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High-D-lactic acid-yield recombinant bacterium and application thereof

A technology of recombinant bacteria and lactic acid, applied in the field of bioengineering, can solve the problems of poor optical purity of products, low yield and conversion rate, difficult synthesis of lactic acid, etc.

Inactive Publication Date: 2017-03-22
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In the prior art, there are relatively few studies on the production of D-lactic acid by microbial methods, and there are many problems. For example, traditional lactic acid fermentation strains can only synthesize L-lactic acid and D-lactic acid at the same time, and the optical purity of the product is relatively poor. For the synthesis of polylactic acid
The existing engineering strains have low yield and conversion rate and high production cost, which is not conducive to the realization of industrialization development

Method used

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  • High-D-lactic acid-yield recombinant bacterium and application thereof
  • High-D-lactic acid-yield recombinant bacterium and application thereof
  • High-D-lactic acid-yield recombinant bacterium and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Cloning of exogenous genes

[0049] Obtain the D-lactate dehydrogenase gene (ldhA) derived from K.pneumoniae (Genbank accession number is CP000964.1, or as shown in SEQ ID NO.1), which is obtained by PCR amplification using K.pneumoniae as a template The ldhA gene. Primers were designed as follows:

[0050] 5'-CGAGCTCTATAATCACTGGAGAAAAGT-3'

[0051] 5'-GCTCTAGAGAATCAGACGATGGCGTTCG-3', and then use the recovery kit to recover the target fragment.

Embodiment 2

[0052] Example 2 Construction of expression vector

[0053] The D-lactate dehydrogenase ldhA gene fragment obtained in Example 1 and the plasmid pBAD18 were digested with SacI and XbaI, and the digested products were recovered, and then ligated. The molar ratio of the vector and the ldhA gene fragment was 1:5, at 16°C The recombinant plasmid pBAD18-ldhA was constructed after ligation for more than 6 hours, and transformed into E.coli DH5α, and then spread on a medium containing 100 μg·mL -1 On LB solid medium with kanamycin, positive clones were screened by PCR. After the recombinant plasmid pBAD18-ldhA was extracted from the positive clone, it was identified by restriction enzyme digestion and sequencing.

Embodiment 3

[0054] Example 3 Obtaining of D-lactic acid-producing recombinant bacteria

[0055] Inoculate wild-type Klebsiella pneumoniae K. peneumoniae into 20 mL of LB liquid medium, add 50 μg·mL -1 Kanamycin, cultured to a certain concentration of bacteria. Use a sterilized centrifuge tube to centrifuge at 5500 rpm at 4°C for 5 minutes, remove the supernatant, suspend with sterile water stored at 4°C, and centrifuge again, repeating twice. Then resuspend with sterile water, aliquot and store at -80°C to obtain competent cells.

[0056] The expression vector obtained in Example 2 was transformed by electric shock and transferred into K. peneumoniae competent cells to obtain recombinant Klebsiella pneumoniae.

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Abstract

The invention discloses a high-D-lactic acid-yield recombinant bacterium and application thereof, belonging to the field of bioengineering. The recombinant bacterium provided by the invention is recombinant Klebsiella pneumoniae obtained by overexpressing D-lactate dehydrogenase gene ldhA in Klebsiella pneumoniae. According to the production method of D-lactic acid, progressive amplification culture is carried out after the recombinant bacterium is activated, thereby implementing pilot-scale production on the 500L fermentation tank level. The production method of D-lactic acid is simple and easy to implement, and implements high-efficiency synthesis of D-lactic acid on the pilot-scale level; the yield reaches 210 g / L, the D-lactic acid is free of L-lactic acid; and the recombinant bacterium has the characteristics of high yield and lower production cost, and has favorable market prospects and economic benefits.

Description

technical field [0001] The invention relates to a high-yield D-lactic acid recombinant bacterium and its application, belonging to the field of bioengineering. Background technique [0002] With the rapid development of modern society, human social life requires more convenience. In order to meet this demand, plastic products such as disposable foam plastic boxes, plastic bags, tableware, and water cups have entered people's daily life on a large scale. While these plastic products bring convenience to human beings, they also exacerbate the resulting "white pollution" problem. The development and manufacture of degradable plastic products has become the key to solving this problem. [0003] Polylactic acid is a new type of biodegradable material with good mechanical properties, physical properties and biocompatibility, which is mainly obtained by polymerizing lactic acid monomers through polycondensation. According to the different lactic acid monomers, polylactic acid ca...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/74C12P7/56C12R1/22
CPCC12N9/0006C12N15/74C12P7/56C12Y101/01028
Inventor 赵广冯新军程涛咸漠
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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