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Recombinant mutant microorganism with malonate producing ability and method for producing malonate using the same
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A technology of microorganisms and malonic acid, applied in biochemical equipment and methods, recombinant DNA technology, introduction of foreign genetic material using vectors, etc., can solve problems such as low metabolite production efficiency
Active Publication Date: 2022-08-09
KOREA ADVANCED INST OF SCI & TECH +1
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[0003] However, the malonyl-CoA pathway is known to have lower metabolite production efficiency than the aspartate pathway (VinodKumar et al., Biotechnology Advances 31:945–961, 2013; Valdehuesa et al., Appl. Microbiol. Biotechnol. , 97:3309–3321, 2013)
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Embodiment 1
[0064] Example 1: Construction of the vector
[0065] 1-1: pTac15k PTAP vector for β-alanine production from carbon sources
[0066] Using the chromosomal DNA of Escherichia coli W3100 as a template, PCR was performed with primers having the following SEQ ID NOs: 1 and 2 to construct a ppc gene fragment encoding phosphoenolpyruvate carboxylase.
[0071] Next, the constructed ppc fragment was treated with SbfI restriction enzyme, and the plasmid pTac15kPTA (Song et al., Metab. Eng., 30: 121-129, 2015), which expresses glutamate derived from glutamate, was treated with SbfI restriction enzyme The panD gene encoding aspartate-α-decarboxylase of Corynebacterium strain (ATCC 13032) and the aspA gene encoding aspartase derived from Escherichia coli W3110. Thereafter, the ppc fragment and pTac15k PTA were...
Embodiment 2
[0087] Example 2: Measurement of Malonic Acid-Producing Ability of Recombinant Mutant Microorganisms
[0088] 2-1: Mutant microorganism producing malonic acid from β-alanine
[0089] The p100-99A pa4123pa0132 or p100-99A yneIpa0132 plasmid constructed in Example 1-2 was introduced into the Escherichia coli W3110 strain, which was then cultured in a medium supplemented with 8 g / L β-alanine, and the strain was analyzed for Malonic acid production (Table 1).
[0090] Selected on LB plate medium supplemented with 30 μg / ml kanamycin and 30 μg / ml ampicillin having malonate-producing ability due to the introduction therein of a vector for malonate production from β-alanine Recombinant mutant microorganisms. Transformed microorganisms were inoculated into 10 ml of LB medium and pre-cultured at 37°C for 12 hours, and then 3 ml of the pre-culture was inoculated and cultured in 50 ml of modified MR medium in a 350-ml flask.
[0091] The composition of the modified MR medium (pH 6.5)...
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Abstract
The present invention relates to a recombinant mutant microorganism having the ability to produce malonate and a method for producing malonate using the same, and more particularly, to a mutant microorganism having the ability to produce malonate, wherein a gene encoding a semialdehyde dehydrogenase is Introduction into a microorganism having the ability to produce malonate semialdehyde, and a method for producing malonate using the microorganism. The mutant microorganisms according to the present invention can be used to efficiently produce malonate from β-alanine or various carbon sources via malonate semialdehyde as an intermediate metabolite by biologically expressing an enzyme having semialdehyde dehydrogenase activity .
Description
technical field [0001] The present invention relates to a recombinant mutant microorganism having the ability to produce malonate and a method for producing malonate using the same, and more particularly, to a mutant microorganism having the ability to produce malonate, wherein a gene encoding a semialdehyde dehydrogenase is Introduction into a microorganism having the ability to produce malonate semialdehyde, and a method for producing malonate using the microorganism. Background technique [0002] Malonic acid, a C3-dicarboxylic acid, is a chemical used in the production of various fragrances, flavors, and pharmaceuticals. Malonic acid is in high demand, but conventional chemical methods for producing it have limitations due to high cost and environmentally harmful production processes. Known methods for the production of malonate by biological routes include methods for the production of malonate from malonyl-CoA via malonyl-CoA hydrolase activity (WO 2013134424). [00...
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