title: Manipulation of GES and ERG20 for geraniol overproduction in Saccharomyces cerevisiae.
abstract: Manipulation of monoterpene synthases to maximize flux towards targeted products from GPP (geranyl diphosphate) is the main challenge for heterologous monoterpene overproduction, in addition to cell toxicity from compounds themselves. In our study, by manipulation of the key enzymes geraniol synthase (GES) and farnesyl diphosphate synthase (Erg20), geraniol (a valuable acyclic monoterpene alcohol) overproduction was achieved in Saccharomyces cerevisiae with truncated 3-hydroxy-3-methylglutaryl-coenzyme reductase (tHMGR) and isopentenyl diphosphate isomerase (IDI1) overexpressed. The expressions of all above engineered genes were under the control of Gal promoter for alleviating product toxicity. Geraniol production varied from trace amount to 43.19mg/L (CrGES, GES from Catharanthus roseus) by screening of nine GESs from diverse species. Further through protein structure analysis and site-directed mutation in CrGES, it was firstly demonstrated that among the high-conserved amino acid residues located in active pocket, Y436 and D501 with strong affinity to diphosphate function group, were critical for the dephosphorylation (the core step for geraniol formation). Moreover, the truncation position of the transit peptide from the N-terminus of CrGES was found to influence protein expression and activity significantly, obtaining a titer of 191.61mg/L geraniol in strain with CrGES truncated at S43 (t3CrGES). Furthermore, directed by surface electrostatics distribution of t3CrGES and Erg20WW(Erg20F96W-N127W), co-expression of the reverse fusion of Erg20ww/t3CrGES and another copy of Erg20WWpromoted the geraniol titer to 523.96mg/L at shakes flask level, due to enhancing GPP accessibility led by protein interaction of t3CrGES-Erg20WWand the free Erg20WW. Eventually, a highest reported titer of 1.68g/L geraniol in eukaryote cells was achieved in 2.0L fed-batch fermentation under carbon restriction strategy. Our research opens large opportunities for other microbial production of monoterpenes. It also sets a good reference for desired compounds overproduction in microorganisms in terms of manipulation of key enzymes by protein engineering and metabolic engineering.
authors.affiliation: Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin 300072, PR China; SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, PR China. Electronic address: guozhenj@tju.edu.cn.
authors.forename: Guo-Zhen
authors.initials: GZ
authors.lastname: Jiang
authors.affiliation: Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin 300072, PR China; SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, PR China.
authors.forename: Ming-Dong
authors.initials: MD
authors.lastname: Yao
authors.affiliation: Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin 300072, PR China; SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, PR China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin 300072, PR China; SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, PR China.
authors.forename: Liang
authors.initials: L
authors.lastname: Zhou
authors.affiliation: Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin 300072, PR China; SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, PR China.
authors.forename: Tian-Qing
authors.initials: TQ
authors.lastname: Song
authors.affiliation: Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin 300072, PR China; SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, PR China.
authors.forename: Hong
authors.initials: H
authors.lastname: Liu
authors.affiliation: Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin 300072, PR China; SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, PR China. Electronic address: wenhai.xiao@tju.edu.cn.
authors.forename: Wen-Hai
authors.initials: WH
authors.lastname: Xiao
authors.affiliation: Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin 300072, PR China; SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, PR China.
authors.forename: Ying-Jin
authors.initials: YJ
authors.lastname: Yuan
mesh_terms: D000080462:Acyclic Monoterpenes; D030008:Catharanthus / Q000201:enzymology / Q000235:genetics*; D051228:Geranyltranstransferase / Q000096:biosynthesis / Q000235:genetics; D060847:Metabolic Engineering; D010744:Phosphoric Monoester Hydrolases / Q000096:biosynthesis / Q000235:genetics; D010940:Plant Proteins / Q000096:biosynthesis / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis / Q000235:genetics; D013729:Terpenes / Q000378:metabolism*
chemical_list: D000080462:Acyclic Monoterpenes; D010940:Plant Proteins; D029701:Saccharomyces cerevisiae Proteins; D013729:Terpenes; D051228:Geranyltranstransferase; D010744:Phosphoric Monoester Hydrolases; C007836:geraniol
keywords: Enzyme species; Geraniol; Geraniol synthase; Metabolic engineering; Synthetic biology; Truncation position