title: Quantitative target analysis and kinetic profiling of acyl-CoAs reveal the rate-limiting step in cyanobacterial 1-butanol production.
abstract: Cyanobacterial 1-butanol production is an important model system for direct conversion of CO2to fuels and chemicals. Metabolically-engineered cyanobacteria introduced with a heterologous Coenzyme A (CoA)-dependent pathway modified fromClostridiumspecies can convert atmospheric CO2into 1-butanol. Efforts to optimize the 1-butanol pathway inSynechococcus elongatusPCC 7942 have focused on the improvement of the CoA-dependent pathway thus, probing the in vivo metabolic state of the CoA-dependent pathway is essential for identifying its limiting steps. In this study, we performed quantitative target analysis and kinetic profiling of acyl-CoAs in the CoA-dependent pathway by reversed phase ion-pair liquid chromatography-triple quadrupole mass spectrometry. Using13C-labelled cyanobacterial cell extract as internal standard, measurement of the intracellular concentration of acyl-CoAs revealed that the reductive reaction of butanoyl-CoA to butanal is a possible rate-limiting step. In addition, improvement of the butanoyl-CoA to butanal reaction resulted in an increased rate of acetyl-CoA synthesis by possibly compensating for the limitation of free CoA species. We inferred that the efficient recycling of free CoA played a key role in enhancing the conversion of pyruvate to acetyl-CoA.
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1Yamadaoka, Suita, Osaka 565-0871 Japan.
authors.forename: Shingo
authors.initials: S
authors.lastname: Noguchi
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1Yamadaoka, Suita, Osaka 565-0871 Japan.
authors.forename: Sastia P
authors.initials: SP
authors.lastname: Putri
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, 5531 Boelter Hall, 420 Westwood Plaza, Los Angeles, CA 90095 USA.
authors.forename: Ethan I
authors.initials: EI
authors.lastname: Lan
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1Yamadaoka, Suita, Osaka 565-0871 Japan.
authors.forename: Walter A
authors.initials: WA
authors.lastname: Laviña
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1Yamadaoka, Suita, Osaka 565-0871 Japan.
authors.forename: Yudai
authors.initials: Y
authors.lastname: Dempo
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1Yamadaoka, Suita, Osaka 565-0871 Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Bamba
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, 5531 Boelter Hall, 420 Westwood Plaza, Los Angeles, CA 90095 USA.
authors.forename: James C
authors.initials: JC
authors.lastname: Liao
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1Yamadaoka, Suita, Osaka 565-0871 Japan.
authors.forename: Eiichiro
authors.initials: E
authors.lastname: Fukusaki
keywords: 1-Butanol; Acyl-CoAs; Cyanobacteria; Kinetic profiling; Liquid chromatography-mass spectrometry (LC–MS); Quantitative target analysis