Engineering bacteria and application in coproduction of tanshinol and alanine

A technology of amino acid and transaminase, applied in the field of bioengineering, can solve problems such as high cost and complicated operation, and achieve the effects of simple production process, easy-to-obtain raw materials, and strong optical specificity

Active Publication Date: 2018-11-23
卓虹超源生物科技(郑州)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These two methods prepare 3, the cost of 4-dihydroxyphenylpyruvate intermediate is higher, and operation is complicated

Method used

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  • Engineering bacteria and application in coproduction of tanshinol and alanine
  • Engineering bacteria and application in coproduction of tanshinol and alanine
  • Engineering bacteria and application in coproduction of tanshinol and alanine

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] hpaD and mhpB on Escherichia coli BL21 (DE3) were single or double knockout. Among them, the gene knockout plasmid used in the present invention is pCasRed and pCRISPR-gDNA (hpaD sgRNA) and the homology arm (hpaDdonor) are introduced into Escherichia coli BL21 (DE3), and Cas9 / sgRNA induces the host to generate double strands at the hpaD gene locus Break, the recombinase Red integrates the hpaD donor into the hpaD gene to achieve gene knockout, and sequence verification. hpaD sgRNA, hpaD donor, mhpB sgRNA, and mhpB donor are respectively shown in the sequence listing SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12. mhpB was knocked out in the same way.

[0068] Configure a solution with a pH of 7, 4 g / L of levodopa or D-danshensu, 200 g / L of wet bacteria, and measure the concentration after standing at 35 ° C for 10 hours. Table 1 shows the reaction system, levodopa and D-danshensu - The remaining amount of Danshensu.

[0069] Table 1 Residual concentra...

Embodiment 2

[0073] For the screening of L-α-amino acid transaminase, multiple L-α-amino acid transaminase genes were cloned from Escherichia coli and Lactobacillus, and expressed in Escherichia coli BL21 (DE3). When acid is the acceptor, compare the activity of various enzymes, and measure L-α according to the method described in the literature (Asymmetric synthesis of aromatic L-amino acids catalyzed by transaminases. Bioengineering Journal, 2012, 28 (11): 1346-1358.) - the activity of amino acid transaminase, the results are shown in Table 2. Therefore, it is the best to choose L-α-amino acid transaminase ect1 derived from Escherichia coli for transamination and deamination of levodopa.

[0074] Induced expression method: the recombinant Escherichia coli is transferred to LB fermentation medium (peptone 10g / L, yeast powder 5g / L, NaCl 10g / L) in the amount of 2% by volume ratio, when the cell OD 600 After reaching 0.6-0.8, add IPTG with a final concentration of 0.4mM, induce expression a...

Embodiment 3

[0078] Recombinant Escherichia coli construction: first, the genes encoding L-α-amino acid transaminase, α-hydroxycarboxylic acid dehydrogenase and glucose dehydrogenase were connected to the plasmid. A recombinant plasmid co-expressing the three genes was obtained, and the plasmid was transformed into Escherichia coli HM, and positive transformants were obtained by screening on an antibiotic plate, that is, recombinant Escherichia coli was obtained.

[0079] After the expression of recombinant Escherichia coli is induced and expressed, the bacterial cells are collected, and in a reaction volume of 100ml, the cell wet weight is 40g / L, levodopa is 40g / L, pyruvate is 30g / L, glucose is 30g / L, pH 8.0, and reacted at 35°C , time 12 hours. After the conversion, the yield and configuration of Danshensu were determined by liquid chromatography.

[0080] Table 3 Comparison of various recombinant bacteria

[0081]

[0082]

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Abstract

The invention discloses engineering bacteria and an application in coproduction of tanshinol and alanine, which belong to the technical field of biological engineering. The three-enzyme co-expressed gene engineering bacteria are established in the invention, the coproduction of the tanshinol and alanine is realized, and by knocking out or enhancing relevant genes on the expression of an escherichia coli genome, the transfer of a substrate can be promoted, and the decomposition of products can be reduced. A gene engineering machine provided by the invention can generate optically-pure D-tanshinol and L-tanshinol, and can also co-produce pyruvic acid at the same time; and moreover, the production process is simple, raw materials are easy to obtain, fewer impurities are contained, and the industrialized application prospect is good.

Description

technical field [0001] The invention relates to an engineering bacterium and its application in the joint production of danshensu and alanine, belonging to the technical field of bioengineering. Background technique [0002] Danshensu extracted from Danshen, scientific name R-(+)-3-(3,4-dihydroxyphenyl)-2-hydroxypropionic acid, D-(+)-β-(3,4-dihydroxyphenyl ) lactic acid, English name: Danshensu, D-DSS, R-DSS, (R)-(+)-3-(3,4-Dihydroxyphenyl)-lactic acid, (R)-(+)-3-(3 , 4-Dihydroxyphenyl)-2-hydroxypropanoic acid, is a dextrophenolic acid compound. Natural levodanshensu does not currently exist. [0003] Danshensu is an important active ingredient in the water extract of Danshen, and its structure was obtained and identified from the water extract of Danshen in 1980 in China (Study on the water-soluble active ingredients of Danshen, Ⅱ.D(+)β(3,4-di The structure of hydroxyphenyl) lactic acid, Shanghai First Medical College Journal, 1980, 05 (7), 384-385), various studies have...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P7/42C12P13/06C12R1/19
CPCC12N9/0006C12N9/1096C12P7/42C12P13/06Y02A50/30
Inventor 蔡宇杰熊天真蒋静丁彦蕊白亚军郑晓晖
Owner 卓虹超源生物科技(郑州)有限公司
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