(643e) Model-Guided Metabolic Engineering of Increased 2-Phenylethanol Production in Plants

Model-Guided
Metabolic Engineering of Increased 2-Phenylethanol Production in Plants
Shaunak
Ray¹*, Joseph Lynch², Clint Chapple², Natalia
Dudareva², John A. Morgan^1,2
1Davidson School of Chemical Engineering, Purdue University,
West Lafayette, Indiana 47907
²Department of Biochemistry, Purdue University, West Lafayette,
Indiana 47907

            2-Phenylethanol
(2-PE) is a naturally occurring aromatic with properties that make it a potential
oxygenate for gasoline. In plants, biosynthesis of 2-PE competes with the
phenylpropanoid pathway for the common precursor phenylalanine. The
phenylpropanoid pathway in plants directs approximately 30% of carbon flux
towards the biosynthesis of lignin, a major constituent of plant cell walls
that impedes the process of biofuel production. We therefore propose a genetic
engineering strategy, whereby a portion of the carbon flux towards lignin
biosynthesis is diverted from phenylalanine (Phe) towards the production of an
economically valuable product, 2-PE. Transgenic Arabidopsis thaliana
were generated that overexpress aromatic aldehyde synthase (AAS) in tandem with
phenylacetaldehyde reductase (PAR) introducing a pathway for the production of
2-PE. Excised 5-week old stems and leaves were exogenously fed different
concentrations of ¹³C₆-ring labeled Phe, and both amount
and isotopic enrichment of downstream metabolites were quantified using
LC-MS/MS and GC-MS at multiple time points. A kinetic model of the
phenylpropanoid pathway was constructed, and the parameters were identified
through non-linear optimization with training datasets, and validated with data
from an independent experiment. In silico analysis predicted that the
endogenous cytosolic Phe pools limit the 2-PE production in these transgenic
plants. This prediction was tested by combining the overexpression of PAR and
AAS with: (1) the overexpression of a feedback-insensitive
3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase known to have
hyper-induced phenylalanine biosynthesis in Arabidopsis, and (2) with
the double mutant pal1 pal2 known to have significant reduction in
activity of the first committed enzyme in phenylpropanoid biosynthesis, phenylalanine
ammonia lyase (PAL). These transformations led to significantly
increased accumulation of 2-PE in transgenic Arabidopsis. The use of
kinetic modeling combined with time-course in vivo metabolite profiling
is shown to be a promising approach to rationally engineer plants that
accumulate high-value commodity chemicals.