title: Metabolically engineered Saccharomyces cerevisiae for enhanced isoamyl alcohol production.
abstract: Higher chain alcohols have gained much attention as next generation transport fuels because of their higher energy density and low moisture absorption capacity compared to ethanol. In the present study, we attempted to engineer Saccharomyces cerevisiae for the synthesis of isoamyl alcohol via de novo leucine biosynthetic pathway coupled with Ehrlich degradation pathway. To achieve high-level production of isoamyl alcohol, two strategies are used in the current study: (1) reconstruction of a chromosome-based leucine biosynthetic pathway under the control of galactose-inducible promoters; (2) overexpression of the mitochondrial 2-isopropylmalate (α-IPM) transporter to boost the transportation of α-IPM from mitochondria to the cytosol. We found engineered yeast cells with a combinatorially assembled leucine biosynthetic pathway coupled with the Ehrlich degradation pathway resulted in high-level production of isoamyl alcohol; however, there was still a significant amount of isobutanol co-formed during the fermentation process. Further introducing an α-IPM transporter not only boosted the isoamyl alcohol biosynthetic pathway activity but also reduced isobutanol to a much lower level. Taken together, our work represents the first study to construct a chromosome-based leucine biosynthetic pathway for isoamyl alcohol production. Furthermore, the utilization of the mitochondrial compartment coupled with the transporter engineering serves as an effective approach to minimize the by-product formation and to improve the isoamyl alcohol production.
authors.affiliation: Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore, 117585, Singapore. jifengy@gmail.com.|Temasek Laboratories, National University of Singapore, T-Lab Building 5A, Engineering Drive 1, Singapore, 117411, Singapore. jifengy@gmail.com.|Biotransformation Innovation Platform, Agency for Science, Technology and Research, Singapore, 138673, Singapore. jifengy@gmail.com.
authors.forename: Jifeng
authors.initials: J
authors.lastname: Yuan
authors.affiliation: Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore, 117585, Singapore.
authors.forename: Xue
authors.initials: X
authors.lastname: Chen
authors.affiliation: Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore, 117585, Singapore.
authors.forename: Pranjul
authors.initials: P
authors.lastname: Mishra
authors.affiliation: Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore, 117585, Singapore. Chibun.ching@singaporetech.edu.sg.|Temasek Laboratories, National University of Singapore, T-Lab Building 5A, Engineering Drive 1, Singapore, 117411, Singapore. Chibun.ching@singaporetech.edu.sg.|Singapore Institute of Technology, 10 Dover Drive, Singapore, 138683, Singapore. Chibun.ching@singaporetech.edu.sg.
authors.forename: Chi-Bun
authors.initials: CB
authors.lastname: Ching
mesh_terms: D000440:Butanols / Q000378:metabolism; D060847:Metabolic Engineering / Q000379:methods*; D053858:Metabolic Networks and Pathways / Q000235:genetics; D000439:Pentanols / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*
chemical_list: D000440:Butanols; D000439:Pentanols; C040507:isobutyl alcohol; C029683:isopentyl alcohol
keywords: 2-isopropylmalate transporter; Ehrlich degradation pathway; Isoamyl alcohol; Leucine biosynthetic pathway; Pathway assembly; Saccharomyces cerevisiae