Improved biotin, thiamine, and lipoic acid biosynthesis by engineering the global regulator IscR.

Biotin, thiamine, and lipoic acid are industrially important molecules naturally synthesized by microorganisms via biosynthetic pathways requiring iron-sulfur (FeS) clusters. Current production is exclusively by chemistry because pathway complexity hinders development of fermentation processes. For biotin, the main bottleneck is biotin synthase, BioB, a S-adenosyl methionine-dependent radical enzyme that converts dethiobiotin (DTB) to biotin. BioB overexpression is toxic, though the mechanism remains unclear. We identified single mutations in the global regulator IscR that substantially improve cellular tolerance to BioB overexpression, increasing Escherichia coli DTB-to-biotin biocatalysis by more than 2.2-fold. Based on proteomics and targeted overexpression of FeS-cluster biosynthesis genes, FeS-cluster depletion is the main reason for toxicity. We demonstrate that IscR mutations significantly affect cell viability and improve cell factories for de novo biosynthesis of thiamine by 1.3-fold and lipoic acid by 1.8-fold. We illuminate a novel engineering target for enhancing biosynthesis of complex FeS-cluster-dependent molecules, paving the way for industrial fermentation processes.

Wiring cell growth to product formation.

Microbial cell factories offer new and sustainable production routes for high-value chemicals. However, identification of high producers within a library of clones remains a challenge. When product formation is coupled to growth, millions of metabolic variants can be effectively interrogated by growth selection, dramatically increasing the throughput of strain evaluation. While growth-coupled selections for cell factories have a long history of success based on metabolite auxotrophies and toxic antimetabolites, such methods are generally restricted to molecules native to their host metabolism. New synthetic biology tools offer the opportunity to rewire cellular metabolism to depend on specific and non-native products for growth.