Production of Bio-Based Chemicals In vivo and In vitro
- Author(s): Opgenorth, Paul;
- Advisor(s): Bowie, James U;
- et al.
Over the last century the petro chemical industry has provided an abundant and cheap source of hydrocarbons that have impacted and transformed many facets of our lives. Petrochemicals not only provide fuel that revolutionized transportation industry but also provided cheap petrochemical feedstock molecules that form a basis of many textiles, plastics, adhesives, detergents, and lubricants that are indispensable in modern life. Our dependence on the petrochemical industry and the sheer quantity of petrochemical based products that are consumed have led to a number of adverse and unintended effects on our environment. Recent attention to unintended effects of the petrochemical industry such as climate change, air pollution, and landfill overflow have sparked renewed interest in green chemistry and research in renewables that could potentially decrease our dependence on the petrochemical industry.
Over the last two decades, with the advent of simple and cheap genetic engineering, a new field called metabolic engineering has emerged. A large focus of this field is centered around re-engineering the metabolism of simple organisms to produce bulk and specialty chemicals. We genetically engineered a lithoautotrophic organism, Ralstonia eutropha, to produce drop-in ready biofuels (isobutanol and 3-methyl-1-butanol) in an electro-bio reactor using CO2 as the sole carbon source and electricity as the sole energy input. This method integrates electrochemical formate production and biological CO2 fixation and higher alcohol synthesis. The liquid fuels generated are a relatively stable way to store energy and possess energy densities about 100 times higher than current-day batteries.
A different approach to metabolic engineering coined synthetic biochemistry seeks to reconstitute complex metabolic pathways outside of cells. The Synthetic Biochemistry approach eliminates the myriad unwanted side reactions that occur in cells and product toxicity, enabling near 100% theoretical yields. Unfortunately, eliminating the cellular environment also eliminates key regulatory networks involved in maintaining appropriate carbon flux and cofactor balance. Synthetic biochemistry seeks to replace these complex regulatory networks in vitro by creating self-balancing pathways that automatically regulate the production and consumption of cofactors while maintaining constant carbon flux through the pathway. To this end, we developed the concept of a molecular purge valve. Generally, the purge valve is made up of a NADPH dependent oxidase, a NADH dependent oxidase and a NADH specific oxidase (noxE). We first demonstrated this proof of concept by engineering the G. stearothermophilus pyruvate dehydrogenase complex to flip its cofactor specificity from NADH to NADPH and using it in the purge valve to transform pyruvate to either polyhydroxybutyrate bioplastic or isoprene at near quantitative yields. The concept of using a purge valve to maintain carbon flux in an inherently imbalanced pathway was expanded through the production of polyhydroxybutyrate from glucose using a synthetic pathway called the PBG cycle. The PBG cycle is made up of enzymes from the pentose phosphate pathway, the bifido shunt , and standard glycolysis (EMP). This pathway utilizes the purge valve concept at the two reductase enzymes of the pentose phosphate pathway and produced the bioplastic polyhydroxybutyrate from glucose. The PBG pathway ran semi-continuously for 55 hours and produced 86% yield from 110 mM glucose. The pathway had a maximum productivity of 0.7g/L PHB and maintained 50% of its productivity over the course of the 55 hours at room temperature. The PBG cycle serves as a demonstration that synthetic biochemistry could be an alternative to in vivo metabolic engineering for the production of bulk and specialty chemicals and could potentially be one way to replace some of the petrochemicals we use today with renewable bio-based chemicals.