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Biocatalyst for production of d-lactic acid

A technology of lactic acid and production method, applied in D-lactic acid production, effective D-lactic acid production, microorganisms producing D-lactic acid, high-efficiency production of high-purity lactic acid, D-lactic acid production field, can solve aspA inactivation, Difficult to decompose aspartic acid or glutamine, research on the production of fumaric acid, etc., to achieve the effect of reducing the content of pyruvate, inhibiting by-products, and reducing purification costs

Active Publication Date: 2010-02-24
MITSUI CHEM INC
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  • Description
  • Claims
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Problems solved by technology

[0029] In addition, the effect of aspA inactivation has only been disclosed 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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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0130] [Example 1] Utilize MT-10364 bacterial strain to produce lactic acid

[0131] The composition of the medium used for the culture is shown in Table 1 below.

[0132] Table 1

[0133]

[0134] This culture medium contains 0.34% of reducing sugar derived from grain extract after acid hydrolysis, 0.31% of D-lactic acid, 0.31% of L-lactic acid, 0.33% of free amino acid and trace amounts of various organic acids.

[0135] As pre-cultivation, 25ml LB Broth, Miller culture solution (Difco244620) was installed in the Erlenmeyer flask to carry out the planting of Escherichia coli MT-10934, stirred and cultivated overnight at a speed of 120rpm, and then cultured in 475g of the above-mentioned composition In a culture tank with a volume of 1 L (manufactured by ABLE Corporation, BMJ-01), the whole amount of bacteria was planted. Under the conditions of atmospheric pressure, aeration rate of 0.5vvm, stirring speed of 150rpm, culture temperature of 31°C, and pH of 6.7 (adjusted w...

Embodiment 2

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

[0142] In order to obtain the promoter of serine hydroxymethyl transferase (glyA), the genomic DNA of Escherichia coli was used as a template, and sequence number 1 and sequence number 2 were used as probes to amplify by PCR method, and use restriction The resulting fragment was cut with endonuclease EcoRI, thereby obtaining a fragment encoding the glyA promoter of about 850 bp. Moreover, in order to obtain the structural gene of ldhA, the genomic DNA of Escherichia coli was used as a template, and sequence number 3 and sequence number 4 were used as probes to amplify by PCR method and cut with restriction enzymes EcoRI and HindIII The obtained fragment, thereby obtaining a 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, ligated with DNA ligase, and then...

Embodiment 3

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

[0146] As pre-cultivation, carry out the planting of the lactic acid producing bacteria MT-10934 / pGlyldhA bacterial strain obtained in Example 2 in an Erlenmeyer flask equipped with 25ml LB Broth, Miller culture solution (Difco244620), and utilize the same method as Example 1 to cultivate . After the cultivation, the content and optical purity of lactic acid were measured 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 that the carbon source in the grain extract was utilized. However, even if all the 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 subject of the present invention is to provide a method for producing D-lactic acid in high yield, and to provide a method for producing D-lactic acid with high selectivity, in which optical purity is high and a by-product organic acid is small. A microorganism, wherein activity of pyruvate formate-lyase (pfl-) is inactivated or decreased, and further activity of Escherichia coli-derived NADH-dependent D-lactate dehydrogenase (ldhA) is enhanced, is cultured to produce a remarkable amount of D-lactic acid in a short time. With regard to a method for enhancing ldhA activity, by linking, on agenome, a gene encoding 1dhA with a promoter of a gene which controls expression of a protein involved in a glycolytic pathway, a nucleic acid biosynthesis pathway or an amino acid biosynthesis pathway, suitable results are obtained compared to the method for enhancing expression of the gene using an expression vector. In addition, a microorganism in which a did gene is substantially inactivatedor decreased is cultured to produce high quality D-lactic acid with reduced concentration of pyruvic acid. Furthermore, it is possible to suppress by-production of succinic acid and fumaric acid whilemaintaining high D-lactic acid productivity by using the above-mentioned microorganism having a TCA cycle, wherein activity of malate dehydrogenase (mdh) is inactivated or decreased, and further activity of aspartate ammonia-lyase (aspA) is inactivated or decreased.

Description

[0001] This application is a divisional application of the application dated September 17, 2004, the application number is 200480027610.5 (the international application number is PCT / JP2004 / 014037), and the invention title is "Biocatalyst for the production of D-lactic acid". technical field [0002] 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 efficient method for producing D-lactic acid with a small accumulation of pyruvic acid. [0003] The present invention also relates to a method for producing D-lactic acid, characterized in that the method uses a microorganism with inactivated or reduced activity of FAD-dependent D-lactate dehydrogenase. [0004] In addition, the present invention also relates to a D-lactic ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P7/56C12R1/19
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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