CN112980711A - Construction method of recombinant yarrowia lipolytica for efficiently and completely synthesizing hydroxytyrosol by using strong promoter - Google Patents
Construction method of recombinant yarrowia lipolytica for efficiently and completely synthesizing hydroxytyrosol by using strong promoter Download PDFInfo
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Abstract
The invention discloses a construction method of recombinant yarrowia lipolytica for efficiently and completely synthesizing hydroxytyrosol by using a strong promoter, which integrates TYR1, ARO10, ADH and HpaBC target genes onto a chromosome of the yarrowia lipolytica, and efficiently drives the specific expression of the exogenous genes in the yarrowia lipolytica by using a novel strong TDH3 promoter mutant, so that the recombinant expression efficiency is improved, the capability of the recombinant yarrowia lipolytica for synthesizing hydroxytyrosol is effectively improved, and the yield of the hydroxytyrosol is improved. The construction method of the recombinant yarrowia lipolytica is simple to operate and low in cost, and the strain can be used for fully synthesizing hydroxytyrosol by taking glucose as a substrate, so that the problems of insufficient yield and high price of hydroxytyrosol are effectively solved.
Description
Technical Field
The invention belongs to the technical field of synthesis of hydroxytyrosol, and particularly relates to a construction method of recombinant yarrowia lipolytica for efficiently and completely synthesizing hydroxytyrosol by using a strong promoter.
Background
Hydroxytyrosol (HT), also known as 3, 4-dihydroxyphenylethanol, has the molecular formula: c8H10O3Molecular weight: 154.1632. hydroxytyrosol is a natural phenolic compound mainly existing in olive, has strong antioxidant activity, can remove free radicals in human body, protect lipid from oxidative damage, and has anti-tumor and anti-inflammatory effects. In addition, hydroxytyrosol has good bioavailability and no known toxicity. Therefore, it has great application prospect in food additives, functional foods and even medicines.
Currently, there are three major sources of hydroxytyrosol. The first is to extract from olive leaves and waste residues thereof, or extract from olive oil or olive oil factory wastewater, but the method has the defects of low yield, strong acidity, low product purity, long production period and the like. Secondly, hydroxytyrosol is synthesized by a chemical synthesis method, but chemical reagents are generally toxic and difficult to separate, so the method has certain safety risk and serious environmental pollution, and is not suitable for industrial production of hydroxytyrosol. Thirdly, hydroxytyrosol is synthesized by a biosynthesis method, and the biosynthesis method has the advantages of stability, safety, low pollution and the like, is suitable for industrial large-scale production, and is a method which is popular at present and has a continuous development prospect. The biosynthesis method comprises semisynthesis and total synthesis, and the semisynthesis has the advantages of simple path and high yield, but the price of a substrate is higher, and the production cost is increased.
CN104805110A, CN107201331A, CN107723306A, CN107586794A and the like report some methods for improving the yield of hydroxytyrosol by using recombinant Escherichia coli. Some introduce exogenous protein, but the expression level of the target protein is low, and the production efficiency of the hydroxytyrosol is difficult to improve; some methods over-express related enzymes of hydroxytyrosol synthesis pathway, but the method can increase the burden of cell metabolism, and have many potential defects when applied to industrial production. Therefore, the development of recombinant strains which can produce hydroxytyrosol with high yield and are suitable for large-scale industrial production has great significance.
Disclosure of Invention
The invention aims to solve the problems of low yield of hydroxytyrosol, high production cost, serious environmental pollution, complex process, incapability of being suitable for industrial large-scale production and the like in the existing method, and provides a construction method of recombinant yarrowia lipolytica yeast which takes glucose as a substrate, utilizes a strong promoter to efficiently and completely synthesize hydroxytyrosol and is simple to operate, so that the construction method is expected to be applied to industrial production and effectively solves the problems of insufficient yield and high price of hydroxytyrosol.
Glucose is used as a carbon source, prephenate is generated through glycolysis pathway and shikimic acid pathway, and the prephenate generates p-hydroxyphenylpyruvate (4-HPP) under the action of prephenate dehydrogenase (TYR 1); generating 4-hydroxyphenylacetaldehyde (4-HPPA) under the action of phenylpyruvic acid decarboxylase (ARO 10); generating Tyrosol (Tyrosol) under the action of Alcohol Dehydrogenase (ADH); finally, Hydroxytyrosol (HT) is generated under the action of 4-hydroxyphenylacetic acid-3-monooxygenase (HpaBC). The biosynthetic pathway is shown below:
aiming at the purposes and the biosynthesis pathway, the construction method of the recombinant yarrowia lipolytica yeast comprises the following steps:
1. construction of plasmid pJN44-TYR1 '-ARO 10' -ADH '-HpaBC'
Synthesis of mutant P 'containing TDH3 promoter'TDH3The expression cassette P 'of the prephenate dehydrogenase gene TYR 1'TDH3-TYR1-Ter, phenylpyruvate decarboxylase gene ARO10 expression cassette P'TDH3ARO10-Ter, alcohol dehydrogenase gene ADH expression cassette P'TDH3Expression cassette P 'of ADH-Ter, 4-hydroxyphenylacetic acid-3-monooxygenase gene HpaBC'TDH3The four expression cassettes are connected to pJN44 vector in turn to form expression plasmid pJN44-TYR1 '-ARO 10' -ADH '-HpaBC'.
2. Construction of recombinant yarrowia lipolytica
Extracting a yarrowia lipolytica genome, and obtaining the upstream and downstream homologous arms of a POX3 integration site on the genome through PCR (polymerase chain reaction) so as to construct a plasmid pURA-POX 3L & R; an expression cassette containing a target gene in the plasmid pJN44-TYR1 '-ARO 10' -ADH '-HpaBC' is connected to the plasmid pURA-POX 3L & R to construct the plasmid pURA-POX 3L & R-TYR1 '-ARO 10' -ADH '-HpaBC', and the obtained plasmid is transformed into the yarrowia lipolytica yeast to obtain the recombinant yarrowia lipolytica yeast.
In the step 1, the Gene ID of the prephenate dehydrogenase Gene TYR1 is 2909038, the Gene ID of the phenylpyruvate decarboxylase Gene ARO10 is 2910787, the Gene ID of the alcohol dehydrogenase Gene ADH is 2910787, and the Gene ID of the 4-hydroxyphenylacetic acid-3-monooxygenase Gene HpaBC is 60901904. SpeI and XbaI cleavage sites were added to both ends of the four expression cassettes, respectively, and P 'was digested with restriction enzymes SpeI and XbaI, respectively'TDH3-TYR1-Ter、P’TDH3-ARO10-Ter、P’TDH3-ADH-Ter、P’TDH3the-HpaBC-Ter is subjected to double enzyme digestion and then is connected to an pJN44 vector to construct a plasmid pJN44-TYR1 '-ARO 10' -ADH '-HpaBC'.
In the step 1, the TDH3 promoter gene sequence is used as a template, and an error-prone PCR is carried out to obtain a TDH3 promoter mutant P'TDH3Gathering; p 'is prepared by an overlapping PCR method'TDH3Fusion with EGFP to construct P'TDH3-an EGFP expression cassette; the expression cassette is inserted into pJN44, and then the expression is carried out on yarrowia lipolytica to construct a TDH3 promoter mutant library; detecting the fluorescence intensity of the yeast enhanced green fluorescent protein pEGFP of the recombinant bacteria by a fluorescence microplate reader, and screening out a TDH3 promoter mutant with a gene sequence of any one of SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.44, preferably SEQ ID No. 2.
Ter in the expression cassette is selected from a T7 terminator, and the gene sequence of the Ter is as follows: caaaaaacccctcaagacccgtttagaggccccaaggggttatgctag are provided.
The invention has the following beneficial effects:
1. the invention integrates target genes of TYR1, ARO10, ADH and HpaBC on the chromosome of yarrowia lipolytica. Through the effective expression of the enzymes in the recombinant yarrowia lipolytica, the strain can be used for fully synthesizing hydroxytyrosol by taking glucose as a substrate, the synthesis cost is low, and the operation is simple.
2. The invention utilizes the novel strong TDH3-2 promoter to efficiently drive the specific expression of the exogenous gene in the yarrowia lipolytica, improves the construction and conversion efficiency of the recombinant expression vector, further effectively improves the capability of the recombinant yarrowia lipolytica in synthesizing the hydroxytyrosol, and improves the yield of the hydroxytyrosol.
Detailed Description
The present invention will be described in further detail with reference to examples, but the scope of the present invention is not limited to these examples.
Example 1
1. Construction of plasmid pJN44-TYR1 '-ARO 10' -ADH '-HpaBC'
(1) Determination of key genes for biological total synthesis of hydroxytyrosol
The Gene sequence of the prephenate dehydrogenase Gene TYR1 (Gene ID: 2909038), the Gene sequence of the phenylpyruvate decarboxylase Gene ARO10 (Gene ID: 2910787), the Gene sequence of the alcohol dehydrogenase Gene ADH (Gene ID: 2910787) and the Gene sequence of the 4-hydroxyphenylacetic acid-3-monooxygenase Gene HpaBC (Gene ID: 60901904) are found at NCBI, and enzyme cutting sites Spe I and Xba I are respectively designed at both ends of the Gene sequences. The fragment was then sent to Nanjing King sry Biotech, codon optimized according to the codon preference of yarrowia lipolytica and the gene sequence was synthesized, the synthesized fragment was ligated to pUT 18.
Digesting the gene fragment on pUT18 by Spe I and Xba I, processing for 2h at 37 ℃, purifying and glue recovering the DNA fragment of the target gene and the vector; the recovered 5 DNA fragments were sequentially ligated to pJN44 vector (containing TEF promoter and T7 terminator) by NEB DNA ligase (NEB Corp., product No.: M0367S), and finally constructed to obtain recombinant plasmid pJN44-TYR1-ARO 10-ADH-HpaBC.
The recombinant plasmid pJN44-TYR1-ARO10-ADH-HpaBC was then transformed into yarrowia lipolytica for free expression according to the yeast transformation kit instructions (Zymo Research corporation, USA). Through a control experiment of fermentation culture of the recombinant yarrowia lipolytica yeast and the original strain, the result shows that hydroxytyrosol is not detected in the original strain, and hydroxytyrosol is detected in the recombinant yarrowia lipolytica yeast and the content of the hydroxytyrosol is 480 mg/L. Namely, TYR1, ARO10, ADH and HpaBC were expressed freely in yarrowia lipolytica that does not produce hydroxytyrosol, and the synthetic yield of hydroxytyrosol was measured to be 480mg/L, as shown in Table 1.
(2) Obtaining and screening of promoter mutants
Using TDH3 promoter as a template, a primer pTDH3-F/pTDH3-R (base sequence of pTDH 3-F: base sequence of CGCGGATCCCTACTTTTATAGTTAGTCTTTTTT, pTDH 3-R: CCCAAGCTTATGGCGATGTCAAC) was designed, and a TDH3 promoter mutant was obtained by error-prone PCR. The error-prone PCR reaction system is as follows: the total volume is 50 mu L, and the content is 8mmol/L MgCl2、1mmol/L MnCl250mmol/L KCl, 10mol/L Tris-Cl, pH 8.3(25 ℃), 5U Taq DNA polymerase, dATP, dGTP, dCTP, dTTP concentrations of 0.3, 1 and 1mmol/L respectively. The error-prone PCR reaction conditions were: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 1min, annealing at 55 ℃ for 1min, extension at 72 ℃ for 1min, and 30 cycles. And carrying out agarose gel electrophoresis detection on the PCR amplification product, and recovering qualified products by using gel. And carrying out double enzyme digestion on the error-prone PCR recovery product and a promoter-driven plasmid pJN43-pEGFP expressed by EGFP and driven by a promoter constructed by a gene recombination technology by using restriction endonucleases BamH I and HindIII, connecting the gel recovery product by using NEB ligase at 25 ℃ for 30 minutes, transforming the gel recovery product into a torulopsis lipolytica competent cell, transforming a mutant strain, and constructing a TDH3 promoter mutant library. After transformation, coating the cells on a corresponding auxotroph culture medium, carrying out inverted culture at 30 ℃ for 3-4 days until single clones appear, identifying transformants by colony PCR, and selecting positive clones. Taking a recombinant bacterium which is not subjected to error-prone PCR (polymerase chain reaction) and transferred by a target gene as negative control, detecting the fluorescence intensity of yeast enhanced green fluorescent protein pEGFP (phospho-enzyme labeling) of the recombinant bacterium by a fluorescent microplate reader, sorting strains with fluorescence higher than that of the control recombinant bacterium into a 96-well plate which is replaced by 200 mu L of a resistant LB (lysogeny broth) culture medium containing 100mg/mL ampicillin (Amp), carrying out shaking culture at 37 ℃ until the strains grow, carrying out propagation culture on bacteria liquid, measuring the absorbance value and the fluorescence value in a fermentation liquid, and screening out 4 strainsStrong TDH3 promoter mutant: TDH3-1, TDH3-2, TDH3-3 and TDH3-4, wherein the gene sequences are shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4 in sequence.
Synthesis of TYR1 expression cassette (P ') with promoter mutant'TDH3-TYR1-Ter), the expression cassette is connected with expression cassettes of ARO10, ADH, HpaBC in sequence to form expression pellet pJN44-TYR 1' -ARO 10-ADH-HpaBC. The obtained plasmid is transformed into yarrowia lipolytica to carry out free expression, and the yield of hydroxytyrosol produced by strains driven by TDH3-1, TDH3-2, TDH3-3 and TDH3-4 promoter mutants is 855mg/L, 1350mg/L, 1020mg/L and 940mg/L respectively, which is shown in table 1, wherein the TDH3-2 promoter mutant is optimal.
(3) Episomal expression of strains with strong promoters
Synthesis of TYR1 expression cassette (P ') containing TDH3-2 promoter mutant'TDH3-2-TYR1-Ter), ARO10 expression cassette (P'TDH3-2ARO10-Ter), ADH expression cassette (P'TDH3-2ADH-Ter), HpaBC expression cassette (P'TDH3-2-HpaBC-Ter), and the four expression cassettes were sequentially ligated to pJN44 vector to construct plasmid pJN44-TYR1 '-ARO 10' -ADH '-HpaBC'. The obtained plasmid is transformed into yarrowia lipolytica yeast for free expression, and the synthetic yield of hydroxytyrosol is found to be 1920 mg/L.
2. Construction of recombinant yarrowia lipolytica driven by strong promoter and capable of efficiently and completely synthesizing hydroxytyrosol
Extracting yarrowia lipolytica genome, obtaining the upstream and downstream homologous arms of the POX3 integration site on the genome through PCR, and further constructing a plasmid pURA-POX 3L&And R is shown in the specification. The expression cassette containing the target gene in pJN44-TYR1 '-ARO 10' -ADH '-HpaBC' was digested and ligated to pURA-POX 3L&On R, constructing to obtain plasmid pURA-POX 3L&R-TYR1 '-ARO 10' -ADH '-HpaBC'. Following the yeast transformation kit protocol (Zymo Research corporation, USA), activated yarrowia lipolytica single colonies were inoculated into 2mL YPD medium and cultured overnight at 30 ℃ and 180rpm until OD of the bacterial liquid600At about 1.8; 1mL of the cell suspension was placed in 1.5mL of a clean centrifuge tube, centrifuged (4000 Xg, 4min), the medium was discarded, and 500. mu.L of solution 1 (containing50mmo1/L glucose, 5mmo1/L trihydroxymethyl aminomethane (Tris. HCl) and 1.0mmo1/L Ethylene Diamine Tetraacetic Acid (EDTA), uniformly mixing by vortex, centrifuging (4000 Xg, 4min), removing supernatant, adding 50 μ L solution 2 (mixed solution of 0.4mo1/L Na0H aqueous solution and 2% sodium dodecyl sulfate aqueous solution in a volume ratio of 1: 1), uniformly mixing by vortex, adding 5 μ L plasmid pURA-POX 3L&R-TYR1-ARO10-ADH-HpaBC, shaking and mixing uniformly, then adding 500 mu L of solution 3 (prepared by mixing 5mo1/L potassium acetate aqueous solution, glacial acetic acid and water according to the volume ratio of 60:11.5: 28.5), vortex and mixing uniformly, culturing at 30 ℃ and 180rpm for 4 hours, and then coating on a corresponding auxotrophic SD screening plate; after bacterial colonies grow out, selecting bacterial colony monoclonal, inoculating the bacterial colony monoclonal in a test tube containing 5mL of seed culture solution, and culturing at 28 ℃ and 200rpm/min for 24h to obtain first-stage seed solution; wherein the seed culture solution is prepared by adding 2g of glucose, 2g of peptone and 1g of yeast powder into 100mL of deionized water and sterilizing. Transferring the first-stage seed solution to a 250mL shake flask containing 50mL fermentation medium according to the inoculation amount of 1%, culturing at 30 ℃ and 200rpm/min for 120h, and collecting bacterial solution; wherein the fermentation medium is prepared by adding 4g of glucose, 0.5g of ammonium sulfate, 0.2g of leucine-deficient yeast nitrogen source (Drop-out mix synthetic yeast extract w/o yeast nitrogen base) and 0.17g of yeast nitrogen source into 100mL of deionized water and sterilizing. The content of hydroxytyrosol in the bacterial liquid was 2035mg/L by HPLC detection, as shown in Table 1.
TABLE 1 Total hydroxytyrosol yield by introducing exogenous genes driven by different promoters into yarrowia lipolytica
Note: in the table ≈ o: represents an expression cassette comprising a TEF promoter; and (delta): represents an expression cassette containing a TDH3-1 promoter mutant; o: a cassette containing a TDH3-2 promoter mutant; □: represents an expression cassette containing a TDH3-3 promoter mutant; it: the expression cassette containing the TDH3-4 promoter mutant is shown.
Sequence listing
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Claims (5)
1. A construction method of recombinant yarrowia lipolytica for efficiently and completely synthesizing hydroxytyrosol by using a novel strong promoter is characterized by comprising the following steps:
(1) construction of plasmid pJN44-TYR1 '-ARO 10' -ADH '-HpaBC'
Synthesis of mutant P 'containing TDH3 promoter'TDH3The expression cassette P 'of the prephenate dehydrogenase gene TYR 1'TDH3-TYR1-Ter, phenylpyruvate decarboxylase gene ARO10 expression cassette P'TDH3ARO10-Ter, alcohol dehydrogenase gene ADH expression cassette P'TDH3Expression cassette P 'of ADH-Ter, 4-hydroxyphenylacetic acid-3-monooxygenase gene HpaBC'TDH3-HpaBC-Ter, which are sequentially ligated to pJN44 vector to form expression plasmid pJN44-TYR1 '-ARO 10' -ADH '-HpaBC';
the TDH3 promoter mutant P'TDH3The sequence of (A) is any one of SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No. 4;
ter in the expression cassette is selected from a T7 terminator, and the gene sequence of the Ter is as follows: caaaaaacccctcaagacccgtttagaggccccaaggggttatgctag, respectively;
(2) construction of recombinant yarrowia lipolytica
Extracting a yarrowia lipolytica genome, and obtaining the upstream and downstream homologous arms of a POX3 integration site on the genome through PCR (polymerase chain reaction) so as to construct a plasmid pURA-POX 3L & R; an expression cassette containing a target gene in the plasmid pJN44-TYR1 '-ARO 10' -ADH '-HpaBC' is connected to the plasmid pURA-POX 3L & R to construct the plasmid pURA-POX 3L & R-TYR1 '-ARO 10' -ADH '-HpaBC', and the obtained plasmid is transformed into the yarrowia lipolytica yeast to obtain the recombinant yarrowia lipolytica yeast.
2. The method for constructing recombinant yarrowia lipolytica for efficient total synthesis of hydroxytyrosol by using strong promoter as claimed in claim 1, wherein the method comprises the following steps: in the step (1), the Gene ID of the prephenate dehydrogenase Gene TYR1 is 2909038, the Gene ID of the phenylpyruvate decarboxylase Gene ARO10 is 2910787, the Gene ID of the alcohol dehydrogenase Gene ADH is 2910787, and the Gene ID of the 4-hydroxyphenylacetic acid-3-monooxygenase Gene HpaBC is 60901904.
3. The method for constructing recombinant yarrowia lipolytica for highly efficient total synthesis of hydroxytyrosol by using strong promoters as claimed in claim 1 or 2, wherein the method comprises the following steps: in the step (1), SpeI and XbaI enzyme cutting sites are respectively added at two ends of the four expression cassettes, and P 'is respectively subjected to restriction enzyme SpeI and XbaI'TDH3-TYR1-Ter、P’TDH3-ARO10-Ter、P’TDH3-ADH-Ter、P’TDH3the-HpaBC-Ter is subjected to double enzyme digestion and then is connected to an pJN44 vector to construct a plasmid pJN44-TYR1 '-ARO 10' -ADH '-HpaBC'.
4. The method for constructing recombinant yarrowia lipolytica for efficient total synthesis of hydroxytyrosol by using strong promoter as claimed in claim 1, wherein the method comprises the following steps: in the step (1), TDH3 promoter gene sequence is used as a template, and an easy-to-error PCR is carried out to obtain TDH3 promoter mutant P'TDH3Gathering; p 'is prepared by an overlapping PCR method'TDH3Fusion with EGFP to construct P'TDH3-an EGFP expression cassette; the expression cassette is inserted into pJN44, and then the expression is carried out on yarrowia lipolytica to construct a TDH3 promoter mutant library; the fluorescence intensity of the yeast enhanced green fluorescent protein pEGFP of the recombinant bacteria is detected by a fluorescence microplate reader, and a TDH3 promoter mutant with a gene sequence of any one of SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.44 is screened out.
5. The method for constructing recombinant yarrowia lipolytica for efficient total synthesis of hydroxytyrosol by using strong promoter as claimed in claim 1, wherein the method comprises the following steps: in the step (1), the TDH3 promoter mutant P'TDH3The base sequence of (A) is shown as SEQ ID No. 2.
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| CN116064344B (en) * | 2022-08-04 | 2024-06-07 | 浙江熙正霖生物科技有限公司 | Construction method and application of recombinant escherichia coli for producing hydroxytyrosol by fermentation |
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