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.Electronic supplementary materialThe online version of this article (doi:10.1007/s11306-015-0940-2) contains supplementary material, which is available to authorized users.
Highlights
Cyanobacterial biofuel production is considered as an attractive approach for providing sustainable energy resources due to its capability to utilize solar energy and CO2 as sole energy and carbon sources, respectively (Machado and Atsumi 2012; Jin et al 2014)
This condensation reaction is thermodynamically unfavorable since it requires an acetyl-Coenzyme A (CoA) pool to drive the reaction forward, which was thought to be unsuitable for the effective conversion of acetyl-CoA to acetoacetyl-CoA under photosynthetic condition (Lan and Liao 2011)
We investigated in vivo the metabolic state in the CoAdependent 1-butanol biosynthesis pathway of cyanobacteria by quantitative target analysis and kinetic profiling of acyl-CoAs in the CoA-dependent pathway through (RP-IPLC)/QqQ-MS
Summary
Cyanobacterial biofuel production is considered as an attractive approach for providing sustainable energy resources due to its capability to utilize solar energy and CO2 as sole energy and carbon sources, respectively (Machado and Atsumi 2012; Jin et al 2014). We were able to successfully engineer the cyanobacteria Synechococcus elongates PCC 7942 to produce 1-butanol under anoxic and dark conditions via a modified CoA-dependent pathway (Lan and Liao 2011) based on the acetone-butanol-ethanol fermentation in Clostridium species (Gheshlaghi et al 2009). 1-butanol productivity was dramatically improved under photosynthetic condition by substituting the oxygen-sensitive butanal dehydrogenase with an oxygen-tolerant CoA-acylating propionaldehyde dehydrogenase (Lan et al 2013). The maximum titer in cyanobacteria is still low compared to the 1-butanol-producing Escherichia coli with the heterologous CoA-dependent pathway; the 1-butanol titer is 317 mg/L from CO2 in 12 days using Synechococcus elongatus and 30 g/L from glucose in 7 days using Escherichia coli (Lan et al 2013; Shen et al 2011). Intracellular metabolic profiling of the intermediates in the CoA-dependent pathway is expected to be essential for gaining clues for further optimization of the 1-butanol biosynthesis pathway
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