Double-gene knockout engineering bacteria and construction method and application thereof in fermentation production of 1,3-propylene glycol

A construction method and dual-gene technology, applied in the biological field, can solve the problems of increased synthesis, inability to increase significantly, and increased lactic acid synthesis, and achieve the effects of increased proportion, reduced production cost, and important application prospects.

Inactive Publication Date: 2016-09-14
SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have reported that knocking out the lactate dehydrogenase gene in the lactic acid synthesis pathway alone can greatly reduce the lactic acid synthesis, but at the same time the 2,3-butanediol synthesis is greatly increased, indicating that the lactic acid synthesis pathway is cut off, and the metabolic flow mainly shifts to 2, The synthesis of 3-butanediol cannot significantly increase the proportion of 1,3-propanediol in the total metabolites; while knocking out the key catalytic enzyme genes in the 2,3-butanediol synthetic metabolic pathway can greatly reduce 2,3-butanediol synthesis, but at the same time lactic acid synthesis is greatly increased

Method used

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  • Double-gene knockout engineering bacteria and construction method and application thereof in fermentation production of 1,3-propylene glycol
  • Double-gene knockout engineering bacteria and construction method and application thereof in fermentation production of 1,3-propylene glycol
  • Double-gene knockout engineering bacteria and construction method and application thereof in fermentation production of 1,3-propylene glycol

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Construction of a Klebsiella pneumoniae mutant strain in which the lactate dehydrogenase gene, a key gene in the lactic acid synthesis pathway, is inactivated. The starting strain wild Klebsiella pneumoniae, the construction process is as follows figure 1 shown.

[0035] (l) Cloning of lactate dehydrogenase gene LDH

[0036] According to the DNA sequence of lactate dehydrogenase gene LDH (GenBank number: CP000647.1) design primers, PCR amplification gene sequence, primer sequence is as follows: upstream primer LDH-F: 5'-CCTCGGACATTTCCTGTTAAT-3') and downstream primer LDH- R: (5'-GGCAAACGCTGCAGCGAGCAG-3'). Using wild-type Klebsiella pneumoniae (preserved in China Center for Type Culture Collection, deposit number: CCTCC M 2011075) genomic DNA as a template, under the guidance of primers LDH-F and LDH-R, PCR amplified lactate The nucleic acid sequence of the hydrogenase gene LDH, the PCR amplification conditions are: first 95°C for 3min; then 94°C for 1min, ...

Embodiment 2

[0046] Example 2: Construction of a Klebsiella pneumoniae mutant strain in which the acetolactate synthase gene, a key gene in the 2,3-butanediol synthetic metabolic pathway, is inactivated. Build process like figure 2 shown.

[0047] The starting strain is the Klebsiella pneumoniae mutant strain of the lactate dehydrogenase gene inactivation obtained in Example 1, and the construction process is as follows: figure 2 shown.

[0048] (l) Cloning of acetolactate synthase gene ALS

[0049]According to the DNA sequence of the acetolactate synthase gene ALS (GenBank number: CP006738.1), primers were designed and the gene sequence was amplified by PCR. The primer sequences were as follows: upstream primer ALS-F: 5'-atggacaaacagtatccggtacgc-3' and downstream primer ALS-R : 5'-ttacagaatctgactcagatgcag-3'. Acetolactate was amplified by PCR using the genomic DNA of wild-type Klebsiella pneumoniae (preserved in the China Center for Type Culture Collection, preservation number: CCTC...

Embodiment 3

[0058] Example 3: Detection of lactate dehydrogenase and acetolactate synthase activities of a Klebsiella pneumoniae mutant strain in which both the lactate dehydrogenase gene and the acetolactate synthase gene are simultaneously inactivated.

[0059] Carry out the activity detection of lactate dehydrogenase gene and acetolactate synthase to the Klebsiella pneumoniae mutant strain of the lactate dehydrogenase gene and acetolactate synthase gene inactivated simultaneously of embodiment 2, take wild-type Klebsiella pneumoniae Bacteria is a control, and the specific method comprises the following steps:

[0060] 1. Activity detection of the key enzyme lactate dehydrogenase in the lactic acid metabolic pathway:

[0061] The lactate dehydrogenase gene and the acetolactate synthase gene of the key genes of the lactic acid and 2,3-butanediol metabolic pathway constructed in Example 2 were simultaneously inactivated Klebsiella pneumoniae mutant strains were subjected to lactate dehydr...

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Abstract

The invention discloses double-gene knockout engineering bacteria and a construction method and an application thereof in fermentation production of 1,3-propylene glycol. A D-lactic dehydrogenase gene and an alpha-acetolactate synthetase gene in a genome of a wild type strain for production of 1,3-propylene glycol are knocked out to obtain the engineering strain; the wild type strain for production of 1,3-propylene glycol takes glycerol as a raw material for fermentation production of 1,3-propylene glycol. The engineering bacteria obtained after simultaneous knockout of the two genes of lactic dehydrogenase and acetolactate synthetase are applied in the process of fermentation-process production of 1,3-propylene glycol; the accounting proportion of 1,3-propylene glycol in a fermentation liquid in metabolites is increased, synthesis of lactic acid and 2,3-butylene glycol are simultaneously greatly reduced, and other by-products are not significantly increased. In the process of microbiological fermentation-process production of 1,3-propylene glycol, the role in improving the accounting proportion of 1,3-propylene glycol synthesized by the engineering bacteria and reducing the proportion of the synthesized by-products are played, the production cost is facilitated to be reduced, and the engineering bacteria have important application prospects.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an engineering bacterium capable of simultaneously knocking out lactate dehydrogenase and acetolactate synthase genes, its construction method and its application in the fermentation production of 1,3-propanediol. Background technique: [0002] 1,3-Propanediol (PDO) is an important chemical raw material, which can be used as an organic solvent in high pressure lubricants, dyes, inks, antifreeze and other industries. PDO can be used to synthesize polyester and polyurethane, heterocycles, pharmaceutical intermediates, etc., and is mainly used to synthesize polytrimethylene terephthalate (PTT). PTT is a new fiber-forming polyester polymer material that has achieved industrial scale after polyethylene terephthalate (PET) in the 1950s and polybutylene terephthalate (PBT) in the 1970s. A very promising new polyester material. In 1998, PTT was rated as one of the six new petro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N1/21C12P7/18C12R1/22
CPCC12N9/0006C12N9/1022C12N15/74C12N2800/101C12N2800/30C12P7/18C12Y101/01028C12Y202/01006
Inventor 周胜秦启伟王著希黄友华
Owner SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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