Recombinant bacterium for synthesizing D-lactic acid by adopting biodiesel by-product, namely, glycerol and applications of recombinant bacterium

A biodiesel and recombinant bacteria technology, applied in the field of genetic engineering, can solve the problem of high raw material prices, achieve the effects of improving product conversion rate, reducing production costs, and improving fermentation conditions

Inactive Publication Date: 2017-02-22
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

On the other hand, the existing technology is mainly to synthesize D-lactic acid by fermenting starchy raw materials such as corn. Due to the influence of national policies, the price of raw materials is relatively high.

Method used

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  • Recombinant bacterium for synthesizing D-lactic acid by adopting biodiesel by-product, namely, glycerol and applications of recombinant bacterium
  • Recombinant bacterium for synthesizing D-lactic acid by adopting biodiesel by-product, namely, glycerol and applications of recombinant bacterium
  • Recombinant bacterium for synthesizing D-lactic acid by adopting biodiesel by-product, namely, glycerol and applications of recombinant bacterium

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] The knockout of embodiment 1 gene

[0047] In this example, primers were designed using about 500 base fragments upstream and downstream of the 1,3-propanediol dehydrogenase gene (dhaT) (Gene ID: 7946507) of the wild strain of Klebsiella pneumoniae (K. penumoniae) as a template 1,3-propanediol dehydrogenase gene (dhaT) upstream and downstream fragments were amplified by PCR respectively, and then the target gene fragment was recovered by using a recovery kit.

[0048] The upstream amplification primer sequence is: Kp-dhaT-Up-5': 5'-CGGGGTACCATGAGCTATCGTAT GTTTG-3' and Kp-dhaT-Up-3': 5'-GATGAGGCGGATCGCCTGCGATCACAAACTT CACTTTG-3'; the downstream amplification primer sequence is Kp -dhaT-Down-5': 5'-CAAAGTGAAGTTTGTGATCGCAGGCGATCCGCCTCATC-3' and Kp-dhaT-Down-3': 5'-GTCCGAGCTCTCAGAATGCCTGGCGGAAAATCG-3'.

[0049] After recovering the amplified fragment above, use the recovered fragment as a template to perform bridge PCR to obtain the fragment ΔdhaT for knocking out the 1,3-...

Embodiment 2

[0054] The cloning of embodiment 2 exogenous gene

[0055] In this example, the D-lactate dehydrogenase gene (ldhA) (Gene ID: 6938538) derived from K.pneumoniae was obtained, which was obtained by PCR amplification using K.pneumoniae as a template (primer: 5'-CGAGCTCTATAATCACTGGAGAAAAGT- 3' and 5'-GCTCTAGAGAATCAGACGATGGCGTTCG-3'), and then use the recovery kit to recover the target fragment.

Embodiment 3

[0056] The construction of embodiment 3 expression vector

[0057] The D-lactate dehydrogenase ldhA gene fragment obtained in Example 2 and the plasmid pBAD18 were digested with SacI and XbaI, and the digested products were recovered, and then ligated. The vector and the ldhA gene fragment were in a molar ratio of 1:5, 16 ℃ for more than 6h, the ligation product was transformed into E.coliDH5α, and then coated with 100μg·mL -1 On the LB solid plate of 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, and was used to overexpress D-lactate dehydrogenase.

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Abstract

The invention discloses a recombinant bacterium for synthesizing D-lactic acid by adopting the biodiesel by-product, namely, glycerol and applications of the recombinant bacterium, belonging to the field of genetic engineering. For the recombinant bacterium provided by the invention, the dehydrogenase gene of D-lactic acid, namely, ldhA is introduced into the host, namely, recombinant Klebsiella pneumoniae with the 1,3-propanediol oxidoreductase gene dhaT and the aldehyde reductase / alcohol dehydrogenase gene yqhD knocked out, and thus the engineering strain is obtained. D-lactic acid is synthesized by taking the biodiesel by-product, namely, glycerol as the substrate, in the shake flask level, the yield and conversion rate respectively achieve 12.7g / L and 50.4%, the optical purity is 100%, and the recombinant bacterium has the advantages that the optical purity and conversion rate are high, and the production cost is low.

Description

technical field [0001] The invention relates to a recombinant bacterium for synthesizing D-lactic acid by using biodiesel by-product glycerol and its application, belonging to the technical field of genetic engineering. Background technique [0002] With the development of society and the shortage of fossil energy, the synthesis of biofuels from renewable raw materials has become a worldwide research hotspot. Biodiesel is an important biofuel, and its production is increasing year by year. The production of biodiesel produces a large amount of glycerol as a by-product. It is estimated that about 1 ton of crude glycerol is produced for every 10 tons of biodiesel produced. The large-scale surplus of glycerol has caused its price to remain at a low level, and it has become a carbon source material with abundant resources and low price that needs to be developed urgently. [0003] D-lactic acid is an important platform compound, which is widely used in industries such as medi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P7/56C12R1/22
CPCC12N9/0006C12P7/56C12Y101/01002C12Y101/01028C12Y101/01202
Inventor 赵广冯新军咸漠
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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