Enzyme assembly guided by SPFH‐induced functional inclusion bodies for enhanced cascade biocatalysis

Abstract

Enzyme clustering into compact agglomerates could accelerate the processing of intermediates to enhance metabolic pathway flux. However, enzyme clustering is still a challenging task due to the lack of universal assembly strategy applicable to all enzymes. Therefore, we proposed an alternative enzyme assembly strategy based on functional inclusion bodies. First, functional inclusion bodies in cells were formed by the fusion expression of stomatin/prohibitin/flotillin/HflK/C (SPFH) domain and enhanced green fluorescent protein, as observed visually and by transmission electron microscopy. The formation of SPFH‐induced functional inclusion bodies enhanced intermolecular polymerization as revealed by further analysis combined with Förster resonance energy transfer and bimolecular fluorescent complimentary. Finally, the functional inclusion bodies significantly improved the enzymatic catalysis in living cells, as proven by the examples with whole‐cell biocatalysis of phenyllactic acid by Escherichia coli, and the production of N‐acetylglucosamine by Bacillus subtilis. Our findings suggest that SPFH‐induced functional inclusion bodies can enhance the cascade reaction of enzymes, to serve as a potential universal strategy for the construction of efficient microbial cell factories.

Enzyme Assembly for Compartmentalized Metabolic Flux Control

“…Recently, our research group performed a preliminary study on the biological function of CatIBs in vivo. When investigating the properties of the stomatin/prohibitin/flotillin/HflK/C (SPFH) domain of the HflK protein, we found that the SPFH domains could promote the formation of functional IBs, and these functional IBs could obviously improve enzymatic catalysis in vivo, as proven by the examples with whole-cell biocatalysis of phenyllactic acid by E. coli and the biosynthesis of N-acetylglucosamine by B. subtilis [56]. Bimolecular fluorescent complimentary (BiFC) and Förster resonance energy transfer (FRET) analysis showed that the mechanism of this effect may be that the formation of CatIBs can promote intermolecular polymerization, which is consistent with what was speculated by Arié et al [52].…”