The Optimization Of Electrotransformation Conditions And The Construction Of Expression System For Cytophaga Hutchinsonii

With the development of human society, oil, coal and other fossil fuels on the earth will be gradually dried up, people will be faced with the challenges posed by the lack of resources. Cellulose is the oldest and the most abundant natural polymer on the earth and is mankind’s most valuable natural renewable resources. It is considered as new biomass resources that should be urgently developed and has become the hot focus, at the same time more and more attention is paid to the efficient use of the lignocellulose.There is a wide variety of microorganisms which can degrade cellulose in the nature. The mechanism of degradation of cellulose is also showing diversity. The aerobic bacterium Cytophaga hutchinsonii demonstrates efficient and complete degradation of crystalline cellulose, but its mechanism which is significantly different from that of fungi and anaerobic bacteria is considered to have a special kind of degradation mechanism.To further elucidate the degradation mechanism of cellulose of Cytophaga hutchinsonii, this study establishes the electroporation method for the bacterium for the first time, on this basis of which the mutant is acquired through gene knockout technology and its characterizations are preliminarily studied. In addition, in order to meet the requirement for the system of native expression a promoter gene library is established and then some of the active promoters are screened. Finally, expression system is initially established by using the promoters screened as expression elements. The main contents and results are as follows:1The establishment and optimization of electroporation methods of C. hutchinsoniiThe electroporation method of C. hutchinsonii is established for the first time aiming at its existing deficiencies of genetic transformation methods. Through optimizing culture conditions, growth state, shock buffer, the electric field strength and plasmid concentration, the recovery time, etc., the optimal conditions of electroporation is obtained and the electrotransformation efficiency for the plasmid pEPO293constrcted in our lab can reach5×104transformants/μg DNA. The method is simple, stable and has good reproducibility, providing a new tool for the research of the exogenous gene into C. hutchinsonii and its gene knockout.2Construction of gene deletion mutant of C. hutchinsonii and its characterization researchThe CHU₃732gene deletion mutants are successfully screened through electroporation of a gene knockout suicide plasmid pLYL03₃732for the outer membrane protein gene of C. hutchinsonii （CHU₃732）. A preliminary study for the characterizations of the mutant found that its ability to degrade the cellulose significantly decreased; the ability of spreading on plates containing0.5%and1%of agar defects, but the cellulose adsorption rate between wild-type and mutant shows no significant difference. It’s not clear that the mechanism of how the gene affects the movement and cellulose degradation. By the initial localization of this protein, no its structure is exposed to the outside of cell wall, suggesting that it is not directly involved in the adsorption of cellulose and may participate in movements and the transportation of degradation of substances.3The construction of promoters gene library for C. hutchinsonii and screening of strong promotersA transposon probe capture plasmid pUCTn19is constructed by use of promoterless gfp gene as the reporter gene. After being electrotransformed into C. hutchinsonii, transposon on the plasmid will be randomly inserted into the C. hutchinsonii genome, producing a large number of mutants. Strong promoters from the promoter library are screened by detecting the fluorescence intensity. Finally, four strong transcriptional activity promoter fragments are obtained, laying the foundation for the establishment of its own expression system or complement system.4The preliminary construction of self-expression system of C. hutchinsonii Activity expression of cellulase of C. hutchinsonii in Escherichia coli is difficult, we use screened OmpA promoter as an expression element within C. hutchinsonii to overexpress the cellulase CHU₁280, and isolate and purify it by fusion expression with the His tag from the cells, and the successful expression of soluble fusion protein provides a new way to further study the characteristics of the cellulase.