(516f) Model-Guided Metabolic Engineering of Increased 2-Phenylethanol Production in Plants

2-Phenylethanol (2-PE) is a naturally occurring aromatic with properties that make it a candidate oxygenate for petroleum-derived 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. Therefore, we propose a genetic engineering strategy at the phenylalanine branch point, whereby a portion of the carbon flux towards lignin biosynthesis is diverted 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) from tomato introducing a pathway for the production of 2-PE. The primary stem was selected as the experimental system for analyzing competition of Phe between phenylpropanoid metabolism and the engineered 2-PE pathway. Excised 5-week-old stems were exogenously fed different concentrations of ¹³C₆-ring labeled Phe. Both the amount and isotopic enrichment of downstream intermediates was quantified using LC-MS/MS at multiple time points after feeding. A kinetic model of the phenylpropanoid network 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 of the results from our model predicted that the endogenous cytosolic Phe pools limit the 2-PE production in these transgenic plants. This prediction was tested by combining overexpression of PAR and PAAS with overexpression of a feedback-insensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase, the latter of which has been previously shown to have hyper-induced phenylalanine biosynthesis in Arabidopsis. This transformation led to a 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.