Basic Study On Expression System Of Heterologous Protein In Methylosinus Trichosporium OB3b

Methane-oxidizing bacteria (Methanotrophs) are a group of gram-negative bacteria that grow by using methane as source of carbon and energy. Methanotrophs contain a special enzyme system named methane monooxygenase (MMO), which not only can be used to produce many valuable chemicals, but also remediate the polutants of the enverionment. Therefore, methanotrophs and MMO are considered to be a kind of milti-functional biocatalyst. However, many problems limited their industrial applications including low MMO activity and lack of efficient NADH regeneration system. Currently, To construct methanotrophs by gene engeneering is popularly used, but study on expression of heterologous protein are little reported until now. So the aim of this thesis is to do such research in M. trichosporium OB3b and establish the base for further application of methanotroph.Gene manipulation of methanotroph could only be achieved by conjugative transfer and homologous recombination. In this study, the concentration of E. coli S17-1 and M. trichosporium OB3b in conjugation experiment were optimized and the results showed the optimal OD600 of E. coli S17-1 is 0.6-0.7 and OD540 of M. trichosporium OB3b is 0.3-0.4. About 500bp downstream gene of pMMO gene cluster, as expected insertion site of heterologous gene, was amplified from chromosome DNA of M. trichosporium OB3b, and that was then coloned into pTJS140 with kanamycin resistent gene cassette inserted for the construction of suitcide plasmid pSHK. Restriction enzyme analysis showed pSHK was successfully transform into E. coli S17-1. E. coli S17-1 (pSHK) and M. trichosporium OB3b conjugated on the optimized conditions and conjugants were screened by kanamycin. Finally, although trying many times, expected mutant could not be detected. The result showed pSHK could not insert into the expected region, that maybe result from low recombination frequence due to short homologous arm of pSHK designed or maybe the key role played by this 500bp gene for the growth of M. trichosporium OB3b.About 5200bp upstream gene region of sMMO was choosed to as insertion site, GFP expression plasmid pJSG was constructed by inserting a gfp ORF in pTJS175, then which was transformed into E. coli S17-1. By conjugation, pJSG was successfully transformed into M. trichosporium OB3b. PCR analysis showed pJSG was inserted into the chromosome DNA of M. trichosprium OB3b by single homologous recombination and the whole plasmid was located in upstream of sMMO gene cluster. Fluorecence analysis showed the mutant could express GFP successfully. Further cultivation by adding various concentration of Cu2+ and the results showed GFP expression was regulated by promoter of PmmoX and fluorecence could only be detected in low concentration of Cu2+.Based on this study, preliminary expression system of heterologous protein of M. trichosporium OB3b was established, that provide platform for further research on gene engineering and expression of heterologous protein in M. trichosporium OB3b.