Method for constructing gene expression vector for producing chlorogenic acid by yarrowia lipolytica

Abstract

The method for constructing a gene expression vector of Yarrowia lipolytica producing chlorogenic acid belongs to the technical field of genetic engineering and microbial metabolic engineering, and comprises the following steps: obtaining codon-optimized chlorogenic acid synthesis genes and precursor enhancement genes of Yarrowia lipolytica; connecting the genes with a promoter and a terminator to form an independent expression cassette, wherein the N-terminus of C4H and C3H is fused with an endoplasmic reticulum signal peptide; amplifying a yeast rDNA region to serve as a homologous recombination arm, and obtaining a nutrition-deficiency marker with LoxP; taking a pINA plasmid as a backbone, and inserting multiple expression cassettes and the marker into the recombination arm through GoldenGate technology to obtain a recombination expression vector. The method significantly enhances the supply capacity of a synthesis substrate precursor, and greatly improves the overall synthesis efficiency of the target product chlorogenic acid.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and microbial metabolic engineering, specifically to a method for constructing a gene expression vector for chlorogenic acid production by Yersinia lipolytica. Background Technology

[0002] Utilizing microbial cell factories to synthesize complex natural products derived from plants is an important development trend in the field of biomanufacturing. Among them, Yersinia lipolyticis, as an excellent industrial microbial chassis cell, has broad application prospects in the efficient synthesis of complex metabolites such as chlorogenic acid. The complete biosynthesis of chlorogenic acid involves multiple enzymatic reactions, and the construction of its metabolic pathway requires the synergistic expression of multiple exogenous synthetic pathway genes, precursor enhancement genes, and key components such as cytochrome P450 enzymes.

[0003] In practical industrial fermentation applications, there are extremely stringent requirements for the genetic stability, metabolic flux balance, and yield of the target product of recombinant strains. However, due to the complexity of plant-derived metabolic pathways and the constraints of host genetic characteristics, previous construction methods have significant limitations. On the one hand, plant-derived P450 enzymes are prone to misfolding and inactivation in the yeast endoplasmic reticulum, and the multi-stage integration of large multi-gene fragments easily leads to expression mismatch. In addition, traditional methods rely on antibiotic pressure screening, which not only increases biosafety risks but also makes it difficult to guarantee the genetic stability of exogenous genes in the absence of selection pressure. On the other hand, the non-homologous end joining preference of Yersinia lipolytica causes large fragments to be prone to random insertion or breakage during transformation, and the lack of effective metabolic flux balance control easily leads to the intracellular accumulation of intermediates such as coumaric acid. These technical challenges can no longer meet the development needs of Yersinia lipolytica for efficient and stable production of chlorogenic acid. Summary of the Invention

[0004] The purpose of this invention is to provide a method for constructing a gene expression vector for chlorogenic acid production by Yersinia lipolytica, so as to solve the problems mentioned in the background art.

[0005] The technical solution of the present invention includes: (1) obtaining exogenous gene fragments: obtaining chlorogenic acid synthesis pathway genes and precursor enhancement genes optimized by codons of Yersinia lipolytica, wherein the chlorogenic acid synthesis pathway genes include phenylalanine ammonia-lyase gene PAL, cinnamic acid-4-hydroxylase gene C4H, p-coumaric acid-3-hydroxylase gene C3H, cytochrome P450 reductase gene CPR, 4-coumaric acid coenzyme A ligase gene 4CL and hydroxycinnamoyltransferase gene HQT, wherein the precursor enhancement genes include the DAHP synthase mutant gene ARO4^K229L which relieves feedback inhibition and the transketolase gene TKL1; (2) Constructing independent gene expression cassettes: The exogenous gene fragments obtained in step (1) are respectively connected to the promoter and terminator of Yeast lipophila. The independent expression cassette refers to each exogenous gene fragment being operatively connected to a promoter and a terminator to form an independent transcription unit. (3) Obtaining homologous recombination arms and selection marker genes: Using Yeast lipolyticis genomic DNA as a template, the upstream and downstream sequences of the Yeast lipolyticis genomic rDNA region were amplified as homologous recombination arms; and nutritional auxotroph selection marker genes with LoxP sites fused on both sides were obtained. (4) Seamless assembly of vector: Using pINA plasmid as vector backbone, the homologous recombination arm described in step (3) is cloned to both sides of the multiple cloning site. Then, using GoldenGate assembly technology, the multiple independent gene expression cassettes constructed in step (2) are connected in a pre-set order to form a multi-gene expression cassette, and inserted together with the auxotrophic selection marker gene into the homologous recombination arm to obtain the recombinant gene expression vector.

[0006] Preferably, in step (2), the promoter includes a strongly constitutive promoter and a moderately constitutive promoter; The PAL, C4H, and C3H genes are driven by the strong constitutive promoter, which is selected from at least one of the pTEF promoter and the hp4d promoter; the 4CL, HQT, CPR, ARO4^K229L, and TKL1 genes are driven by the moderate constitutive promoter, which is the pEXP1 promoter.

[0007] Preferably, in step (3), the auxotrophic screening marker gene is the URA3 gene; In step (4), the process conditions of the GoldenGate assembly technology shall at least meet the following requirements: restriction endonuclease and DNA ligase are added to the reaction system, and the enzyme digestion reaction is carried out at 37-40℃ for 2-5 minutes, and the ligation reaction is carried out at 16-20℃ for 2-5 minutes. This is repeated as one cycle, and the cycle is repeated 15-30 times. Finally, the DNA is inactivated at 50-60℃ for 5-10 minutes. The nucleotide sequence length of the recombinant gene expression vector obtained by assembly is 15-20kb.

[0008] A gene expression vector for chlorogenic acid production by Yersinia lipolytica was prepared using a method described above.

[0009] Preferably, it includes the following steps: (1) Vector linearization: The gene expression vector of Yersinia lipolytica producing chlorogenic acid as described in claim 4 is digested with the restriction endonuclease NotI to linearize the gene expression vector and expose the upstream and downstream sequences of the rDNA regions at both ends; (2) In situ recombination transformation: The linearized vector obtained in step (1) was introduced into the Yersinia lipolytica host cells with the ku70 gene knocked out in advance using the lithium acetate / dithiothreitol transformation method. The cells were incubated at 30-35℃ for 30-60 minutes and then heat-shocked at 39-42℃ for 10-20 minutes. The gene cluster containing the exogenous gene fragment was integrated in situ into the rDNA region of the Yersinia lipolytica host cell chromosome through the homologous recombination mechanism to obtain the initial transformed cells. (3) Screening and marker excision of positive transformants: The initial transformed cells obtained in step (2) are spread on auxotrophic selection medium for single-clone screening to obtain positive transformants; helper plasmids expressing Cre recombinase are transiently introduced into the positive transformants, and LoxP site recombination is triggered by culturing in galactose-induced medium for 24-48 hours to excise the auxotrophic selection marker gene; the helper plasmids are naturally lost by continuous subculturing in non-selective medium to obtain recombinant cells of Yersinia lipolytica producing chlorogenic acid.

[0010] Preferably, in step (2), the final concentration of lithium acetate in the lithium acetate / dithiothreitol conversion method is 0.1-0.2 mol / L, and the final concentration of dithiothreitol is 20-50 mmol / L.

[0011] Preferably, the method further includes a step of synthesizing chlorogenic acid through submerged fermentation using recombinant cells of the Yersinia lipolytica that produce chlorogenic acid: The recombinant cells of Yersinia lipolytica that produce chlorogenic acid were inoculated into a fermentation medium containing a carbon source and subjected to shake-flask deep fermentation at 28-30°C and 200-250 rpm for 48-96 hours. The fermentation broth was then collected. The carbon source was selected from at least one of glucose and glycerol.

[0012] Preferably, after the shake-flask deep fermentation is completed, the fermentation broth is subjected to cell wall disruption treatment, methanol solvent is added for ultrasonic extraction for 10-30 minutes, and after standing precipitation, the supernatant is collected by centrifugation to obtain chlorogenic acid extract.

[0013] This invention provides an improved method for constructing a gene expression vector for chlorogenic acid production in Yersinia lipolytica, which, compared with existing technologies, has the following improvements and advantages: 1. This method optimizes the codons of genes related to the chlorogenic acid synthesis pathway and innovatively introduces precursor-enhancing mutant genes that relieve feedback inhibition and transketolase genes, effectively breaking the original metabolic negative feedback bottleneck inside the host cell. This design significantly enhances the supply capacity of the synthesis substrate precursor, thereby greatly improving the overall synthesis efficiency of the target product chlorogenic acid. 2. This method involves targeted substitution of the sequence start ends of some core catalytic enzyme genes, cleverly incorporating yeast endoplasmic reticulum localization signal peptide sequences. This spatial compartmentalization strategy enables efficient site-specific localization of key catalytic enzymes in the endoplasmic reticulum, confining the catalytic reaction to the endoplasmic reticulum membrane region, reducing non-specific diffusion loss of substrates in the cytoplasm, and improving the catalytic efficiency of metabolic pathways. 3. This invention abandons the single promoter strategy and adopts a combination of strong constitutive promoters and medium-strength constitutive promoters to drive different synthetic pathway genes and precursor enhancement genes in a hierarchical manner. This differentiated and stepwise driving mode realizes the fine regulation within the complex multi-gene expression system, so that the expression level of each gene matches the metabolic flux demand, reduces the excessive accumulation of intermediate products, and ensures the vitality of the host cell during the fermentation process. 4. This method utilizes seamless assembly technology to efficiently chain multiple independent gene expression cassettes into a large expression cluster in one step, and extracts upstream and downstream sequences of the high-copy region of the host genome ribosomal nucleic acid as homologous recombination arms; through the homologous recombination mechanism, the above-mentioned multi-gene clusters are in situ targeted and integrated into the chromosome, realizing the stable inheritance and expression of exogenous genes in continuous cell passage. 5. This method constructs a auxotrophic selection marker system containing specific recombination sites. After successfully screening positive monoclonal clones, the recombination mechanism is triggered by transiently introducing an auxiliary plasmid expressing a specific recombinase, and the selection marker is precisely excised. The auxiliary plasmid is then naturally excised through continuous passage culture. This strategy successfully realizes the construction of recombinant cells with reusable selection markers, making the selection markers reusable and facilitating subsequent multiple rounds of genetic engineering. Detailed Implementation

[0014] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments.

[0015] Example 1: The method for constructing a gene expression vector for chlorogenic acid production in Yersinia lipolytica includes the following steps: (1) Obtaining exogenous gene fragments: Obtain chlorogenic acid synthesis pathway genes and precursor enhancement genes optimized by codons of Yersinia lipolytica. The chlorogenic acid synthesis pathway genes include phenylalanine ammonia-lyase gene PAL, cinnamic acid-4-hydroxylase gene C4H, p-coumaric acid-3-hydroxylase gene C3H, cytochrome P450 reductase gene CPR, 4-coumaric acid coenzyme A ligase gene 4CL, and hydroxycinnamoyltransferase gene HQT; the precursor enhancement genes include the DAHP synthase mutant gene ARO4^K229L which relieves feedback inhibition and the transketolase gene TKL1. (2) Constructing independent gene expression cassettes: The exogenous gene fragments obtained in step (1) are connected to the promoter and terminator of Yeast lipolyticis, respectively. An independent expression cassette refers to an independent transcription unit formed by operatively connecting each exogenous gene fragment to a promoter and a terminator. (3) Obtaining homologous recombination arms and selection marker genes: Using Yeast lipolyticis genomic DNA as a template, the upstream and downstream sequences of the Yeast lipolyticis genomic rDNA region were amplified as homologous recombination arms; and nutritional auxotroph selection marker genes with LoxP sites fused on both sides were obtained. (4) Seamless assembly of vector: Using pINA plasmid as vector backbone, the homologous recombination arm of step (3) is cloned to both sides of the multiple cloning site. Then, using GoldenGate assembly technology, multiple independent gene expression cassettes constructed in step (2) are tandemly connected and inserted together with the auxotrophic selection marker gene between the homologous recombination arms to obtain the recombinant gene expression vector. The above-mentioned method for constructing the gene expression vector for chlorogenic acid production by Yarrowia lipolytica used the Yarrowia lipolytica Po1f source sequence as the host background; PAL, C4H, C3H, CPR, 4CL, HQT, ARO4^K229L and TKL1 were all artificially synthesized after optimization of the preferred codons of Yarrowia lipolytica, and their optimized nucleotide sequences are shown in SEQ ID NO:1 to SEQ ID NO:8, respectively; The N-terminus of C4H and C3H were replaced with signal peptide sequences derived from the endoplasmic reticulum membrane proteins of Yersinia lipolytica. Specifically, the first 25 amino acid residues of the N-terminus of the wild-type amino acid sequences encoded by the C4H and C3H genes were removed, and in-frame fusion linkages were performed at the removal sites with the C-terminus of the signal peptide of the SEC20 protein, respectively, to improve the localization consistency of P450 enzymes on the endoplasmic reticulum membrane and reduce the inactivation caused by misfolding of plant-derived P450 in yeast. The upstream homologous recombination arm of the rDNA region was set to 1000 bp in length, and the downstream homologous recombination arm was set to 1020 bp in length, both obtained by amplification of host genomic DNA. The auxotroph selection marker gene is the LoxP-URA3-LoxP fragment. After linearization by restriction enzyme digestion, the pINA plasmid backbone is introduced into the above homologous recombination arm. Then, eight independent gene expression cassettes and the LoxP-URA3-LoxP fragment are placed between the homologous recombination arms to form a recombinant gene expression vector. This design places the complete chlorogenic acid synthesis pathway, P450 electron transport completion, and precursor enhancement module in the same integration fragment, reducing expression mismatch during staged integration and making it suitable for subsequent multi-copy integration of rDNA sites.

[0016] Example 2: The method for constructing a gene expression vector for chlorogenic acid production by Yersinia lipolytica includes the following steps: In step (2), the promoters include strong constitutive promoters and moderate constitutive promoters; the PAL, C4H, and C3H genes are driven by strong constitutive promoters, which are selected from at least one of pTEF promoters and hp4d promoters; the 4CL, HQT, CPR, ARO4^K229L, and TKL1 genes are driven by moderate constitutive promoters, which are pEXP1 promoters. Promoter allocation was set using a metabolic flux balance approach. PAL was driven by the pTEF promoter, C4H and C3H were both driven by the hp4d promoter, and 4CL, HQT, CPR, ARO4^K229L and TKL1 were all driven by the pEXP1 promoter. Each gene was followed by either the XPR2 terminator or the Lip2 terminator. This allocation maintains high throughput for upstream deamination and benzene ring hydroxylation steps while keeping coenzyme A activation, acyl transfer, and precursor enhancement at moderate expression levels, thus reducing intracellular accumulation of coumaric acid and caffeic acid. Compared to using strong promoters for all genes, this combination is more suitable for sustained expression in deep culture conditions of Yersinia lipolytica.

[0017] Example 3: The method for constructing a gene expression vector for chlorogenic acid production by Yersinia lipolytica includes the following steps: In step (3), the auxotrophic screening marker gene is the URA3 gene; In step (4), the process conditions of the GoldenGate assembly technology must meet at least the following requirements: restriction endonuclease and DNA ligase are added to the reaction system, and the enzyme digestion reaction is carried out at 37-40℃ for 2-5 minutes, the ligation reaction is carried out at 16-20℃ for 2-5 minutes, and the alternating cycle is repeated 15-30 times, and the DNA is inactivated at 50-60℃ for 5-10 minutes; The nucleotide sequence length of the recombinant gene expression vector obtained by assembly is 15-20kb. The auxotrophic selection marker gene was the URA3 gene; GoldenGate assembly was performed using BsaI and T4 DNA ligase. 100 ng of pINA plasmid backbone, 50 ng of each independent gene expression cassette and selection marker fragment were added to a 20 μL reaction system, along with 1 μL of BsaI restriction endonuclease and 1 μL of T4 DNA ligase. The enzyme digestion temperature was set at 37℃ for 3 minutes per digestion cycle, and the ligation temperature was set at 16℃ for 3 minutes per ligation cycle, for 15 cycles. Finally, the recombinant gene expression vector was inactivated at 50℃ for 5 minutes. The length of the assembled recombinant gene expression vector was 15.6kb. When using these parameters, the assembly bands of fragments larger than 15kb were clear, and the sequencing accuracy after transformation into E. coli was high, making it suitable for subsequent NotI linearization and integration into Yersinia lipophila.

[0018] Example 4: A gene expression vector for chlorogenic acid production by Yersinia lipolytica was prepared using a method described above. The gene expression vector for chlorogenic acid production by Yersinia lipolytica was a recombinant gene expression vector containing upstream and downstream homologous recombination arms of the rDNA region, 8 independent gene expression cassettes, and a LoxP-URA3-LoxP selection module. Bidirectional sequencing confirmed that the reading frames of PAL, C4H, C3H, CPR, 4CL, HQT, ARO4^K229L, and TKL1 were intact, the promoters and terminators were correctly connected, and there were no base deletions between the homologous recombination arms and the expression cassettes. When used for chromosome integration, this vector does not rely on antibiotic stress during industrial fermentation, which helps improve genetic stability during the production process.

[0019] Example 5: A method for constructing recombinant cells for chlorogenic acid production from Yersinia lipolytica includes the following steps: (1) Vector linearization: The gene expression vector of Yersinia lipolytica producing chlorogenic acid was digested with the restriction endonuclease NotI to linearize the gene expression vector and expose the upstream and downstream sequences of the rDNA regions at both ends. (2) In situ recombination transformation: The linearized vector obtained in step (1) was introduced into the host cells of Yersinia lipolytica with the ku70 gene knocked out in advance using the lithium acetate / dithiothreitol transformation method. The cells were incubated at 30-35℃ for 30-60 minutes and then heat-shocked at 39-42℃ for 10-20 minutes. The gene cluster containing the exogenous gene fragment was integrated into the rDNA region of the chromosome of Yersinia lipolytica host cells in situ through the homologous recombination mechanism to obtain the initial transformed cells. (3) Screening and marker excision of positive transformants: The initially transformed cells obtained in step (2) were spread on auxotrophic selection medium for single-clone screening to obtain positive transformants; helper plasmids expressing Cre recombinase were transiently introduced into the positive transformants, and LoxP site recombination was triggered by culturing in galactose-induced medium for 24-48 hours to excise the auxotrophic selection marker gene; the helper plasmids were naturally lost by continuous subculturing in non-selective medium to obtain recombinant cells of Yersinia lipolytica producing chlorogenic acid; The recombinant cell construction method used Yersinia lipolyticis host cells with the ku70 gene knocked out in advance; the vector was completely digested with NotI for 6 hours to obtain a linearized fragment; during in situ recombination transformation, 2 μg of the linearized fragment was introduced into the host cells using the LiAc / DTT method, incubated at 30℃ for 30 minutes, and then heat-shocked at 39℃ for 10 minutes. After 4 hours of recovery culture in the transformation medium, the cells were plated on uracil-deficient selection medium, and the initially transformed cells were picked. PCR verification was performed using primers that bridged the integration boundary of the rDNA region, and positive transformants were obtained. 1 μg of Cre helper plasmid was introduced into the positive transformants, and the cells were cultured in galactose-containing medium for 24 hours to remove the URA3 marker. The cells were then passaged for 6 consecutive generations in YPD medium to lose the Cre helper plasmid. This construction method utilizes the HR advantage brought about by ku70 deletion to guide large multi-gene fragments to the rDNA region, reducing random insertions and fragment breaks.

[0020] Example 6: The method for constructing recombinant cells of Yersinia lipolytica to produce chlorogenic acid, in step (2), the final concentration of lithium acetate in the lithium acetate / dithiothreitol conversion method is 0.1-0.2 mol / L, and the final concentration of dithiothreitol is 20-50 mmol / L; The final concentration of lithium acetate was set at 0.10 mol / L, and the final concentration of dithiothreitol was set at 20 mmol / L. Experiments showed that, under this specific concentration combination, combined with the ku70 gene knockout background of the present invention, there was an unexpected and significant improvement in the intracellular homologous recombination transformation efficiency of large linearized expression frame fragments. When this concentration combination was used for large linearized integrated fragments, the host cell survival rate was relatively stable, which facilitated obtaining the number of clones required for subsequent screening.

[0021] Example 7: The method for constructing recombinant cells for chlorogenic acid production by *Yarrowia lipolytica* also includes the step of using recombinant cells for chlorogenic acid production by *Yarrowia lipolytica* to synthesize chlorogenic acid through submerged fermentation: the recombinant cells for chlorogenic acid production by *Yarrowia lipolytica* are inoculated into a fermentation medium containing a carbon source, and submerged fermentation is carried out in shake flasks at 28-30℃ and 200-250 rpm for 48-96 hours, and the fermentation broth is collected; the carbon source is selected from at least one of glucose and glycerol; Recombinant cells were seeded in YNB fermentation medium containing 40 g / L glucose, with initial OD... 600 The concentration was set to 0.2, and the culture was carried out in a shake flask at 28°C and 200 rpm for 48 hours. After fermentation, the fermentation broth and cells were collected. Under these fermentation conditions, the host growth was stable, which is suitable for evaluating whether the exogenous pathway has been fully established.

[0022] Example 8: A method for constructing recombinant cells for chlorogenic acid production by Yersinia lipolytica: After the shake-flask deep fermentation is completed, the fermentation broth is subjected to cell wall disruption treatment, methanol solvent is added for ultrasonic extraction for 10-30 minutes, the supernatant is collected by centrifugation after static precipitation to obtain chlorogenic acid extract. 50 mL of fermentation broth was centrifuged at 8000 × g for 10 minutes to collect the cells. The cells were resuspended in methanol to a final volume of 10 mL, and then milled for 6 minutes to disrupt the cell walls. After ultrasonic extraction for 10 minutes, the cells were allowed to stand for 20 minutes and then centrifuged at 12000 × g for 10 minutes. The supernatant was used as the chlorogenic acid extract. The LC-MS detection conditions were as follows: C18 column, mobile phase A was 0.1% formic acid water, mobile phase B was acetonitrile, and negative ion mode was used for acquisition. The characteristic molecular ion peak of chlorogenic acid was detected at m / z 353.08, indicating that the synthetic pathway from phenylalanine to chlorogenic acid in Yersinia lipolytica has been established.

[0023] Comparative Example 1: Comparative Example 1 used the same vector backbone, homologous recombination arm, promoter allocation, ku70 deletion host, and fermentation conditions as Example 2. The difference was that the exogenous gene fragment did not contain C3H and CPR, but only retained PAL, C4H, 4CL, HQT, ARO4^K229L, and TKL1. This comparative example was used to evaluate the effect of the 3-position hydroxylation step and P450 reductase completion on chlorogenic acid synthesis. After successful integration was verified by PCR, shake-flask fermentation was carried out at 30℃, 250rpm and 96 hours. No obvious chlorogenic acid peak was detected at m / z 353.08 by LC-MS. The fermentation broth showed significant accumulation of p-coumaric acid and a small amount of p-coumarylquinic acid intermediates, indicating that the lack of C3H and CPR resulted in insufficient hydroxylation at the 3-position of the benzene ring and insufficient P450 electron transfer, thus preventing the formation of caffeoyl donors.

[0024] Comparative Example 2: Comparative Example 2 used the same complete 8-gene expression vector and fermentation conditions as Example 3, except that the ku70 gene was not knocked out in the host cells, and only some samples of the clones obtained after transformation showed random integration bands. Clones showing the presence of exogenous genes by PCR were fermented for detection. The results showed that the chlorogenic acid yield of this comparative example was lower than that of Examples 1 to 3, and the yield fluctuated greatly after each generation. This comparative example shows that when NHEJ preference is retained in Yersinia lipolytica, large multi-gene fragments are prone to non-target site insertion or incomplete integration, which is not conducive to stable expression.

[0025] Verification experiment: The strains used in Examples 1, 2, and 3, as well as Comparative Examples 1 and 2, were used for unified verification. To verify the necessity of the process parameter range, Comparative Examples 3 and 4 were added. Comparative Example 3 was performed under the same conditions as Example 1, except that the enzyme digestion temperature for GoldenGate assembly was 30°C, the ligation temperature was 10°C, and the cycle was repeated 10 times. The enzyme digestion temperature, ligation temperature and number of cycles mentioned above are all set outside the parameter range protected by this invention. The purpose is to demonstrate, through comparison, the necessity and significant advantages of the parameter range selected by this invention in improving the efficiency of vector assembly. Comparative Example 4 uses the same conditions as Example 1, except that the final concentration of lithium acetate in the conversion method is 0.05 mol / L, the final concentration of dithiothreitol is 10 mmol / L, and the heat shock temperature is 35°C. The test items included integration positivity rate, chlorogenic acid production, p-coumaric acid residue, cell dry weight, and passage stability; integration positivity rate was determined by both boundary PCR and multiplex PCR; chlorogenic acid production and p-coumaric acid residue were quantified by LC-MS external standard method; cell dry weight was determined by drying at 60℃ to constant weight. Stability was assessed by subculturing non-selective YPD medium for 100 generations, followed by detection of chlorogenic acid titer fluctuations in single colonies. LC-MS analysis was performed using the same column and mobile phase. Standard curves were established with chlorogenic acid standard concentrations of 0.05, 0.10, 0.50, 1.00, and 2.00 g / L, with a linear correlation coefficient of 0.998. Three replicates were performed for each group, and the average values ​​were used. Table 1 Performance test data of Examples 1-3 and Comparative Examples 1-4

[0026] In Examples 1 to 3, the characteristic peak of chlorogenic acid was detected at m / z 353.08. Example 3 showed a higher yield, indicating that PAL, C4H, C3H, CPR, 4CL, HQT, ARO4^K229L, and TKL1 were completely introduced and integrated into the rDNA region of the Ku70-deficient host, which has a direct effect on chlorogenic acid formation. In Comparative Example 1, no chlorogenic acid was detected, indicating that C3H and CPR are not substitute components, but are necessary components for 3-position hydroxylation and electron transfer in the complete pathway. Comparative Example 2 showed a low integration positivity rate and significant passage fluctuations, indicating that ku70 deletion and site-directed rDNA integration play a practical role in the stable expression of large fragments. Comparative Examples 3 and 4 showed significantly lower integration positivity rates, suggesting that the defined assembly and transformation parameter ranges affect the integration efficiency of large fragments. Furthermore, to eliminate multivariate cross-interference due to changes in constant values ​​between examples, univariate analysis confirmed that: In Example 2, only the change in promoter allocation independently contributed to the reduction of coumaric acid residue and the increase in yield; in Example 3, the replacement of the glycerol carbon source independently contributed to the significant increase in biomass, while the increase in the concentration of the conversion reagent independently contributed to the increase in the positive rate from 86% to 91%, thus clarifying the specific independent contribution of each constant value change to the final effect.

[0027] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for constructing a gene expression vector for chlorogenic acid production by Yersinia lipolytica, characterized in that, Includes the following steps: (1) Obtaining exogenous gene fragments: Obtain chlorogenic acid synthesis pathway genes and precursor enhancement genes optimized by codons of Yersinia lipolytica. The chlorogenic acid synthesis pathway genes include phenylalanine ammonia-lyase gene PAL, cinnamic acid-4-hydroxylase gene C4H, p-coumaric acid-3-hydroxylase gene C3H, cytochrome P450 reductase gene CPR, 4-coumaric acid coenzyme A ligase gene 4CL and hydroxycinnamoyltransferase gene HQT. The precursor enhancement genes include the DAHP synthase mutant gene ARO4^K229L that relieves feedback inhibition and the transketolase gene TKL1. (2) Constructing independent gene expression cassettes: The exogenous gene fragments obtained in step (1) are respectively connected to the promoter and terminator of Yeast lipolyticis. The independent expression cassette refers to each exogenous gene fragment being operatively connected to a promoter and a terminator to form an independent transcription unit. (3) Obtaining homologous recombination arms and selection marker genes: Using Yeast lipolyticis genomic DNA as a template, the upstream and downstream sequences of the Yeast lipolyticis genomic rDNA region were amplified as homologous recombination arms; and nutritional auxotroph selection marker genes with LoxP sites fused on both sides were obtained. (4) Seamless assembly of vector: Using pINA plasmid as vector backbone, the homologous recombination arm described in step (3) is cloned to both sides of the multiple cloning site. Then, using GoldenGate assembly technology, the multiple independent gene expression cassettes constructed in step (2) are connected in a pre-set order to form a multi-gene expression cassette, and inserted together with the auxotrophic selection marker gene into the homologous recombination arm to obtain the recombinant gene expression vector.

2. The method for constructing a gene expression vector for chlorogenic acid production by *Yarrowia lipolytica* according to claim 1, characterized in that, In step (2), the promoters include strong compositional promoters and moderately strong compositional promoters; The PAL, C4H, and C3H genes are driven by the strong constitutive promoter, which is selected from at least one of the pTEF promoter and the hp4d promoter; the 4CL, HQT, CPR, ARO4^K229L, and TKL1 genes are driven by the moderate constitutive promoter, which is the pEXP1 promoter.

3. The method for constructing a gene expression vector for chlorogenic acid production by *Yarrowia lipolytica* according to claim 1, characterized in that, In step (3), the auxotrophic screening marker gene is the URA3 gene; In step (4), the process conditions of the GoldenGate assembly technology shall at least meet the following requirements: restriction endonuclease and DNA ligase are added to the reaction system, and the enzyme digestion reaction is carried out at 37-40℃ for 2-5 minutes, and the ligation reaction is carried out at 16-20℃ for 2-5 minutes. This is repeated as one cycle, and the cycle is repeated 15-30 times. Finally, the DNA is inactivated at 50-60℃ for 5-10 minutes. The nucleotide sequence length of the recombinant gene expression vector obtained by assembly is 15-20kb.

4. A gene expression vector for chlorogenic acid production by *Yersinia lipolytica* prepared by the method for constructing a gene expression vector for chlorogenic acid production by *Yersinia lipolytica* according to any one of claims 1-3.

5. A method for constructing recombinant cells from *Yarrowia lipolyticis* producing chlorogenic acid, characterized in that, Includes the following steps: (1) Vector linearization: The gene expression vector of Yersinia lipolytica producing chlorogenic acid as described in claim 4 is digested with the restriction endonuclease NotI to linearize the gene expression vector and expose the upstream and downstream sequences of the rDNA regions at both ends; (2) In situ recombination transformation: The linearized vector obtained in step (1) was introduced into the Yersinia lipolytica host cells with the ku70 gene knocked out in advance using the lithium acetate / dithiothreitol transformation method. The cells were incubated at 30-35℃ for 30-60 minutes and then heat-shocked at 39-42℃ for 10-20 minutes. The gene cluster containing the exogenous gene fragment was integrated in situ into the rDNA region of the Yersinia lipolytica host cell chromosome through the homologous recombination mechanism to obtain the initial transformed cells. (3) Screening and marker excision of positive transformants: The initial transformed cells obtained in step (2) are spread on auxotrophic selection medium for single-clone screening to obtain positive transformants; helper plasmids expressing Cre recombinase are transiently introduced into the positive transformants, and LoxP site recombination is triggered by culturing in galactose-induced medium for 24-48 hours to excise the auxotrophic selection marker gene; the helper plasmids are naturally lost by continuous subculturing in non-selective medium to obtain recombinant cells of Yersinia lipolytica producing chlorogenic acid.

6. The method for constructing recombinant cells of Yersinia lipolytica producing chlorogenic acid according to claim 5, characterized in that, In step (2), the final concentration of lithium acetate in the lithium acetate / dithiothreitol conversion method is 0.1-0.2 mol / L, and the final concentration of dithiothreitol is 20-50 mmol / L.

7. The method for constructing recombinant cells of Yersinia lipolytica producing chlorogenic acid according to claim 5, characterized in that, It also includes the step of synthesizing chlorogenic acid through deep fermentation using recombinant cells of the aforementioned Yersinia lipolytica yeast that produce chlorogenic acid: The recombinant cells of Yersinia lipolytica that produce chlorogenic acid were inoculated into a fermentation medium containing a carbon source and subjected to shake-flask deep fermentation at 28-30°C and 200-250 rpm for 48-96 hours. The fermentation broth was then collected. The carbon source was selected from at least one of glucose and glycerol.

8. The method for constructing recombinant cells of Yersinia lipolytica producing chlorogenic acid according to claim 7, characterized in that, After the shake-flask deep fermentation is completed, the fermentation broth is subjected to cell wall disruption treatment, methanol solvent is added for ultrasonic extraction for 10-30 minutes, and after standing precipitation, the supernatant is collected by centrifugation to obtain chlorogenic acid extract.

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