Plasmid, recombinant engineering bacteria and preparation method of hyaluronic acid with uniform molecular weight

Abstract

A plasmid, recombinant engineering bacteria and a preparation method of hyaluronic acid with uniform molecular weight belong to the technical field of gene engineering. A first expression plasmid containing a Pasteurella multocida hyaluronan synthase gene (SEQ NO.1 in the sequence table) and a second recombinant expression plasmid containing hyaluronic acid precursor synthesis related genes (SEQ NO.2, SEQ NO.3, SEQ NO.4 and SEQ NO.5 in the sequence table) cloned from Bacillus subtilis are introduced into gram positive safe microorganisms, and an expression regulation and control system consisting of the two recombinant expression plasmids respectively controls hyaluronan synthase and hyaluronic acid precursor synthesis related enzyme, so as to obtain hyaluronic acid with uniform molecular weight by a biosynthesis method. The method provided by the invention not only improves the capability of the host strains to synthesize hyaluronic acid, but also can obtain several varieties of sufficient pure hyaluronic acid with uniform molecular weight.

Description

technical field [0001] The invention relates to a method in the technical field of genetic engineering of microorganisms, in particular to a biosynthetic method for preparing several hyaluronic acids with uniform molecular weights for safe engineering strains by using Gram-positive safe microorganisms constructed in the form of plasmids and genome integration. Background technique [0002] Hyaluronic acid (Hyaluronic Acid, Hyaluronan, HA) is a linear unbranched macromolecule formed by repeated cross-linking of disaccharide units composed of β-D-glucuronic acid (GlcUA) and β-D-N-acetylglucosamine (GlcNAc). Acid mucopolysaccharides. The average molecular weight (MW) range of natural hyaluronic acid is 2×10 4 -7×10 7 Da. HA with different molecular weights has different biological functions: high molecular weight HA (HMW-HA, MW>2×10 6 ) has high viscoelasticity, plasticity, permeability and good biocompatibility. Clinically, hyaluronic acid is mainly injected into the kn...

AI Technical Summary

Problems solved by technology

Although the above technology can realize the heterologous synthesis of hyaluronic acid in Bacillus subtilis, if more refined hyaluronic acid, that is, homogeneous molecular weight hyaluronic acid, is efficiently prepared, and homogeneous hyaluronic acid with different molecular weights is obtained through artificial regulation, now Not yet possible with technology

Images