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Biocatalyst for producing d-lactic acid

A lactic acid and production method technology, applied in the field of microorganisms producing D-lactic acid, can solve problems such as inactivation of aspA, research on the production of fumaric acid, difficulty in decomposing aspartic acid or glutamine, etc., and achieve a decrease in the content of pyruvate , Inhibition of by-products and reduction of purification costs

Active Publication Date: 2009-11-18
MITSUI CHEM INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0028] In addition, the effect of aspA inactivation has only been disclosed in the past in the case of Yersinia pestis (Dreyfus, L.A., et.al., J.Bacteriol., Vol.136(2), pp757-764(1978))
However, the gist of this thesis is that the inactivation of aspA makes it difficult to decompose aspartic acid or glutamine in cells, and did not study the amount of fumaric acid produced

Method used

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  • Biocatalyst for producing d-lactic acid
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  • Biocatalyst for producing d-lactic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0129] [Example 1] Production of lactic acid using strain MT-10364

[0130] The composition of the culture medium used for the culture is shown in Table 1 below.

[0131] Table 1

[0132]

[0133] This medium contains 0.34% of reducing sugars, 0.31% of D-lactic acid, 0.31% of L-lactic acid, 0.33% of free amino acids, and trace amounts of various organic acids after acid hydrolysis derived from grain extracts.

[0134] As a pre-culture, Escherichia coli MT-10934 was planted in an Erlenmeyer flask containing 25ml of LB Broth, Miller culture solution (Difco244620), stirred overnight at 120rpm, and then cultured with 475g of the above composition In a 1L culture tank (a culture device BMJ-01 manufactured by ABLE), the entire amount of bacteria is planted. Under the conditions of atmospheric pressure, an aeration rate of 0.5 vvm, a stirring speed of 150 rpm, a culture temperature of 31° C., and a pH of 6.7 (adjusted by NaOH), the culture was cultured until the glucose was completely ...

Embodiment 2

[0141] [Example 2] Construction of ldhA expression vector and lactic acid producing bacteria

[0142] In order to obtain the serine hydroxymethyl transferase (glyA) promoter, the genomic DNA of Escherichia coli was used as the template, and the sequence number 1 and sequence number 2 were used as probes. PCR was used for amplification and restriction The resulting fragment was cleaved with the endonuclease EcoRI, thereby obtaining an approximately 850 bp fragment encoding the glyA promoter. Furthermore, in order to obtain the structural gene of ldhA, the genomic DNA of Escherichia coli was used as a template, and the sequence number 3 and sequence number 4 were used as probes, amplified by PCR, and cut with restriction enzymes EcoRI and HindIII The obtained fragment, thereby obtaining an ldhA structural gene fragment of about 1.0 kbp. The above two fragments were mixed with the fragment obtained by cutting plasmid pUC18 with restriction enzymes EcoRI and HindIII, and ligated with ...

Embodiment 3

[0145] [Example 3] Lactic acid production using lactic acid producing strain MT-10934 / pGlyldhA

[0146] As a pre-culture, the lactic acid-producing bacteria MT-10934 / pGlyldhA strain obtained in Example 2 was planted in an Erlenmeyer flask containing 25ml of LB Broth, Miller culture solution (Difco244620), and cultured in the same manner as in Example 1. . After the incubation, the content and optical purity of lactic acid were determined by HPLC according to conventional methods. The results are shown in Table 3.

[0147] table 3

[0148]

[0149] In the above results, the reason why the amount of total lactic acid exceeds the amount of glucose added at the beginning of the culture is the use of the carbon source in the grain extract. However, even if all reducing sugars, organic acids, and amino acids in the grain extract are used, a conversion rate of 90% or more can be achieved.

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Abstract

The object of the present invention is to provide an efficient production method of D-lactic acid, and also provide a highly selective production method of D-lactic acid with high optical purity and less organic acid by-products. Culture of microorganisms with inactivated or reduced activity of pyruvate formate lyase and enhanced activity of NADH-dependent D-lactate dehydrogenase (ldhA) derived from Escherichia coli, inactivation of FAD-dependent D-lactate dehydrogenase Or microorganisms with reduced activity, or microorganisms with TCA cycle, malate dehydrogenase inactivation or reduced activity, and aspartate ammonia-lyase inactivation or reduced activity in addition to the above-mentioned characteristics of microorganisms, producing D- lactic acid. IdhA activity is enhanced by genomically linking a gene encoding ldhA to a promoter of a gene controlling expression of proteins involved in glycolysis, nucleic acid biosynthesis, or amino acid biosynthesis.

Description

Technical field [0001] The present invention relates to a microorganism capable of producing D-lactic acid with high selectivity and high efficiency and a method for producing D-lactic acid using the microorganism. Specifically, it relates to a method for efficiently producing high-purity lactic acid, and particularly relates to an effective method for producing D-lactic acid with a small amount of pyruvate produced and accumulated. [0002] The present invention also relates to a method for producing D-lactic acid, which is characterized in that the method uses a microorganism whose FAD-dependent D-lactate dehydrogenase is inactivated or has decreased activity. [0003] In addition, the present invention also relates to a microorganism that produces D-lactic acid and a method for producing D-lactic acid using the microorganism. The microorganism does not produce succinic acid or fumaric acid as impurities when producing D-lactic acid. Background technique [0004] Biodegradable ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P7/56C12N1/21C12N15/09
Inventor 和田光史及川利洋望月大资德田淳子川岛美由贵安乐城正阿部玲子三宅仁基高桥均泽井秀树耳塚孝森重敬东庸介
Owner MITSUI CHEM INC
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