Construction And Application Of Episomal Expression System In Candida Glycerinogenes

Episomal expression of genes has a simpler operation than integrated expression,but the absence of episomal plasmids in many industrial strains seriously affects the modification of strains,especially the expression of exogenous genes.Candida glycerinogenes is a industrial strain with autonomous intellectual property rights,and it has the multiple resistance properties and excellent fermentation performance,which is a potential industrial synthetic biology chassis cell,but the strain has no natural episomal plasmids and the integration of diploids is tedious and complicated,thus affecting the deep anabolic modification.This project aims to establish the episomal expression system of C.glycerinogenes and apply it to the episomal expression synthesis of caffeic acid and geraniol.The main studies are as follows:(1)To construction of episomal plasmids for C.glycerinogenes,a total of 10 episomal plasmids with GAPp,GFP,AOX1 t and URA5 as promoter,reporter gene,terminator and screening marker gene,respectively,were constructed using autonomous replication sequences(ARS)from different yeast strains,and investigated the performance of the constructed episomal plasmids in C.glycerinogenes,such as transformation efficiency,fluorescence expression intensity,copy number and presence status in the cell.The results showed that all of 212 ARS from Saccharomyces cerevisiae plasmid p YX212,pan ARS from Kluyveromyces lactis,PPARS1 and PPARS2 from Pichia pastoris and IOARS from Issatchenkia orientalis have autonomous replication capacity in C.glycerinogenes,and they all could be used for the construction of episomal plasmids in C.glycerinogenes.p TGAPp-GFP-AOX1t-pan ARSURA5,the episomal plasmid constructed by pan ARS,has the strongest expression performance and it did not affect strain tolerance performance.The plasmid stability test showed that the plasmid loss rate was only 9% at the fifteenth generation of the strain,which has high plasmid stability.(2)To further improve the performance of the obtained episomal plasmid p TGAPp-GFPAOX1t-pan ARS-URA5,its different structural components were optimized.It was shown that different endogenous inducible promoters and terminators from different sources could successfully express the exogenous gene GFP,with GAPp showing the highest regulatory intensity and URA5 t having the highest termination efficiency.Addition of Kozak sequences on both sides of the GFP start codon effectively increasesed the intensity of fluorescence expression.When the URA5 promoter was truncated by 250 bp,it increasesed the plasmid copy number and improved the expression intensity of GFP.Using different strategies to optimize the use of ARS,it was found that pan ARS was most beneficial for exogenous gene expression when it was located upstream of the target gene expression element,and that the plasmid copy number could be increased when pan ARS was coupled with PPARS2 using a dual-ARS strategy.In addition,a dual episomal plasmid system was successfully constructed in C.glycerinogenes using pan ARS and IOARS.(3)To evaluate the performance of the episomal plasmids obtained from the study for practical application in C.glycerinogenes,the effect of episomal plasmids expressing exogenous genes was examined.It was shown that successful episomal expression of TAL,HPAB and HPAC,pathway genes for the synthesis of caffeic acid,yielded up to 29.1 mg·L-1 of caffeic acid.The exogenous gene Cr GES for the synthesis of geraniol was expressed using episomal plasmids with a yield of up to 23.2 mg·L-1.The application results further demonstrated the effectiveness of the constructed episomal expression system of C.glycerinogenes,and provided a new molecular manipulation tool for future research and synthetic biology modification of C.glycerinogenes,as well as a reference for other strains without episomal plasmids to construct the episomal expression system.