Quantitative Target Analysis and Kinetic Profiling of Acyl-CoAs Reveal the Rate-limiting Step in Cyanobacterial 1-butanol Production

Overview

Journal Metabolomics

Publisher Springer

Specialty Endocrinology

Date 2016 Jan 15

PMID 26766939

Citations 7

Authors

Shingo Noguchi

Sastia P Putri

Ethan I Lan

Walter A Lavina

Yudai Dempo

Takeshi Bamba

James C Liao

Eiichiro Fukusaki

Affiliations

Soon will be listed here.

Abstract

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