Enhanced performance of the methylerythritol phosphate pathway by manipulation of redox reactions relevant to IspC, IspG, and IspH

초록

The 2C-methyl-D-erythritol 4-phosphate (MEP) pathway is a carbon-efficient route for synthesis of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the building blocks of isoprenoids. However, practical application of a native or recombinant MEP pathway for the mass production of isoprenoids in Escherichia coli has been unsatisfactory. In this study, the entire recombinant MEP pathway was established with plasmids and used for the production of an isoprenoid, protoilludene. E. coli harboring the recombinant MEP pathway plasmid (ME) and a protoilludene synthesis pathway plasmid (AO) produced 10.4 mg/L of protoilludene after 48 h of culture. To determine the rate-limiting gene on plasmid ME, each constituent gene of the MEP pathway was additionally overexpressed on the plasmid AO. The additional overexpression of IPP isomerase (IDI) enhanced protoilludene production to 67.4 mg/L. Overexpression of the Fpr and FIdA protein complex, which could mediate electron transfer from NADPH to Fe-S cluster proteins such as IspG and IspH of the MEP pathway, increased protoilludene production to 318.8 mg/L. Given that it is required for IspC as well as IspG/H, the MEP pathway has high demand for NADPH. To increase the supply of NADPH, a NADH kinase from Saccharomyces cerevisiae (tPos5p) that converts NADH to NADPH was introduced along with the deletion of a promiscuous NADPH-dependent aldehyde reductase (YjgB) that consumes NADPH. This resulted in a protoilludene production of 512.7 mg/L. The results indicate that IDI, Fpr-FIdA redox proteins, and NADPH regenerators are key engineering points for boosting the metabolic flux toward a recombinant MEP pathway. (C) 2017 Elsevier B.V. All rights reserved.

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