Harnessing the Periplasm of Bacterial Cells To Develop Biocatalysts for the Biosynthesis of Highly Pure Chemicals

Abstract

Although biocatalytic transformation has shown great promise in chemical synthesis, there remain significant challenges in controlling high selectivity without the formation of undesirable byproducts. For instance, few attempts have been successful in constructing biocatalyst for synthesis of pure flavin mononucleotide (FMN) due to riboflavin (RF) accumulating inside 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 strain. This construct is able to over-produce FMN with high specificity (92.4% of total excreted flavins). Such biosynthesis approach allows isolation of the final biosynthesis step from the cytoplasm so as to eliminate undesirable by-products, providing a new route to develop biocatalysts for high-purity chemicals. 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 product with high specificity. We demonstrate the successful implementation of this strategy in microbial production of highly pure flavin mononucleotide (FMN).

Production of riboflavin and related cofactors by biotechnological processes

“…Another method aimed to produce highly pure FMN by compartmentalizing the final FMN biosynthesis step into the periplasm [143]. The synthesis of FMN outside the cytoplasm helped to eliminate the undesirable accumulation of RF and FAD in the spent medium.…”

Modular Engineering of the Flavin Pathway in Escherichia coli for Improved Flavin Mononucleotide and Flavin Adenine Dinucleotide Production

“…9 Some reports concentrated on the production of highly pure FMN via approaches such as cofactor trapping 10 and compartmentalization of the final FMN biosynthesis step in the periplasm. 11 The optimal periplasmically engineered strain accumulated 70.8 mg/L FMN, which accounted for 92.4% of total excreted flavins. However, while the cofactor trapping approach led to FMN production with high purity, it had a low yield and low titer.…”

Enhancement of malate production through engineering of the periplasmic rTCA pathway in Escherichia coli

“…The metabolic engineering of cytoplasmic biosynthetic pathways to create industrial strains of E. coli is commonplace, whereas the engineering of sophisticated biosynthetic pathways in the periplasm has been ignored. The periplasm has been engineered for substrate spectrum expansion (Choi et al, 2016;Shin et al, 2012), final products storage (Jeschek et al, 2016;Lee et al, 2017), and by-products elimination (Yang et al, 2018). Indeed, the periplasm has many potential advantages for metabolic engineering, including the sequestration of diverse metabolites, such as peptidoglycan, and the hydrolysis reaction (Mogensen & Otzen, 2005).…”