Title |
Quantitative target analysis and kinetic profiling of acyl-CoAs reveal the rate-limiting step in cyanobacterial 1-butanol production
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Published in |
Metabolomics, January 2016
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DOI | 10.1007/s11306-015-0940-2 |
Pubmed ID | |
Authors |
Shingo Noguchi, Sastia P. Putri, Ethan I. Lan, Walter A. Laviña, Yudai Dempo, Takeshi Bamba, James C. Liao, Eiichiro Fukusaki |
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 (13)C-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. |
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