title: Efflux transporter engineering markedly improves amorphadiene production in Escherichia coli.
abstract: Metabolic engineering aims at altering cellular metabolism to produce valuable products at high yields and titers. Achieving high titers and productivity can be challenging if final products are largely accumulated intracellularly. A potential solution to this problem is to facilitate the export of these substances from cells by membrane transporters. Amorphadiene, the precursor of antimalarial drug artemisinin, is known to be secreted from Escherichia coli overexpressing the biosynthetic pathway. In order to assess the involvement of various endogenous efflux pumps in amorphadiene transport, the effects of single gene deletion of 16 known multidrug-resistant membrane efflux transporters were examined. The outer membrane protein TolC was found to be intimately involved in amorphadiene efflux. The overexpression of tolC together with ABC family transporters (macAB) or MFS family transporters (emrAB or emrKY) enhanced amorphadiene titer by more than threefold. In addition, the overexpression of transporters in the lipopolysaccharide transport system (msbA, lptD, lptCABFG) was found to improve amorphadiene production. As efflux transporters often have a wide range of substrate specificity, the multiple families of transporters were co-expressed and synergistic benefits were observed in amorphadiene production. This strategy of screening and then rationally engineering transporters can be used to improve the production of other valuable compounds in E. coli. Biotechnol. Bioeng. 2016;113: 1755-1763. © 2016 Wiley Periodicals, Inc.
authors.affiliation: Chemical and Pharmaceutical Engineering, Singapore-MIT Alliance, 4 Engineering Drive 3, Singapore, Singapore.|Biotransformation Innovation Platform (BioTrans), Agency for Science, Technology and Research (A*STAR), Singapore, Singapore.
authors.forename: Congqiang
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Chemical and Pharmaceutical Engineering, Singapore-MIT Alliance, 4 Engineering Drive 3, Singapore, Singapore.|Biotransformation Innovation Platform (BioTrans), Agency for Science, Technology and Research (A*STAR), Singapore, Singapore.
authors.forename: Xixian
authors.initials: X
authors.lastname: Chen
authors.affiliation: Chemical and Pharmaceutical Engineering, Singapore-MIT Alliance, 4 Engineering Drive 3, Singapore, Singapore.|Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts.
authors.forename: Gregory
authors.initials: G
authors.lastname: Stephanopoulos
authors.affiliation: Chemical and Pharmaceutical Engineering, Singapore-MIT Alliance, 4 Engineering Drive 3, Singapore, Singapore. heng-phon_too@nuhs.edu.sg.|Biotransformation Innovation Platform (BioTrans), Agency for Science, Technology and Research (A*STAR), Singapore, Singapore. heng-phon_too@nuhs.edu.sg.|Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, 8 Medical Drive, Blk MD7, Level 4, Singapore, 117597, Singapore. heng-phon_too@nuhs.edu.sg.
authors.forename: Heng-Phon
authors.initials: HP
authors.lastname: Too
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D001692:Biological Transport; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D055786:Gene Knockout Techniques; D008070:Lipopolysaccharides / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism; D060847:Metabolic Engineering / Q000379:methods*; D009154:Mutation; D000081223:Polycyclic Sesquiterpenes; D012717:Sesquiterpenes / Q000032:analysis / Q000378:metabolism*
chemical_list: D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D008070:Lipopolysaccharides; D026901:Membrane Transport Proteins; D000081223:Polycyclic Sesquiterpenes; D012717:Sesquiterpenes; C515348:amorpha-4,11-diene; C073126:tolC protein, E coli
keywords: TolC; amorphadiene; artemisinin; efflux pump; lipopolysaccharide transport system; transporter engineering