Construction Of Saccharomyces Cerevisiae With High Isoprene Yield By Comprehensive Engineering

Isoprene,as a versatile bulk chemical,has wide industrial applications.Due to the difficulty of isoprene collection from natural producers which are mostly plants,its supply mostly comes from petroleum industry,which suffers from fluctuations of the petroleum market.Biosynthesis of chemicals has advantages over chemical routes in environmental friendliness,mild conditions and renewable feedstock.Saccharomyces cerevisiae with clear genetic background and exquisite genetic manipulation system,has been widely used as cell factories for a variety of terpenes.In this study,we attempted to improve isoprene biosynthesis in S.cerevisiae by combining metabolic engineering and protein engineering,in order to lay foundation for isoprene bioproduction.A strengthened and balanced metabolic flux towards the target product is vital for effective biosynthesis.In isoprene synthesis pathway of engineered S.cerevisiae,isoprene synthase(ISPS)is a rate-limiting enzyme.Although an ISPS mutant ISPSM4(F340L/A570T)had been obtained by directed evolution in previous work,it was still not efficient enough for rapid conversion of accumulated precursor to isoprene.By saturation and combinatorial mutagenesis of the two sites identified in directed evolution,a superior isoprene synthase mutant ISPSLN(F340L/A570N)was created,leading to almost 3-fold improvement in isoprene production.Subsequent introduction of ISPSLN to strains with strengthened precursor supply in either cytoplasm or mitochondria implied an imperfect match between the synthesis and conversion of the isopentenyl pyrophosphate(IPP)/dimethylallyl diphosphate(DMAPP)pool.To reconstruct metabolic balance between the upstream and downstream flux,additional copies of diphosphomevalonate decarboxylase gene(MVD1)and isopentenyl-diphosphate delta-isomerase gene(IDI1)were introduced into the cytoplasmic and mitochondrial engineered strains.The diploid strain created by mating the above haploid strains produced 11.9 g/L of isoprene.To facilitate efficient genetic manipulation for following experiments,we constructed plasmids for CRISPR/Cas9 system-mediated gene editing.Through tRNA processing,multi gRNAs flanked by tRNA were cut by RNase II,releasing the single gRNAs.Using this system,1-3 loci(GPD1,GPD2 and MPC3)were successfully targeted and knocked out,with an disruption efficiency of 100%in all cases.This CRISPR/Cas9 system was then employed to engineer the diploid strains with dual regulation of cytoplasmic and mitochondrial pathways.As a common precursor for cytoplasmic and mitochondrial isoprene biosynthesis pathways,the transportation of pyruvate from cytoplasm to mitochondria affects the carbon distribution between the two compartments.Upregulation of the mitochondrial pyruvate carrier 3(MPC3)decreased isoprene production in the dual-regulation strain,whereas knock-out and down-regulation of this gene both increased isoprene production by about 20%.This result implied that the pyruvate conversion efficiency of the cytoplasm pathway was higher that of the mitochondria pathway.However,diploid strains constructed by mating two strains which individually harbored the cytoplasmic pathway and down-regulated or knocked out MPC3 showed lower isoprene production as compared with the diploid strains harboring the mitochondria/cytoplasm dual pathways,which is possibly due to the loss of pyruvate to mitochondria via the second mitochondrial pyruvate carrier complex in S.cerevisiae,MPC1/MPC2.In fed-batch fermentation,14.3 g/L isoprene was obtained using the dual-regulated S.cerevisiae with MPC3 knockout,which was highest ever reported in eukaryotic cells.Considering that ethanol is the major by-product in the growing process of S.cerevisiae,the carbon yield of the target product may be further improved if we could rationally control the production of ethanol.Here we investigated the applicability of the ethanol-induced system ALCR from Aspergillus nidulans in S.cerevisiae.It turned out the PALCA-driven GFP was constitutively expressed in S.cerevisiae,although its expression could be enhanced by the presence of AlcR.To obtain an ethanol-induced promoter,PALCA was truncated from 5’ and 3’ ends to created a number of truncated promoters,and directed evolution was conducted to engineer PALCA.However,all mutant promoters obtained were not ethanol-sensitive but rather inactivated.Although this work failed to achieve the desired promoter,it provides valuable experience for constructing ethanol-induced system in S.cerevisiae.To sum up,by combining protein engineering and metabolic engineering,an S.cerevisiae strain with not only superior isoprene synthase,but also balance between upstream and downstream pathway,as well as better distribution of carbon flux in mitochondria and cytoplasm was obtained.Finally,the isoprene synthesis efficiency of the engineered yeast was obviously improved,paving the way for bio-based production of isoprene.