Recombinant Yarrowia lipolytica engineering strain for producing chlorogenic acid and application of recombinant Yarrowia lipolytica engineering strain

Abstract

The invention discloses a recombinant Yarrowia lipolytica engineering strain for producing chlorogenic acid and application of the recombinant Yarrowia lipolytica engineering strain, and belongs to the technical field of genetic engineering and bioengineering. According to the invention, a hydroxylation module and a condensation module are constructed in the Yarrowia lipolytica, heterologous synthesis of CGA is realized, supply of NADPH is promoted through optimization of an endogenous pentose phosphate pathway, oxygen intake is increased and a shikimic acid pathway is enhanced by using hemoglobin VHb of heterologous Vitreoscilla, and the yield of chlorogenic acid is further increased. The yield of chlorogenic acid of the constructed engineering bacterium can reach 13.13 g / L, a foundation is laid for subsequent biosynthesis of compounds such as chicoric acid, and the engineering bacterium has potential value and significance for development of synthetic biology.

Description

Technical Field

[0001] The invention relates to a recombinant Yarrowia lipolytica engineering strain for producing chlorogenic acid and application thereof, belonging to the technical field of genetic engineering and bioengineering. Background Art

[0002] Chlorogenic acid (CGA) is a water-soluble polyphenolic phenylpropionate compound produced by plants through the shikimic acid pathway during aerobic respiration. It was first discovered in green coffee beans. Chlorogenic acid has important biological activities such as antioxidant, anti-inflammatory, antibacterial, anti-tumor, and regulation of glucose metabolism and lipid metabolism. It shows great application prospects in food preservation, nutrition, health care and pharmaceutical fields.

[0003] At present, the production of CGA mainly relies on the extraction from plant tissues such as honeysuckle, coffee beans, and Eucommia ulmoides leaves, which is limited by plant availability, low levels in plant tissues, and complex similar components. CGA contains multiple hydroxyl and carboxyl groups, which are unstable in chemical synthesis. Chemical synthesis involves many reaction steps, expensive reagents, and more by-products, which makes it unsuitable to use chemical synthesis for large-scale production of CGA. Therefore, it is necessary to develop a sustainable and green production method for CGA. The synthesis of chlorogenic acid by microbial cell factories has attracted widespread attention due to its high purity and high efficiency. However, it has been reported that Escherichia coli is used to synthesize chlorogenic acid, and the host cells do not meet the safety requirements of food production, and the yield of chlorogenic acid synthesized in Saccharomyces cerevisiae is low. Therefore, how to use a safer biological method to synthesize chlorogenic acid and obtain a higher yield and improve fermentation production efficiency is an urgent problem to be solved. Summary of the invention

[0004] The invention provides an engineered strain of Yarrowia lipolytica for efficiently synthesizing chlorogenic acid. Based on a starting strain, hydroxycinnamoyl-CoA quinate transferase, 4-coumaric acid:CoA ligase, quinate / shikimate 5-dehydrogenase, cytochrome P450 98A3 and cytochrome P450 reductase are expressed.

[0005] In one embodiment, the engineered Yarrowia lipolytica has at least one of the following improvements:

[0006] (1) knocking out the endogenous ABC transporter gene Ticw of Yarrowia lipolytica; the nucleotide sequence of the gene Ticw is shown in the NCBI reference sequence: XM_501196.3;

[0007] (2) Using promoter P TDH , P TEF , P MNDH2 or P GPD1 Initiate the expression of 4CL, HQT, C3H, CPR or YdiB genes;

[0008] (3) Strengthening the endogenous NADPH biological regeneration pathway;

[0009] (4) Hemoglobin VHb from Vitreoscilla was introduced to increase oxygen uptake.

[0010] In one embodiment, the hydroxycinnamoyl-CoA quinoyltransferase is derived from Nicotiana tabacum or Cynara scolymus; the 4-coumarate:CoA ligase is derived from Arabidopsis thaliana or Petroselinum crispum; the cytochrome P450 98A3 is derived from Arabidopsis thaliana; and the cytochrome P450 reductase is derived from Arabidopsis thaliana.

[0011] In one embodiment, the engineered Yarrowia lipolytica is based on the starting strain, and expresses 4-coumaric acid: CoA ligase At4CL from Arabidopsis thaliana and hydroxycinnamoyl-CoA quinate transferase NtHQT from tobacco, and expresses the bifunctional enzyme EcYdiB from Escherichia coli, and expresses the hydroxylase PaHpaB from Pseudomonas aeruginosa and the reductase SeHpaC from Salmonella enterica.

[0012] In one embodiment, the engineered Yarrowia lipolytica is based on the starting strain, and expresses 4-coumaric acid: CoA ligase At4CL from Arabidopsis thaliana and hydroxycinnamoyl-CoA quinate transferase NtHQT from tobacco, and expresses the bifunctional enzyme EcYdiB from Escherichia coli, and expresses the hydroxylase AtC3H from Arabidopsis thaliana and the reductase AtCPR1 from Arabidopsis thaliana.

[0013] In one embodiment, the engineered Yarrowia lipolytica is based on the starting strain, and expresses 4-coumaric acid: CoA ligase At4CL from Arabidopsis thaliana and hydroxycinnamoyl-CoA quinate transferase CsHQT from artichoke, and expresses the bifunctional enzyme EcYdiB from Escherichia coli, and expresses the hydroxylase AtC3H from Arabidopsis thaliana and the reductase AtCPR1 from Arabidopsis thaliana.

[0014] In one embodiment, the engineered Yarrowia lipolytica is based on the starting strain, and expresses 4-coumaric acid: CoA ligase Pc4CL from pseudo-parsley and hydroxycinnamoyl-CoA quinate transferase NtHQT from tobacco, and expresses the bifunctional enzyme EcYdiB from Escherichia coli, and expresses the hydroxylase AtC3H from Arabidopsis thaliana and the reductase AtCPR1 from Arabidopsis thaliana.

[0015] In one embodiment, the strengthening of the endogenous NADPH biological regeneration pathway comprises overexpressing the Yarrowia lipolytica endogenous genes ZWF1 and MAE1.

[0016] In one embodiment, the nucleotide sequence of gene ZWF1 is shown as SEQ ID NO.14; the nucleotide sequence of gene MAE1 is shown as SEQ ID NO.15.

[0017] In one embodiment, the nucleotide sequence encoding the hemoglobin VHb is shown as SEQ ID NO.16.

[0018] In one embodiment, the 4-coumaric acid:CoA ligase At4CL and hydroxycinnamoyl-CoA quinate transferase CsHQT are linked to short peptides RIAD and / or RIDD.

[0019] In one embodiment, the nucleotide sequence encoding the short peptide RIAD is shown as SEQ ID NO.17; the nucleotide sequence encoding the short peptide RIDD is shown as SEQ ID NO.18.

[0020] In one embodiment, the starting strain includes but is not limited to Yarrowia lipolytica Po1f.

[0021] In one embodiment, the starting strain is strain CGAp01 constructed by overexpressing shikimic acid pathway genes based on Yarrowia lipolytica Po1f; the strain CGAp01 is disclosed in the paper "Remodelling metabolism for high-level resveratrol production in Yarrowia lipolytica".

[0022] In one embodiment, the shikimate pathway genes include ARO1, ARO2, ARO4 K221L and ARO7 G139S .

[0023] In one embodiment, genes EcYdiB, At4CL, and NtHQT are integrated at the AXP site, and genes AtC3H and AtCPR1 are integrated at the A3 site.

[0024] In one embodiment, genes EcYdiB, At4CL, and CsHQT are integrated at the AXP site, and genes AtC3H and AtCPR1 are integrated at the A3 site.

[0025] In one embodiment, genes AtC3H and AtCPR1 are at the A3 site, genes EcYdiB, At4CL, CsHQT F371A Integrated at the AXP locus.

[0026] In one embodiment, the AtC3H gene or the AtCPR1 gene is also integrated and expressed at multiple copy sites.

[0027] In one embodiment, the multi-copy loci include but are not limited to ZETA or 26S rDNA.

[0028] The present invention also provides a hydroxycinnamoyl-CoA quinate transferase mutant, which has a 371st amino acid mutation relative to the hydroxycinnamoyl-CoA quinate transferase CsHQT parent.

[0029] In one embodiment, the parent has the amino acid sequence shown in NCBI No.: XP_024980016.1.

[0030] In one embodiment, the mutation is to mutate phenylalanine (F) at position 371 to alanine (A).

[0031] The present invention also provides a gene encoding the mutant.

[0032] In one embodiment, the nucleotide sequence of the gene is shown as SEQ ID NO.23.

[0033] The present invention also provides a fusion enzyme comprising 4-coumaric acid: coenzyme A ligase At4CL and the hydroxycinnamoyl coenzyme A quinate transferase mutant.

[0034] In one embodiment, the 4-coumaric acid:CoA ligase is linked to a short peptide RIAD; and the hydroxycinnamoyl-CoA quinate transferase mutant is linked to a short peptide RIDD.

[0035] The present invention also provides a recombinant microorganism expressing the mutant or the fusion enzyme.

[0036] In one embodiment, the microorganism includes but is not limited to Yarrowia lipolytica.

[0037] The present invention also provides the use of the Yarrowia lipolytica engineered bacteria in producing chlorogenic acid.

[0038] In one embodiment, the engineered Yarrowia lipolytica is fermented in a culture medium at 30° C. for 72 to 96 hours, or 72 to 144 hours.

[0039] In one embodiment, the culture medium includes but is not limited to YPD medium and inorganic salt medium.

[0040] In one embodiment, the inorganic salt culture medium contains glucose, ammonium sulfate, potassium salt and magnesium salt; the potassium salt includes but is not limited to potassium dihydrogen phosphate; the magnesium salt includes but is not limited to magnesium sulfate.

[0041] The present invention also claims to protect the application of the engineered Yarrowia lipolytica in the production of chlorogenic acid-containing products in the fields of food, medicine and chemical industry.

[0042] In one embodiment, the application is for preparing a vaccine or a medicine containing chlorogenic acid, or preparing a cosmetic containing chlorogenic acid.

[0043] Beneficial effects:

[0044] The present invention realizes heterologous synthesis of CGA by constructing a hydroxylation module and a condensation module in Yarrowia lipolytica. In order to improve the efficiency of hydroxylation, the present invention systematically evaluates various P450 cytochrome P450 98A3 (C3H) and cytochrome P450 reductase (CPR) enzymes, and based on the extensive catalytic activity of HQT and 4CL enzymes, screens the optimal form of the combination of enzymes to reduce the generation of by-products, thereby improving the utilization rate of carbon sources. CsHQT is optimized by semi-rational design to enhance CGA synthesis, and exogenous gene expression is regulated to minimize caffeic acid (CA) by-products and balance quinic acid (QA) and p-coumaric acid (p-CA) accumulation. In addition, an NADPH regeneration system is introduced to improve the efficiency of the hydroxylation module. By analyzing the transcriptome during the degradation process of CGA in lipolytic yeast, the Ticw gene (YALI0_B21824g) is identified and knocked out to slow down the degradation of CGA, and finally a CGA yield of 612.83 mg / L is achieved in a shake flask. Finally, the titer of CGA reached 13.13 g / L by fed-batch fermentation in a 5 L bioreactor. The present invention lays a foundation for the microbial production of CGA and other p-CA and CGA derivatives. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 Schematic diagram of the metabolism of heterologous chlorogenic acid synthesis in Yarrowia lipolytica.

[0046] Figure 2 This is the chromatogram of chlorogenic acid produced by the engineered strain under YPD culture.

[0047] Figure 3 Schematic diagram of the docking results of CsHQT enzyme and quinic acid molecule.

[0048] Figure 4 This is a graph showing the chlorogenic acid production of different recombinant Yarrowia lipolytica strains under YPD culture.

[0049] Figure 5 The synthesis of chlorogenic acid by the optimized strain CGA157 in a 5 L fermenter.

[0050] Figure 6 This is the effect of different connecting peptides or short peptides on the synthesis of chlorogenic acid in recombinant bacteria. DETAILED DESCRIPTION

[0051] (I) Culture medium

[0052] LB medium: peptone 10g / L, yeast powder 5g / L, sodium chloride 10g / L. Add 20g / L agar powder to prepare LB solid medium.

[0053] YNB medium: Yeast Nutrition Base 67.4 g / L, glucose 20 g / L, according to the nutritional deficiency type of the strain to be cultured, appropriate amino acids (5 g / L uracil, 5 g / L leucine) are added to the medium.

[0054] YPD medium: peptone 20 g / L, yeast powder 10 g / L, glucose 20 g / L.

[0055] Inorganic salt culture medium: ammonium sulfate 5g / L, potassium dihydrogen phosphate 3.5g / L, magnesium sulfate 5g / L, glucose 40g / L.

[0056] Trace metal solution: 4.5g / L CaCl 2 ·2H 2 O, 4.5g / L ZnSO 4 7H 2 O, 3g / L FeSO 4 7H 2 O, 1g / LH 3 BO 3 、1g / L MnCl 2 ·4H 2 O,0.4g / L Na 2 MoO 4 ·2H 2 O,0.3g / L CoCl 2 6H 2 O,0.1g / LCuSO 4 ·5H 2O, 0.1g / LKI, 15g / LEDTA. Adjust pH to 4, filter sterilize, and store at 4℃ away from light.

[0057] Vitamin solution: 50 mg / L biotin, 200 mg / L p-aminobenzoic acid, 1 g / L niacin, 1 g / L calcium pantothenate, 1 g / L pyridoxine hydrochloride, 1 g / L thiamine hydrochloride, 25 g / L inositol. Adjust the pH to 6.5, filter and sterilize, and store at 4°C away from light.

[0058] (II) Transformation steps of Yarrowia lipolytica: Pick a single yeast colony on the plate and inoculate it into 3-4mL YPD liquid medium, and culture it at 30℃ for 16-22h as the first-level seed liquid. Transfer the first-level seed liquid to 10mL YPD liquid medium at a 2% inoculation amount, and culture it at 30℃ for 4-6h as the second-level seed liquid. Take 500μL of the second-level seed liquid and centrifuge it at 5000rpm for 4min, and discard the supernatant. Then prepare it in a sterile EP tube. Transformation buffer: 90μL 50% sterilized PEG4000, 5μL 2M lithium acetate and 5μL boiled linear single-stranded DNA, shake and mix. Put it in the transformation buffer, add 0.4-0.6μg circular plasmid or 3-10μg linear DNA fragment, and shake and mix. Incubate in a 30℃ water bath for 30-45min, shaking for 15s every 10min. In a 39℃ water bath for 10min. Spread the corresponding selective plates and culture in a 30°C incubator for about 2-3 days.

[0059] (III) Chlorogenic acid HPLC determination: Shimadzu high performance liquid chromatography was used for determination. HPLC conditions: chromatographic column: InertSustain C18 250mm×4.6mm column (particle size 5μm); mobile phase A, ultrapure water containing 1‰ trifluoroacetic acid; mobile phase B, acetonitrile containing 1‰ trifluoroacetic acid; mobile phase ratio conditions, 0-10min, 10-40% B, 10-30min, 40-80% B, 30-35min, 80-80% B, 35-37min, 80-10% B, 37-40min, 10-10% B; flow rate: 1mL / min; column temperature: 40℃; injection volume: 10μL; detector wavelength: 290nm.

[0060] (IV) The strain information is shown in Table 1.

[0061] Table 1 Strains involved in the present invention

[0062]

[0063] Table 2 Primer sequences

[0064]

[0065]

[0066] Example 1 Construction of a recombinant Yarrowia lipolytica strain that synthesizes chlorogenic acid

[0067] Using Yarrowia lipolytica Po1f (published in the paper "Remodelling metabolism for high-level resveratrol production in Yarrowia lipolytica") as the starting strain, a D17::P TEF -FjTAL-T XPR2 ,F1-3::P TEF -Aro1-T XPR2 -P TEF -Aro2-T XPR2 -P TEF -ARO4 K221L -T XPR2 -PTEF-ARO7 G139S -T XPR2 The specific steps are as follows: based on strain ST422, the expression cassette P TEF -FjTAL-T XPR2 (shown in SEQ ID NO.24) was integrated at the D17 site to replace the original "P TEF -FjTAL-T XPR2 -P TEF -Pc4CL1-T XPR2 -P TEF -VvSTS-T XPR2 ", the primer pairs for amplifying the homology arms of the D17 locus were "D17-armup-F / D17-armup-R and D17-armdown-F / D17-armdown-R", and the constructed chassis strain was named CGAp01.

[0068] Furthermore, the A3 and AXP sites of strain CGAp01 were used as integration sites for hydroxycinnamoyl-CoA quinate transferase from tobacco (Nicotiana tabacum) or artichoke (Cynara scolymus), 4-coumaric acid:CoA ligase from Arabidopsis thaliana or parsley (Petroselinum crispum), quinate / shikimate 5-dehydrogenase from Escherichia coli, cytochrome P450 98A3 from Arabidopsis thaliana, and cytochrome P450 reductase from Arabidopsis thaliana to construct an engineered strain CGA01.

[0069] The specific construction method is as follows: synthesize the gene encoding CsHQT (nucleotide sequence as shown in SEQ ID NO.1), and amplify the gene CsHQT fragment with primers CsHQT-F / CsHQT-R; synthesize the gene encoding NtHQT (nucleotide sequence as shown in SEQ ID NO.2), and amplify the gene NtHQT fragment with primers NtHQT-F / NtHQT-R; synthesize the gene encoding At4CL (nucleotide sequence as shown in SEQ ID NO.3), and amplify the gene At4CL fragment with primers At4CL-F / At4CL-R; synthesize the gene encoding Pc4CL (nucleotide sequence as shown in SEQ ID NO.4), and amplify the gene Pc4CL fragment with primers Pc4CL-F / Pc4CL-R; synthesize the gene encoding EcYdiB (nucleotide sequence as shown in SEQ ID NO. NO.5), and amplified the gene EcYdiB fragment with primers EcYdiB-F / EcYdiB-R; synthesized the gene encoding CgYdiB (nucleotide sequence as shown in SEQ ID NO.6), and amplified the gene CgYdiB fragment with primers CgYdiB-F / CgYdiB-R; synthesized the gene encoding AtC3H (nucleotide sequence as shown in SEQ ID NO.7), and amplified the gene AtC3H fragment with primers AtC3H-F / AtC3H-R; synthesized the gene encoding PtrC3H (nucleotide sequence as shown in SEQ ID NO.8), and amplified the gene PtrC3H fragment with primers PtrC3H-F / PtrC3H-R; synthesized the gene encoding AtCPR1 (nucleotide sequence as shown in SEQ ID NO.9), and amplified the gene AtCPR1 fragment with primers AtCPR1-F / AtCPR1-R; synthesized the gene encoding AtCPR2 (nucleotide sequence as shown in SEQ ID NO.10), and the AtCPR2 gene fragment was amplified with primers AtCPR2-F / AtCPR2-R; the gene encoding SmCPR (nucleotide sequence is shown in SEQ ID NO.11) was synthesized, and the SmCPR gene fragment was amplified with primers SmCPR-F / SmCPR-R; the gene encoding HpaB (nucleotide sequence is shown in SEQ ID NO.12) was synthesized, and the HpaB gene fragment was amplified with primers HpaB-F / HpaB-R; the gene encoding HpaC (nucleotide sequence is shown in SEQ ID NO.13) was synthesized, and the HpaC gene fragment was amplified with primers HpaC-F / HpaC-R. The promoter P was amplified with primers PTEF-F / PTEF-R. TEF. The upstream and downstream homology arms of the A3 site were amplified using primers A3-armup-F / A3-armup-R and A3-armdown-F / A3-armdown-R, respectively, and the upstream and downstream homology arms of the AXP site were amplified using primers AXP-armup-F / AXP-armup-R and AXP-armdown-F / AXP-armdown-R, respectively. The above fragments were assembled using OE-PCR, and the C3H gene fragment (AtC3H fragment or PtrC3H fragment) and the CPR gene fragment (AtCPR1 fragment or SmCPR fragment) were connected to the A3 homology arm, and the HQT gene fragment (CsHQT fragment or NtHQT fragment), 4CL gene fragment (At4CL fragment or Pc4CL fragment) and YdiB gene (EcYdiB fragment, EcYdiB fragment) were connected to the A3 homology arm. S67A The fragment or CgYdiB fragment) was connected to the AXP homology arm. The PCR product was recovered by ethanol precipitation. About 1 μg of the integrated fragment and about 300 ng of sgRNA were transformed into the Yarrowia lipolytica engineered strain CGAp01 using the lithium acetate method, coated on the screening solid culture medium, and cultured at 30°C for 3 days until colonies appeared. The correct clones were named CGA01-CGA14. Pick a single colony and transfer it to 5 mL of the corresponding YNB culture medium. After 24 hours, transfer it to 25 mL of YPD culture medium at a 1% inoculation amount. After culturing for 120 hours, take 500 μL of fermentation broth, add 500 μL of methanol, centrifuge the resuspension at 12000×g for 10 minutes, filter through a 0.22 μm organic filter membrane, and perform HPLC analysis. The analysis results show that the CGA yields of the recombinant engineered bacteria CGA01 to CGA14 are shown in Table 3.

[0070] Table 3 Effects of genes from different sources on CGA yield

[0071]

[0072]

[0073] Example 2 Semi-rational modification to improve the catalytic efficiency of the enzyme CsHQT

[0074] In order to improve the catalytic efficiency of the enzyme CsHQT, the protein structure of CsHQT was predicted and molecular docking was performed with quinic acid. The sites of the CsHQT enzyme were mutated according to the docking results. These residues include I46, H163, V312, R366, F371, N377, N379, W381, T409 and Y411, which are located at different positions of the binding pocket. The F371A-F / F371A-R primers were used to amplify the CsHQT F371AThe mutant was constructed by referring to the method of constructing strain CGA08 in Example 1, and the upstream and downstream homology arms of AXP, EcYdiB, At4CL, CsHQT F371A The gene fragments were connected. The PCR products were recovered by ethanol precipitation. About 1 μg of the integrated fragment and about 300 ng of sgRNA were transformed into the Yarrowia lipolytica engineered strain CGAp01 using the lithium acetate method, spread on the screening solid medium, and cultured at 30°C for 3 days until colonies appeared. The correct clone was named CGAm-5.

[0075] The same strategy was used to construct recombinant bacteria expressing the CsHQT mutations shown in Table 4. A single colony was picked and transferred into 5 mL of the corresponding YNB medium. After 24 hours, it was transferred into 25 mL of YPD medium at a 1% inoculation amount. After 120 hours of culture, 500 μL of fermentation broth was taken, 500 μL of methanol was added, and the resuspension was centrifuged at 12000×g for 10 minutes, filtered through a 0.22 μm organic filter membrane, and analyzed by HPLC. The results showed that the mutation at the F371A site had the most significant effect on the yield, with the CGA yield reaching 74.04 mg / L.

[0076] Table 4 Effects of genes from different sources on CGA yield

[0077]

[0078] Example 3 Fusion enzyme improves enzyme catalytic efficiency

[0079] To improve CsHQT F371A The catalytic efficiency of At4CL was improved to reduce the potential loss of unstable intermediates. The gene fragment encoding RIAD was amplified and synthesized using RIAD-F / RIAD-R primers (the nucleotide sequence is shown in SEQ ID NO.17), and the gene fragment encoding RIDD was amplified and synthesized using RIDD-F / RIDD-R primers (the nucleotide sequence is shown in SEQ ID NO.18). Referring to the method of Example 1, the gene fragment encoding RIAD and the gene fragment encoding RIDD were combined with the upstream and downstream homologous arms of AXP, EcYdiB, At4CL gene fragments, CsHQT F371AThe mutant gene fragments were connected. The PCR product was recovered by ethanol precipitation. About 1 μg of the integrated fragment and about 300 ng of sgRNA were transformed into the Yarrowia lipolytica engineered strain CGAp01 using the lithium acetate method, spread on the screening solid culture medium, and cultured at 30°C for 3 days until colonies appeared. The correct clone was named CGAl-13. Pick a single colony and transfer it to 5 mL of the corresponding YNB culture medium. After 24 hours, transfer it to 25 mL YPD culture medium at a 1% inoculation amount. After 120 hours of culture, take 500 μL of fermentation broth, add 500 μL of methanol, centrifuge the resuspension at 12000×g for 10 minutes, filter through a 0.22 μm organic filter membrane, and perform HPLC analysis. The analysis results showed that the yield of strain CGAl-13 reached 81.56 mg / L.

[0080] Example 4 Optimizing cis-acting elements to balance enzyme expression

[0081] To optimize the expression level of the enzyme, four endogenous promoters P GPD1 , P TDH , P TEF , P MNDH2 The promoters of C3H and CPR were replaced and the genes were integrated into the A3 site. The promoters of HQT, 4CL and YdiB were replaced and the genes were integrated into the AXP site. The promoter fragments P were amplified from the Yarrowia lipolytica genome using primers PGPD1-F / PGPD1-R. GPD1 , and the promoter fragment P was amplified using primers PTDH-F / PTDH-R TDH , and the promoter P was amplified using primers PTEF-F / PTEF-R TEF The promoter fragment P was amplified using primers PMNDH2-F / PMNDH2-R. MNDH2 , using primers TXPR2-F / TXPR2-R to amplify the terminator T XPR2. A3-armup-F / A3-armup-R and A3-armdown-F / A3-armdown-R amplified the upstream and downstream homology arms of the A3 site, respectively, and AXP-armup-F / AXP-armup-R and AXP-armdown-F / AXP-armdown-R amplified the upstream and downstream homology arms of the AXP site, respectively. The above fragments were assembled using OE-PCR. The PCR products were recovered by ethanol precipitation. About 1 μg of the integrated fragment and about 300 ng of sgRNA were transformed into the Yarrowia lipolytica engineered strain CGAp01 using the lithium acetate method, spread on the screening solid culture medium, and cultured at 30°C for 3 days until colonies appeared. The correct clone was named CGA75. Pick a single colony and transfer it to 5mL of the corresponding YNB medium. After 24h, transfer it to 25mL YPD medium at a 1% inoculation amount. After culturing for 120h, take 500μL of fermentation broth, add 500μL of methanol, centrifuge the resuspension at 12000×g for 10min, filter through a 0.22μm organic filter membrane, and perform HPLC analysis. The analysis results show that the yield of chlorogenic acid of the recombinant engineered bacteria E32T13 is 123.89mg / L.

[0082] Example 5 Construction of recombinant engineered bacteria CGA93

[0083] In order to increase the synthesis flux of quinic acid and increase the copy number of the YdiB gene, the upstream and downstream homologous arms of the C2 site were amplified using C2-armup-F / C2-armup-R and C2-armdown-F / C2-armdown-R, respectively. The above fragments and the YdiB gene fragment described in Example 1 were assembled using OE-PCR. The PCR product was recovered by ethanol precipitation. About 1 μg of the integrated fragment and about 300 ng of sgRNA were transformed into the Y. lipolytica engineered strain CGA75 constructed in Example 4 using lithium acetate, spread on the screening solid culture medium, and cultured at 30°C for 3 days until colonies appeared. The correct clone was named CGA93. Pick a single colony and transfer it to 5mL of the corresponding YNB medium. After 24h, transfer it to 25mL YPD medium at a 1% inoculation amount. After 120h of culture, take 500μL of fermentation broth, add 500μL of methanol, centrifuge the resuspension at 12000×g for 10min, filter through a 0.22μm organic filter membrane, and perform HPLC analysis. The analysis results show that the yield of chlorogenic acid of the recombinant engineered bacteria CGA93 is 175.73mg / L.

[0084] Example 6 Expression of endogenous pentose phosphate pathway to enhance NADPH supply

[0085] To promote the supply of NADPH, the endogenous genes ZWF1 (nucleotide sequence shown in SEQ ID NO.14) and MAE1 (nucleotide sequence shown in SEQ ID NO.15) of Yarrowia lipolytica were overexpressed. The ZWF1 gene fragment was amplified from the Yarrowia lipolytica genome using primers ZWF1-F / ZWF1-R, and the MAE1 gene fragment was amplified from the Yarrowia lipolytica genome using primers MAE1-F / MAE1-R, and the Vhb gene was synthesized (the nucleotide sequence is shown in the sequence table SEQ ID NO.16), and the Vhb gene fragment was amplified using primers Vhb-F / Vhb-R; the upstream and downstream homologous arms of the E3 site were amplified using E3-armup-F / E3-armup-R and E3-armdown-F / E3-armdown-R, and the upstream and downstream homologous arms of the D6 site were amplified using D6-armup-F / D6-armup-R and D6-armdown-F / D6-armdown-R. The above fragments were assembled using OE-PCR. The PCR products were recovered by ethanol precipitation. About 1 μg of the integration fragment and about 300 ng of sgRNA were transformed into the Yarrowia lipolytica engineered strain CGA93 constructed in Example 5 using lithium acetate, spread on the screening solid culture medium, and cultured at 30°C for 3 days until colonies appeared. The correct clone was named CGA108. Pick a single colony and transfer it to 5 mL of the corresponding YNB culture medium. After 24 hours, transfer it to 25 mL of YPD culture medium at a 1% inoculation amount. After 120 hours of culture, take 500 μL of fermentation broth, add 500 μL of methanol, centrifuge the resuspension at 12000×g for 10 minutes, filter through a 0.22 μm organic filter membrane, and perform HPLC analysis. The analysis results show that the yield of chlorogenic acid in the recombinant engineered bacteria CGA108 is 333.16 mg / L.

[0086] Example 7 Knockout of endogenous ABC transporter gene Ticw

[0087] The upstream and downstream homologous arms of the TicW gene (YALI0_B21824g) site were amplified using TicW-armup-F / TicW-armup-R and TicW-armdown-F / TicW-armdown-R, respectively. The above fragments were assembled using OE-PCR. The PCR product was recovered by ethanol precipitation. About 1 μg of the integrated fragment and about 300 ng of sgRNA were transformed into the Y. lipolytica engineered strain CGA108 constructed in Example 6 using lithium acetate, spread on the screening solid culture medium, and cultured at 30°C for 3 days until colonies appeared. The correct clone was named CGA109. Pick a single colony and transfer it to 5mL of the corresponding YNB medium. After 24h, transfer it to 25mL YPD medium at a 1% inoculation amount. After 120h of culture, take 500μL of fermentation broth, add 500μL of methanol, centrifuge the resuspension at 12000×g for 10min, filter through a 0.22μm organic filter membrane, and perform HPLC analysis. The analysis results show that the yield of chlorogenic acid in the recombinant engineered bacteria CGA109 is 351.33mg / L.

[0088] Example 8 Multi-copy integration of key genes to enhance hydroxylation efficiency

[0089] Liquid chromatography and mass spectrometry identified that the fermentation broth of strain CGA109 still contained a large amount of p-coumaroylquinic acid that was not converted into chlorogenic acid. Previous studies have identified two multi-copy integration sites (ZETA and 26s rDNA) in Y. lipolytica. And 26S rDNA has 100-200 copies in Y. lipolytica. Therefore, a multi-copy integration strategy was used to further increase the synthesis flux of chlorogenic acid.

[0090] Since the Yarrowia lipolytica constructed in this application is a leucine and uracil deficient strain, rDNA-F / rDNA-R primers were used with pYlST1206 plasmid (disclosed in the paper "A set of efficient and stable multi-copy integration toolkit in Yarrowia lipolytica") as a template to amplify the multi-copy plasmid backbone, and the leucine tag, the key gene AtC3H and the multi-copy site rDNA homology arm were connected to construct the multi-copy plasmid pYLXP'-rDNA-AtC3H.

[0091] Using ZETA-F / ZETA-R primers and pYlST889 plasmid (published in the paper "Remodelling metabolismfor high-level resveratrol production in Yarrowia lipolytica") as a template, amplify the multi-copy plasmid backbone, connect the uracil tag, the key gene CPR and the multi-copy site ZETA homology arm to construct the multi-copy plasmid pYLXP'-ZETA-AtCPR. Multi-copy integration was performed at the 26S rDNA site of the strain CGA109 constructed in Example 7, and 24 single colonies were randomly selected on the transformation plate and transferred to 5mL of the corresponding YNB medium. After 24h, it was transferred to 25mL YPD medium at a 1% inoculation amount. After 120h of culture, 500μL of fermentation broth was taken, 500μL of methanol was added, and the resuspension was centrifuged at 12000×g for 10min, filtered through a 0.22μm organic filter membrane, and HPLC analysis was performed. The analysis results showed that the yield of chlorogenic acid in the obtained recombinant engineered bacteria CGA133 was 490.9 mg / L. Multi-copy integration was performed at the ZETA site of the recombinant engineered bacteria CGA133, and 24 single colonies were randomly selected on the transformation plate and transferred to 5 mL of the corresponding YNB medium. After 24 hours, they were transferred to 25 mL of YPD medium at a 1% inoculation amount. After 120 hours of cultivation, 500 μL of fermentation broth was taken, 500 μL of methanol was added, and the resuspension was centrifuged at 12000×g for 10 minutes, filtered through a 0.22 μm organic filter membrane, and HPLC analysis was performed. The analysis results showed that the yield of chlorogenic acid in the recombinant engineered bacteria CGA157 was 612.83 mg / L.

[0092] Example 9 Optimization of chlorogenic acid production in a 5L fermenter

[0093] The chlorogenic acid production capacity of strain CGA157 constructed in Example 8 was verified in a 5L fermenter. A single colony was picked from the plate and inoculated into 4mL of YPD liquid medium and cultured at 30°C for 18-24h. The primary seed solution was transferred to a shake flask containing 200mL of YNB liquid medium and cultured at 30°C and 220rpm for 24h as the secondary seed solution. The secondary seed solution was inoculated into a 5L fermenter, with an initial inoculation amount of 8%-10% and an initial OD 600 The pH value was controlled at 0.6-0.8 and equipped with electrodes to detect dissolved oxygen level, pH and temperature. The fermenter initially contained 2.2L YPD medium or 2.2L inorganic salt medium, and an additional 3mL trace metal solution and 3mL vitamin solution were added to promote bacterial growth. When the initial glucose was exhausted, glucose was added at a constant flow rate by controlling the feed and maintaining the glucose concentration in the range of 0.1g / L to 1g / L. In addition, NH3 ·H 2 O to maintain the pH of the fermentation broth at 4.5-5.0 and control the dissolved oxygen at 10-20% by adjusting the stirring speed and ventilation volume. Samples were taken every 6 h, and 3 samples were used as parallels to measure glucose concentration and OD 600 After 144 hours of fermentation, the yield can reach more than 7.7g / L; after 144 hours of fermentation in inorganic salt medium, the chlorogenic acid yield reached 13.13g / L, OD 600 Reached 180.6.

[0094] Comparative Example:

[0095] The specific implementation is the same as in Example 3, except that a method for connecting At4CL and CsHQT via TP linker, GS linker or EAAAK of different lengths is also constructed. F371A The fusion enzyme was expressed in strain CGAp01. Figure 6 As shown, no chlorogenic acid production was detected in the fermentation broth of the constructed recombinant bacteria.

[0096] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A hydroxycinnamoyl-CoA quinate transferase mutant, characterized in that: Compared with the hydroxycinnamoyl-CoA quinate transferase CsHQT parent, the phenylalanine at position 371 of the amino acid sequence was mutated to alanine.

2. A gene encoding the mutant according to claim 1.

3. A fusion enzyme, characterized in that Contains 4-coumaric acid: CoA ligase and the hydroxycinnamoyl CoA quinate transferase mutant according to claim 1.

4. The fusion enzyme according to claim 3, characterized in that The 4-coumaric acid: CoA ligase is connected with a short peptide RIAD; the hydroxycinnamoyl CoA quinate transferase mutant is connected with a short peptide RIDD.

5. A recombinant microorganism expressing the mutant according to claim 1 or the fusion enzyme according to any one of claims 3 to 4.

6. An engineered strain of Yarrowia lipolytica, characterized in that: Based on the starting strain, hydroxycinnamoyl-CoA quinate transferase, 4-coumaric acid:CoA ligase, quinate / shikimate 5-dehydrogenase, cytochrome P450 98A3 and cytochrome P450 reductase are expressed; the hydroxycinnamoyl-CoA quinate transferase is derived from tobacco (Nicotiana tabacum) or artichoke (Cynara scolymus), or has the amino acid sequence shown in NCBI No.: XP_024980016.

1.

7. The engineered yeast Yarrowia lipolytica according to claim 6, characterized in that There is also at least one of the following improvements: (1) Knockout the endogenous ABC transporter gene Ticw of Yarrowia lipolytica; (2) Using promoter P TDH , P TEF , P MNDH2 or P GPD1 Initiate the expression of one or more genes among 4CL, HQT, C3H, CPR, and YdiB; (3) Strengthening the endogenous NADPH biological regeneration pathway; (4) Hemoglobin VHb from heterologous Vitreoscilla was introduced.

8. The engineered yeast Yarrowia lipolytica according to claim 7, characterized in that The enhanced endogenous NADPH biological regeneration pathway includes overexpressing the endogenous genes ZWF1 and MAE1 of Yarrowia lipolytica.

9. The engineered yeast Yarrowia lipolytica according to any one of claims 6 to 8, characterized in that The starting strain includes but is not limited to Yarrowia lipolytica Po1f.

10. The engineered Yarrowia lipolytica according to any one of claims 6 to 9, characterized in that: The AtC3H gene and / or AtCPR1 gene are also integrated and expressed at multiple copy sites.

11. A method for preparing chlorogenic acid, characterized in that: The engineered Yarrowia lipolytica according to any one of claims 6 to 10 is fermented in a culture medium at 28 to 30° C. for at least 72 hours.

12. Use of the engineered Yarrowia lipolytica according to any one of claims 6 to 10 in the production of chlorogenic acid-containing products in the fields of food, medicine and chemical industry.

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