Primary Research On Engineering Global Transcription Regulation In Yarrowia Lipolytica

Evolution of complex phenotypes in cells requires simultaneously tuning expression of large amounts of genes,which can be achievable by reprogramming global transcription.Lipophilicity is an important complex trait in oleaginous yeast Yarrowia lipolytica.It is necessary to explore the changes of which genes' expression levels will tune cellular lipophilic properties via the strategy of global transcription engineering.This study achieved a strategy of global transcription engineering in Y.lipolytica by modifying the sequences of a key transcriptional factor(TF),SPT15-like(Yl-SPT15).The combinatorial mutagenesis of this gene was achieved by DNA assembly of up to five expression cassettes of its error-prone PCR libraries.A heterologous beta-carotene biosynthetic pathway was constructed to research the effects of combined Yl-SPT15 mutants on beta-carotene and lipid production.As a result,a series of mutant strains were obtained.The highest beta-carotene production was 4.7 times the initial,and the lowest was 0.13 times,nearly 40-fold changing range.Genotype verification,comparative transcriptome analysis,and detections of the amounts of total,free fatty acids and proteins were made for the selected strains,indicating effective tuning of cells' lipophilic properties and complex phenotype.This study exploited the key pathways including RNA polymerase,ketone bodies metabolism,fatty acid synthesis and degradation that drastically determined cells' variable lipophilicity,and got some gene targets.This study has examined the effects of combinatorial mutagenesis of Yl-SPT15 on cells' capacity of producing beta-carotene and lipids.The lipophilic properties in Y.lipolytica could be effectively tuned by simultaneously regulating genome-wide multi-gene expression levels.The exploited gene targets and pathways could guide design and reconstruction of yeast cells for tunable and optimal production of other lipophilic products.