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