Elsevier

Journal of Biotechnology

Volume 248, 20 April 2017, Pages 1-8
Journal of Biotechnology

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

https://doi.org/10.1016/j.jbiotec.2017.03.005Get rights and content

Highlights

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    A new insight into the engineering of the MEP pathway is provided with manipulation of its peripheral metabolic systems.

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    Overexpression of the redox recycling system composed of FldA and Fpr is required for optimal function of IspG and IspH.

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    Heterologous overexpression of the NADH kinase tPos5p and deletion of the non-essential NADPH-dependent aldehyde reductase YjgB enhance performance of the MEP pathway.

ABSTRACT

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 FldA 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-FldA redox proteins, and NADPH regenerators are key engineering points for boosting the metabolic flux toward a recombinant MEP pathway.

Section snippets

INTRODUCTION

Isoprenoids are a large family of over 55,000 valuable natural compounds with potential as pharmaceuticals, insecticides, fragrances, and ideal fuel alternatives (McGarvey and Croteau, 1995, Peralta-Yahya and Keasling, 2010). They are derived from the universal building blocks, isopentenyl diphosphate (IPP), and dimethylallyl diphosphate (DMAPP). These building blocks are synthesized from two well-known biosynthetic pathways, namely the mevalonate (MVA) pathway and the 2C-methyl-D-erythritol

Bacterial strains and culture conditions

E. coli DH5α (F-,Φ80d lacZΔM15, Δ(lacZYA-argF) U169, deoR, recA1, endA1, hsdR17(rK− mK+), phoA, supE44, λ−, thi-1; ATCC 98040) was used as the parent strain. For knockout of yjgB from the chromosome of E. coli DH5α, a FRT-flanked kanamycin resistance cassette was amplified from plasmid pKD13 (Baba et al., 2006) with a primer pair of yjgB-KO-F/yjgB-KO-R and transformed into DH5α cells harboring plasmid pRedET for homologous recombination according to the instructions of the Quick and Easy Red/ET E

Recombinant MEP pathway for production of protoilludene

To evaluate the capacity of an engineered MEP pathway in E. coli, a strong isoprenoid synthesis pathway draining IPP/DMAPP should be coupled with the MEP pathway to prevent an intracellular accumulation of toxic IPP/DMAPP. Our previous report showed that a high-copy, plasmid-based protoilludene synthesis operon (AO in Fig. 2A), encoding E. coli farnesyl diphosphate synthase (IspA) and Omphalotus olearius protoilludene synthase (Omp7) under a strong trc promoter, was highly efficient for

CONCLUSIONS

We have provided a new insight into the engineering of the MEP pathway with manipulation of its peripheral metabolic systems. When direct manipulations of a target pathway are confronted with limits in increasing the pathway performance, the manipulation of peripheral metabolic systems could be an alternative approach of metabolic engineering. In this study, an entire recombinant MEP pathway was constructed on the plasmid ME, where additional overexpression of IDI from the plasmid AOI was

ACKNOWLEDGMENTS

This work was supported by C1 Gas Refinery Program (NRF-2016M3D3A1A01913246) and grants (NRF-2016M1A2A2924237; NRF-2016R1A2B2010678) from the National Research Foundation, MSIP, Korea; a grant (BK20150417) from Natural Science Foundation of Jiangsu Province of China; and a grant (406815002) from the scientific research start-up funding of HuaiYin Institute of Technology. Jia Zhou was supported by scholarships from the BK21 Plus Program, MEST, Korea.

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