Cloning And Research Of Carotenoid Biosynthesis Related Genes From Methylobacterium SP.MB200

Methylotrophic bacteria are a group of microorganisms can utilizing C1compounds such as methanol and methylamine and so on as their only carbon and energy source, and the unique metabolic pathways make them have widely applications, such as production of single cell protein, amino acids and carotenoids, etc. So the research of biosynthesis pathways of theses organisms is significant both in theory and practical application.In this work, a total of33colorless mutants were screened from the11552mutants library of M. sp. MB200by transposon mutagenesis, and eight genes, are relative to color, were cloned from the colorless mutants by molecular cloning techniques. Bioinformatic analysis of these eight genes respectively, and found that three genes were related to carotenoid biosynthetic pathways. GGPP synthase may catalyze the farnesyl pyrophosphate (FPP) to form GGPP, CrtB catalyze the GGPP to form phytoene, and CrtI is functive for desaturation of phytoene in two ways, it catalyzes the phytoene to form the red lycopene with four desaturations, or to form the yellow neurosporene with three desaturations, therefor CrtI plays an important role in the carotenoid biosynthetic pathway. This experiment chose crtI gene for further study. Bioinformatics analysis found the crtI gene cloned from the mutant MBW31, the gene size is1539bp, encoding512amino acids, it shared93%identity with the crtI gene from M. populi BJ001、M. chloromethanicum CM4and M. extorquens AM1at the nucleotide level, shared96%similarity with CrtI from M. extorquens PA1、M. chloromethanicum CM4and M. populi BJ001in the amino acid level.A recombinant plasmid pCM80-crtI was futher constructed by ligating crtI to vector pCM80, then was imported into M. sp. MB200to obtain recombinant strain MB200/pCM80-crtI. The activity of enzyme CrtI from MB200/pCM80-crtI was increased42%than that of the wild strain M. sp. MB200. In addition, the extracted pigments were detected by spectrophotometry and HPLC, no product of lycopene was found, which indicated that the role of CrtI in the carotenoid biosynthetic pathway may be catalyzing the phytoene to form neurosporene with three desaturations, rather than catalyzing the phytoene to form lycopene with four desaturations. The experimental results provided a theoretical reference to improve the biosynthetic and metabolic pathways of carotenoid in Methylobacterium.