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A genetically engineered bacterium producing l-malic acid and its construction method and application

A technology of malate synthase and malate dehydrogenase, which is applied in the field of biotechnology and fermentation engineering, can solve the problems of mycelium easy to ball, many by-products, complex condition control, etc., and achieve the effect of good industrial application prospects

Active Publication Date: 2019-05-21
BEIJING UNIV OF CHEM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Mold fermentation has the advantages of high yield and high production intensity, but has the disadvantages of many by-products, mycelium is easy to ball, and the condition control is complicated.

Method used

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  • A genetically engineered bacterium producing l-malic acid and its construction method and application
  • A genetically engineered bacterium producing l-malic acid and its construction method and application
  • A genetically engineered bacterium producing l-malic acid and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1, Construction of recombinant bacteria E.coliΔMA6 (pMCS-GA4+pUC-MA3)

[0065] 1. Construction of recombinant expression vector pMCS-GA4

[0066] 1. The DNA shown in Sequence 1 in the sequence list is artificially synthesized, which contains the BurMR1_0153 expression cassette, the upstream is the SacI site, and the downstream is the XbaI site, wherein the 9th-51st nucleotide is the promoter sequence, and the 70th-942nd Nucleotide is BurMR1_0153 gene sequence.

[0067] 2. The artificially synthesized DNA shown in Sequence 2 in the sequence list contains the fucK expression cassette, the upstream is the XbaI site, and the downstream is the HindIII site, wherein the 9th-51st nucleotide is the promoter sequence, and the 70th-1518th nucleotide Nucleotides are fucK gene sequences.

[0068] 3. The artificially synthesized DNA shown in Sequence 3 in the sequence list contains the fucA expression cassette, the upstream is the HindIII site, and the downstream is the Xh...

Embodiment 2

[0174] Example 2, the application of E.coliΔMA6 (pMCS-GA4+pUC-MA3) in the production of malic acid

[0175] 1. Cultivation of recombinant bacteria E.coliΔMA6 (pMCS-GA4+pUC-MA3) in shake flasks to produce malic acid

[0176] 1. The recombinant bacterium E.coliΔMA6 (pMCS-GA4+pUC-MA3) prepared in Example 1 was cultivated for 16 hours at 37° C. at 200 rpm in LB liquid medium containing ampicillin and kanamycin, as seed liquid.

[0177] 2. The seed solution was inoculated into the MMX1 liquid medium according to the inoculation amount of 4% by volume, and the liquid volume in the 500ml shake flask was 50ml, and cultivated at 37°C and 200rpm for 72h, during which the fermentation broth was collected.

[0178] The preparation method of MMX1 liquid medium: each liter of medium contains 6g xylose, 2g NH 4 Cl, 5.0g (NH 4 ) 2 SO 4 , 6.0g KH 2 PO 4 , 8.214g MOPS, 0.5g NaCl, 1mL trace element solution, 0.1g ampicillin and 0.05g kanamycin, and the rest is water.

[0179] Preparation...

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PUM

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Abstract

The invention provides a genetically engineered bacterium for producing malic acid efficiently by means of xylose fermentation and a construction method and application of the genetically engineered bacterium. In the recombinant bacterium, hosphopentose isomerase, L-fuculokinase, L-fucose-1-phosphate aldolase, aldehyde dehydrogenase, glycolate oxidase and malate synthase are overexpressed, and meanwhile xylulokinase, malate dehydrogenase and fumarate hydratase are knocked out, so that a malic acid synthesis route is obtained. According to the recombinant bacterium, the malic acid yield and the xylose conversion rate in shake flask culture and fermentation tank culture can reach ideal levels, and a good industrialized application prospect is achieved.

Description

technical field [0001] The invention belongs to the field of biotechnology and fermentation engineering, and relates to a genetic engineering bacterium producing L-malic acid and its construction method and application, in particular to a construction method of an Escherichia coli genetic engineering bacterium efficiently utilizing xylose to ferment and produce malic acid And the method for producing L-malic acid. Background technique [0002] Malic acid, also known as 2-hydroxysuccinic acid, is an important four-carbon compound. The malic acid molecule contains an asymmetric carbon atom, so it can exist in three forms, namely D-malic acid, L-malic acid and the mixture DL-malic acid. As an intermediate of the tricarboxylic acid cycle, L-malic acid is ubiquitous in the living body, has a slightly irritating pleasant sour taste, and can be directly absorbed and utilized by the human body. D-malic acid needs to be converted into L-malic acid by the action of racemase and oxid...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P7/46C12N15/70C12N1/21C12R1/19
CPCC12N9/0006C12N9/0008C12N9/1025C12N9/1205C12N9/88C12N9/90C12P7/46C12Y101/01037C12Y101/03015C12Y102/01003C12Y203/03012C12Y207/01017C12Y207/01052C12Y401/02C12Y402/01002C12Y503/01006
Inventor 李正军洪鹏辉刘笑杰陈静李良康
Owner BEIJING UNIV OF CHEM TECH
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