Engineering bacterium, and application thereof in production of pyruvic acid

A technology of pyruvic acid and lactic acid bacteria, applied in the field of bioengineering, can solve problems such as affecting yield and purification effect, and achieve the effects of good industrial application prospects, simple production process, and easy availability of raw materials

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

Engineering bacteria or wild bacteria containing lactate oxidase (WO9500656, CN200710156905.8, CN201110092393.X) can produce pyruvate, but hydrogen peroxide will be produced in this proces...

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Construction of recombinant Escherichia coli: Firstly, the genes encoding lactate dehydrogenase and NADH oxidase were respectively connected to the double gene expression plasmid pETDuet-1. Various double-gene co-expression recombinant plasmids were obtained, the plasmids were transformed into Escherichia coli BL21 (DE3), and positive transformants were obtained by screening with ampicillin plate, that is, recombinant Escherichia coli was obtained.

[0056] 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 and culture at 20°C for 8h. After induction of expression, the cells were collected by centrifugation at 20° C., 8000 rpm, and 20 minutes.

[0057] The collected cells were analyzed for transformation, and the results are shown...

Embodiment 2

[0062] According to the strain construction method described in Example 1 (different resistance plates were used to screen positive transformants for various plasmids according to the instructions) and the induced expression method, various types of cells were collected for transformation analysis, and the results are shown in Table 2. The whole cell transformation system is: cell wet weight 50g / L, L-lactic acid 10g / L, pH 7.0, temperature 30°C, shaker speed 250 rpm; transformation time 1 hour.

[0063] The comparison of various expression plasmids of table 2 for transforming and producing pyruvate

[0064] strain

[0065] It can be seen from the above table that the effect is best when pCOLADuet-1 is used for co-expression.

Embodiment 3

[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, btsT and ybdD on Escherichia coli BL21(DE3) were single Or double knockout, wherein, the gene knockout plasmid used in the present invention is pCasRed and pCRISPR-gDNA (btsT sgRNA) and the homology arm (btsTdonor) are introduced into Escherichia coli BL21 (DE3), and Cas9 / sgRNA induces the host in the btsT gene A double-strand break occurred at the site, and the recombinase Red integrated the btsT donor into the btsT gene to achieve gene knockout, which was verified by sequencing. bstT sgRNA, bstT donor, ybdD sgRNA, and ybdD donor are respectively shown in the sequence listing SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32.

[0068] After gene knockout, the pCOLADuet-1-llnox-llldh plasmid was introduced into the corresponding strains for transformation comparison. ...

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PUM

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Abstract

The invention discloses an engineering bacterium, and an application thereof in the production of pyruvic acid, and belongs to the technical field of bioengineering. The engineering bacterium simultaneously expresses exogenous L-lactate dehydrogenase and NADH oxidase, is obtained by knocking out a pyruvic acid absorption gene from host Escherichia coli, and can achieve enhanced expression of a lactic acid transporter gene, a pyruvic acid transporter gene and an NAD synthesis gene. The double-enzyme co-expression engineering bacterium is constructed on the basis of reconstructing Escherichia coli transportation and a coenzyme synthesis system in order to realize efficient production of pyruvic acid and reduce the generation of impurities.

Description

technical field [0001] The invention relates to an engineering bacterium and its application in producing pyruvate, belonging to the technical field of bioengineering. Background technique [0002] Pyruvate, also known as 2-oxopropionic acid or acetylformic acid, is a weakly acidic organic acid. Pyruvate is widely used in chemical industry, food, medicine and other fields. [0003] At present, the production of pyruvate mainly includes chemical and biological methods. The chemical method produces pyruvic acid by oxidizing lactic acid under the action of a catalyst, but the cost is high and the pollution is serious (CN201510848841.2, etc.). Biological methods mainly include lactic acid conversion method and direct fermentation method. Fermentation of pyruvate with glucose as raw material (WO8901523, CN201310722490.1, CN201510205724.4, etc.) usually takes a long time to ferment, and the cost of extracting pyruvate from the impurity fermentation broth system is relatively hi...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P7/40C12R1/19
CPCC12N9/0006C12N9/0036C12P7/40C12Y101/01027C12Y106/99003
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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