(160ai) Analyzing in-Vivo Enzyme Activity to Optimize Recombinant Microbial Fermentation

Development of recombinant microbial hosts for high-level chemical production mandates the optimization of enzyme expression level and fine-tuning of process parameters to maximize titers, productivities, and yields. In the process of optimizing these intricate and interconnected conditions, a critical attribute that emerges central to all such efforts is balancing effective enzyme activity across all biosynthesis modules such that metabolic burden is minimized. Among other compounds, we have successfully produced psilocybin and norbaeocystin through fermentation in E. coli at titers greater than 1 g/L. To accomplish this, we altered pH, temperature, induction time, selection pressure, and media composition to compare final yields, post genetic optimization. We also maintained an excess of substrate throughout the fermentation processes, allowing evaluation of effective enzyme activity following zero order kinetics as a function of enzyme concentration through the different stages of growth. Therefore, by analyzing individual enzyme activity throughout the process we were able to identify stages where the in-vivo catalytic activity was enhanced, diminished, or remained unchanged. This analysis was used to inform our understanding of key genetic and process parameters and also facilitated targeting specific process stages to circumvent reduced productivity and therefore representing multiple conditions by a unique metric. The results of this analysis enabled the smooth technology transfer of two of our production strains to commercial manufacturing facilities.