Strategies for Metabolic Engineering of Environmental Microorganisms - Application to Degradation of Organophosphate Contaminants

Jay D. Keasling
Department of Chemical Engineering
University of California
Berkeley, CA 9470-1462
phone: (510) 642-4862 fax: (510) 643-1228 email: keasling@socrates.berkeley.edu

The goal of this work is to develop the experimental and theoretical methods to introduce multiple, heterologous, biodegradation pathways into a single organism and to optimize the flux through those pathways for the remediation of toxic or recalcitrant organic contaminants. The objectives of this work are: (1) to find and clone a gene that encodes an enzyme capable of degrading diethylphosphate, (2) to clone and express a pathway for complete mineralization of p-nitrophenol phosphate, (3) to clone and express a phosphotriesterase capable of hydrolyzing parathion, (4) to develop a co-culture biofilm capable of degrading parathion (as a proof-of-concept), and (5) to combine all of the genes in a single organism for complete mineralization of parathion or paraoxon.

Parathion (O,O-diethyl-O-p-nitrophenyl phosphorothioate), an organophosphate pesticide which has been widely used and is highly toxic, was chosen as the model compound for this project. Parathion is also structurally and functionally similar to many chemical warfare agents (including VX and soman).

Metabolic engineering of isoprenoid production

The objectives of this work are (i) to maximize the production of the isoprenoid precursor isopentenyl diphosphate in E. coli by expressing the genes for either the mevalonate-dependent or the mevalonate-independent synthesis pathway using the metabolic engineering tools developed in this laboratory; (ii) to maximize production of the primary precursors to the terpenoids: geranyl diphosphate, farnesyl diphosphate, and geranylgeranyl diphosphate; (iii) to introduce into E. coli the genes for specific classes of terpenoids and optimize production of these "natural" terpenoids; and (iv) to use laboratory evolution of terpene cyclases to produce novel terpenoids or to change the distribution of products made by terpenoid biosynthetic enzymes.

To accomplish this work, we are (i) cloning the genes encoding the enzymes in the non-mevalonate IPP biosynthetic pathway and express these genes under the control of inducible promoters on high, medium, and low-copy plasmids; (ii) cloning the genes for synthesis of DMAPP, GPP, FPP, and GGPP and express these genes under the control of inducible and constitutive promoters on high, medium, and low-copy plasmids; (iii) cloning the genes for various plant and fungal terpenes and express these genes under the control of inducible and constitutive promoters on high, medium, and low-copy plasmids; and (iv) mutating the terpene cyclases genes using mutagenic PCR and gene shuffling. For the maximization of IPP, DMAPP, and GGPP production, we will express the genes for lycopene synthesis and look for deep red colonies (containing large quantities of lycopene).

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