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 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.

SUBMITTER: Noguchi S 

PROVIDER: S-EPMC4700068 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature