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

• Published in: Metabolomics 2016-02, Vol.12 (2), p.26-10, Article 26
• Main Authors: Noguchi, Shingo, Putri, Sastia P., Lan, Ethan I., Laviña, Walter A., Dempo, Yudai, Bamba, Takeshi, Liao, James C., Fukusaki, Eiichiro
• Format: Article
• Language: English
• Subjects: Biochemistry; Biomedical and Life Sciences; Biomedicine; Cell Biology; Clostridium; Cyanobacteria; Developmental Biology; Life Sciences; Molecular Medicine; Original; Original Article; Synechococcus elongatus
• ISSN: 1573-3882; 1573-3890
• DOI: 10.1007/s11306-015-0940-2

Cyanobacterial 1-butanol production is an important model system for direct conversion of CO 2 to fuels and chemicals. Metabolically-engineered cyanobacteria introduced with a heterologous Coenzyme A (CoA)-dependent pathway modified from Clostridium species can convert atmospheric CO 2 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.