甲基营养菌 Methylobacterium sp MB200 中crtI基因的克隆及表达

八氢番茄红素脱氢酶( CrtI)催化八氢番茄红素经过4次脱氢合成番茄红素,或者经过3次脱氢合成链孢红素,在类胡萝卜素的生物合成中发挥重要的作用.以甲基营养菌Methylobacterium sp MB200为原始菌株,首先采用转座子突变技术构建部分突变体库共11552株,筛选得到33株颜色发生变化的目的突变体,随后利用分子克隆技术从目的突变体中获得crtI基因的完整ORF,长为1539 bp,编码512个氨基酸.与来自M.populi BJ001、M.chloromethanicum CM4和M.extorquens AM1的crtI一致性均为93％.将crtI与载体pCM80连接得到重组质粒pCM80-crtI,导入原始菌株中得到重组菌MB200/pCM80-crtI.测定原始菌株与重组菌株的CrtI酶活,结果发现,重组菌株CrtI的酶活与原始菌株相比约提高了40％.实验结果为完善甲基营养菌中类胡萝卜素的生物合成代谢途径提供了理论参考.

Cloning and Expression crtI Gene in Methylobacterium sp MB200

The partial mutants library of M.sp MB200 was constructed by transposon mutagenesis and 11552 mutants were obtained which could survived in medium with methanol as the sole carbon source.Rescreening results showed that 33 strains were colorless mutants.The crtI gene was got from a colorless mutant by molecular cloning techniques.It shared 93% identity with the crtI gene of M.populi BJ001,M.chloromethanicum CM4 and M.extorquens AM1 at the nucleotide level reported in Genbank.The gene size is 1539 bp,encoding the phytoene desaturase(CrtI) that catalyzes phytoene to form lycopene with four desaturations,or catalyzes phytoene to form neurosporene with three desaturations,therefore it plays an important role in the biosynthetic pathway of carotenoid.Here,a recombinant plasmid pCM80-crtI was constructed by ligating the crtI to the vector pCM80,then imported into M.sp MB200 to obtain recombinant strain MB200/pCM80-crtI.The activity of enzyme from MB200/pCM80-crtI was more by 40% than M.sp MB200.The experimental results provided a theoretical reference to improve the biosynthetic and metabolic pathways of carotenoid in Methylobacterium.