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Engineering bacterium containing NADH (nicotinamide adenine dinucleotide) kinase gene and application thereof

A gene and kinase encoding technology, applied in the field of engineering bacteria containing NADH kinase gene, can solve problems such as multiple production costs

Active Publication Date: 2014-04-23
BEIJING UNIV OF CHEM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the key problem restricting the large-scale industrial production of PHB is its much higher production cost than petroleum-derived plastic materials.

Method used

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  • Engineering bacterium containing NADH (nicotinamide adenine dinucleotide) kinase gene and application thereof
  • Engineering bacterium containing NADH (nicotinamide adenine dinucleotide) kinase gene and application thereof
  • Engineering bacterium containing NADH (nicotinamide adenine dinucleotide) kinase gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1, the construction of recombinant bacteria E.coli JM109 (pBHR68pos5)

[0039] 1. Construction of recombinant expression vector pBHR68pos5

[0040] 1. Acquisition of expression vector p19pdc

[0041] A piece of double-stranded DNA is artificially synthesized, and its nucleotide sequence is sequence 1 in the sequence listing, wherein the nucleotides 28-208 from the 5' end of sequence 1 in the sequence listing are pyruvate decarboxylase promoters, and sequence 1 from 5 Nucleotides 213-248 at the 'end are multiple cloning sites, and nucleotides 249-334 at the 5' end of Sequence 1 are transcription terminators.

[0042] The above-mentioned artificially synthesized double-stranded DNA fragment shown in Sequence 1 was ligated into the T vector pMD19-T Simple (purchased from Japan Takara Company) by TA cloning method to obtain the expression vector p19pdc.

[0043] 2. Acquisition of NADH kinase gene pos5

[0044] Genomic DNA of Saccharomyces cerevisiae s288c (ATC...

Embodiment 2

[0069] Example 2, the application of recombinant bacteria E.coli JM109 (pBHR68pos5) in the preparation of PHB

[0070] 1. Fermentation culture

[0071] Obtaining seed solution: culture the recombinant strain E.coli JM109 (pBHR68pos5) prepared in Example 1 in LB-Amp liquid medium for 14 hours (37°C shaker, 200rpm) as the seed solution;

[0072] Fermentation: Inoculate the seed liquid into MSG-Amp liquid medium with an inoculation amount of 4% by volume, and ferment on a shaking table at 37°C at 200 rpm for 48 hours, and collect the fermentation product to obtain PHB.

[0073] The control recombinant bacterium E.coli JM109 (pBHR68) was fermented and cultured according to the above method, and the control fermentation product was collected.

[0074] 2. Detection of PHB content

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Abstract

The invention discloses an engineering bacterium containing an NADH (nicotinamide adenine dinucleotide) kinase gene and an application thereof. The invention provides a method for constructing a recombinant bacterium. The method is characterized by guiding an NADH kinase coding gene pos5 and a PHB (poly-beta-hydroxybutyrate) synthetic gene phbCAB into a target bacterium to obtain the recombinant bacterium. Experiments prove that the NADH kinase gene in the recombinant bacterium can promote transformation of NADH in cells to NADPH (nicotinamide adenine dinucleotide phosphate), thus improving synthesis of NADPH-dependent poly-3-hydroxybutyrate. Compared with former strains, the engineering bacterium has higher PHB production efficiency.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an engineering bacterium containing NADH kinase gene and its application. Background technique [0002] NAD(H) and NADP(H) are important coenzymes in organisms, they mainly participate in various redox reactions as electron carriers. Coenzymes are involved in a wide range of reactions, and changes in their concentration can cause changes in intracellular substance and energy metabolism, thereby affecting the synthesis of many metabolites. More and more attention has been paid to improving the efficiency of biosynthesis by regulating the coenzyme concentration and redox state ratio in cells. NADH kinase can use ATP and the like as a phosphate group donor to catalyze the phosphorylation of NADH to form NADPH, which plays a key role in the coenzyme metabolism in organisms. There are three NADH kinase genes in Saccharomyces cerevisiae, UTR1, YEF1 and POS5 (Genomic characterization of P...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12P7/62C12R1/19
Inventor 李正军陈国强吴琼张洁
Owner BEIJING UNIV OF CHEM TECH
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