Metabolic and protein engineering approaches for microbial production of c3 and c4 commodity chemicals

Several reports of establishing efficient biological platforms for manufacture of high value and commodity chemicals have surfaced over the last decade. Metabolic and protein engineering has enabled the construction of novel metabolic pathways, as well as manipulation of cellular metabolism. The microbial production of C3 and C4 alcohols and diols hold significant commercial interest due to their global market size and applications. In this work we report the microbial production of 1,2-propanediol, 1-propanol and 1,4-butanediol. To achieve this, synthetic pathways in Escherichia coli were constructed and its glucose/ xylose catabolism was engineered. By systematically redirecting carbon flux, introducing a NADH regeneration system and developing a cell adaptation method 1,2-propanediol production was achieved at 94% theoretical maximum yield from glucose. We then utilize a diol dehydratase to achieve the dehydration of 1,2-propanediol, leading to 1-propanol production via a novel metabolic pathway in E. coli. By constructing a fusion dehydratase and utilizing a dual strain strategy we achieve de novo production of 1-propanol at 2.91 g/L. In order to establish a novel metabolic route for biological production of 1,4-butanediol a diol dehydratase is first engineered to achieve non-native catalysis. By developing a rational design approach we screen the mutant library in silico via docking simulations. By characterizing the mutant dehydratase’s activity in vitro we confirm the engineered non-native activity. The de novo production of 1,4-butanediol is achieved by engineering E. coli’s xylose catabolism and overcoming the pathway bottleneck with the expression of an engineered dehydratase.