Engineering Modularity of Ester Biosynthesis Across Biological Scales

Metabolic engineering and synthetic biology enable controlled manipulation of whole-cell biocatalysts to produce valuable chemicals from renewable feedstocks in a rapid and efficient manner, helping reduce our reliance on the conventional petroleum-based chemical synthesis. However, strain engineering process is costly and time-consuming that developing economically competitive bioprocess at industrial scale is still challenging. To accelerate the strain engineering process, modular cell engineering has been proposed as an innovative approach that harnesses modularity of metabolism for designing microbial cell factories. It is important to understand biological modularity and to develop design principles for effective implementation of modular cell engineering. In this dissertation, the modularity of ester biosynthesis was engineered from the molecular to the microbial community levels. Specifically, three important features of modularity (i.e., robustness, efficiency, and compatibility) were engineered and quantitatively analyzed across different scales. At the molecular (enzymatic) level, thermostability and promiscuity of alcohol acyltransferases were engineered to develop a robust designer ester biosynthesis. At the metabolic network (cellular) level, metabolism of Escherichia coli was rewired to overproduce isoamyl acetate through metabolic engineering and synthetic biology strategies. Also, by harnessing the engineered robust alcohol acyltransferase, a non-model thermophilic bacterium Clostridium thermocellum was engineered to produce medium chain esters directly from recalcitrant lignocellulosic biomass at elevated temperatures. Finally, at the microbial community level, a syntrophic E. coli co-culture was engineered for isobutyl butyrate production from a mixture of glucose and xylose. The successful engineering of the modularity of ester biosynthesis not only sheds light into the modular design principles of biological systems, but also seeks to develop industrially relevant ester production platforms.