CN-119736189-B - Method for improving styrene stress resistance of gluconobacter oxydans

Abstract

The invention discloses a method for improving the styrene stress resistance of gluconobacter oxydans, and belongs to the technical fields of genetic engineering and microbial engineering. According to the invention, firstly, styrene is added into a culture medium in a gradient way, so that the adaptive evolution of the wild type gluconobacter oxydans to the styrene is carried out, and finally, the obtained evolved strain ST remarkably improves the tolerance of the gluconobacter oxydans to the styrene. And secondly, the stress capability of the gluconobacter oxydans on styrene is improved by knocking out flagellin FlgE in the gluconobacter oxydans, and the growth performance of the gene-deleted gluconobacter oxydans ST-delta flgE prepared by the method is far stronger than that of wild gluconobacter oxydans after being cultured for 10 hours under 15g/L of styrene.

Inventors

• ZHANG XIAN
• CHEN YAN
• RAO ZHIMING
• Sha Aobo
• XU MEIJUAN
• YANG TAOWEI

Assignees

• 江南大学

Dates

Publication Date

20260505

Application Date

20241205

Claims (5)

1. 1. The engineering bacteria of the gluconobacter oxydans is characterized in that the engineering bacteria of the gluconobacter oxydans knock out flagellin genes flgE in the bacillus oxydans (Gluconobacter oxydans) ST, the bacillus oxydans ST is preserved in China center for type culture collection (China center for type culture collection) with the preservation number of CCTCC NO: M20242435, and the nucleotide sequence of the flagellin genes flgE is shown as SEQ ID NO. 2.
2. 2.A method for improving the styrene stress resistance of gluconobacter oxydans is characterized in that the method is used for inhibiting or weakening the expression of flagellin FlgE in the gluconobacter oxydans, the nucleotide sequence of a flagellin gene flgE is shown as SEQ ID NO.2, the gluconobacter oxydans comprises gluconobacter oxydans 621H or gluconobacter oxydans ST, and the gluconobacter oxydans ST is preserved in China center for type culture collection (China center for type culture collection) at the 11 th month of 2024, and the preservation number is CCTCC NO. M20242435.
3. 3. The method according to claim 2, characterized in that it is a knock-out of the flagellin gene flgE in gluconobacter oxydans.
4. 4. The application of flagellin FlgE in improving the styrene stress resistance of gluconobacter oxydans is characterized in that the application is to knock out the flagellin FlgE gene of the gluconobacter oxydans, the amino acid sequence of the flagellin FlgE is shown as SEQ ID NO.1, and the nucleotide sequence of the gene for encoding the flagellin FlgE is shown as SEQ ID NO. 2.
5. 5. The engineering bacterium of claim 1, or the application of the method of any one of claims 2-3 in microbial treatment of wastewater containing styrene or biocatalysis with styrene as a substrate.

Description

Method for improving styrene stress resistance of gluconobacter oxydans Technical Field The invention relates to a method for improving styrene stress resistance of gluconobacter oxydans, belonging to the technical fields of genetic engineering and microbial engineering. Background Gluconobacter oxydans (Gluconobacter oxydans) is one of the most commonly used microorganisms in industrial biotechnology, and the cell outer membrane is rich in alcohol, aldehyde dehydrogenase and oxidoreductase in cytoplasm, so that alcohol, aldehyde and carbohydrate can be rapidly and incompletely oxidized, and the substrate can be reacted in the periplasmic space without being transported into the cell, so that the Gluconobacter oxydans has been widely used for synthesizing compounds such as gluconic acid, miglyol, vitamin C and the like at present and has wide market in the industries of food additives, animal feeds, cosmetics, pharmacy and the like. At present, in the catalytic production process of the gluconobacter oxydans, various stresses from the external environment are inevitably faced, as most catalytic substrates are indissolvable in water, organic cosolvent is often required to be added for improving the solubility of the substrates, the gluconobacter oxydans is gram-negative bacteria, the tolerance to solvents is poor, the normal conversion is not easy to carry out, and the physiological activity and the viability of the gluconobacter oxydans are seriously influenced by the added solvents or environmental stresses caused by the organic substrates, so that the efficiency of biocatalysis reaction is seriously limited. Furthermore, solvent stress exposes Gluconobacter oxydans to lethal or sublethal environments, which have a great impact on cell survival. In order to achieve the win-win of yield and efficiency, it is extremely important to improve the tolerance of Gluconobacter oxydans to organic substrates such as styrene and solvents. Therefore, by means of adaptive evolution combined with genetic modification, the tolerance of the gluconobacter oxydans to styrene can be improved, and the leap of the tolerance property of the gluconobacter oxydans can be realized. Disclosure of Invention Aiming at the defects of the prior art, the invention provides a method for improving the styrene stress resistance of gluconobacter oxydans, which aims to solve the technical problems that the gluconobacter oxydans has poor solvent tolerance, is unfavorable for the normal transformation, and the added solvent or organic substrate causes environmental stress, so that the physiological activity and the viability of the gluconobacter oxydans are seriously influenced, and the efficiency of the biocatalysis reaction is seriously limited. The first technical scheme provided by the invention is that a strain of Gluconobacter oxydans (Gluconobacter oxydans) ST is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M20242435 in year 2024 and month 11. The invention also provides an adaptive evolution method of the gluconobacter oxydans which tolerates high-concentration styrene. The adaptive evolution method of the high-concentration resistant styrene comprises the step of domesticating the glucose-oxidizing acetobacter by adding styrene into a culture medium in a gradient way. In certain embodiments, the starting strain from which styrene adaptively evolved is Acetobacter oxydans 621H. In certain embodiments, the styrene adaptive evolution strain is cultured at a temperature of 30 ℃ and a rotational speed of 220rpm for 36-48 hours. In certain embodiments, the initial styrene concentration in the styrene adaptation is 0.2g/L, the styrene concentration increases sequentially with a 0.2g/L gradient, and the final maximum concentration of gluconacetobacter oxydans-tolerant styrene is 15g/L. The second technical scheme provided by the invention is a microbial preparation containing the gluconobacter oxydans ST of the first technical scheme. In certain embodiments, the concentration of the gluconobacter oxydans ST in the microbial preparation is not less than 1 x 10 6 CFU/mL or 1 x 10 6 CFU/g. The third technical scheme provided by the invention is that the engineering bacteria of the gluconobacter oxydans are obtained by knocking out flagellin genes flgE from the engineering bacteria of the gluconobacter oxydans ST in the first technical scheme, wherein flagellin FlgE is a protein related to regulation of cell movement and biofilm formation. In certain embodiments, the flagellin FlgE has the amino acid sequence shown in SEQ ID No. 1. In certain embodiments, the nucleotide sequence of the gene encoding the flagellin FlgE is shown in SEQ ID No. 2. In some embodiments, the gene deletion is to synthesize a fragment of 2000bp from 1000bp homology arms on the upstream and downstream of flagellin FlgE gene through fusion PCR, then connect the fragment with knockout expression vector to construct recombinant