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Method for preparing high optical purity D-lactic acid by fermentation

A technology of optical purity and lactic acid, which is applied in the field of microbial fermentation engineering, can solve the problem that acetic acid cannot be used as industrial lactic acid, and achieve the effect of ensuring the quality of polymerization and efficient synthesis

Inactive Publication Date: 2009-09-30
SUZHOU PURIZYMES BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The former example illustrates the operability of the lactic acid synthesis pathway in Escherichia coli at the gene level; the latter example illustrates that when E. The metabolic pathway that is completely metabolized is meaningful for the synthesis of lactic acid; but acetic acid obviously cannot be used as a raw material for industrial lactic acid production

Method used

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  • Method for preparing high optical purity D-lactic acid by fermentation
  • Method for preparing high optical purity D-lactic acid by fermentation

Examples

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Effect test

Embodiment 1

[0033] Example 1 - Deletion of the gene encoding pyruvate dehydrogenase

[0034] Using synthetic oligonucleotide primers P1 and P2 as primers, P1 and P2 are:

[0035] P1: 5′-ATA GGATCC TATCGAAATCAAAGTACCGGACATCGGGG-3′

[0036] P2: 5′-CATCA GGATCC AGACGGCGAATGTCAGA-3′

[0037] Using Escherichia coli 947 chromosomal DNA as a template, the pyruvate dehydrogenase gene was amplified from the genome by PCR (polymerase chain reaction, Maties, 1989), and the PCR product was cloned into the BamHI site of pUC18 to obtain the recombinant plasmid pUC -PDH. Use EcoRV to remove the 963bp fragment and combine it with dif Ec -Gm R Ligation, to obtain the gene deletion sequence for pyruvate dehydrogenase, pdh′-dif-Gm R -dif-pdh', ie: pdh'::Gm R dif . This gene deletion sequence was transformed into E. coli. Transformants were selected for on selective media. Then subculture on non-selective medium, and select the transformant whose selectable marker disappears. The chromosomal D...

Embodiment 2

[0038] Example 2—Deletion of the gene encoding pyruvate formate lyase

[0039] Using synthetic oligonucleotide primers P3 and P4 as primers, P3 and P4 are:

[0040] P3: 5′-ATA GGATCC TGATTACCGCTGGCAACAACGA-3′

[0041] P4: 5′-CATCA GGATCC ATTGGCAACCAGGCAAGCGA-3′

[0042] Using Escherichia coli 947 chromosomal DNA as a template, the pyruvate formate lyase gene was amplified from the genome by PCR, and the PCR product was cloned into the BamHI site of pUC18 to obtain the recombinant plasmid pUC-PFL. Use EcoRV to remove the 1773bp fragment and combine it with dif Ec -Gm R Ligation, to obtain the gene deletion sequence for pyruvate formate lyase, pfl'-dif-Gm R -dif-pfl', ie: pfl'::Gm R dif . This gene deletion sequence was transformed into E. coli 947 (Δpdh). Transformants were selected for on selective media. Then subculture on non-selective medium, and select the transformant whose selectable marker disappears. The chromosomal DNA of the transformant was extracted, ...

Embodiment 3

[0043] Example 3—Deletion of the gene encoding FAD-dependent D-lactate dehydrogenase

[0044] Using synthetic oligonucleotide primers P5 and P6 as primers, P5 and P6 are:

[0045] P5: 5′-ATA GGATCC GCGATGTCTTCCATGACAACAACTG-3′

[0046] P6: 5′-CATCA GGATCC GGATTCATGCTGTTGGTCGGATC-3′

[0047] Using Escherichia coli 947 chromosomal DNA as a template, the pyruvate formate lyase gene was amplified from the genome by PCR, and the PCR product was cloned into the BamHI site of pUC18 to obtain the recombinant plasmid pUC-DLD. The 700bp fragment was digested with StuI and EcoRV and combined with dif Ec -Gm R Ligation, to obtain the gene deletion sequence of FAD-dependent D-lactate dehydrogenase, dld′-dif-Gm R -dif-dld', ie: dld'::Gm R dif . This gene deletion sequence was transformed into E. coli 947 (Δpdh, Δpfl). Transformants were selected for on selective media. Then subculture on non-selective medium, and select the transformant whose selectable marker disappears. The ...

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Abstract

The invention provides a method for preparing high optical purity D-lactic acid by fermentation. The method is characterized in that D-lactic acid high-yield recombinant strain is fermented for 30 to 50 hours in stages at a temperature of between 20 and 45 DEG C to prepare the D-lactic acid with yield level reaching 12 percent and optical purity of the D-lactic acid reaching more than 99.5 percent. By expressing pyruvate dehydrogenase encoding gene and NAD dependency D-lactic acid dehydrogenase encoding gene of the D-lactic acid high recombinant strain under the control of fermentation parameter, the production process of the D-lactic acid can be controlled only by changing the fermentation temperature, so the aim and the effect of synthesizing optical purity D-lactic acid in high-efficiency by using glucose as a raw material are achieved.

Description

technical field [0001] The present invention relates to a bacterial strain for producing lactic acid by microbial fermentation method and its fermentation production technology, especially a D-lactic acid bacterial strain capable of high-efficiency and high-selectivity production of high optical purity and high chemical purity and its corresponding manufacturing technology. No impurities such as succinic acid and fumaric acid are produced during the production of D-lactic acid; in addition, the invention also relates to the fermentation and metabolism control method of the high-yielding D-lactic acid bacteria, which belongs to the technical field of microbial fermentation engineering. Background technique [0002] Lactic acid is one of the three major organic acids recognized in the world. According to the optical purity, lactic acid can be divided into L-lactic acid, D-lactic acid and DL-mixed lactic acid. Because D-lactic acid and DL-mixed lactic acid affect the normal func...

Claims

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Application Information

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IPC IPC(8): C12P7/56C12R1/19C12R1/07C12R1/01C12R1/645
Inventor 王正祥石贵阳
Owner SUZHOU PURIZYMES BIOTECH
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