title: Combining Gal4p-mediated expression enhancement and directed evolution of isoprene synthase to improve isoprene production in Saccharomyces cerevisiae.
abstract: Current studies on microbial isoprene biosynthesis have mostly focused on regulation of the upstream mevalonic acid (MVA) or methyl-erythritol-4-phosphate (MEP) pathway. However, the downstream bottleneck restricting isoprene biosynthesis capacity caused by the weak expression and low activity of plant isoprene synthase (ISPS) under microbial fermentation conditions remains to be alleviated. Here, based on a previously constructed Saccharomyces cerevisiae strain with enhanced precursor supply, we strengthened the downstream pathway through increasing both the expression and activity of ISPS to further improve isoprene production. Firstly, a two-level expression enhancement system was developed for the PGAL1-controlled ISPS by overexpression of GAL 4. Meanwhile, the native GAL1/7/10 promoters were deleted to avoid competition for the transcriptional activator Gal4p, and GAL80 was disrupted to eliminate the dependency of gene expression on galactose induction. The IspS expression was obviously elevated upon enhanced Gal4p supply, and the isoprene production was improved from 6.0mg/L to 23.6mg/L in sealed-vial cultures with sucrose as carbon source. Subsequently, a novel high-throughput screening method was developed based on precursor toxicity and used for ISPS directed evolution towards enhanced catalytic activity. Combinatorial mutagenesis of the resulting ISPS mutants generated the best mutant ISPSM4, introduction of which into the GAL4-overexpressing strain YXM29 achieved 50.2mg/L of isoprene in sealed vials, and the isoprene production reached 640mg/L and 3.7g/L in aerobic batch and fed-batch fermentations, respectively. These results demonstrated the effectiveness of the proposed combinatorial engineering strategy in isoprene biosynthesis, which might also be feasible and instructive for biotechnological production of other valuable chemicals.
authors.affiliation: Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, PR China.
authors.forename: Fan
authors.initials: F
authors.lastname: Wang
authors.affiliation: Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, PR China.
authors.forename: Xiaomei
authors.initials: X
authors.lastname: Lv
authors.affiliation: Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, PR China.
authors.forename: Wenping
authors.initials: W
authors.lastname: Xie
authors.affiliation: Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, PR China.
authors.forename: Pingping
authors.initials: P
authors.lastname: Zhou
authors.affiliation: Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, PR China.
authors.forename: Yongqiang
authors.initials: Y
authors.lastname: Zhu
authors.affiliation: Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, PR China.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Yao
authors.affiliation: Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, PR China.
authors.forename: Chengcheng
authors.initials: C
authors.lastname: Yang
authors.affiliation: Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, PR China.
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Yang
authors.affiliation: Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, PR China; Key Laboratory of Biomass Chemical Engineering of Ministry of Education, Zhejiang University, Hangzhou 310027, PR China. Electronic address: yelidan@zju.edu.cn.
authors.forename: Lidan
authors.initials: L
authors.lastname: Ye
authors.affiliation: Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, PR China. Electronic address: yuhongwei@zju.edu.cn.
authors.forename: Hongwei
authors.initials: H
authors.lastname: Yu
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000378:metabolism*; D053898:Biosynthetic Pathways / Q000235:genetics; D002070:Butadienes / Q000302:isolation & purification / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D019020:Directed Molecular Evolution / Q000379:methods*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D024861:Genetic Enhancement / Q000379:methods; D045782:Hemiterpenes / Q000302:isolation & purification / Q000378:metabolism*; D060847:Metabolic Engineering / Q000379:methods*; D053858:Metabolic Networks and Pathways / Q000235:genetics; D010420:Pentanes / Q000302:isolation & purification / Q000378:metabolism*; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation / Q000235:genetics
chemical_list: D002070:Butadienes; D004268:DNA-Binding Proteins; C052130:GAL4 protein, S cerevisiae; C055002:GAL80 protein, S cerevisiae; D045782:Hemiterpenes; D010420:Pentanes; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C005059:isoprene; D019883:Alkyl and Aryl Transferases; C093854:isoprene synthase
keywords: Directed evolution; GAL4 regulation; Isoprene; Isoprene synthase; Saccharomyces cerevisiae