Development And Application Of Genetic Manipulation Tools For Rhodosporidium Toruloides

R.toruloides is an important non-model oil-producing microorganism.Under the limitation of nitrogen,the accumulation of oil production can reach 70%of its dry cell weight.Various fatty acid derived fuels and chemicals,such as fatty acids ethyl ester and polyunsaturated fatty acids,have been produced in R.toruloides by the introduction of different modifying enzymes or metabolic pathways.Also,it has a wide range of available carbon and nitrogen sources,natural resistance to toxic substances such as acetic acid in the cellulose hydrolysate.Therefore,it is considered to be a strain with broad industrial application prospects.Although the genome sequencing and gene function annotation of several strains of this yeast have been performed,the genetic manipulation tools are still relatively lacking,such as the lack of available auxotrophic and resistance selection markers,promoters and terminators.It is still difficult to carry out metabolic engineering and synthetic biology research.These limit the development and application of R.toruloides to some extent.A promoter is a basic functional element in metabolic engineering and an effective tool for regulating metabolic pathways,and it could significantly affect the initiation,duration and expression of transcription of the target gene.A set of promoters with varying strengths can be used to optimize the flux of a target metabolic pathway by tuning the expression of each individual pathway gene.Here,a group of constitutive promoters with strengths varying over 2 orders of magnitude were identified from R.toruloides by analyzing its transcriptome data under different growth conditions.These promoters were first cloned and characterized using an enhanced green fluorescent protein（EGFP）as a reporter under eight conditions.and 31 promoters were identified with strength varied from 0.1to 19.0 folds of the commonly used strong promoter of the glyceraldehyde-3-phosphate dehydrogenase gene(P_GPD1).The resultant promoters were then used to optimize the linoleic acid biosynthetic pathway in R.toruloides in 5 different media.Among them,corncob hydrolysate was used as the fermentation substrate with the highest yield,which increased the linoleic acid production from 292.7 mg/L to 485.2 mg/L.This work has enriched the promoter library of R.toruloides,and helped develop R.toruloides as a platform organism for applications in biomanufacturing and synthetic biology.With the deepening of metabolic engineering research,it is often necessary to modify multiple genes in a host bacterium simutanously to realize the comprehensive regulation of cells.For R.toruloides,which has weak homologous recombination and relatively lack of gene manipulation tools,the short sequence and self cleavable 2A peptide is very necessary in the construction of polycistronic expression vector.This work aimed to characterize various 2A peptide sequences for metabolic engineering applications in R.toruloides.First of all,by expressing a fluorescence reporter cassette(P_GPD1::flag-LEU2::2A::T_SV40-EGFP::T_35S),we reached the conclusion that twenty-two 2A peptides exhibited a cleavage efficiency of 23-94%calculated by EGFP fluorescence and western blot.It’s worth noting that the cleavage efficiency of 2A peptide from Equine rhinitis B virus（ERBV-1）was the highest and closed to 100%.Then,co-expression of three/four proteins was successfully achieved mediated by two/three ERBV-1 sequence.Next,by applying three ERBV-1 sequences to theα-linolenic acid metabolism pathway of R.toruloides,the production ofα-linolenic acid was increased by 160.8%.These results suggested that 2A peptides was suitable for expression and translation of multiple proteins in metabolic engineering applications in R.toruloides.Episomal expression vectors typically have higher copy numbers to achieve strong gene expression compared to chromosomal integration.In addition,it’s more convenient and flexible for DNA manipulation applications.However,the current study has not yet determined the episomal plasmid of R.toruloides,and the expression of heterologous genes or CRISPR/Cas9-based genome editing needs to be completed by integration.Thus,this work aims at exploring the autonomously replicating sequence（ARS）and centromeric（CEN）elements of the unconventional yeast-R.toruloides.Consequently,a stable episomal plasmid was constructed based on ARS/CEN and was applied to CRISPR/Cas9 editing in R.toruloides.All in all,our work enriches the existing tool library of R.toruloides and successfully applied these tools for strain transformation,with important implications for its metabolic engineering applications and synthetic biology research,and also important guidance and significance for mining,characterizing and transforming new biological elements in new species.