the fluorescence intensity is higher than that of a control recombinant bacterium (the wild POX2 promoter regulates the expression of yEGFP)Bacterial strain) is obviously improved, and then 4 beneficial mutants POX2-1, POX2-2, POX2-3 and POX2-4 with the promoter strength higher than that of a wild-type POX2 promoter are obtained. In order to verify that the yEGFP fluorescence intensity of the recombinant bacteria is indeed caused by mutation of a promoter sequence, the mutant POX2 promoter sequence is cloned and sequenced, and the gene sequences of POX2-1, POX2-2, POX2-3 and POX2-4 are sequentially shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No. 4.
The original promoter of the ACC1 expression cassette on plasmid pJN44-TAL/2-ER/4CL/CHS/ACC1 is subjected to double enzyme digestion by using restriction enzymes BamHI and Hind III, BamHI and Hind III cleavage sites are added at two ends of POX2-1, POX2-2, POX2-3 and POX2-4 promoter mutant fragments respectively, and then POX2-1, POX2-2, POX2-3 and POX2-4 promoter mutant fragments are respectively connected to pJN44-TAL/2-ER/4CL/CHS/ACC1 vectors to replace the original promoter of the ACC1 expression cassette. Then according to the operation of yeast transformation kit (Zymo Research corporation, USA), respectively transforming into yarrowia lipolytica for free expression, correspondingly obtaining recombinant bacteria, culturing the recombinant bacteria, adding tyrosine substrate into the cultured bacteria liquid, and reacting for 120h at 30 ℃ and 200 rpm/min. The content of phloretin in the bacterial liquid is measured by HPLC, the yield can respectively reach 663.4mg/L, 632.8mg/L, 612.9mg/L and 642.6mg/L, and the mutant POX2-1 promoter is introduced to drive ACC1 gene expression, so that the effect is optimal.
Similarly, POX2-1, POX2-2, POX2-3 and POX2-4 promoter mutant fragments are respectively connected to pJN44-TAL/2-ER/4CL/CHS/ACC1 vectors to replace the original promoter of a 4CL expression cassette, corresponding recombinant bacteria are obtained through transformation, after fermentation culture under the same conditions, the yield of the recombinant bacteria can respectively reach 528.9mg/L, 566.7mg/L, 587.6mg/L and 532.7mg/L, and after comparison, the POX2-3 promoter after mutation is introduced to drive 4CL gene expression, and the effect is optimal.
Carrying out double digestion on the original promoters of an ACC1 expression cassette and a 4CL expression cassette on plasmid pURA-GUT2L & R-TAL/2-ER/4CL/CHS/ACC1 by using restriction enzymes BamHI and Hind III, adding BamHI and Hind III cleavage sites at both ends of the POX2-1 and POX2-3 promoter mutant fragments obtained by screening, respectively, then connecting the POX2-1 and POX2-3 promoter mutant fragments to pURA-GUT2L & R-TAL/2-ER/4CL/CHS/ACC1 vectors, respectively replacing the original promoters of ACC1 and 4CL expression cassettes, then transforming the recombinant plasmid into yarrowia lipolytica by operating according to the yeast transformation kit instruction (Zymo Research corporation, USA), using homologous recombination to integrate the expression cassette containing five target genes into the chromosome of yarrowia lipolytica yeast, further to obtain the recombinant yarrowia lipolytica yeast capable of stably synthesizing phloretin. Culturing the obtained recombinant yarrowia lipolytica, adding a tyrosine substrate into the cultured bacterial liquid, and reacting for 120h at 30 ℃ and 200 rpm/min. HPLC detection shows that the final yield of phloretin can reach 783.5 mg/L.
Example 2
In this example, plasmid pURA-GUT2L & R-TAL/2-ER/4CL/CHS/ACC1 was constructed according to the method of example 1, then the original promoters of ACC1 expression cassette and 4CL expression cassette on plasmid pURA-GUT2L & R-TAL/2-ER/4CL/CHS/ACC1 were digested with restriction enzymes BamHI and HindIII, BamHI and HindIII cleavage sites were added to both ends of POX2-1 and POX2-2 promoter mutant fragments obtained by screening in example 1, then POX2-1 and POX2-2 promoter mutant fragments were ligated to pURA-GUT2L & R-TAL/2-ER/4CL/CHS/ACC1 vectors, respectively replacing the original promoters of ACC1 and 4CL expression cassettes, and then the original promoters of the yeast transformation kit (yeast Research, USA), the recombinant plasmid is transformed into yarrowia lipolytica, and an expression cassette containing five target genes is integrated to the chromosome of the yarrowia lipolytica by utilizing the homologous recombination effect, so that the recombinant yarrowia lipolytica capable of stably synthesizing phloretin is obtained. Culturing the obtained recombinant yarrowia lipolytica, adding a tyrosine substrate into the cultured bacterial liquid, and reacting for 120h at 30 ℃ and 200 rpm/min. HPLC detection shows that the final yield of phloretin can reach 763.1 mg/L.
Example 3
In this example, plasmid pURA-GUT2L & R-TAL/2-ER/4CL/CHS/ACC1 was constructed according to the method of example 1, then the original promoters of ACC1 expression cassette and 4CL expression cassette on plasmid pURA-GUT2L & R-TAL/2-ER/4CL/CHS/ACC1 were double digested with restriction enzymes BamHI and HindIII, BamHI and HindIII cleavage sites were added to both ends of POX2-4 and POX2-3 promoter mutant fragments obtained by screening in example 1, then POX2-4 and POX2-3 promoter mutant fragments were ligated to pURA-GUT2L & R-TAL/2-ER/4CL/CHS/ACC1 vectors, respectively replacing the original promoters of ACC1 and 4CL expression cassettes, and then the original promoters of ACC1 and 4CL expression cassettes were replaced according to the yeast transformation kit (yeast Research, USA), the recombinant plasmid is transformed into yarrowia lipolytica, and an expression cassette containing five target genes is integrated to the chromosome of the yarrowia lipolytica by utilizing the homologous recombination effect, so that the recombinant yarrowia lipolytica capable of stably synthesizing phloretin is obtained. Culturing the obtained recombinant yarrowia lipolytica, adding a tyrosine substrate into the cultured bacterial liquid, and reacting for 120h at 30 ℃ and 200 rpm/min. HPLC detection shows that the final yield of phloretin can reach 754.2 mg/L.
Example 4
In this example, plasmid pURA-GUT2L & R-TAL/2-ER/4CL/CHS/ACC1 was constructed according to the method of example 1, then the original promoters of ACC1 expression cassette and 4CL expression cassette on plasmid pURA-GUT2L & R-TAL/2-ER/4CL/CHS/ACC1 were digested with restriction enzymes BamHI and HindIII, BamHI and HindIII cleavage sites were added to both ends of POX2-4 and POX2-2 promoter mutant fragments obtained by screening in example 1, then POX2-4 and POX2-2 promoter mutant fragments were ligated to pURA-GUT2L & R-TAL/2-ER/4CL/CHS/ACC1 vectors, respectively replacing the original promoters of ACC1 and 4CL expression cassettes, and then the original promoters of yeast transformation kit (yeast Research, USA), the recombinant plasmid is transformed into yarrowia lipolytica, and an expression cassette containing five target genes is integrated to the chromosome of the yarrowia lipolytica by utilizing the homologous recombination effect, so that the recombinant yarrowia lipolytica capable of stably synthesizing phloretin is obtained. Culturing the obtained recombinant yarrowia lipolytica, adding a tyrosine substrate into the cultured bacterial liquid, and reacting for 120h at 30 ℃ and 200 rpm/min. HPLC detection shows that the final yield of phloretin can reach 739.5 mg/L.
Sequence listing
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tttgacgtgc aaggggcgaa ttggggcgag aaacacgtcg tggacatggt gcaaggcccg 120
cagggttgat tcgacgcttt tccgcgaaga aaacaagtcc aaataccccc gtttattctc 180
cctcggctct cggtatttca catgaaaact ataacctaga ctacacgggc aaccttaact 240
ccagagtata cttatatacc aaagggatgg gtcctcaaaa atcacacaag caacgacgcc 300
<210> 4
<211> 300
<212> DNA
<213> Yarrowia lipolytica yeast
<400> 4
tttcccttat acttttcccc acagtcacat gttatggagg ggtctagatg gaggcctagt 60
tttgacgtgc aaggggcgaa ttggggcgag aaacacgtcg tggacatggt gcaaggcccg 120
cagggttgat tcgacgcttt tccgcgaaaa aaacaagtcc aaataccccc gtttattctc 180
cctcggctct cggtacctca catgaaaact ataacctaga ctacacgggc aaccttaacc 240
ccagagtata cttatatacc aaagggatgg gtcctcaaaa atcacacaag caacgacgcc 300
<210> 5
<211> 300
<212> DNA
<213> Yarrowia lipolytica yeast
<400> 5
tttcccttat acttttcccc acagtcacat gttatggagg ggtctagatg gaggcctaat 60
tttgacgtgc aaggggcgaa ttggggcgag aaacacgtcg tggacatggt gcaaggcccg 120
cagggttgat tcgacgcttt tccgcgaaaa aaacaagtcc aaatatcctc gtttattctc 180
cctcggctct cggtatttca catgaaaact ataacctaga ctacacgggc aaccttaacc 240
ccagagtata cttatatacc aaagggatgg gtcctcagga atcacacaag caacgacgcc 300