Abstract
Prenyltransferase NovQ is a vital class involved in the biosynthesis of secondary metabolites such as clorobiocin and novobiocin. To investigate the relationship between structure and catalytic properties of NovQ, here, we have analyzed the substrate-binding site, namely PT barrel, and revealed that menadione hydroquinol formed intermolecular interactions with the residue Glu281 near the center of the active pocket. In this study, Glu281 was substituted with 9 diverse amino acids and catalytic properties of mutants were observed in vitro. Among them, E281Q showed 2.05-fold activities towards the aromatic substrate and prenyl donor, while others obtained catalytic efficiency between 8.4 and 88.6% of that of wild-type NovQ. Furthermore, the effects of catalytic conditions and substrate status on the activity of NovQ and its mutants were considered to obtain the optimized prenylated reaction. When the evolutionary NovQ variant E281Q was overexpressed in the host constructed to synthesize dimethylallyl diphosphate through the engineered mevalonate (MVA) pathway, we harvested up to 4.7 mg/L prenylated menadione at C-3 position by exogenously supplying the aromatic substrate. The construction of the microbial platform based on NovQ opens a new orientation to further biosynthesize various vitamin K2 with other ABBA prenyltransferases in E. coli.
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14 April 2020
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Funding
This work was supported by the Key research and development plan of Anhui Province (1804b06020342), Natural Science Foundation of Anhui Province (1908085MB48 and 1908085MB43), China National Key Research and Development Program (2019YFA0904300) and Major Projects of Science and Technology in Anhui Province (17030801036).
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Ni, W., Zheng, Z., Liu, H. et al. Combining mutagenesis on Glu281 of prenyltransferase NovQ and metabolic engineering strategies for the increased prenylated activity towards menadione. Appl Microbiol Biotechnol 104, 4371–4382 (2020). https://doi.org/10.1007/s00253-020-10470-w
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DOI: https://doi.org/10.1007/s00253-020-10470-w