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Method for improving activity of rate-limiting enzyme in methanol metabolism process of escherichia coli

A technology of methanol metabolism pathway and Escherichia coli, which is applied in the field of assembly and regulation of Escherichia coli methanol metabolism pathway, can solve the problems of low utilization efficiency, low efficiency and backwardness of strain genetic manipulation, etc., and achieve the effect of realizing methanol metabolism and improving the effect of methanol metabolism

Active Publication Date: 2017-10-20
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these microorganisms can use methanol as a raw material, their utilization efficiency is low. The reason is that, on the one hand, most of the methanolotrophic bacteria are aerobic, and on the other hand, the genetic manipulation of the strains is inefficient and backward.

Method used

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  • Method for improving activity of rate-limiting enzyme in methanol metabolism process of escherichia coli
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  • Method for improving activity of rate-limiting enzyme in methanol metabolism process of escherichia coli

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Embodiment 1

[0027] Example 1: Escherichia coli Methanol Metabolic Pathway Assembly

[0028] The invention assembles the methanol metabolism pathway dependent on NAD methanol dehydrogenase and RuMP pathway in Escherichia coli. Mdh2 derived from Bacillus methanolicus MGA3 was selected by comparing methanol dehydrogenase Mdh from different sources with 3-hexose-6-phosphate synthase Hps and 3-hexose-6-phosphate isomerase Phi and Hps-Phi, after the gene was synthesized, the gene was cloned into the plasmid pETDuet, and the plasmid pETDuet-Mdh2 and the plasmid pETDuet-Mdh2-RuMP were constructed. The plasmid was introduced into Escherichia coli BL21(DE3) by conventional methods, and the obtained strains were named M and MR, respectively, and were stored in the form of glycerol tubes, wherein the strain M only overexpressed the gene Mdh2, and was connected to the enzyme cutting site of the vector The dots are NcoI and BamHI; strain MR overexpresses genes Mdh2 and Hps-Phi, and the restriction sit...

Embodiment 2

[0029] Embodiment 2: Enhancing the enzymatic activity of methanol dehydrogenase

[0030] In order to enhance the activity of methanol dehydrogenase Mdh, three schemes have been adopted in this embodiment:

[0031] Scheme 1 is to enhance the enzyme activity of methanol dehydrogenase through directed evolution, but the enzyme activity is not significantly improved;

[0032] The second scheme is to enhance the enzymatic activity of methanol dehydrogenase by adding activator protein, in which the activator protein adopts two sources: one is to add the homologous activator protein nudF from E.coliMG1655 to construct the strain BL21(DE3) / pETDuet-nudF -Mdh2 and BL21(DE3) / pETDuet-nudF-Mdh2-RuMP, the strains were named nudF-M and nudF-MR, respectively;

[0033] The second is to add heterologous activating protein ACT from Bacillus.methanolicusPB1 to construct strain BL21(DE3) / pETDuet-ACT-Mdh2, and the strain is named ACT-M;

[0034] Scheme 3 is to replace methanol dehydrogenase Mdh2....

Embodiment 3

[0036] Embodiment 3: the detection of methanol dehydrogenase enzymatic activity

[0037] In the present invention, the enzyme activity of detecting methanol dehydrogenase Mdh adopts the color reaction of Nash reagent (i.e. acetylacetone reagent) and formaldehyde, and detects the activity of methanol dehydrogenase (i.e. the ability of methanol to be oxidized to formaldehyde) by the amount of formaldehyde generated. .

[0038] M9 medium: 17.1g / LNa 2 HPO 4 12H 2 O, 3g / LKH 2 PO 4 , 10g / LNH 4 Cl, 0.5g / LNaCl, trace elements, and 10g / LGlucose.

[0039] Nash reagent: 150g / L ammonium acetate, 3ml / L acetic acid, 2ml / L acetylacetone.

[0040] The strain in the glycerol tube was connected to the shaking tube to rejuvenate the strain, cultivated overnight at 37°C, 200rpm, collected the bacteria, centrifuged at 5000g, 7min, resuspended in M9, transferred to 9ml of M9 medium, added 0.5M methanol to start the reaction, different Time sampling, mixed with Nash reagent 1:1, reacted at 5...

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Abstract

The invention discloses a method for improving the activity of rate-limiting enzyme in a methanol metabolism process of escherichia coli. After genes Mdh and Hps-Phi are overexpressed by the escherichia coli, the enzyme activity of methanol dehydrogenase is improved by adding activator protein nudF, so that the metabolic rate of methyl alcohol is increased; the genes Mdh and Hps-Phi are from Mdh2 and Hps-Phi of bacillus methanol MGA3; the activator protein nudF is from E.coli MG1655. According to the method, a methanol metabolism path is assembled in the escherichia coli, so that methanol metabolism is realized; furthermore, the enzyme activity of the methanol dehydrogenase Mdh is improved through molecular improvement, thus realizing enhancement of the methanol metabolism.

Description

technical field [0001] The invention relates to the assembly and regulation of the methanol metabolism pathway of Escherichia coli, in particular to a method for enhancing the activity of the rate-limiting enzyme methanol dehydrogenase in the methanol metabolism pathway, thereby increasing the metabolism of methanol in the Escherichia coli. Background technique [0002] With the rapid development of metabolic engineering and the rise of biosynthesis, the ability of humans to transform microorganisms as cell factories for biomanufacturing has been significantly improved. At present, the raw materials of bio-manufacturing are still mainly grains. With the growing food crisis and rising prices, bio-manufacturing using grains as raw materials is facing a severe test. Therefore, in order to solve the problem of raw material sources and realize the sustainable development of bio-manufacturing, the "methanol economy" is gradually developing into a reasonable alternative energy sour...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/70C12N1/21C12R1/19
CPCC12N9/0006C12N15/70C12Y101/01244
Inventor 陈可泉陆晓璐王昕张博文毛静文
Owner NANJING UNIV OF TECH
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