Glycerol kinase mutant gene glpK of Bacillus subtilis and application thereof
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A Bacillus subtilis, mutation gene technology, applied in the field of engineering bacteria, to achieve the effect of improving the ability to use glycerol and increasing the accumulation level of riboflavin
Active Publication Date: 2018-11-30
ZHENGZHOU UNIVERSITY OF LIGHT INDUSTRY
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[0004] The present invention proposes a Bacillus subtilis glycerol kinase mutant gene glP And applicat
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[0034] Step 1: Screen the starting strains of the Bacillus subtilis system without antibiotic markers B. subtili s 168 Δupp and the construction of basic operation plasmid pSS
[0035] Bacillus subtilis starting strain used in the present invention B. subtili s 168 Δupp from B. subtilis 168. The basic operation plasmid pSS is derived from pUC18. For the detailed construction process of the two, please refer to Shi, T., Wang, G., Wang, Z., Fu, J., Chen, T., Zhao, X., 2013. Establishment of a Markerless Mutation Delivery System in Bacillus subtilis Stimulated by a Double-Strand Break in the Chromosome. PLoS one. 8, e81370.
[0036] The present invention does not limit the construction method of the basic operation plasmid pSS and the selection of the resistance gene.
[0037] Step 2: Engineering Strains B. subtilis M270I build process
[0038] (1) to the strain B. subtili s 168 Δupp Introduce genetic mutations without trace on the genome glP (G810T) The o...
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Abstract
The invention especially relates to the glycerol kinase mutant gene glpK of Bacillus subtilis and application thereof, belonging to the field of engineering bacteria. The base sequence of the mutant gene glpK is as shown in SEQ ID No. 1. An engineered bacterium constructed in the invention and containing the glycerol kinase mutant gene glpK (G810T) of Bacillus subtilis has biosafety and clear genetic background; and the included mutant gene glpK (G810T) can greatly improve the glycerol utilization of Bacillus subtilis, and under the condition of glycerol basic salt shake-flask culture, the included mutant gene glpK (G810T) increases the specific growth rate of the bacterium by 11% and allows maximum biomass to be 16% or more.
Description
technical field [0001] The invention relates to the field of engineering bacteria, in particular to a glycerol kinase mutant gene glpK of Bacillus subtilis and its application. Background technique [0002] Glycerol molecules contain a higher carbon content (52.8%), and high reducing properties (reducing power is twice that of glucose), which can produce a higher yield of reducing compounds, so it can be used as a cheap alternative to the microbial fermentation industry carbon source. Microorganisms ferment glycerol to produce different chemicals such as 1,3-propanediol, dihydroxyacetone phosphate, 1,2-propanediol, propionic acid, 2,3-butanediol, lactic acid, formic acid, acetic acid, ethanol, butyric acid, Chemicals such as butanol, succinic acid, pyruvate, phosphoenolpyruvate, etc. Genetically engineered bacteria with bacteria as host bacteria have the advantages of short fermentation cycle, simple raw material requirements, and mature genetic engineering technology. In...
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