CN115851474A - Method for synthesizing resveratrol through multi-copy integration and high-density fermentation - Google Patents
Method for synthesizing resveratrol through multi-copy integration and high-density fermentation Download PDFInfo
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Abstract
The invention discloses a method for synthesizing resveratrol by multi-copy integration and high-density fermentation, belonging to the technical field of genetic engineering and bioengineering. The invention further improves the synthesis of p-coumaric acid by constructing a strain with p-coumaric acid synthesis capacity and introducing a phenylalanine cracking way and central carbon metabolism modification. Then integrating key genes synthesized by resveratrol by constructing and optimizing a multi-copy integration method, and realizing the optimization of the resveratrol by a fermentation processHigh level of synthesis. OD of 144h of fermentation 600 The yield of the resveratrol reaches 256 +/-1.35, the yield of the resveratrol reaches 28.9 +/-0.17 g/L, and a foundation is laid for industrial production of the resveratrol or high-density fermentation of other polyphenol compounds by yarrowia lipolytica.
Description
Technical Field
The invention relates to a method for synthesizing resveratrol by multi-copy integration and high-density fermentation, belonging to the technical field of genetic engineering and bioengineering.
Background
Yarrowia lipolytica (Yarrowia lipolytica) is one of the most studied and most widely used non-conventional yeasts at present, and is Generally Recognized As Safe (GRAS) state. Compared with the conventional yeast Saccharomyces cerevisiae, the yeast has better tolerance and broad-spectrum substrate utilization. In recent years, with the application and rapid development of synthetic biology and gene editing technology in yarrowia lipolytica, research on synthetic biology using yarrowia lipolytica as a basal cell has also been rapidly developed. Yarrowia lipolytica, because of its high content of acetyl-CoA and malonyl-CoA precursors, has been used to synthesize plant secondary metabolites, such as flavonoids and polyketides, polyunsaturated fatty acids and isoprenoids, etc. Although yarrowia lipolytica is an excellent natural product production host, it is also a typical two-form yeast. It has been shown that yarrowia lipolytica is in filamentous form, which is detrimental to product synthesis and only in yeast form is an excellent production host. However, under severe environmental stress, yarrowia lipolytica often turns into filamentous form, which is unfavorable for production. Furthermore, instability of the condition parameters during fermentation is also a major cause of the transformation of yarrowia lipolytica from a yeast morphology to a filamentous morphology. Therefore, the optimized technology to make the yarrowia lipolytica in the yeast form is an effective means to improve the yield of the metabolite.
Resveratrol (3, 5,4' -trihydroxy-trans-stilbene) is a flavonoid polyphenol naturally found in grapes, berries, japanese giant knotweed and peanuts. Clinical research shows that resveratrol can improve diabetes, cardiac vascular diseases and nervous system diseases effectively. In addition, resveratrol is also used as a raw material for foods and cosmetics. At present, the main synthesis mode of resveratrol is to extract resveratrol from plants, but the plant extraction method is greatly influenced by seasons, and compared with the method for synthesizing resveratrol by adopting a microbial fermentation method, the method has the advantage of being more environment-friendly. However, insufficient supply of precursors and inefficient synthesis of downstream synthesis pathways are major reasons limiting the high level of resveratrol synthesis by microorganisms.
Disclosure of Invention
The invention further improves the synthesis of p-coumaric acid by constructing a strain with p-coumaric acid synthesis capacity and introducing a phenylalanine cracking way and central carbon metabolism modification. Then, key genes for synthesizing the resveratrol are integrated by constructing and optimizing a multi-copy integration method, and the high-level synthesis of the resveratrol is realized by optimizing a fermentation process.
The invention provides a recombinant yarrowia lipolytica, which takes yarrowia lipolytica PO1F as an initial strain, integrates tyrosine ammonia lyase gene FjTAL shown in SEQ ID NO.l at a D17 locus on a genome, and integrates and expresses YLARO1 gene and YLARO4 gene at an F1-3 locus K221L And YLARO7 G139S A gene; and expresses the genes AtPAL, atC4H, atATTR 2 of the phenylalanine cleavage pathway derived from Arabidopsis thaliana and endogenous YLCYB5, and expresses phosphoketolase CaFPK derived from Clostridium acetobutylicum and phosphotransacetylase BsPTA derived from Bacillus subtilis.
In one embodiment, the YLARO1 and YLARO4 K221L And YLARO7 G139S The nucleotide sequence of (A) is shown in SEQ ID NO. 2-4, and the promoter TEFin shown in SEQ ID NO.7 enhances expression.
In one embodiment, the recombinant yarrowia lipolytica further expresses the gene YLARO3 for feedback inhibition with elimination of phenylalanine K225L 。
In one embodiment, the recombinant yarrowia lipolytica also expresses the Pc4CL1-EAAAK-VvSTS fusion gene in multiple copies under the control of a strong promoter.
In one embodiment, the strong promoter regulated fusion gene Pc4CL1-EAAAK-VvSTS is integrated in multiple copies at the rDNA and/or ZETA sites.
In one embodiment, the strong promoter is 8xUASTEFin or 12xUASTEFin.
In one embodiment, the genes AtPAL, atC4H, atrr 2 and the endogenous YlCYB5 gene are integrated at the C3 locus of the genome under the promoter TEFin; the nucleotide sequences of the gene AtPAL, atC4H, atATR2 and YLCYB5 are respectively shown as SEQ ID NO.9-NO. 12.
In one embodiment, the CaFPK, bsPTA and YLARO3 K225L The gene sequence of (a) is integrated to the AXP locus of a genome under the promotion of a strong promoter TEFin, and the genes CaFPK, bsPTA and YLARO3 K225L The nucleotide sequences of (A) are respectively shown in SEQ ID NO. 13-15.
The invention also provides a method for producing resveratrol by high-density fermentation of yarrowia lipolytica, wherein the high-density fermentation is to culture the expanded yarrowia lipolytica seed liquid in a fermentation culture medium, the pH value in the fermentation process is controlled to be 3.5-6.5, and the dissolved oxygen is 0-30%.
In one embodiment, the fermentation process is also fed with feed; the supplementary material is prepared by the following raw materials in a carbon-nitrogen ratio (5-20): 1, and controlling the sugar concentration in the fermentation environment to be 0.5-1 g/L.
In one embodiment, the high density fermentation specifically comprises the steps of:
(1) Inoculating the engineering bacteria to a solid YPD culture medium, and culturing for 2-3 days at 30 ℃ to obtain slant strains;
(2) The slant strain is subjected to shake culture at 220rpm and 30 ℃ for 16-24h, and the pH is natural. Then inoculating the seeds into a secondary shake flask with the inoculation amount of 5 percent for secondary seed culture, culturing at 30 ℃ and 220rpm for 16-24h to obtain secondary seed liquid with natural pH;
(3) Inoculating the secondary seed liquid into a 5L fermentation tank filled with 2.5L fermentation medium according to the proportion of 5-8%, wherein the initial ventilation amount is 3.5-10L/min, the initial stirring speed is 300rpm, the culture temperature is 28-30 ℃, and the pH value is controlled by 8mol/L KOH to be 3.5-6.5 to start the culture.
In one embodiment, the fermentation mediumComprises the following components: 6g/L (NH 4) 2 SO 4 ，5g/L KH 2 PO 4 ，1g/LMgSO 4 ·7H 2 O,40g/L glucose.
In one embodiment, the feeding of 800g/L glucose or 60-400g/L (NH) is started after the initial glucose consumption in the fermentation medium of step (3) 4 ) 2 SO 4 。
In one embodiment, the method comprises controlling the pH to 3.5-6.5 by feeding 8mol/L KOH, maintaining the dissolved oxygen at 10% by controlling stirring and air flow, and controlling the glucose concentration in the fermentation tank to 0.5-1 g/L by feeding 800g/L glucose for 120-168 h.
In one embodiment, the method maintains dissolved oxygen at 10-30% by controlling stirring and air flow, controls pH at 4.5 by feeding 8M/L KOH, controls the concentration of sucrose at 0.5-1 g/L in the fermentation tank by feeding 800g/L glucose, and ferments for 120-168 h.
In one embodiment, the process is carried out by feeding a mixture comprising glucose and (NH) in a carbon to nitrogen ratio of from 5 to 20 4 ) 2 SO 4 The culture medium of (1) is prepared by controlling the sugar concentration in a fermentation tank to be 0.5-1 g/L, maintaining the dissolved oxygen at 20% by controlling stirring and air flow, controlling the pH to be 4.5 by feeding 8M/L KOH, and fermenting for 120-168 h.
In one embodiment, the medium used for seed culture is a YPD medium comprising: 10g/L yeast extract, 20g/L peptone, 20g/L glucose and natural pH value.
The invention also claims the application of the recombinant bacteria or the method for producing resveratrol by high-density fermentation in the aspects of producing resveratrol or other polyphenol compounds in the fields of food, medicine and chemical industry.
Has the advantages that: the invention constructs a recombinant strain capable of synthesizing resveratrol, integrates key genes for synthesizing resveratrol by introducing a phenylalanine cracking way and central carbon metabolism modification and optimizing a multi-copy integration method, further improves the supply of malonyl coenzyme A and realizes high-level synthesis of resveratrol. On the basis, the process optimization is carried out on a 5L tank,realizes the high-density fermentation process of the yarrowia lipolytica to efficiently synthesize the resveratrol, so that the OD of 144h is fermented 600 The yield of the resveratrol reaches 256 +/-1.35, the yield of the resveratrol reaches 28.9 +/-0.17 g/L, and a foundation is laid for industrial production of the resveratrol or high-density fermentation of other polyphenol compounds by yarrowia lipolytica.
Drawings
FIG 1 is a fermentation curve of yarrowia lipolytica engineering bacteria ST992 in a 5L tank for fermentation production of resveratrol.
Detailed Description
(I) culture Medium
YPD medium: peptone 20g/L, yeast powder 10g/L, and glucose 20g/L.
Fermentation medium: 6g/L (NH 4) 2 SO 4 ,5g/L KH 2 PO 4 ,1g/L MgSO 4 ·7H 2 O,40g/L glucose;
a supplemented medium: 800g/L glucose, 8mol/L KOH.
Mixing C/N culture medium: the carbon source is glucose, and the nitrogen source is ammonium sulfate; when the C/N ratio (by mass ratio) is 5:1, the glucose is 800g/L, and the ammonium sulfate is 302g/L; when C/N is 10:1, the glucose is 800g/L, and the ammonium sulfate is 151g/L; when C/N is 15:1, the glucose is 800g/L, and the ammonium sulfate is 101g/L; when C/N is 20: at 1, the concentration of glucose was 800g/L and that of ammonium sulfate was 75g/L.
(II) extracting resveratrol and p-coumaric acid: and (3) taking 1ml of fermentation liquor and an isometric methanol solution, fully shaking and uniformly mixing, centrifuging, collecting supernatant, and performing membrane filtration for liquid phase detection.
(III) measuring resveratrol and p-coumaric acid by HPLC: the measurement was carried out by Shimadzu high performance liquid chromatography. HPLC conditions are as follows: a chromatographic column: inertSustain C18 mm × 4.6mm column (particle size 5 μm); mobile phase A, ultrapure water containing 1 ‰ trifluoroacetic acid; mobile phase B, acetonitrile containing 1% trifluoroacetic acid; flow phase ratio conditions, 0-10min,10-40% B,10-20min,40-60% by weight, 20-22min,60-90% by weight, B,22-23min,90-10% by weight, B,23-25min,10-10% by weight, B; flow rate: 1mL/min; column temperature: at 40 ℃; sample introduction amount: 10 mu L of the solution; detector wavelength: 305nm.
Example 1 construction of a recombinant yarrowia lipolytica Strain with high yield of p-coumaric acid
Neutral site D17 of strain Po1f was selected as the integration site for the integration of key genes to the initial pathway of coumaric acid. Using the synthesized FjTAL sequence (the nucleotide sequence is shown as SEQ ID NO. 1) as a template, and carrying out PCR amplification on the FjTAL fragment by using a primer pair F01/R01; the FG1/RG1 was PCR-amplified with primers using vector pYLxp (published in the paper "Combining 26s rDNA and the Cre-loxP System for Iterative Gene Integration and Efficient Marker Current in Yarrowia lipolytica") as a template, and the product was purified. Recombining the fragment FjTAL and the vector pYLxp framework by a Gibson assembly method to obtain a recombinant vector, transforming the recombinant vector into Escherichia coli JM109, extracting a plasmid, and performing sequencing verification to obtain the correct recombinant vector pYLxp-FjTAL.
Primers F02/R02 and F03/R03 are designed, and the genome of yarrowia lipolytica PO1F is used as a template to amplify the upper and lower homologous arms uD17 and dD17 of the D17 site. PCR amplification is carried out on pYLxp-FjTAL by using a primer F04/R04, and an open reading frame fragment pFjTAL with a promoter and a terminator is obtained. The fragments uD17, dD17, pFjTAL and pYLxp vector skeletons are recombined by a Gibson assembly method to obtain a correct recombinant vector pYLxp-d17-PTEFIN-FjTAL-T XPR2 (ii) a And carrying out PCR amplification on pYLxp-d17-FjTAL by using a primer F05/R05, and purifying a PCR product to obtain a linearized fragment. The obtained pYLxp-d17-FjTAL fragment is transferred into yarrowia lipolytica PO1f to obtain a strain d17-FjTAL.
Overexpression of the endogenous Gene YLARO4 of yarrowia lipolytica for Release of feedback inhibition for increasing the supply of coumaric acid K221L (the nucleotide sequence is shown as SEQ ID NO. 3) and YLARO7 G139S (the nucleotide sequence is shown as SEQ ID NO. 4), and simultaneously over-expresses an endogenous gene YLARO1 (the nucleotide sequence is shown as SEQ ID NO. 2). Amplifying a gene YLARO4 (the nucleotide sequence is shown as SEQ ID NO. 5) from the genome of the yarrowia lipolytica PO1F by using a primer F06/R06; PCR amplification was performed using the primer FG/RG using the vector pYLxp as a template, and the product was purified. And recombining the fragment YLARO4 and the vector pYLXP by a Gibson assembly method to obtain the correct recombinant vector pYLXP-YLARO4. The primer F07/R07 is used to pass through a Gibson group by using a vector pYLxp-YLARO4 as a templateThe recombinant vector pYLxp-YLARO4 containing the mutant gene is obtained by the packaging method K221L . Amplifying a gene YLARO7 (the nucleotide sequence is shown as SEQ ID NO. 6) from the genome of the yarrowia lipolytica PO1F by using a primer F08/R08; the fragment YLARO7 and the vector pYLXP are recombined by a Gibson assembly method to obtain a correct recombinant vector pYLXP-YLARO7. The vector pYLxp-YLARO7 is amplified by a primer F09/R09, and a recombinant vector pYLxp-YLARO7 carrying a mutant gene is obtained by a Gibson assembly method G139S . A gene ARO1 (the nucleotide sequence is shown as SEQ ID NO. 2) is amplified from the genome of the yarrowia lipolytica PO1F by using a primer F010/R010, and the correct recombinant vector pYLxp-ARO1 is obtained by recombining the fragment ARO1 and the vector pYLxp by a Gibson assembly method. Primers F011/R011 and F012/R012 are designed, and the genome of yarrowia lipolytica PO1F is taken as a template, and the upper and lower homologous arms uF1-3 and dF1-3 of the neutral site F1-3 are amplified from the genome. Primers F013/R013, F014/R014 and F015/R015 were designed to be specific to pYLxp-YLARO4 K221L ,pYlxp-YlARO7 G139S pYLxp-YLARO1 is subjected to PCR amplification, and PCR products are purified to respectively obtain fragments pYLARO4 K221L ，pYlARO7 G139S And pYLARO1. The fragment uF1-3, dF1-3, pYLARO4 was assembled by Gibson K221L ，pYlARO7 G139S The pYLARO1 and the vector pYLxp are recombined to obtain a correct recombinant vector pYLxp-471; designing a primer F016/R016, carrying out PCR amplification on the pYLxp-471 by using the primer, and purifying a PCR product to obtain a pYLxp-471 linearized fragment. Transferring the obtained pYLxp-471 linearized fragment into a yarrowia lipolytica recombinant strain d17-FjTAL to obtain a strain d17-FjTAL, wherein F1-3 is shown in the specification, P TEF in-ARO4 K221L -T XPR2 -P TEFin -ARO7 G139S -T XPR2 -P TEFin -ARO1-T XPR2 Named ST401. Culturing the engineering strain in a seed culture medium for 16-18h to obtain a seed solution, inoculating the seed solution in an YPD culture medium, wherein the OD reaches 0.6-0.8 after inoculation, and fermenting at 30 ℃ and 220rpm for 96h. Liquid chromatography verifies that the engineering bacteria ST401 can produce p-coumaric acid, and the content of the p-coumaric acid in ST401 fermentation liquor is 850 +/-23.4 mg/L.
The primer sequences used are listed in table 1.
TABLE 1 primer sequences
EXAMPLE 2 construction of a Strain synthesizing p-Coumaric acid at a high level
The supply of coumaric acid was further increased based on the engineered bacterium ST401 constructed in example 1. Taking synthesized AtPAL, atC4H, atATR2 and endogenous YLCYB5 (gene sequences are respectively shown as SEQ ID NO. 8-11) as templates, and respectively amplifying corresponding gene segments by PCR by using primer pairs F1/R1, F2/R2, F3/R3 and F4/R4; using vector pYLxp (disclosed in the paper "Combining 26s rDNA and the Cre-loxP System for Iterative Gene Integration and Efficient Marker amplification in Yarrowia lipolytica") as a template, PCR amplification was performed using primer FG/RG, and the product was purified to obtain a vector backbone. The gene fragments are respectively recombined with pYLxp vector skeletons by a Gibson assembly method to obtain recombinant vectors, the recombinant vectors are transformed into Escherichia coli JM109, plasmids are extracted and sequence verification is carried out, and correct recombinant vectors pYLxp-AtPAL, pYLxp-AtC4H, pYLxp-AtATATR 2 and pYLxp-YLCYB5 are respectively obtained. Primers F5/R5 and F6/R6 are designed, and the genome of the yarrowia lipolytica PO1F is used as a template to amplify the upper and lower homologous arms uC3 and dC3 of the C3 site. The recombinant vectors pYLxp-AtPAL, pYLxp-AtC4H, pYLxp-AtATATR 2 and pYLxp-YLCYB5 are respectively subjected to PCR amplification by using primers F7/R7, F8/R8, F9/R9 and F10/R10 to obtain open reading frame fragments pAtPAL, pAtC4H, pAtATR2 and pYLCYB5 with promoters and terminators. Recombining the vector skeletons of the fragments uC3, dC3, pAtPAL, pAtC4H, pAtATR2, pYLCYB5 and pYLxp by a Gibson assembly method to obtain a correct recombinant vector pYLxp-C3-AtPAL-AtC 4H-AtATATR 2-YLCYB5; synthesized BsPTA, caFPK and YLARO3 K225L (the gene sequences are respectively shown as SEQ ID NO. 12-14) as a template, and PCR is carried out by using primer pairs F11/R11, F12/R12 and F13/R13 to obtain corresponding gene segments; to be provided withThe vector pYLxp is used as a template, FG/RG is subjected to PCR amplification by using a primer pair, and a product is purified to obtain a vector framework. Recombining the gene fragments with a pYLxp vector framework to obtain recombinant vectors by a Gibson assembly method, transforming the recombinant vectors into Escherichia coli JM109, extracting plasmids and sequencing to verify to obtain correct recombinant vectors pYLxp-CaFPK, pYLxp-BsPTA and pYLxp-YLARO3 respectively K225L . Primers F14/R14 and F15/R15 are designed, and the genome of yarrowia lipolytica PO1F is used as a template to amplify the upper and lower homologous arms uAXP and dAXP of the AXP site. Primers F16/R16, F17/R17 and F18/R18 are used for respectively targeting recombinant vectors pYLxp-CaFPK, pYLxp-BsPTA and pYLxp-YLARO3 K225L PCR amplification is carried out to obtain open reading frame fragments pCaFPK, pBsPTA and pYLARO3 with promoters and terminators K225L . Fragments uAXP, dAXP, pCaFPK, pBsPTA, pYLARO3 by Gibson Assembly K225L Recombining with pYLxp vector skeleton to obtain correct recombinant vector pYLxp-AXP-CaFPK-BsPTA-YLARO3 K225L (ii) a Primers F19/R19 and F20/R20 were used to pair pYLxp-C3-AtPAL-AtC4H-AtATR2-YLCYB5 and pYLxp-AXP-CaFPK-BsPTA-YLARO3, respectively K225L And performing PCR amplification, and purifying the PCR product to obtain a linearized fragment. The two linearized fragments obtained were transferred into yarrowia lipolytica ST401 constructed in example 1 to obtain strain ST470. Culturing the engineering strain in seed culture medium for 16-18h to obtain seed solution, inoculating the seed solution in YPD culture medium, and fermenting at 30 deg.C and 220rpm for 96h until OD reaches 0.6-0.8. Liquid chromatography verifies that the engineering bacteria ST470 can produce p-coumaric acid, and the content of the p-coumaric acid in ST470 fermentation liquor is 1235 +/-12.5 mg/L.
TABLE 2 primer sequences
Example 3 multicopy integration high level Synthesis of resveratrol
The downstream synthesis path further limits the synthesis of resveratrol, and integrates the downstream synthesis path into rDNA and ZETA sites on a genome in order to improve the synthesis efficiency of resveratrol, and because the yarrowia lipolytica is a URA and LEU deficient strain, a multicopy plasmid carrying a uracil URA tag and a leucine tag LEU is constructed by utilizing a weak promoter Leu. Primers F21/R21 and F22/R22 are designed to respectively amplify rDNA upstream and downstream homologous arms on the genome of the yarrowia lipolytica PO1F to obtain segments of urDNA and drDNA. Primers F23/R23 and F24/R24 are designed, and a fragment promoter pleu and a tag URA are amplified from a genome. Primers F25/R25 and F26/R26 are designed, and fragments 8xUASTEFin-Pc4CL1-EAAAK-VvSTS-Txpr2 and 12xUASTEFin-Pc4CL1-EAAAK-VvSTS-Txpr2 are amplified and characterized from sequences SEQ ID NO.15 and SEQ ID NO. 16. The fragments urDNA, drDNA, pleu and URA are recombined with 8xUASTEFin-Pc4CL1-EAAAK-VvSTS-Txpr2 or 12xUASTEFin-Pc4CL1-EAAAK-VvSTS-Txpr2 and a pYLxp vector skeleton respectively by a Gibson assembly method to obtain correct recombinant vectors prDNAYLxp-Pleeu-URA-Txpr 2-8xUASTEFin-Pc4CL 1-EAAAK-STS Vv-Txpr 2 and prYLxp-Pleu-URA-Txpr 2-12xUASTEFin-Pc4CL 1-VvEAK-STS-Txpr 2. The prDNAYlxp-urDNA-Pleu-URA-Txpr2-8xUASTEFin-Pc4CL1-EAAAK-VvSTS-Txpr2-drDNA and the prDNAYlxp-urDNA-Pleu-URA-Txpr2-12xUASTEFin-Pc4CL1-EAAAK-VvSTS-Txpr2-drDNA are respectively introduced into the engineering bacteria ST470 by linearization to obtain strains ST889 (URDNA-Pleu-URA-Txpr 2-8xUASTEFin-Pc4CL 1-EAK-VvSTS-Txpr 2-drDNA) and ST990 (URDNA-Pleu-URA-Txpr 2-12 xSTEFin-AAK-1-EAK-Vv-Txpr 2-drDNA). Respectively culturing engineering strains ST889 and ST990 in a seed culture medium for 16-18h to obtain seed liquid, inoculating the seed liquid into YPD culture medium, allowing OD to reach 0.6-0.8 after inoculation, and fermenting at 30 deg.C and 220rpm for 96h. Wherein ST889 can produce 632.1 + -10.8 mg/L resveratrol, and ST990 can produce 768.1 + -22.3 mg/L resveratrol.
As the resveratrol yield of Pc4CL1-EAAAK-VvSTS regulated by the promoter 12xUASTEFin is higher than the regulation level of the promoter 8xUASTEFin, primers F27/R27 and F28/R28 are designed to respectively amplify the upstream and downstream homologous arms of zeta on the genome of the lipolytica yarrowia PO1F, and fragments uzeta and dzeta are obtained. Primers F29/R29 and F30/R30 were designed to amplify the promoter pleu and the tag LEU from the genome. The fragments uzeta, dzeta, pleur and LEU are recombined with the 12xUASTEFin-Pc4CL1-EAAAK-VvSTS-Txpr2 fragment and the pYLxp carrier skeleton by a Gibson assembly method to obtain the correct recombinant carrier przetaYLxp-uzeta-Pleu-URA-Txpr2-12xUASTEFin-Pc4CL1-EAAAK-VvSTS-Txpr2-dzeta. The przetaYlxp-uzeta-Pleu-URA-Txpr2-12xUASTEFin-Pc4CL1-EAAAK-VvSTS-Txpr2-dzeta is linearly introduced into an engineering bacterium ST990 to obtain a strain ST991, the engineering strain is cultured in a seed culture medium for 16-18h to obtain a seed solution, then the seed solution is inoculated in a YPD culture medium, the OD reaches 0.6-0.8 after inoculation, the seed solution is fermented at 30 ℃,220rpm for 96h, and the ST991 can produce 946 +/-20.1 mg/L resveratrol.
The upper and lower homologous arms of DGA1 of PO1F genome of yarrowia lipolytica are respectively amplified by using primers F31/R31 and F32/R32, and products are purified to respectively obtain fragments uDGA1 and dDGA1. The fragments uDGA1, dDGA1 and pYLxp vector skeletons are recombined by a Gibson assembly method to obtain a recombinant vector pYLxp-DGA1. The pYLxp-DGA1 linearized fragment is amplified by a primer F33/R33, and the obtained pYLxp-DGA1 fragment is transferred into the yarrowia lipolytica ST991 which integrates a fusion gene 12xUASTEFin-Pc4CL1-EAAAK-VvSTS-Txpr2 on rDNA and zeta sites, so as to obtain an engineering strain ST992. Culturing the engineering strain in a seed culture medium for 16-18h to obtain a seed solution, inoculating the seed solution in a YPD culture medium, wherein OD reaches 0.6-0.8 after inoculation, fermenting at 30 ℃ and 220rpm for 96h, and the yield of resveratrol reaches 1256 +/-15.6 mg/L.
TABLE 3 primer sequences
Example 4 resveratrol yield in different pH conditions of 5L tank fermentation
The yarrowia lipolytica engineering bacterium ST992 constructed in the example 3 is subjected to streak culture in a YPD solid medium plate through an inoculating loop, and is placed in a constant temperature incubator at 30 ℃ for 2-3 days to obtain an activated engineering strain. Inoculating the activated strain into YPD medium containing 50mL/250mL for primary seed culture, shake-culturing at 220rpm and 30 deg.C for 16-24h, and allowing pH to be natural to obtain OD 600 Is the first-class seed liquid of 5-8. Then inoculating the first-stage seed solution with inoculation amount of 5% into 1L shake flask containing YPD medium liquid volume of 200mL for second-stage seed culture, culturing at 30 deg.C and 220rpm for 16-24h to obtain OD 600 Is a secondary seed liquid of 5-8, and has natural pH; then inoculating the secondary seed liquid into a 5L fermentation tank containing 2.5L of fermentation medium by 8 percent of inoculation amount, controlling the pH value to 3.5-6.5 by using 4-8mol/L KOH to start culture, adjusting the aeration and the rotating speed to maintain when the initial dissolved oxygen is reduced to 10 percent, starting feeding the feed medium when the glucose in the fermentation liquid is reduced to 0.5-1 g/L, controlling the glucose concentration in the fermentation liquid to 0.5-1 g/L, and sampling and detecting resveratrol and p-coumaric acid within 146-168 h of fermentation period.
A control was set up for the protocol described above, which differs from the protocol described above in that: the pH was controlled to 3.5,4.5,5.5,6.5 by auto-feeding 8mol/L KOH, respectively.
The yield of resveratrol of the yarrowia lipolytica engineering bacteria under different pH conditions is different, wherein the yield of the resveratrol is low and only reaches 7.1 +/-0.04 g/L and 8.0 +/-0.12 g/L when the pH is 3.5 and 6.5. Under the conditions of pH 4.5 and pH 5.5, the yield of the resveratrol can reach more than 10g/L, wherein under the condition of pH 4.5, the yield of the resveratrol is the highest and can reach 13.4 +/-0.12 g/L.
Example 5 production of resveratrol by fermentation in 5L tank under different dissolved oxygen conditions
The yarrowia lipolytica engineering bacterium ST992 constructed in the example 3 is subjected to streak culture in a YPD solid culture medium through an inoculating loop, and is placed in a constant-temperature incubator for culture at 30 ℃ for 2-3 days to obtain an activated strain. Inoculating the activated strain into 250mL shake flask containing YPD culture medium with liquid content of 50mL for first-stage seed culture, shake-flask culturing at 220rpm and 30 deg.C for 16-24h, and allowing pH to be natural to obtain OD 600 Is 5-8 of seed liquid. Inoculating the seed solution into 1L secondary shake flask containing 200mL/YPD medium at 5% inoculum size for secondary seed culture at 30 deg.C and 220rpm for 16-24 hr to obtain OD 600 Is a secondary seed liquid of 5-8, and has natural pH; then inoculating 8% of inoculum size into a 5L fermentation tank filled with 2.5L of fermentation medium, wherein the initial aeration rate is 3.5L/min, the initial stirring speed is 300rpm, the culture temperature is 30 ℃, the pH is controlled by 8mol/L KOH to be 4.5, the culture is started, when the glucose is reduced to 0.5g/L-1g/L, 800g/L glucose is fed, the glucose concentration in the fermentation liquid is controlled to be 0.5g/L-1g/L, when the initial dissolved oxygen is reduced to 0% -30%, the dissolved oxygen is maintained by stirring and aeration, the fermentation period is 146h-168h, sampling is carried out, resveratrol is detected, and p-coumaric acid is detected.
A control was set up for the protocol described above, which differs from the protocol described above in that: the dissolved oxygen is maintained at 0%,10%,20% and 30% respectively by controlling the stirring (300-1200 rpm) and the air flow (3.5-10L/min).
The resveratrol yield of the yarrowia lipolytica under different dissolved oxygen conditions is different, wherein the resveratrol yield under the 0% dissolved oxygen condition is the lowest and is only 5.1 +/-0.04 g/L. Under the conditions of 10 percent, 20 percent and 30 percent dissolved oxygen, the yield of the resveratrol is respectively 13.3 +/-0.16 g/L,19.7 +/-0.06 g/L and 16.4 +/-0.21 g/L.
Example 6 resveratrol yield in 5L tank fermentation under different C/N conditions
The yarrowia lipolytica engineering bacterium ST992 constructed in the example 3 is subjected to streak culture in a YPD solid culture medium through an inoculating loop, and is placed in a constant-temperature incubator for culture for 2-3 days at 30 ℃ to obtain an activated strain. Inoculating the activated strain into 250mL shake flask containing YPD culture medium with liquid content of 50mL for first-stage seed culture, shake-flask culturing at 220rpm and 30 deg.C for 16-24h, and allowing pH to be natural to obtain OD 600 Is the first-class seed liquid of 5-8. Then inoculating the first-stage seed solution with 5% inoculum size into 1L shake flask containing 200mL YPD culture medium for second-stage seed culture at 30 deg.C and 220rpm for 16-24h to obtain OD 600 Is a secondary seed liquid of 5-8, and has natural pH; then inoculating the secondary seed liquid into a 5L fermentation tank with 2.5L fermentation medium liquid loading volume at 8% inoculation amount, with initial aeration rate of 3.5L/min, initial stirring at 300rpm, culturing at 30 deg.C, controlling pH with 8mol/L KOH to 4.5, culturing until initial dissolved oxygen is reduced to 20%, maintaining dissolved oxygen by stirring and aeration until glucose is reduced to 0.5-1 g/L, feeding mixed glucose and ammonium sulfate with different C/N ratioCulturing the medium, controlling the glucose concentration in the fermentation liquid to be 0.5-1 g/L, fermenting for 146-168 h, sampling and detecting resveratrol and p-coumaric acid.
A control was set using the protocol described above, which differs from the protocol described above in that: feeding mixed C/N media of different C/N ratios (5; specifically, the C/N ratio of the medium is 5, 10, 1, 15.
The yield of resveratrol from yarrowia lipolytica under different C/N conditions is different, and under the conditions that the C/N is 5, 10, 1, 15 and 1, 20.
Example 7 fermentation of resveratrol at 5L tank fermentor level
The yarrowia lipolytica engineering bacterium ST992 constructed in the embodiment 3 is subjected to streak culture in a YPD solid medium plate through an inoculating loop, and is placed in a constant temperature incubator for culture at 30 ℃ for 2-3 days to obtain an activated engineering strain. Inoculating the activated strain into YPD medium containing 50mL/250mL for primary seed culture, shake-culturing at 220rpm at 30 deg.C for 16-24h, and naturally adjusting pH to obtain OD 600 Is the first-class seed liquid of 5-8. Then inoculating the first-stage seed solution with inoculation amount of 5% into 1L shake flask containing YPD medium liquid volume of 200mL for second-stage seed culture, culturing at 30 deg.C and 220rpm for 16-24h to obtain OD 600 Is a secondary seed liquid of 5-8, and has natural pH; then inoculating the secondary seed liquid into a 5L fermentation tank with the liquid loading capacity of 2.5L of fermentation medium by 8 percent of inoculation amount, wherein the initial aeration rate is 3.5L/min, the initial stirring speed is 300rpm, the culture temperature is 30 ℃, the culture is started by controlling the pH value to 4.5 by 8mol/L KOH, when the initial dissolved oxygen is reduced to 20 percent, the dissolved oxygen is maintained to be 20 percent by adjusting and controlling the stirring speed (300-1200 rpm) and the air flow (3.5-10L/min), when the glucose is reduced to 0.5g/L-1g/L, feeding a mixed culture medium with the C/N ratio of the glucose and the ammonium sulfate being 10, and controlling the glucose concentration in the fermentation liquid to be 0.5g/L-1g/L, and periodically sampling and detecting the resveratrol and the p-coumaric acid.
As shown in FIG. 1, the growth of the cells was in a lag phase and slow in 1 to 10 hours. In the logarithmic growth phase in 11-60h, the thalli grow rapidly in this period. 60 to 130 hours atIn the stationary phase, the growth of the cells becomes slow. Finally, fermentation was carried out for 144h 600 256 plus or minus 1.35, 28.9 plus or minus 0.17g/L of resveratrol output and 18.45 plus or minus 1.41mg/L of p-coumaric acid.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. A recombinant Yarrowia lipolytica is characterized in that a tyrosine ammonia lyase gene FjTAL shown in SEQ ID NO. l is integrated and expressed on a Yarrowia lipolytica genome, and a YLARO1 gene and YLARO4 gene shown in SEQ ID NO. 2-4 are integrated and expressed K221L And YLARO7 G139S A gene; and expresses the genes AtPAL, atC4H, atATTR 2 of the phenylalanine cleavage pathway derived from Arabidopsis thaliana and endogenous YLCYB5, and expresses phosphoketolase CaFPK derived from Clostridium acetobutylicum and phosphotransacetylase BsPTA derived from Bacillus subtilis.
2. The recombinant yarrowia lipolytica of claim 1, wherein the strong promoter regulated fusion gene Pc4CL1-EAAAK-VvSTS is integrated in multiple copies at the rDNA and/or ZETA sites.
3. The recombinant yarrowia lipolytica yeast of claim 1 or 2, wherein said strong promoter is 8 xuastfin or 12 xuastfin.
4. The recombinant yarrowia lipolytica yeast according to any one of claims 1 to 3, wherein said genes AtPAL, atC4H, atATR2 and endogenous YLCYB5 gene are integrated at the C3 locus of the genome, driven by the strong promoter TEFin.
5. The recombinant yarrowia lipolytica of any one of claims 1 to 4, wherein said CaFPK, bsPTA and YLARO3 K225L The gene of (a) is regulated and integrated into the AXP site of the genome by the strong promoter TEFin.
6. A method for producing resveratrol by high-density fermentation is characterized in that the recombinant yarrowia lipolytica yeast in any claim 1-5 is fermented in a fermentation culture medium, the pH value in the fermentation process is controlled to be 4.5-5.5, and the dissolved oxygen is controlled to be 10% -30%.
7. The method of claim 6, wherein the fermentation process is further fed with a feed; the supplementary material is prepared by the following raw materials in a carbon-nitrogen ratio (5-20): 1, and controlling the sugar concentration in the fermentation environment to be 0.5-1 g/L; the mixed C/N medium contains glucose and (NH) 4 ) 2 SO 4 。
8. The method of claim 7, wherein the high density fermentation comprises the steps of:
(1) Carrying out amplification culture on the recombinant yarrowia lipolytica according to any one of claims 1 to 5 step by step to obtain an amplified seed solution;
(2) Inoculating the seed liquid expanded in the step (1) into a fermentation culture medium, and controlling the initial ventilation amount to be 3.5-7L/min to ferment at 28-30 ℃;
the fermentation medium contains: (NH) 4 ) 2 SO 4 ,KH 2 PO 4 ,MgSO 4 ·7H 2 O and glucose.
9. The method according to claim 7 or 8, wherein the pH is controlled to be 4.5-5.5, the glucose concentration is controlled to be 0.5-1 g/L, and the fermentation is carried out for 120-168 h.
10. Use of the recombinant yarrowia lipolytica yeast according to any one of claims 1 to 5 or the method according to any one of claims 6 to 9 for the production of resveratrol or other polyphenols in the fields of food, medicine and chemical engineering.
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