S34: Homologous Metabolic Engineering (HoME): A native avenue for the efficient biosynthesis of butanol in E. coli

Interest in the use of long-chain linear alcohols as biofuels has rapidly developed due to their higher energy density, low hygroscopicity, and compatibility with current fuel infrastructure. This potential has not been realized however due to issues associated with the industrial use of native producers (e.g. Clostridia) and the rather unsuccessful efforts in engineering non-native production in industrial organisms. Our work addresses the aforementioned limitations by exploiting an alternative strategy focusing on the use of native E. coli enzymes to assemble a novel n-butanol biosynthetic route without the use of foreign genes, an approach that we term Homologous Metabolic Engineering (HoME).

n-butanol is the only higher-chain linear alcohol found in nature as primary fermentation product and it is produced in clostridial species through a six-step pathway originating from acetyl-CoA. Through sequence and structure comparisons between the clostridial enzymes and those encoded in the E. coli genome, we identified potential E. coli surrogates of the clostridial pathway. Functional expression of the surrogate pathway was attained in part by manipulating gene regulators to induce its constitutive expression, and overexpression. Synthesis of byproducts competing for both carbon (AcCoA) and reducing equivalents was also impaired. The high efficiency of the pathway assembled using the HoME methodology was demonstrated by producing higher-chain linear n-alcohols at levels several folds better than previously reported. When grown under optimized conditions in batch fermentations and in the absence of rich nutrients, the engineered strain produced n-butanol at high titer (~14 g/L) and yield (0.33 g n-butanol/g total glucose consumed).