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Engineering bacterium capable of co-producing Danshensu and alanine, and application thereof

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

Active Publication Date: 2018-12-07
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 bacterium capable of co-producing Danshensu and alanine, and application thereof
  • Engineering bacterium capable of co-producing Danshensu and alanine, and application thereof
  • Engineering bacterium capable of co-producing Danshensu and alanine, and application thereof

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Experimental program
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Effect test

Embodiment 1

[0067] According to the method described in the literature Large scale validation of an efficient CRISPR / Cas-based multigene editing protocol in Escherichia coli. Microbial Cell Factories, 2017, 16(1):68, hpaD and mhpB on Escherichia coli BL21(DE3) were single-sampled or double knockout. Wherein, the gene knockout plasmid used in the present invention is pCasRed and pCRISPR-gDNA (hpaD sgRNA) and homology arm (hpaDdonor) together into Escherichia coli BL21 (DE3), Cas9 / sgRNA induces the host to produce double-stranded at the hpaD gene site Fragmentation, recombinase Red integrates the hpaD donor into the hpaD gene to achieve gene knockout, and sequence verification. hpaD sgRNA, hpaD donor, mhpB sgRNA, mhpB donor are shown in SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 25, and SEQ ID NO: 26 in the sequence table, respectively. mhpB was knocked out in the same way.

[0068] Prepare a solution with pH 7, levodopa or D-danshensu 4g / L, wet bacterial volume 200g / L, and measure the conc...

Embodiment 2

[0073] Screening of L-α-amino acid transaminase, a variety of L-α-amino acid transaminase genes were cloned from Escherichia coli and Lactobacillus, and expressed in Escherichia coli BL21 (DE3).

[0074] Induction expression method: The recombinant Escherichia coli was transferred to LB fermentation medium (peptone 10g / L, yeast powder 5g / L, NaCl 10g / L) in a volume ratio of 2%. 600 After reaching 0.6-0.8, IPTG with a final concentration of 0.4 mM was added to induce expression and culture at 20°C for 8 h.

[0075] After induction of expression, cells were collected by centrifugation at 20°C, 8000 rpm, and 20 minutes. The activity of the crude enzyme solution was measured by disrupting the cell, and the activities of various enzymes were compared when pyruvate was used as the ammonia acceptor. 1358.) described the method to measure the activity of L-α-amino acid transaminase, and the results are shown in Table 1. Therefore, it is optimal to select L-α-amino acid aminotransfera...

Embodiment 3

[0079] Comparison of the enzymatic properties of α-hydroxycarboxylic acid dehydrogenases. Usually this type of enzymes may also have the ability to reduce pyruvate to produce lactate, so an enzyme that cannot reduce or very weakly reduces pyruvate is better. Using pyruvate as a substrate, the reducing ability of different enzymes was compared. According to the literature (Serratia marcescens H3010 fermentative D-lactate dehydrogenase gene cloning, expression, purification and enzymatic properties study. Industrial Microbiology, 2012, The method described in 42(04):30-37.) measures the activity of reducing pyruvate with NAD as a coenzyme, and the experimental results are shown in Table 3.

[0080] Table 3 Comparison of pyruvate reduction activities of various α-hydroxycarboxylic acid dehydrogenases

[0081] Recombinant bacteria

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Abstract

The invention discloses an engineering bacterium capable of co-producing Danshensu and alanine, and an application thereof, and belongs to the technical field of bioengineering. The novel three-enzymeco-expression genetic engineering bacterium is constructed. (D / L)-alpha-hydroxycarboxylase dehydrogenase selected in the invention has the characteristics of poor substrate specificity and strong optical specificity, and can be used to produce optically pure D-Danshensu and L-Danshensu and also produce the L-alanine, and a method for producing the L-alanine has the advantages of simplicity, easyobtaining and few impurities of raw materials, and good industrial application prospect.

Description

technical field [0001] The invention relates to an engineering bacterium capable of co-producing danshensu and alanine and its application, and belongs 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 d-phenolic acid compound. There is currently no natural L-danshensu. [0003] Danshensu is an important active ingredient in the water extract of Salvia miltiorrhiza. The structure was obtained and identified from the water extract of Salvia miltiorrhiza in China in 1980 (Research on the water-soluble active components of Salvia miltiorrhiza, Ⅱ. The structure of hydroxyphenyl) lactic acid, Journal of Shanghai First Medical College, 1980, 05 (7), 384-385), vari...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P7/42C12P13/06C12R1/19
CPCC12N9/0006C12N9/0026C12N9/1096C12P7/42C12P13/06C12Y101/01027
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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