Harnessing the periplasm of bacteria to develop biocatalyst for biosynthesis of highly pure chemicals.

Although biocatalytic transformation has shown great promise in chemical synthesis, there remain significant challenges in controlling high selectivity without the formation of undesirable by-products. For instance, few attempts to construct biocatalysts for de novo synthesis of pure flavin mononucleotide (FMN) have been successful, due to riboflavin (RF) accumulating in the cytoplasm and being secreted with FMN. To address this problem, we show here a novel biosynthesis strategy, compartmentalizing the final FMN biosynthesis step in the periplasm of an engineered Escherichia coli strain. This construct is able to overproduce FMN with high specificity (92.4% of total excreted flavins). Such a biosynthesis approach allows isolation of the final biosynthesis step from the cytoplasm to eliminate undesirable byproducts, providing a new route to develop biocatalysts for the synthesis of highpurity chemicals. IMPORTANCE The periplasm of Gram-negative bacterial hosts is engineered to compartmentalize the final biosynthesis step from the cytoplasm. This strategy is promising for the overproduction of high-value products with high specificity. We demonstrate the successful implementation of this strategy in microbial production of highly pure FMN.