Catalytic Modules in Non-Natural Butanol Biosynthesis: Conversion of the Key Intermediate Crotylalcohol to N-Butanol via a Designed Enzyme Cascade - Abstract
Climate change and dwindling fossil resources drive industrial developments of biomass based processes for chemical and fuel applications. Butanol is poised to be a next generation renewable building block due to its improved energy content and hydrophobicity compared to bio-ethanol. Conventionally, bio-butanol is produced via the anaerobe ABE fermentation that employs Clostridia species as cellular production systems. However, economic viability of this cell based process is limited due to end product toxicities above 2% v/v and accumulation of alternative metabolic products. Alternatively, tailor-made, cell-free enzyme cascades are emerging as alternative production systems, which hold the promise of rapid adaptability to harsh process conditions and improved n-butanol yields. However, the molecular complexity of natural n-butanol biosynthesis currently prohibits realization of a robust cell-free n-butanol production process. Recently, simplified, non-natural n-butanol production pathways have been predicted by computational methods. However, enzyme systems that allow consecutive conversion of predicated intermediates to n-butanol have not been identified. A key biosynthetic module in computationally predicted n-butanol production is the conversion of crotylalcohol to n-butanol. We have designed a non-natural enzyme cascade that allows the three step conversion of crotylalcohol to n-butanol using just two enzymes. The involved enzyme systems, horse liver alcohol dehydrogenase and 2-enoate reductase from Bacillus subtilis,
show pronounced substrate promiscuity, which allows the consolidated conversion of crotylalcohol to n-butanol. Further, the designed enzyme cascade allows production of n-butanol using only the NAD+/NADH redox couple as a unified electron shuttle, which significantly reduces the molecular complexity of the cell-free reaction cascade. This is the first study that could experimentally validate a reaction module of a computationally predicted n-butanol production pathway. The development of designed, cell-free reaction cascades will pave the way towards mass- and cost-efficient n-butanol production at an industrial scale.