System metabolic engineering for the production of lactone, lactam used as monomer for biopolymer synthesis

Abstract

System metabolic engineering contributes to the sustainable chemical industry by environment-friendly production of various chemical through development of microbial cell factory. We aimed to develop $\textit{Escherichia coli}$ for the efficient production of γ-butyrolactone and ε-caprolactam, which are important chemicals used as monomers of biopolymer such as bioplastic and polyamide. First, γ-butyrolactone has evaluated as an excellent material with future prospect since it could be used as a monomer of poly-γ-butyrolactone, a biodegradable plastic. Herein, development of $\textit{E. coli}$ capable of producing γ-butyrolactone with a one-step fermentative strategy was carried out in order to replace the existing petroleum-based production. We proved the activity of CoA transferase helps forming ring structure of lactone compound using $\textit{in vitro}$ and $\textit{in vivo}$ validation. In addition, $\textit{in silico}$ flux analyze was applied for screening genome-based knockdown target. After various optimization of cultivation during scale-up process, the final engineered strain produced with the titer of 6.76 g/L from glucose by fed-batch fermentation, which is the highest ever. Next, the development of $\textit{Escherichia coli}$ was conducted for the production of ε-caprolactam. ε-Caprolactam is widely used as a monomer of polyamide such as nylon 6. Although microbial production of ε-Caprolactam was successfully developed in previous research, it has obviously limitation of low titer. In this study, we constructed novel metabolic pathway for production of ε-Caprolactam composed of glucose-adipic acid flux (upstream) and adipic acid- caprolactam flux (downstream). As a result, we developed adipic acid-overproducing strain with a titer of 102.62 mg/L in flask cultivation. Based on this engineered strain, we introduced downstream pathway and the final strain succeeded in producing with the titer of 2.26 mg/L. It is expected that the recombinant strains developed in this thesis can help us move one-step closer to the bio-based production of polymers such as plastics and nylon.