title: Enhanced performance of the methylerythritol phosphate pathway by manipulation of redox reactions relevant to IspC, IspG, and IspH.
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 10.4mg/L of protoilludene after 48h 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 67.4mg/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 318.8mg/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 512.7mg/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.
authors.affiliation: Faculty of Life Science and Food Engineering, HuaiYin Institute of Technology, Huaian 223003, China; Division of Applied Life Science (BK21 Plus), PMBBRC, Gyeongsang National University, Jinju 52828, South Korea.
authors.forename: Jia
authors.initials: J
authors.lastname: Zhou
authors.affiliation: Division of Applied Life Science (BK21 Plus), PMBBRC, Gyeongsang National University, Jinju 52828, South Korea.
authors.forename: Liyang
authors.initials: L
authors.lastname: Yang
authors.affiliation: Division of Applied Life Science (BK21 Plus), PMBBRC, Gyeongsang National University, Jinju 52828, South Korea.
authors.forename: Chonglong
authors.initials: C
authors.lastname: Wang
authors.affiliation: Industrial Biotechnology Research Center, KRIBB, Daejeon 28116, South Korea. Electronic address: choi4162@kribb.re.kr.
authors.forename: Eui-Sung
authors.initials: ES
authors.lastname: Choi
authors.affiliation: Division of Applied Life Science (BK21 Plus), PMBBRC, Gyeongsang National University, Jinju 52828, South Korea. Electronic address: swkim@gnu.ac.kr.
authors.forename: Seon-Won
authors.initials: SW
authors.lastname: Kim
mesh_terms: D019747:Aldose-Ketose Isomerases / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins; D001709:Biotechnology; D003001:Cloning, Molecular / Q000379:methods*; D004896:Erythritol / Q000031:analogs & derivatives / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D005287:Ferredoxin-NADP Reductase; D005420:Flavoproteins; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D000081223:Polycyclic Sesquiterpenes; D012717:Sesquiterpenes / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D005420:Flavoproteins; C120194:FldA protein, Bacteria; D000081223:Polycyclic Sesquiterpenes; D012717:Sesquiterpenes; C506221:protoilludane; C077565:fpr protein, E coli; D009249:NADP; D010088:Oxidoreductases; C109866:ispH protein, E coli; C114231:1-deoxy-D-xylulose 5-phosphate reductoisomerase; D005287:Ferredoxin-NADP Reductase; D019747:Aldose-Ketose Isomerases; D004896:Erythritol
keywords: Escherichia coli; Fe–S cluster protein; Isoprenoid; MEP pathway; NADPH