Method for preparing alpha-ketoisovalerate by transforming escherichia coli through metabolic engineering
阅读说明：本技术 一种代谢工程改造大肠杆菌制备α-酮异戊酸的方法 (Method for preparing alpha-ketoisovalerate by transforming escherichia coli through metabolic engineering ) 是由 周哲敏 周丽 崔文璟 刘中美 郭军玲 程中一 李雅婷 朱滢 于 2020-05-29 设计创作，主要内容包括：本发明公开了一种代谢工程改造大肠杆菌制备α-酮异戊酸的方法,属于生物工程领域。本发明构建了一种偶联表达乙酰乳酸合成酶、乙酰乳酸异构还原酶和二羟基酸脱水酶的重组菌株,并对该宿主菌株进行改造优化,获得能够发酵法高效生产α-酮异戊酸的重组菌。该重组菌株能够以廉价的葡萄糖为底物,发酵生成附加值更高的α-酮异戊酸,发酵36h,α-酮异戊酸产量可达22.91g/L,葡萄糖转化率达80％。(The invention discloses a method for preparing alpha-ketoisovalerate by transforming escherichia coli through metabolic engineering, and belongs to the field of bioengineering. The invention constructs a recombinant strain for coupling expression of acetolactate synthase, acetolactate isomeroreductase and dihydroxy acid dehydratase, and modifies and optimizes the host strain to obtain the recombinant strain capable of efficiently producing alpha-ketoisovalerate by a fermentation method. The recombinant strain can use cheap glucose as a substrate to ferment and generate alpha-ketoisovalerate with higher added value, the yield of the alpha-ketoisovalerate can reach 22.91g/L after fermentation for 36 hours, and the conversion rate of the glucose reaches 80%.)
1. The recombinant escherichia coli is characterized in that the recombinant escherichia coli is coupled and expresses acetolactate synthase, acetolactate isomeroreductase and dihydroxy acid dehydratase; the acetolactate isomeroreductase, acetolactate synthetase and dihydroxy acid dehydratase are connected in sequence for expression.
2. The recombinant Escherichia coli of claim 1, wherein a terminator is added to the acetolactate isomeroreductase and the dihydroxy acid dehydratase.
3. The recombinant Escherichia coli of claim 2, wherein said acetolactate isomeroreductase has accession number 948286 at NCBI; the acetolactate synthase has accession number 936852 at NCBI; the dihydroxy acid dehydratase has accession number 948277 at NCBI.
4. The recombinant Escherichia coli of claim 3, wherein said recombinant Escherichia coli silences expression of branched-chain amino acid transaminase B gene, isopropylmalate synthase gene; the accession number of the branched chain amino acid transaminase B gene on NCBI is 948278; the leuA accession number on NCBI is 947465.
5. The recombinant Escherichia coli of claim 4, wherein the recombinant Escherichia coli overexpresses a pyridine nucleotide transhydrogenase gene; the pyridine nucleotide transhydrogenase gene is a gene cluster consisting of pntA and pntB, and the accession number of pntA on NCBI is 946628; the accession number for pntB at NCBI is 946144.
6. The recombinant Escherichia coli of any one of claims 1 to 5, wherein the recombinant Escherichia coli is host Escherichia coli B0016-050 or Escherichia coli BL21(DE 3).
7. A method for producing alpha-ketoisovalerate, characterized by using the recombinant Escherichia coli of any one of claims 1 to 6 as a fermentation strain and glucose or sodium pyruvate as a substrate to produce alpha-ketoisovalerate.
8. The method according to claim 6, wherein the strain is cultured at 100 to 200 rpm.
9. The method of claim 6, wherein the recombinant Escherichia coli is fermented to OD600When the concentration is 1.5-2.5, the induction is carried out by IPTG.
10. Use of the recombinant escherichia coli of any one of claims 1 to 6, or the method of any one of claims 7 to 10, for the preparation of α -ketoisovalerate in the fields of drug analysis, food analysis, environmental monitoring, physico-chemical engineering and biopharmaceuticals.
Technical Field
The invention relates to a method for preparing alpha-ketoisovalerate by transforming escherichia coli through metabolic engineering, belonging to the field of bioengineering.
Background
Alpha-ketoisovalerate (alpha-ketoisovalerate) is one of ketoacids, and has wide application and great market development potential in the fields of medicines, foods, cosmetics and the like.
At present, α -ketoisovalerate can be synthesized by a chemical method and a biological conversion method, and the chemical synthesis method is common mainly, however, the chemical method is complicated in process and severe in reaction condition, is not suitable for large-scale industrial production along with generation of toxic by-products, the biological conversion method uses L-valine as a substrate to be converted into α -ketoisovalerate under catalysis of amino acid deaminase and amino acid oxidase, the α -keto acid produced by the amino acid oxidase method is industrialized in China, however, the enzyme conversion method has low efficiency in production of α -ketoisovalerate, and H exists in the reaction process2O2Is difficult to remove. The production by the fermentation method can effectively eliminate the generation of toxic byproducts, has lower cost and is more beneficial to environmental protection.
In addition, the research on the production of alpha-ketoisovalerate by fermentation of escherichia coli is very rare, and most of the current reports use corynebacterium glutamicum as a host bacterium for fermentation accumulation of alpha-ketoisovalerate, and in most cases, alpha-ketoisovalerate is accumulated only as a byproduct in the production process of isobutanol.
Disclosure of Invention
In order to solve the technical problems, the invention aims to provide a method for producing alpha-ketoisovalerate by fermentation through coupled expression of acetolactate synthase (AlsS), acetolactate isomeroreductase (IlvC) and dihydroxy acid dehydratase (IlvD), and provides a recombinant strain for coupled expression of acetolactate synthase (from bacillus subtilis), acetolactate isomeroreductase (from escherichia coli) and dihydroxy acid dehydratase (from escherichia coli), and the recombinant strain is modified and optimized to obtain a recombinant strain capable of efficiently producing alpha-ketoisovalerate through fermentation.
The invention provides a recombinant escherichia coli, which is coupled to express acetolactate synthase, acetolactate isomeroreductase and dihydroxy acid dehydratase.
In one embodiment of the present invention, EcilvC, BsalsS and EcilvD are sequentially linked to express them.
In one embodiment of the present invention, the terminator fragment EcilvC-Term is added to EcilvC and EcilvD.
In one embodiment of the invention, the nucleotide sequence encoding the acetolactate synthase gene is shown in SEQ ID No. 2.
In one embodiment of the invention, the nucleotide sequence encoding the acetolactate isomerase reductase gene is shown as SEQ ID NO. 4.
In one embodiment of the invention, the nucleotide sequence of the gene encoding dihydroxy acid dehydratase is shown as SEQ ID No. 6.
In one embodiment of the present invention, the accession number on the EcilvCNCBI is 948286; BsalsS NCBI has accession number 936852; accession number 948277 on EcilvD NCBI.
In one embodiment of the invention, the recombinant E.coli silences the expression of the branched-chain amino acid transaminase B gene (ilvE), the isopropylmalate synthase gene (leuA); the accession number on the ilvE NCBI is 948278; the leuA NCBI accession number is 947456.
In one embodiment of the present invention, the recombinant escherichia coli overexpresses a pyridine nucleotide transhydrogenase gene (pntAB) derived from escherichia coli; accession numbers 946628 and 946144 are found on the pntAB NCBI.
In one embodiment of the invention, the recombinant Escherichia coli is host Escherichia coli B0016-050 or Escherichia coli BL21(DE 3).
The invention provides a method for producing alpha-ketoisovalerate, which utilizes recombinant escherichia coli to produce alpha-ketoisovalerate for a fermentation strain.
In one embodiment of the invention, glucose or sodium pyruvate or a substance containing sodium pyruvate is used as substrate.
In one embodiment of the present invention, the strain is cultured at 100 to 200 rpm.
In one embodiment of the invention, the OD is in a strain600When the concentration is 1.5-2.5, the induction is carried out by IPTG.
The invention also protects the recombinant Escherichia coli or the application of the method for producing the alpha-ketoisovalerate in preparation of the alpha-ketoisovalerate in drug analysis, food analysis, environmental monitoring, physicochemical engineering and biological pharmacy.
The invention has the beneficial effects that: the invention constructs a recombinant strain for coupling expression of acetolactate synthase, acetolactate isomeroreductase and dihydroxy acid dehydratase, and modifies and optimizes the host strain to obtain the recombinant strain capable of producing alpha-ketoisovalerate by a fermentation method. The recombinant strain can use cheap glucose as a substrate to ferment and generate alpha-ketoisovalerate with higher added value, the yield of the alpha-ketoisovalerate can reach 22.91g/L after fermentation for 36 hours, and the conversion rate of the glucose reaches 80%.
Drawings
FIG. 1 shows different strategies for the coupled expression of AlsS, IlvC and IlvD according to the present invention.
FIG. 2 is a gel electrophoresis image of PCR amplification of BsalsS, EcilvC and EcilvD genes.
FIG. 3 shows the results of SDS-PAGE (FIGS. 3a to 3c) and protein concentration determination (FIGS. 3d to 3e) for the coupled expression of the three enzymes alone and the different enzymes.
FIG. 4 shows the in vitro transformation results of different coupled expressions.
FIG. 5 shows BL21(DE3)/pSDC and BL21(DE3)/pCTSDTAnd (5) fermentation verification results.
FIG. 6 is a gel electrophoresis image of chromosome modification; 6 a: t7RNAP integration and ilvE gene knockout verification; 6 b: carrying out ilvA gene knockout verification; 6 c: and (3) checking leuA gene knockout.
FIG. 7 shows the results of fermentation verification of different knockout strains.
FIG. 8 shows B0016-050T3/pCTSDTAnd B0016-050T3/pCTSDT+ pntAB fermentation verification results and chromosomesThe result of fermentation verification of the upper pntAB replaced by T7 promoter with different strength.
Detailed Description
The detection method of the alpha-ketoisovalerate comprises the following steps:
α the concentration of ketoisovaleric Acid was measured by High Performance Liquid Chromatography (HPLC) under conditions of column Prevail Organic Acid (250mM × 4.6.6 mM,5 m; Grace Davison Discovery Sciences) and mobile phase of KH 25mM at pH 2.52PO4The flow rate of the solution is 1mL/min, the column temperature is 40 ℃, the wavelength of an ultraviolet detector is 210nm, and the sample injection amount is 10 mu L.
Gibbson assembly mode: see Gibson et al, enzymic assembly of DNAmolecules up to a partial and cloned killbased Nat. methods,2009,6(5):343-5.
Plasmid pUC-Term: a chemically synthesized sequence is added between the EcoR V enzyme cutting sites of the commercialized pUC57-Kan vector, and the chemically synthesized sequence is shown as SEQ ID NO. 7.