Metabolic Engineering of
Microorganisms
Jay D. Keasling
University of California
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.
Metabolic engineering offers the opportunity to expand the
role of bioremediation. Traditional metabolic
engineering involves overexpression of a desired protein and
leads to a high metabolic burden on the cell. The
purpose of this work is to develop strategies to help reduce
this burden and make an engineered organism more
environmentally effective.
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
Jay D. Keasling
University of California
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 ãnaturalä 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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