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In this report, we identify the relevant factors to increase production of medium chain n-alcohols through an expanded view of the reverse β-oxidation pathway. We began by creating a base strain capable of producing medium chain n-alcohols from glucose using a redox-balanced and growth-coupled metabolic engineering strategy. By dividing the heterologous enzymes in the pathway into different modules, we were able to identify and evaluate homologs of each enzyme within the pathway and identify several capable of enhancing medium chain alcohol titers and/or selectivity. In general, the identity of the trans-2-enoyl-CoA reductase (TER) and the direct overexpression of the thiolase (FadA) and β-hydroxy-acyl-CoA reductase (FadB) improved alcohol titer and the identity of the FadBA complex influenced the dominant chain length. Next, we linked the anaerobically induced VHb promoter from Vitreoscilla hemoglobin to each gene to remove the need for chemical inducers and ensure robust expression. The highest performing strain with the autoinduced reverse β-oxidation pathway produced n-alcohols at titers of 1.8 g/L with an apparent molar yield of 0.2 on glucose consumed in rich medium (52% of theoretical yield).

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Escherichia coli strain LS5218 is a useful host for the production of fatty acid derived products, but the genetics underlying this utility have not been fully investigated. Here, we report the genome sequence of LS5218 and a list of large mutations and single nucleotide permutations (SNPs) relative to E. coli K-12 strain MG1655. We discuss how genetic differences may affect the physiological differences between LS5218 and MG1655. We find that LS5218 is more closely related to E. coli strain NCM3722 and suspect that small genetic differences between K-12 derived strains may have a significant impact on metabolic engineering efforts.

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