| Rhodotorula glutinis,a member of the Rhodotorula family,is of great value in the biological,food and chemical fields.A strain of R.glutinis X-20 with high selenium enrichment,carotenoid and lipid synthesis capacity was obtained from a long-term selenium-enriched soil in the early stage of the research.however,its evolutionary relationships and metabolic pathways in the Rhodotorula are not well understood.In order to reveal the metabolic characteristics of R.glutinis X-20 and explore its application potential,this thesis analyzes the functional and metabolic pathways of this strain from the perspective of genome-wide analysis,and explores the potential applications of functional genes in R.glutinis X-20 through synthetic biology by means of heterologous synthesis of carotenoids in Saccharomyces cerevisiae,taking the carotenoid synthesis pathway as an example.The following main results were obtained:Whole-genome sequencing was used to resolve the genetic signature of R.glutinis X-20,and a genome of 20 contigs was successfully assembled,with a size of 21.6 Mb.Nr annotation showed 82%similarity between R.glutinis X-20 and Rhodotorula graminis.KEGG annotation showed that 3584 genes were predicted to have 354 metabolic pathways.GO annotation showed that 4558 genes were predicted for molecular functions,cellular components and biological processes.anti SMASH program predicted 3clusters of genes for carotenoid synthesis pathways.Phylogenetic trees revealed a closer relationship between R.glutinis X-20 and R.graminis in the Sporidiobolales family than in other red yeast species.Comparative genomic analysis revealed a high concentration of species-specific genes in R.glutinis X-20for transmembrane transport,enzyme activity and some pathogenicity genes.Studies of the evolutionary rate（Ka/Ks）of single-copy orthologous genes showed that the biosynthesis and cell division evolution were obvious during the evolution of the R.glutinis X-20.Secondary metabolite identification revealed that R.glutinis X-20 had 654 metabolites and 124 lipid secondary metabolites such asβ-carotene,astaxanthin,carotenoid and myristic acid.Taking the carotenoid synthesis pathway as an example,analysis of key genes in this pathway revealed that AL1 may possess the function of phytoene desaturase and AL2 may possess the function of 15-cis-p Hytoene synthase/lycopene beta-cyclase.By expressing the AL1 and AL2 genes of R.glutinis X-20 in Saccharomyces cerevisiae INVSc1,it was demonstrated that AL1 and AL2 function as p Hytoene desaturase and 15-cis-p Hytoene synthase/lycopene beta-cyclase,respectively,and when co-expressed could synthesize 1.59 mg·L-1(0.63mg·g-1DCW)ofβ-carotene.To achieve efficient heterologous synthesis of carotenoids,the AL1 gene expression module and the AL2 gene expression module were co-introduced into an engineered strain W8 optimized for the precursor mevalonate（MVA）pathway using the principle of homologous recombination in Saccharomyces cerevisiae,and 17.89 mg·L-1(2.80 mg·g-1DCW)β-carotene and 9.37 mg·L-1(1.47 mg·g-1DCW)lycopene were synthesized in the engineered strain.By optimising the fermentation process,the engineered strain synthesised 53.88 mg·L-1of lycopene and 64.23 mg·L-1ofβ-carotene,a 5.75-fold and 3.59-fold increase respectively compared to unsupplemented fermentation.Above research suggest that the use of genome sequencing and comparative genomic analysis can effectively explore potential functional genes in R.glutinis X-20,and that gene function validation provides important research ideas for the further exploitation of these strains.The optimization of engineering strains and fermentation process provides application guidance for production practice. |
PDF Full Text Request