title: Enhancing Terpene yield from sugars via novel routes to 1-deoxy-d-xylulose 5-phosphate.
abstract: 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 CO2. A hypothetical novel route from a pentose phosphate to DXP (nDXP) could enable a more direct pathway from C5 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.
authors.affiliation: California Institute of Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, California, USA Joint BioEnergy Institute, Emeryville, California, USA.
authors.forename: James
authors.initials: J
authors.lastname: Kirby
authors.affiliation: California Institute of Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, California, USA Joint BioEnergy Institute, Emeryville, California, USA.
authors.forename: Minobu
authors.initials: M
authors.lastname: Nishimoto
authors.affiliation: California Institute of Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, California, USA.
authors.forename: Ruthie W N
authors.initials: RW
authors.lastname: Chow
authors.affiliation: Joint BioEnergy Institute, Emeryville, California, USA.
authors.forename: Edward E K
authors.initials: EE
authors.lastname: Baidoo
authors.affiliation: Joint BioEnergy Institute, Emeryville, California, USA.
authors.forename: George
authors.initials: G
authors.lastname: Wang
authors.affiliation: Joint Genome Institute, Walnut Creek, California, USA.
authors.forename: Joel
authors.initials: J
authors.lastname: Martin
authors.affiliation: Joint Genome Institute, Walnut Creek, California, USA.
authors.forename: Wendy
authors.initials: W
authors.lastname: Schackwitz
authors.affiliation: California Institute of Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, California, USA.
authors.forename: Rossana
authors.initials: R
authors.lastname: Chan
authors.affiliation: California Institute of Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, California, USA.
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Fortman
authors.affiliation: California Institute of Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, California, USA Joint BioEnergy Institute, Emeryville, California, USA Department of Chemical and Biomolecular Engineering, University of California, Berkeley, Berkeley, California, USA Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA keasling@berkeley.edu.
authors.forename: Jay D
authors.initials: JD
authors.lastname: Keasling
mesh_terms: D001711:Biotransformation; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D005816:Genetic Complementation Test; D060847:Metabolic Engineering; D010428:Pentosephosphates / Q000378:metabolism*; D013729:Terpenes / Q000378:metabolism*; D014994:Xylose / Q000378:metabolism*
chemical_list: C109460:1-deoxylulose 5-phosphate; D010428:Pentosephosphates; D013729:Terpenes; D014994:Xylose