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

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 V_max(2KB) of 1.21 ± 0.03 U mg⁻¹ and a K_m(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.