Study On The Differential Synthesis Of Nostoxanthin In Sphingobium Strains

Sphingobium,a kind of gram-negative bacteria widely distributed in nature,belongs to ?-Proteobacteria,Sphingomonadales,Sphingomonadaceae.Most strains of Sphingobium are able to synthesize nostoxanthin,a polyhydroxy derivative of?-carotene that is only found in some prokaryotes.So far,the biosynthesis and regulation of nostoxanthin in Sphingobium has been less studied.This thesis studied on the differences of nostoxanthin synthesis in the two isolated Sphingobium strains,focusing on their growth and carotenoid production.The results are as follows:1.The isolated strains were identified as Sphingobium based on the phenotypes,16 S r DNA sequences and pigment profiles.Therefore,they were named Sphingobium sp.KIB1 and Sphingobium sp.KIB2.The whole genome sequence revealed a genome size of Sphingobium sp.KIB2 was about 3.9Mb.Meanwhile,the genome consists of 3746 coding genes with the GC content was about 66%.IPP,the precursor of carotenoids,was found to be derived from the MEP(methylerythritol)pathway.All the genes including crt E,crt B,crt I,crt Y,crt Z and crt G were identified and cloned,which were involved in the biosynthesis of nostoxanthin.Crt B,crt I,crt Y existed as a gene cluster crt YIB in the genome.Crt Y and crt I were partly overlapped,indicating the coupling of their transcription.Functional complementation of the six genes confirmed the function of their coding proteins in catalyzing the formation of GGPP,phytoene,lycopene,?-carotene,zeaxanthin and nostoxanthin,respectively.2.Under different culture conditions,Sphingobium sp.KIB2 had much high yields of nostoxanthin than Sphingobium sp.KIB1.Functional complementation revealed that the two strains shared similar enzymatic activities of crt E,crt B,crt I and crt Y.However,the Crt Z from Sphingobium sp.KIB2 exhibited much higher activity than that from Sphingobium sp.KIB1 as indicated by the ratios of zeaxanthin to totle carotenoids produced in E.coli(78% vs 22%).In addition,the Crt G from Sphingobium sp.KIB2 had a lower activity than that of Sphingobium sp.KIB1.Taken together,we concluded that Crt Z was the limiting enzyme for nostaxanthin biosynthesis in Sphingobium and Crt Z from KIB2 had a higher catalytic activity than that from KIB1.3.Treated by the chemical mutagen N-methyl-N-nitro-N-nitrosoguanidine(MNNG)followed by color-based colony screening and confirmed by HPLC detection,the following mutants of Sphingobium sp.KIB2 were obtained: M1 with no carotenoids,M7 accumulating phytoene,M13 accumulating lycopene,M14 accumulating ?-carotene,M22 accumulating zeaxanthin and M10 with high-yield of nostoxanthin.Under different culture conditions,M10 demonstrated higher production of nostoxanthin than wild type.When cultivated with Batch medium containing 30 g/L sucrose,8 g/L yeast extract,M10 produced nostoxanthin at11.898 mg/L,which was 1.9 times of that by its wild type.The differential production of nostoxanthin in Sphingobium was studied in this thesis.Our study provides new insights into the biosynthesis of nostoxanthin,which are valuable in metabolic engineering of Sphingobium for enhanced production of carotenoids.