Photosynthetic production of 2-methyl-1-butanol from CO2 in cyanobacterium Synechococcus elongatus PCC7942 and characterization of the native acetohydroxyacid synthase

Energy & Environmental Science Pub Date: 2012-09-04 DOI: 10.1039/C2EE23148D

Abstract

Direct conversion of CO2 to bio-based fuels and chemicals has emerged as a significant thrust to address the energy and environmental concerns caused by over-reliance on fossil fuels and the increasing level of atmospheric CO2. Here we report the first photosynthetic production of 2-methyl-1-butanol (2MB), an energy-dense fuel molecule, from CO2 in the genetically engineered cyanobacterium Synechococcus elongatus PCC7942. 2MB is synthesized through the isoleucine pathway by decarboxylation of 2-keto-3-methylvalerate followed by reduction and has been produced from glucose by recombinant Escherichia coli with 1-propanol and isobutanol as the major by-products. However, direct photosynthetic production of 2MB from CO2 has not been reported. In this work, introduction of a ketoacid decarboxylase (Kivd), an alcohol dehydrogenase (YqhD), and the citramalate pathway, which produces the isoleucine precursor 2-ketobutyrate (2KB), in S. elongatus PCC7942 successfully redirected the flux to 2MB biosynthesis with significant productivity (an average of 20 mg per L per day). Interestingly, the native isoleucine pathway activity was able to compete with the overexpressed Kivd activity for the same substrate 2KB, such that 1-propanol formation was minimal. Kinetic analysis of the key enzyme in the isoleucine pathway, acetohydroxyacid synthase (AHAS) from S. elongatus PCC7942, yielded a Vmax(2KB) of 1.21 ± 0.03 U mg?1 and a Km(2KB) of 1.9 ± 0.3 mM using the purified protein and demonstrated preferential selectivity towards 2KB. The final titer of 2MB reached 200 mg L?1 in 12 days with minor accumulation of other alcohols. The high in vivo activity of the native S. elongatus PCC7942 AHAS suggests the advantage of utilizing branched-chain amino acid pathways in this organism for the production of fuels and chemicals.

Graphical abstract: Photosynthetic production of 2-methyl-1-butanol from CO2 in cyanobacterium Synechococcus elongatus PCC7942 and characterization of the native acetohydroxyacid synthase
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