Succinic acid-producing mutant microorganism into which high activity malate dehydrogenase is introduced, and method for preparing succinic acid by using same

A technology of malate dehydrogenase and microorganisms, applied in biochemical equipment and methods, using vectors to introduce foreign genetic material, enzymes, etc., can solve the problem of low yield of succinic acid

Pending Publication Date: 2020-06-16
KOREA ADVANCED INST OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the yield of succinic acid was low even when microbially derived MDH was introduced
For this reason, attempts have been made to further metabolically engineer metagenic succinate strains or optimize fermentation processes to enhance succinate production (Ahn et al., Curr. Opin. Biotechnol. 42:54-66), but further improvements are still needed

Method used

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  • Succinic acid-producing mutant microorganism into which high activity malate dehydrogenase is introduced, and method for preparing succinic acid by using same
  • Succinic acid-producing mutant microorganism into which high activity malate dehydrogenase is introduced, and method for preparing succinic acid by using same
  • Succinic acid-producing mutant microorganism into which high activity malate dehydrogenase is introduced, and method for preparing succinic acid by using same

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Construction and mutation of MsMDH, CgMDH, EcMDH, ScMDHc, ScMDHm, ScMDHp and YlMDH overexpression vectors (pET30a:MsMDH, pET30a:CgMDH, pET30a:EcMDH, pET30a:ScMDHc, pET30a:ScMDHm, pET30a:ScMDHp, pET30a:YlMDH) Enzyme overexpression vector (pET30a:MsMDH G11Q , pET30a:CgMDH Q20G , pET30a:MsMDH L101Q , pET30a:CgMDH Q117L , pET30a:MsMDH A224S , pET30a:CgMDH S242A ) construction

[0078] Each overexpression vector was constructed to overexpress the MDH of the succinic acid-producing microorganism in Escherichia coli and thereby obtain the protein. Using the primers of SEQ ID NO: 1 and 2, SEQ ID NO: 3 and 4, SEQ ID NO: 5 and 6, and SEQ ID NO: 7 and 8, and using Mannella succinogenes, Corynebacterium glutamicum, Genomic DNA of Escherichia coli K12 and Yarrowia lipolytica were used as templates for PCR. The resulting PCR product was cut with NdeI and XhoI restriction enzymes, and then cloned into the NdeI and XhoI sites of pET30a (Merck Millipore) to construct overexpressi...

Embodiment 2

[0108] MsMDH, CgMDH, EcMDH, YlMDH and their mutant enzyme proteins (MsMDH G11Q , CgMDH Q20G 、MsMDH L101Q , CgMDH Q117L , CgMDH S242A ) expression and purification

Embodiment 3

[0111] MsMDH, CgMDH, EcMDH, YlMDH, MsMDH G11Q , CgMDH Q20G 、MsMDH L101Q , CgMDH Q117L and CgMDH S242A Measurement of Oxaloacetate Reducing Activity

[0112] NADH is used as a cofactor for the oxaloacetate reducing activity of MDH. As a result, NAD+ is produced. The activity of MDH can be measured hourly by spectrophotometry based on the unique absorbance of NADH at 340 nm. The composition of the final reaction solution for comparative activity measurements was as follows: 0.1 M Tris-HCl (pH 8.0), 100 μM NADH, 100 μM oxaloacetate and finally 3 nM purified MDHs (MsMDH, CgMDH, EcMDH, YlMDH). For the measurement of mutant enzymes (MsMDH G11Q , CgMDH Q20G 、MsMDH L101Q , CgMDH Q117L , CgMDH S242A ) activity The composition of the final reaction solution was the same as that used to measure the comparative activity. Under the above conditions, while changing the concentration of oxaloacetate and the concentration of NADH, the kinetic activity was measured with the final r...

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Abstract

The present invention relates to: a succinic acid-producing mutant microorganism having improved activity of converting oxaloacetic acid to malic acid by the introduction of a gene encoding a malate dehydrogenase in which an amino acid residue interacting with a pyrophosphoric acid region of NADH through an amide functional group of a main chain is glutamine (Gln); and a method for preparing succinic acid by using same. The succinic acid-producing mutant microorganism according to the present invention has a remarkably increased activity of converting oxaloacetic acid to malic acid, and thus when the microorganism is cultured under a defined medium, high-concentration succinic acid can be produced with the highest succinic acid productivity from among that of mutant microorganisms reportedthus far. In addition, by using an advanced fermentation technology, succinic acid can be produced with higher productivity and production concentrations.

Description

【Technical field】 [0001] The present invention relates to a mutant microorganism introduced with highly active malate dehydrogenase for producing succinic acid and a method for producing succinic acid using it. More particularly, the present invention relates to a mutant microorganism exhibiting an improved activity of converting oxaloacetate to malate by introducing a gene encoding malate dehydrogenase, and a method for producing succinic acid using the same, wherein the apple The backbone of acid dehydrogenase contains the amino acid residue glutamine (Gln) that interacts with the pyrophosphate moiety of NADH through an amide function. 【Background technique】 [0002] With the recent emergence of environmental problems, many attempts have been made to replace the production of useful compounds based on conventional fossil fuels. To this end, research has been conducted worldwide to produce bio-based succinic acid from renewable biomass. Succinic acid, a dicarboxylic acid ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N9/04C12P7/46
CPCC12N15/74C12P7/46C12N9/0006C12Y101/01037C12N1/20
Inventor 李相烨金景镇安正镐徐浩钧李宗彦
Owner KOREA ADVANCED INST OF SCI & TECH
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