UC Berkeley

Biology holds an amazing propensity for chemistry. Living systems continuously carry out a vast plethora of chemical reactions within a complex network, known as metabolism, to sustain growth and improve evolutionary fitness. Metabolic engineers seek to utilize this aptitude for chemistry by creating biological catalysts for chemical production from renewable feedstocks. Biological catalysts offer an eco-friendly, and in some cases, superior, alternative to petrochemical-based chemical production.In this work, we examine biological catalysts designed for the production of two C4 commodity chemicals, n-butanol and (R)-1,3-butanediol. These catalysts are strains of Escherichia coli containing constructed biosynthetic pathways. Leveraging an anaerobic growth selection, laboratory adaptive evolution identified several mutant strains with improved phenotypes. We set out to understand the mechanism by which these adaptive mutations confer improved production. Through detailed analysis of n-butanol fermentation, we discovered that the parent strain for our evolution was unable to support sustained anaerobic growth via n-butanol fermentation, potentially due to metabolic burden associated with overexpression of the pathway enzymes. Further experimentation suggested that the mutations arose as a strategy to relieve metabolic burden through decreased expression of our biosynthetic pathway. The results of this study highlight the importance of balanced pathway expression when designing biological catalysts. We then shifted our focus to design a microbial catalyst for production of polyhydroxyalkanoates (PHAs) containing unsaturated monomers. Sites of unsaturation provide functional handles for downstream chemical modification. We devised a metabolic strategy to convert two non-canonical amino acids with unsaturated functional groups to their respective 2-hydroxy acids and activate these acids as coenzyme A thioesters for polymerization within E. coli. We identified and tested candidate enzymes for the appropriate activities in vitro and successfully showed that our identified enzymes can form a functional biosynthetic pathway. These experiments lay the groundwork for creation of a microbial catalyst capable of generating PHAs with unsaturated functional groups using glucose as a carbon source.