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Recombinant bacteria for synthesizing D-lactic acid, construction method of recombinant bacteria and application of recombinant bacteria

A technology of recombinant bacteria and lactic acid, applied in the field of genetic engineering, can solve the problems of expensive antibiotics, high production costs, and adverse effects of product separation in the later stage, and achieve the effect of reducing production costs and improving transformation efficiency

Active Publication Date: 2018-11-13
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

[0005] In order to solve the problem that the glycerol metabolism pathway of Klebsiella pneumoniae has a relatively high level of by-product product accumulation, which reduces the synthesis efficiency of D-lactic acid and adversely affects the separation of later products, and requires the addition of expensive Antibiotics, the problem of high production costs, the present invention provides a recombinant bacterium for synthesizing D-lactic acid and its construction method and application. The present invention uses Klebsiella pneumoniae ATCC25955 as the host strain, and eliminates acetic acid, ethanol, The synthesis of by-products such as 2,3-butanediol realizes the efficient conversion of glucose to D-lactic acid. The fermentation process does not require the addition of antibiotics, and has certain advantages in terms of production cost and conversion efficiency.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1: Construction of recombinant bacteria

[0044] 1. Knockout of gene budB

[0045] In this example, primers were designed using about 500 base fragments upstream and downstream of the acetolactate synthase gene budB (NCBI gene ID: 11848061) of the wild strain of Klebsiella pneumoniae (K. penumoniae) as a template, and PCR amplification Acetolactate synthase gene budB upstream and downstream fragments, and then use the recovery kit to recover the target gene fragments.

[0046] The upstream fragment amplification primers are 5'-GCTCTAGAGTCAGTCAGCTGG AAGCTC-3'(SEQ ID NO.1) and 5'-ATAAGCTTCGCCACCTGGTC-3'(SEQ ID NO.2); the downstream fragment amplification primer sequence is: 5'-GACCAGGTGGCGAAGCTTATCTGCGCATCGT TCGCGCCAT -3' (SEQ ID NO. 3) and 5'-CGAGCTCTTATCGCGATAATCTACCG-3' (SEQ ID NO. 4).

[0047] After the amplified fragments were recovered, bridge PCR was performed using the recovered fragments as templates, and the target fragment ΔbudB was recovered using...

Embodiment 2

[0056] Example 2: Recombinant Klebsiella pneumoniae utilizes glucose to synthesize D-lactic acid

[0057] In this example, the recombinant Klebsiella pneumoniae K. peneumoniaeΔbudBΔadhEΔackA obtained in Example 1 was activated in LB liquid medium, and the activated strain was inoculated into 1.8 L of M9 modified liquid medium at a ratio of 1:150 In a 5L fermenter, cultured at 30°C, 200rpm, and 0.5vvM aeration. By adding ammonia water, the pH of the fermentation broth is maintained at about 6.8. Regular sampling and centrifugation, the supernatant is detected by biosensors for glucose content, and an appropriate amount of 70% glucose solution is added according to the residual sugar concentration to maintain the sugar concentration at 1g / L-5g / L to ensure sufficient carbon sources. Cultivate for 24 hours to terminate fermentation, take 1 mL of fermentation broth, centrifuge at 15,000 rpm for 10 minutes at 4°C, take supernatant, and detect product concentration by high performan...

Embodiment 3

[0059] Example 3: Recombinant Klebsiella pneumoniae utilizes glucose to synthesize D-lactic acid

[0060] In this example, the recombinant Klebsiella pneumoniae K. peneumoniaeΔbudBΔadhEΔackA obtained in Example 1 was activated in LB liquid medium, and the activated strain was inoculated into 1.8 L of M9 modified liquid medium containing 1:100 In a 5L fermenter, cultured at 37°C, 300rpm, and 1.5vvM aeration. The pH of the fermentation broth was maintained at about 7 by adding ammonia water. Regular sampling and centrifugation, the supernatant is detected by biosensors for glucose content, and an appropriate amount of 70% glucose solution is added according to the residual sugar concentration to maintain the sugar concentration at 1g / L-5g / L to ensure sufficient carbon sources. Cultivate for 36 hours to stop the fermentation, take 1 mL of fermentation broth, centrifuge at 15,000 rpm for 10 minutes at 4°C, take the supernatant, and detect the concentration of the product by high ...

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Abstract

The invention discloses recombinant bacteria for synthesizing D-lactic acid, a construction method of the recombinant bacteria and an application of the recombinant bacteria to production of the D-lactic acid, and belongs to the technical field of gene engineering. Klebsiella pneumoniae serve as original strains, and acetolactic acid synthetase genes budB, alcohol dehydrogenase genes adhE and acetokinase genes ackA in a klebsiella pneumoniae genome are knocked out to obtain the recombinant bacteria. The recombinant bacteria have the advantages that the D-lactic acid is synthesized by glucose,antibiotics are omitted in the fermentation process, production cost is reduced, the yield and the conversion rate of the D-lactic acid produced by the klebsiella pneumoniae through the glucose are effectively increased, 150.1g / L D-lactic acid can be obtained at the level of a 5L fermentation tank, and the conversion rate is 96%. The recombinant bacteria are applicable to industrial production ofthe D-lactic acid.

Description

technical field [0001] The invention relates to a recombinant bacterium for synthesizing D-lactic acid and its construction method and application, belonging to the technical field of genetic engineering. Background technique [0002] Lactic acid is a three-carbon compound, which can be divided into L-lactic acid and D-lactic acid in terms of optical properties. There are many studies on L-lactic acid and relatively few studies on D-lactic acid. D-lactic acid is an important platform compound, which is widely used in pesticides, pharmaceuticals, cosmetics and other industries. D-lactic acid with an optical purity greater than 95% can be used as an excellent monomer for polylactic acid. [0003] Polylactic acid is a kind of degradable plastic with excellent properties, which is mainly obtained by polymerization of L-lactic acid. However, pure poly-L-lactic acid or poly-D-lactic acid has certain defects in terms of thermodynamic properties and mechanical properties. Add D-...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P7/56C12R1/22
CPCC12N9/0006C12N9/1022C12N9/1217C12P7/56C12Y101/01001C12Y202/01006C12Y207/02001
Inventor 赵广冯新军咸漠刘修涛刘会洲
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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