Terpene synthesis in the majority of bacterial species, together with plant plastids, takes place via the 1-deoxy-D-xylulose 5-phosphate (DXP) pathway. The first step of this pathway involves the condensation of pyruvate and glyceraldehyde 3-phosphate by DXP synthase (Dxs), with one-sixth of the carbon lost as CO 2 . A hypothetical novel route from a pentose phosphate to DXP (nDXP) could enable a more direct pathway from C 5 sugars to terpenes and also circumvent regulatory mechanisms that control Dxs, but there is no enzyme known that can convert a sugar into its 1-deoxy equivalent. Employing a selection for complementation of a dxs deletion in Escherichia coli grown on xylose as the sole carbon source, we uncovered two candidate nDXP genes. Complementation was achieved either via overexpression of the wild-type E. coli yajO gene, annotated as a putative xylose reductase, or via various mutations in the native ribB gene. In vitro analysis performed with purified YajO and mutant RibB proteins revealed that DXP was synthesized in both cases from ribulose 5-phosphate (Ru5P). We demonstrate the utility of these genes for microbial terpene biosynthesis by engineering the DXP pathway in E. coli for production of the sesquiterpene bisabolene, a candidate biodiesel. To further improve flux into the pathway from Ru5P, nDXP enzymes were expressed as fusions to DXP reductase (Dxr), the second enzyme in the DXP pathway. Expression of a Dxr-RibB(G108S) fusion improved bisabolene titers more than 4-fold and alleviated accumulation of intracellular DXP.T erpenes constitute a very large family of natural products, members of which are produced in virtually all free-living organisms (1). The diverse array of structures within the terpene family is reflected by the variety of applications in society, ranging from nutrition (carotenoids) and medicine (artemisinin, paclitaxel [originally taxol]) to industrial materials (isoprene, linalool) and candidate biofuels (farnesene, bisabolene, pinene) (2). Terpenes can be synthesized via either the mevalonate pathway or the 1-deoxy-D-xylulose 5-phosphate (DXP) pathway, the former predominating in the eukaryotic cytosol and the latter in plastids, while prokaryotes may contain either pathway or, in some cases, both pathways (3).Commercial-scale terpene production has been demonstrated in a variety of organisms, for example, carotenoids (via the DXP pathway) in algae (4, 5), paclitaxel (via DXP) in Taxus sp. (6), and artemisinin (via mevalonate) in Saccharomyces cerevisiae (7). Key metrics in determining the likelihood of commercial viability, particularly when targeting terpenes valued within the range of commodity chemicals or biofuels, are yield, productivity, and titer (8, 9). Of the two metabolic routes, the DXP pathway is considered the better option from the viewpoint of pathway efficiencyfor example, the theoretical maximum yield of isoprene from glucose is around 20% higher when synthesized via DXP instead of mevalonate (9, 10). In considering production of terpenes from hemicellul...

dTo facilitate enzyme and pathway engineering, a selection was developed for improved sesquiterpene titers in Saccharomyces cerevisiae. ␣-Bisabolene, a candidate advanced biofuel, was found to protect yeast against the disruptive action of nonionic surfactants such as Tween 20 (T20). An experiment employing competition between two strains of yeast, one of which makes twice as much bisabolene as the other, demonstrated that growth in the presence of T20 provided sufficient selective pressure to enrich the high-titer strain to form 97% of the population. Following this, various methods were used to mutagenize the bisabolene synthase (BIS) coding sequence, coupled with selection by subculturing in the presence of T20. Mutagenesis targeting the BIS active site did not yield an improvement in bisabolene titers, although mutants were found which made a mixture of ␣-bisabolene and ␤-farnesene, another candidate biofuel. Based on evidence that the 3= end of the BIS mRNA may be unstable in yeast, we randomly recoded the last 20 amino acids of the enzyme and, following selection in T20, found a variant which increased specific production of bisabolene by more than 30%. Since T20 could enrich a mixed population, efficiently removing strains that produced little or no bisabolene, we investigated whether it could also be applied to sustain high product titers in a monoculture for an extended period. Cultures grown in the presence of T20 for 14 days produced bisabolene at titers up to 4-fold higher than cultures grown with an overlay of dodecane, used to sequester the terpene product, and 20-fold higher than cultures grown without dodecane.