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Combining Expression Regulation And Directed Evolution Of Isoprene Synthase To Improve Isoprene Production In Saccharomyces Cerevisiae

Posted on:2018-10-25Degree:MasterType:Thesis
Country:ChinaCandidate:F WangFull Text:PDF
GTID:2311330512967564Subject:Biochemical Engineering
Abstract/Summary:PDF Full Text Request
As the simplest member of isoprenoids,isoprene is widely used in the synthesis of rubber,medicine and flavors.At the moment,isoprene is mainly produced from petroleum-derived C5 fraction.The limited resource and pollution caused by the petrochemical industry have raised increasing concerns.With rapid development of synthetic biology and metabolic engineering,biosynthesis of isoprene with microbial cell factories becomes possible.In this study,Saccharomyces cerevisiae,a eukaryotic model organism,was used as the platform for research of isoprene biosynthesis through combining metabolic regulation and enzyme directed evolution.For construction of isoprene biosynthesis pathway in S.cerevisiae,IspS from Populus alba heterogenously expressed with GAL1 promoter was introduced into a previously engineered strain YXM08 with enhanced precursor supply.In this strain,production of isoprene was induced by galactose.In order to eliminate the dependency of IspS expression on galactose induction,GAL80 was disrupted and a glucose-controlled isoprene synthesis pathway was successfully constructed,generating strain YXM13-ISPS.The isoprene synthesis pathway in S.cerevisiae could be divided into two modules:upstream MVA pathway and downstream isoprene-forming pathway.The imbalance of strong upstream MVA pathway and relatively weak downstream isoprene-forming pathway in YXM 13-ISPS caused accumulation of cytotoxic intermediate DMAPP,resulting in limited biomass and low isoprene production.To achieve the metabolic balance between the pathway modules,we strengthened the isoprene-forming pathway by engineering the expression and catalytic activity of ISPS.Firstly,for improvement of ISPS expression,GAL4 was overexpressed to increase supply of the transcriptional activator Gal4p and meanwhile the native GAL1/7/10 promoters were deleted to avoid competition for Gal4p.A 6-fold improvement in IspS transcriptional level was achieved,leading to improvement of isoprene production from 6.0 mg/L to 23.6 mg/L and decrease of squalene production in the competing pathway from 4.1 mg/L to 2.8 mg/L.Moreover,the biomass was increased by 14%resulted from the accelerated conversion of overproduced DMAPP to isoprene.In addition,a novel high-throughput screening method was developed based on precursor toxicity,validated by introduction of squalene synthesis pathway,and then used for ISPS directed evolution towards enhanced catalytic activity.The best mutants ISPSM4 was obtained through screening of the mutant library and combinatorial mutagenesis.Finally,combining metabolic regulation and enzyme modification by introduction of ISPSM4 into engineered strain with enhanced Gal4p supply,isoprene production reached up to 50.2 mg/L in sealed-vial culture and the dry cell weight was further increased by 7.1%,while squalene production was further dropped to 2.2 mg/L due to the enhanced competitiveness of the isoprene-forming pathway.To demonstrate the effectiveness of strengthening the downstream isoprene-forming pathway,the culture condition was switched from sealed vials to bioreactor and aerobic batch fermentation was performed with YXM13-ISPS,YXM29-ISPS and YXM29-ISPSM4,respectively.Consistent with the results of sealed-vial cultures,YXM29-ISPS(346 mg/L)showed the highest isoprene production and biomass.Therefore,YXM29-ISPSM4 was selected for high density fermentation to achieve high isoprene production.In the end,the OD600 reached 168 and 3.7 g/L isoprene(22.9 mg isoprene/g glucose)was produced after 96 h of fermentation,which is the highest titer ever reported for engineered eukaryotic cells.In this study,metabolic regulation and enzyme directed evolution were combined to strengthen the downstream isoprene-forming pathway,achieving balance between the upstream and downstream pathway modules,further leading to significantly improved isoprene biosynthesis efficiency.This work would provide strong technical support for commercial production of bio-isoprene and possibly also other isoprenoids.
Keywords/Search Tags:isoprene, isoprene synthase, Saccharomyce scerevisiae, metabolic regulation, directed evolution
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