Process for production of lactic acid

A technology of lactic acid and hydroxybenzoic acid, which is applied in the field of lactic acid manufacturing, can solve the problems of lower lactic acid productivity and unknown acetic acid output, and achieve the effect of increasing productivity

Inactive Publication Date: 2008-11-05
KYOWA HAKKO KIRIN CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] It is known that in Escherichia coli, a mutant strain of the ubiquinone biosynthesis gene accumulates 2.5 g / L of D-lactic acid (non-patent literature 11 and 12), and this mutant strain (AN59 strain) is a mutant strain of the ubiE gene ( Non-Patent Document 13), the productivity of lactic acid in strains with reduced activity of other ubiquinone biosynthesis gene products, the acetic acid production rate of ubiquinone biosynthesis gene mutant strains, etc. are unknown

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0114] Production of strains in which the gene encoding UbiA protein or UbiB protein has been deleted

[0115] With the chromosomal DNA of Escherichia coli (E.coli) KM22 strain [Gene, 246, 321-330 (2000)] as a template, use the 25 bases hybridized on the KM22 strain chromosomal DNA according to each 3' end PCR was carried out using DNA consisting of the nucleotide sequences shown in SEQ ID NOs: 5 and 6 designed so that both ends of the kanamycin-resistant gene were used as a primer set. Use LA-Taq for PCR, prepare 25 μl of reaction solution containing 10 ng of chromosomal DNA fragments and 20 pmol of primer DNA according to the instructions attached to LA-Taq, and carry out under the following conditions: after incubation at 94°C for 2 minutes, repeat at 94°C After 30 cycles of 15 seconds at 55°C, 20 seconds at 55°C, and 1 minute at 68°C, it was incubated at 72°C for 10 minutes.

[0116] Next, a DNA fragment in which the nucleotide sequence shown in SEQ ID NO: 7 is added upst...

Embodiment 2

[0123] Production of lactic acid using ubiA gene or ubiB gene deletion strain (1)

[0124] Escherichia coli (E.coli) BW25113ΔubiA strain, Escherichia coli (E.coli) BW25113ΔubiB strain, and the ubiE gene deletion strain (E.coli BW25113ΔubiE) and ubiG produced according to the method described in Example 1 as a control Gene deletion strain (E.coli BW25113ΔubiG), ubiH gene deletion strain (E.coli BW25113ΔubiH), Escherichia coli (E.coli) BW25113 strain were respectively inoculated in test tubes containing 5ml of LB liquid medium containing 10g / l glucose , cultured with shaking at 37°C for 17 hours to obtain a culture. 60 μl of this culture was inoculated into 6 ml of M9 liquid medium containing 3% calcium carbonate and 2% casamino acids (4% glucose, 11.28 g / l M9 Minimal Salts (X5) (manufactured by Difco), 100 μmol / l calcium chloride, 2mmol / l magnesium sulfate, 10μg / ml ferric sulfate) in thick test tubes, shake culture at 37°C for 28 hours. The culture after 28 hours was centrif...

Embodiment 3

[0130] Production of lactic acid using ubiA gene or ubiB gene deletion strain (2)

[0131] Escherichia coli (E.coli) BW25113ΔubiA strain and Escherichia coli (E.coli) BW25113ΔubiB strain were respectively inoculated in test tubes containing 5ml of LB liquid medium containing 10g / l glucose, and shaken at 37°C After culturing for 17 hours, a culture was obtained. 55 μl of this culture was inoculated into 5 ml of M9 liquid medium containing 6% calcium carbonate and 2% casamino acids (5% glucose, 11.28 g / l M9 Minimal Salts (X5) (manufactured by Difco), 100 μmol / l calcium chloride, 2mM magnesium sulfate, 10μg / ml ferric sulfate) in thick test tubes, shake culture at 37°C for 25 hours. After 25 hours, 1041 μl of 30% glucose, 104 μl of 22.56 g / l M9 Minimal Salts (X10) (manufactured by Difco), 104 μl of casamino acids, 2 μl of 1 mol / l magnesium sulfate, 1 μl of 10 mg / ml iron sulfate and 0.06 g of calcium carbonate were used to continue the culture, and the culture was recovered afte...

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Abstract

A microorganism having the reduction or loss of a 4-hydroxybenzoate-polyprenyltransferase activity or a 2-octapernylphenol 2-octaprenyl-6-methoxyphenol;flavin reductase activity and capable of producing lactic acid, particularly a microorganism having chromosomal DNA having the deletion of a part or the entirety of a gene encoding a protein having a 4-hydroxybenzoate-polyprenyltransferase activity or a protein having a 2-octapernylphenol 2-octaprenyl-6-methoxyphenol;flavin reductase activity; and a process for production of lactic acid using the microorganism.

Description

technical field [0001] The present invention relates to a microorganism capable of producing lactic acid and a method for producing lactic acid using the microorganism. Background technique [0002] As microorganisms producing lactic acid, lactic acid bacteria such as Lactobacillus and Lactococcus, filamentous fungi such as Rhizopus, yeast such as Saccharomyces, and Escherichia coli have been reported. It is known that lactic acid bacteria show complex nutritional requirements, and the optical purity of lactic acid is low (non-patent documents 1 and 2); filamentous bacteria often have by-products such as glycerol, ethanol or fumaric acid (non-patent documents 3 and 4); Also when a recombinant strain is used, a large amount of ethanol is by-produced, and the yield with respect to sugar consumption is low (Non-Patent Document 5). [0003] As a method for producing lactic acid using Escherichia coli, a method using a deletion strain of the pyruvate formate lyase gene and the g...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12N1/21C12P7/56
CPCC12N9/0006C12N9/1085C12P7/56C12N15/09C12N1/20
Inventor 伊东干人橓田贵美枝森英郎加藤真辉子
Owner KYOWA HAKKO KIRIN CO LTD
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