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Genetically modified microorganism and method for producing target substance using same

A microorganism and transgenic technology, applied in the field of modified phosphoenolpyruvate carboxylase activity, can solve the problems such as no aspartate conversion efficiency or reaction speed, no record of aspartate dehydrogenase, etc., Achieving the effect of increasing productivity, cutting costs, and improving conversion efficiency

Active Publication Date: 2021-06-01
GREEN EARTH INST CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In Patent Document 2, it is described that even when citric acid or glucose was used instead of succinic acid as a substrate in the culture and fermentation, the production of aspartic acid was confirmed, but the conversion of aspartic acid from these substrates is not shown There are many doubts about whether the technology disclosed in Patent Document 2 can be called a technology suitable for the industrial production of aspartic acid based on any specific data such as acid conversion efficiency or reaction speed
[0008] In addition, in the background art section of Patent Document 2, it is described that aspartic acid can be obtained by using the aspartate dehydrogenase derived from the archaebacterium Archaeoglobus fulgidus disclosed in Patent Document 3. , but in any of Patent Document 2 and Patent Document 3, it is not described that aspartic acid was actually produced by using the aspartate dehydrogenase derived from the Archaeoglobulin fulgidum

Method used

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  • Genetically modified microorganism and method for producing target substance using same
  • Genetically modified microorganism and method for producing target substance using same
  • Genetically modified microorganism and method for producing target substance using same

Examples

Experimental program
Comparison scheme
Effect test

no. 1 approach

[0141] According to the first embodiment of the present invention, the following transgenic microorganisms are provided.

[0142] A genetically modified microorganism that satisfies at least one of the following conditions (I), condition (II) and condition (IV), and meets the following condition (III):

[0143] Condition (I) Compared with the corresponding wild-type microorganism of the transgenic microorganism, succinate dehydrogenase activity or fumarate reductase activity is reduced or inactivated;

[0144] Condition (II) Compared with the wild-type microorganism, the lactate dehydrogenase activity is reduced or inactivated;

[0145] Condition (III) has modified phosphoenolpyruvate carboxylase activity or exogenous phosphoenolpyruvate carboxylase activity, and the modified phosphoenolpyruvate carboxylase activity is relative to wild-type phosphoenolpyruvate carboxylase activity Resistance to feedback inhibition by aspartic acid in pyruvate carboxylase activity that exhibit...

Embodiment approach

[0147] Furthermore, according to the second embodiment of the present invention, the following genetically modified microorganisms are also provided.

[0148] A genetically modified microorganism that satisfies all of the following conditions (I) to (III):

[0149] Condition (I) Compared with the corresponding wild-type microorganism of the transgenic microorganism, succinate dehydrogenase activity or fumarate reductase activity is reduced or inactivated;

[0150] Condition (II) Compared with the wild-type microorganism, the lactate dehydrogenase activity is reduced or inactivated;

[0151] Condition (III) has modified phosphoenolpyruvate carboxylase activity or exogenous phosphoenolpyruvate carboxylase activity, and the modified phosphoenolpyruvate carboxylase activity is relative to wild-type phosphoenolpyruvate carboxylase activity Resistance to feedback inhibition by aspartic acid in pyruvate carboxylase activity that exhibits resistance to exogenous phosphoenolpyruvate c...

Embodiment

[0431]

[0432] Using the Corynebacterium glutamicum ATCC13032 strain as the starting material, a recombinant phosphoenolpyruvate carboxylase gene was produced in which the specified enzyme activity was inactivated by gene disruption and a mutated phosphoenolpyruvate carboxylase gene with specified amino acid substitutions was introduced. Coryneform bacteria. The flow thereof is shown below.

[0433] (1) Preparation of gene deletion strain (Corynebacterium glutamicum ATCC 13032ΔldhΔsdhΔpoxB strain)

[0434] First, the SacB gene fragment was amplified by PCR using the plasmid pNIC-Bsa4 (Source BioScience) as a template and using the primer pairs shown in Table 12 below.

[0435] [Table 12]

[0436]

[0437] Use BamHI and HindIII to treat the amplified DNA fragment and plasmid pHSG299 (TakaraBio Co., Ltd.) with restriction enzymes, and then use the second edition (Ver.2) of the DNA Ligation Kit (DNA Ligation Kit) (Takara Bio Co., Ltd.) to obtain plasmid pGE015.

[043...

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Abstract

The present disclosure pertains to a genetically modified microorganism that satisfies some of specific requirements. The specific requirements include: (I) compared to a wild type microorganism, having reduced or inactivated succinate dehydrogenase activity or fumarate reductase activity; (II) compared to a wild type microorganism, having reduced or inactivated lactate dehydrogenase activity; (III) having a modified phosphoenolpyruvate carboxylase activity and thus showing resistance against the feedback inhibition by aspartic acid in wild type phosphoenolpyruvate carboxylase activity, or having an exogenous phosphoenolpyruvate carboxylase activity and thus showing higher resistance against the feedback inhibition by aspartic acid than wild type phosphoenolpyruvate carboxylase activity shown by a wild type microorganism; and (IV) compared to a wild type microorganism, having reduced or inactivated pyruvate : quinone oxidoreductase.

Description

technical field [0001] The present invention relates to a transgenic microorganism whose prescribed enzyme activities such as lactate dehydrogenase activity, succinate dehydrogenase activity, or fumarate reductase activity have been reduced or lost, and a method for producing a substance using the same. live, and has a modified phosphoenolpyruvate carboxylase activity that exhibits resistance to feedback inhibition by aspartate. [0002] In addition, this application claims priority based on Japanese Patent Application No. JP2019-76629 (Japanese Patent Application No. 2019-76629) filed with the Japan Patent Office on April 12, 2019, the contents of which are for all purposes Instead, it is used in this specification. Background technique [0003] In the substance production technology using microorganisms, attempts have been made to improve the production efficiency of various target substances by using transgenic technology, etc. to strengthen metabolic enzymes in the meta...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N1/15C12N1/19C12N5/10C12N9/88C12N15/60C12P7/42C12P7/46C12P13/06C12P13/20
CPCC12N15/52C12N9/88C12N9/001C12N9/0006C12P13/20C12P13/06C12Y103/99001C12Y101/01027C12Y101/01028C12Y401/01031C12Y103/01006C12R2001/15C12R2001/19C12P7/46C12P7/42C12N15/77C12N15/70C12Y103/05001C12Y103/05004C12Y401/01032C12Y102/05001C12N9/0008C12P13/22C12N5/10C12N15/902
Inventor 中屋敷彻
Owner GREEN EARTH INST CO LTD
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