Quantitative target analysis and kinetic profiling of acyl-CoAs reveal the rate-limiting step in cyanobacterial 1-butanol production

Shingo Noguchi, Sastia P. Putri, Ethan I. Lan, Walter A. Laviña, Yudai Dempo, Takeshi Bamba, James C. Liao, Eiichiro Fukusaki

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18 Citations (Scopus)

Abstract

Cyanobacterial 1-butanol production is an important model system for direct conversion of CO2 to fuels and chemicals. Metabolically-engineered cyanobacteria introduced with a heterologous Coenzyme A (CoA)-dependent pathway modified from Clostridium species can convert atmospheric CO2 into 1-butanol. Efforts to optimize the 1-butanol pathway in Synechococcus elongatus PCC 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. Using 13C-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.

Original languageEnglish
Article number26
Pages (from-to)1-10
Number of pages10
JournalMetabolomics
Volume12
Issue number2
DOIs
Publication statusPublished - Feb 1 2016

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All Science Journal Classification (ASJC) codes

  • Endocrinology, Diabetes and Metabolism
  • Biochemistry
  • Clinical Biochemistry

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