Metabolic engineering of microorganisms for isoprenoid production James Kirby b and Jay D. Keasling * abcde Received 20th February 2008 First published as an Advance Article on the web 25th April 2008 DOI: 10.1039/b802939c Covering: up to the end of 2007 Isoprenoids are ubiquitous in nature and range from essential cell components to unique secondary metabolites. The two isoprenoid biosynthetic pathways have received much attention from a metabolic engineering standpoint, and significant advances have been made in increasing flux through these pathways. Engineering later steps in isoprenoid biosynthetic pathways, specifically those related to the functionalization of terpene backbones, is at an earlier stage of development, both in terms of gene discovery and heterologous expression. Here we review recent advances in the metabolic engineering of microbes for isoprenoid production as well as some novel approaches to gene discovery and expression. Introduction Metabolic engineering of microorganisms through the expres- sion of one or more plant genes, often in combination with genetic alteration of the host cell metabolism, has become an increasingly important route for small molecule synthesis. One group of compounds that has particularly benefited from this approach is the isoprenoids (sometimes referred to as terpenes). Isoprenoids are one of the most structurally diverse groups of natural products, with over 50 000 examples identified to date. 1 However, they are all derived from the same basic units – isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). IPP and DMAPP are combined by prenyl- transferases to form geranyl diphosphate (GPP, the C 10 precursor to monoterpenes), farnesyl diphosphate (FPP, the C 15 precursor to sesquiterpenes and triterpenes) and geranylgeranyl diphosphate (GGPP, the C 20 precursor to diterpenes) (Fig. 1). Isoprenoids are most commonly found in plants, where they have been attributed functions as diverse as maintaining membrane fluidity, electron transport, use in protein prenylation, and protection against assault by microbes, insects and herbivores. 2 They have also found many applications in the areas of medicine (e.g., the anticancer drug Taxol (paclitaxel) from Taxus spp. 3 and the antimalarial drug artemisinin from Artemisia annua 4 ), agriculture (e.g., the insect antifeedant azadirachtin from Azadirachta indica 5 ), and as nutraceuticals (e.g., caroten- oids such as b-carotene 6 and lutein 7 ). In fact, isoprenoids have been used medicinally for over two thousand years, and prior to this in the form of ancient Egyptian embalming oils. 8 One of the earliest known pharmacological manuscripts describes various applications of latex from the succulent Euphorbia resinifera, dried extracts of which were used in ancient Roman times. 9 It was not until 1975 that the likely active component of E. resinifera latex was identified as resiniferatoxin, an isoprenoid deriva- tive. 9,10 Isoprenoids have also been used since the middle ages in James Kirby James Kirby received his B.S. in Industrial Microbiology in 1993 from University College Dublin, Ireland. He completed his Ph.D. in 1997 at the Rowett Research Institute, Scotland, followed by postdoctoral research at the Dept. of Genetics at Trinity College Dublin. He now works as a staff scientist for the California Institute of Quanti- tative Biomedical Research (QB3) with Prof. Jay Keasling. Jay Keasling Jay Keasling received his B.S. in Chemistry & Biology from the University of Nebraska in 1986 and his Ph.D. in Chemical Engineering from the University of Michigan in 1991. Keasling joined the Dept. of Chemical Engineering at the University of California, Berkeley, in 1992, where he is currently professor. He is also a professor in the Dept. of Bioengineering at U.C. Berkeley. a Berkeley Center for Synthetic Biology, 717 Potter Street, Building 977, Mail code 3224, University of California, Berkeley, CA, 94720-3224, USA. E-mail: keasling@berkeley.edu; Fax: +1 (510) 495-2630; Tel: +1 (510) 495-2620 b California Institute of Quantitative Biomedical Research, University of California, Berkeley, CA, 94720, USA c Department of Chemical Engineering, University of California, Berkeley, CA, 94720, USA d Department of Bioengineering, University of California, Berkeley, CA, 94720, USA e Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, USA 656 | Nat. Prod. Rep., 2008, 25, 656–661 This journal is ª The Royal Society of Chemistry 2008 HIGHLIGHT www.rsc.org/npr | Natural Product Reports