CN-120818478-B - High-yield polymerized glutamate engineering bacterium and construction method and application thereof

Abstract

The application relates to a high-yield polymerized glutamate engineering bacterium and a construction method and application thereof, belonging to the technical field of genetic engineering. According to the scheme, the engineering bacteria for biological fermentation are constructed, the high-efficiency directional assembly of the high-molecular polymerized glutamate is realized by utilizing an integrated cell factory biological synthesis technology, the engineering bacteria provided by the application are suitable for continuous production of the polymerized glutamate by industrial biological fermentation, and the prepared polymerized glutamate has narrower molecular weight distribution and better product uniformity.

Inventors

• SONG HAIMEI
• DIAO HONGXIA
• TAI WENJING
• ZHANG CHAOYI
• WANG MENGJIAO
• CUI YANRU
• DENG DEFA
• WANG FAGUO

Assignees

• 青岛海大生物集团股份有限公司

Dates

Publication Date

20260505

Application Date

20250918

Claims (5)

1. 1. An engineering bacterium is characterized by being a recombinant strain which takes bacillus as a chassis microorganism and expresses acyltransferase and glycosyltransferase; the acyltransferase is RhlA, the glycosyltransferase is RhlB, the RhlA and the RhlB are derived from Pseudomonas aeruginosa, the RhlA and the RhlB share one promoter, and a linker is arranged between the RhlA and the RhlB; the engineering bacteria also express pgsB, pgsC and pgsA.
2. 2. The engineering bacterium according to claim 1, wherein the engineering bacterium is a bacillus subtilis, bacillus licheniformis or bacillus amyloliquefaciens.
3. 3. The engineering bacterium according to claim 1, wherein the promoter of pgsB is P43 and/or, The promoter of pgsA is xylA, and/or, The promoter of pgsC is veg.
4. 4. The engineered bacterium of claim 1, wherein the promoters of RhlA, rhlB and pgsA are inducible promoters.
5. 5. The application of the engineering bacteria in preparing poly-rhamnosyl glutamate by a biological fermentation method.

Description

High-yield polymerized glutamate engineering bacterium and construction method and application thereof Technical Field The application relates to a high-yield polymerized glutamate engineering bacterium and a construction method and application thereof, belonging to the technical field of genetic engineering. Background The traditional esterification preparation of polymerized amino acid and saccharides mainly adopts a chemical catalytic esterification method, and has the following defects of (1) a plurality of byproducts (such as sulfonation byproducts of concentrated sulfuric acid catalytic reaction is more than or equal to 15%), low purity of target products (85%), high reaction energy consumption (high temperature is required to be higher than 80 ℃ or high pressure condition), (3) difficulty in controlling molecular weight distribution of products (polydisperse coefficient PDI is more than 1.5), and (4) complex post-treatment caused by chemical catalyst residues. Through searching, a biological fermentation preparation method for preparing poly-glutamic acid sugar ester based on poly-gamma-glutamic acid and related engineering bacteria are not found at present, and therefore, a process capable of overcoming the process defects of the traditional chemical catalytic esterification method is necessary. It should be noted that the above background art is only for illustrating the technical route of the present application and does not necessarily constitute prior art. Disclosure of Invention In order to solve the problems, the application provides a high-yield polymerized glutamate engineering bacterium, a construction method and application thereof, and provides a high-efficiency preparation method of polymerized glutamate sugar ester based on enzyme catalysis self-assembly technology by means of the constructed engineering bacterium and adopting an integrated cell factory biosynthesis technology, which can realize accurate assembly of glutamate and functional oligosaccharide under mild conditions (below 45 ℃ and pH of 5.0-7.0), the selectivity of a product is more than 95%, the cost of raw materials is reduced by 50-60% compared with a chemical catalysis esterification method, and the preparation method is flexible in terminal dosage form and can be realized in liquid or solid dosage form. The application provides an engineering bacterium which is a recombinant strain taking bacillus as a chassis microorganism and expressing acyl transferase and glycosyltransferase. According to the application, bacillus subtilis is used as a chassis strain, synthesis of polyglutamic acid sugar ester is realized through design of two modules, the first module is synthesis of polyglutamic acid (gamma-PGA), and the synthesis of gamma-PGA is formed by catalyzing polymerization of glutamic acid or salt thereof through a gamma-PGA synthetase complex (PgsBCA). PgsB and PgsC together form a catalytic site, while PgsA transports gamma-PGA outside the cell membrane. Different expression levels of pgsB, pgsC and pgsA are achieved by integrating gamma-PGA synthetase complexes of different sources on the genome and by adding promoters of different intensities, the second module being to catalyze the esterification reaction of polyglutamic acid with functional oligosaccharides. The method mainly relates to heterologously expressed acylase and glycosyltransferase, wherein RhlA (acylase) and RhlB (glycosyltransferase) from Pseudomonas aeruginosa are fused through a flexible joint, the substrate transfer distance is shortened, the exogenous acylase and glycosyltransferase are expressed through a plasmid, the two modules are coupled, and finally, the esterification of glutamic acid and functional oligosaccharide into polyglutamic acid sugar ester is realized. Optionally, the engineering bacteria are chassis microorganisms of bacillus subtilis, bacillus licheniformis or bacillus amyloliquefaciens; optionally, the engineering bacteria are microorganisms with bacillus subtilis as a chassis. It should be noted that, the purpose of selecting bacillus subtilis is to express the polyglutamic acid (γ -PGA) synthase complex itself, and through over-expression, the content of the polyglutamic acid (γ -PGA) synthase complex can be further increased, which is beneficial to production, and the same effect can be achieved by using bacillus subtilis directly or by using other engineering bacteria expressing polyglutamic acid (γ -PGA) synthase complex or engineering bacteria not expressing polyglutamic acid (γ -PGA) synthase complex to over-express the polyglutamic acid (γ -PGA) synthase complex, so that the engineering bacteria can be adjusted and selected by those skilled in the art, and the setting and selection of culture conditions according to the selection of different engineering bacteria are also basic operations. Optionally, the acyltransferase comprises one or more of RhlA, ntASAT1, ntASAT, atSAT 1; Optionally, the glycosyltransferase comprise