Novel tools for high-throughput genetic engineering, selection, and screening for improved Biotin production in E. coli cell factories

Abstract: Biotin is an essential co-factor in various metabolic processes and is crucial for the growth and maintenance of living organisms. The market need for biotin is multifaceted and driven by its importance in human health, beauty and cosmetics applications, pharmaceuticals, animal nutrition, and other industries. Current methods of industrial biotin production are unsustainable and generate environmentally hazardous waste, fueling the demand for new, greener strategies. The production of biotin through microbial fermentation has thus become an attractive alternative. Biotin biosynthesis is a complex and energy-intensive process that requires several enzymatic reactions, and biotin titers in microbial production have not reached economically viable levels. Biotin Synthase (BioB), the enzyme that catalyzes the last step of this process in E. coli, the conversion of dethiobiotin to biotin, has been identified as the bottleneck for this pathway. Overexpression of the enzyme generates oxidative stress and inhibits growth, though the exact mechanism has not yet been elucidated. This thesis employed a multiplexed engineering approach via high-throughput genetic engineering, selection, and screening methods to explore whether creating E. coli strains more resistant to oxidative stress would lead to higher biotin production capabilities. Using the expression of a DNA methylase to introduce controlled genetic mutations in Biosyntia's proprietary biotin-producing E. coli strains and selecting strains with improved resistance to oxidative stress yielded a selection of strains with increased resistance, which were screened for their biotin production. The improved resistance to oxidative stress and BioB induction in the strains did not lead to higher biotin production levels.