Exploring in vivo biochemistry with C4 fuel and commodity chemical pathways

The biological diversity found throughout the world contains equally wondrous chemical diversity that can operate with the precision, efficiency, and scale that humanity has yet to attain. This capacity is an untapped resource that must be understood and harnessed to address pressing global needs for food, energy, medicine, and materials. Wielding this power will require a deeper understanding of how a given biological process occurs in the context of a cell. Metabolic pathways are an ideal model system to study biochemical processes in vivo as they are integral to the cell’s survival, they are regulated on multiple interlocking levels, and they have a broad dynamic range with many measurable inputs and outputs.We have studied a synthetic metabolic pathway in E. coli as a means of gaining insight into biological regulatory networks, but also with the goal of optimizing production of the second-generation biofuel n-butanol. Our previous pathway suffered from poor substrate specificity in the final enzyme, leading to off-target products and decreased yield. This enzyme, AdhE2, is a bifunctional aldehyde alcohol dehydrogenase that catalyzes sequential reductions of acyl-CoAs to alcohols through aldehyde intermediates. The enzyme was biochemically characterized to determine its substrate specificity, coordination between active sites, and oligomerization behavior. The enzyme was found to be undesirable for butanol production and new classes of enzymes were explored.To replace AdhE2 we employed bioinformatic methods to identify a family of monofunctional aldehyde dehydrogenases. This family was screened and a highly specific enzyme was identified. The improved butanol production pathway was then a suitable tool for exploring regulatory mechanisms controlling metabolism by employing whole genome mutagenesis and selection. A butanol production strain was engineered such that its growth under anaerobic conditions was directly linked to butanol production. This strain’s genome was mutagenized and subjected to anaerobic growth selection to enrich for mutants producing elevated levels of butanol. We then sequenced the genomes of these strains to identify regulatory mechanisms impacting butanol production.Finally, we expanded upon our butanol production pathway by leveraging the previously identified aldehyde dehydrogenase family for the production of the commodity chemicals 1,3-butanediol and 4-hydroxy-2-butanone. Aldehyde and alcohol dehydrogenases were identified by a variety of methods and screened for production. We developed several strategies to afford control over the ratio of products produced including pathway design and expression level tuning. Directed evolution methods including DNA shuffling and saturation mutagenesis were also used to further tailor aldehyde dehydrogenases for the desired products.In sum we have extensively characterized a number of aldehyde and alcohol dehydrogenases from multiple families. Optimized pathways for production of n-butanol, 1,3-butanediol, and 4-hydroxy-2-butanone were developed. A genetic selection for metabolite production was developed and validated, and evolved strains were characterized to identify important regulatory mechanisms