Engineering bacterium based on cellulose metabolic pathway key enzyme and implementation method of engineering bacterium based on cellulose metabolic pathway key enzyme

Abstract

The invention discloses an engineering bacterium based on a cellulose metabolic pathway key enzyme and an implementation method of the engineering bacterium based on the cellulose metabolic pathway key enzyme. The method comprises the following steps: building a vector as a template by whole-gene synthesis of streptomyces griseorubens genome DNA; respectively carrying out PCR amplification on the template and corresponding primers containing enzyme digestion sites, thereby respectively obtaining a nucleotide sequence of encoding exo-beta-1,4-glucanase, a nucleotide sequence of encoding endo-beta-1,4-glucanase, and a nucleotide sequence of encoding beta-1,4-glucosaccharase; sequentially connecting the amplified sequences to obtain a coexpression vector, thereby finally obtaining a recombinant coexpression vector and a recombinant expression vector; and successively transforming the recombinant coexpression carrier into an escherichia coli expression strain, so as to obtain a transgenic coexpression strain. A large number of in vitro expression synthesis of the exo-beta-1,4-glucanase, the endo-beta-1,4-glucanase and the beta-1,4-glucosaccharase is achieved by using genetic engineering means, and in-situ rapid degradation of cellulose is finally achieved.

Description

technical field [0001] The present invention relates to a gene in the technical field of biogenetic engineering and its engineering strain, specifically a kind of Streptomyces grisea based on exo-β-1,4-glucanase, endo-β-1 , 4‐glucanase and β‐1,4‐glucosidase engineering bacteria and its realization method. Background technique [0002] Lignocellulose is the most abundant natural polymer compound in the plant kingdom, and it is the main dry matter produced by plants through photosynthesis, mainly including cellulose, hemicellulose and lignin. It is estimated that the total dry weight of lignocellulose produced by photosynthesis of green plants in the world every year is 173 billion tons, and the total energy contained is as high as 2×10 18 KJ is equivalent to 10 times the energy consumed by the whole world every year. The biodegradation and depolymerization of lignocellulose is a highly complex process involving the participation of numerous enzyme systems. [0003] Cellulo...

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Problems solved by technology

[0005] The present invention is aimed at exo-β-1,4-glucanase, endo-β-1,4-glucanase and β-1,4-glucosidase in the prior art in Streptomyces grisea ( Most of Streptomyces griseorubens) are deficiencies that are induced and expressed at a very low level. An engineering bacterium based on key enzymes in the cellulose metabolism pathway and its realization method are proposed. Through whole-genome sequencing analysis, the Streptomyces genome was isolated from the Streptomyces genome and cellulose Gene sequences involved in degradation, including those encoding exo‐β‐1,4‐glucanase, endo‐β‐1,4‐glucanase, and β‐1,4‐glucosidase

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