(676f) Kinetic Modeling of Lignin Biosynthesis in Arabidopsis thaliana for Improved Biofuel Production

Kinetic
Modeling of Lignin Biosynthesis in A.
thaliana for Improved Biofuel Production

Rohit
Jaini¹*, Longyun Guo², Peng
Wang², Rachel McCoy², Clint Chapple², Natalia
Dudareva², John A. Morgan^1,2

¹School of Chemical Engineering, Purdue
University, West Lafayette, Indiana 47907, ²Department of
Biochemistry, Purdue University, West Lafayette, Indiana 47907

Lignin
is a hetero-phenolic polymer formed as a result of radical polymerization of monolignols namely coniferyl, syringyl and p-coumaryl alcohols – products of the phenylpropanoid pathway. It imparts structural strength,
vascular integrity and pathogen resistance to plants.  Although lignin is essential for plant
sustenance, it renders valuable lignocellulosic
biofuel feedstock recalcitrant to chemical, mechanical and biological treatment.
Several attempts to manipulate lignin monomer content and composition, to
improve forage digestibility and saccharification
efficiency, have revealed a lack of understanding of the dynamics and
regulatory properties of monolignol biosynthesis. We are
developing a kinetic model to elucidate the underlying mechanisms regulating
carbon flux through the phenylpropanoid network.
Kinetic parameters for the model will be obtained by non-linear least squares
optimization of metabolomics data from Arabidopsis
thaliana stem tissue fed with [ring-¹³C₆]-phenylalanine,
the pathway precursor.

As
a first step, we developed a novel and comprehensive analytical method based on
liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) for quantifying
intermediates of the phenylpropanoid pathway. The
experimental procedure entailed extraction of metabolites from A. thaliana stem tissue using 75% (v/v)
methanol in water as a solvent. The extract was then analyzed using reverse
phase LC-MS/MS by electrospray ionization in the negative ion mode with
multiple reaction monitoring (MRM) of two ions
(Q1–parent/Q3–daughter) per compound. The chromatography method was
improved by tuning for chromatographic parameters such as buffer concentration,
flow rate, pH of mobile phase, and column temperature. Transgenic Arabidopsis
plants with knockdown or overexpression of the ferulate
5-hydroxylase gene were utilized to validate the analytical method. Next, isotopic
feeding experiments with three different concentrations (100, 300 μM & 1 mM) of [ring-¹³C₆]-phenylalanine
were conducted to obtain kinetic parameters for the entry reactions to the phenylpropanoid network, starting at phenylalanine to p-coumaroyl CoA.

The analytical method allowed detection and quantification
of 12 of the 17 compounds of the pathway for which standards were available in
wild type tissue. Signal suppression due to matrix effects and analyte losses during sample preparation and extraction
were accounted for by standard spiking studies to accurately quantify the
pathway metabolites. A set of kinetic parameters were
identified that simultaneously fit fractional label and pool sizes of the
endogenous intermediates obtained using the analytical method. The model showed
good agreement with experimental data allowing reliable parameter estimation. Model
validation was conducted using an independent data set obtained on a pal1/pal2 knockout line. Our study has
laid foundation to extend the kinetic model to the entire metabolic network and
shows promise as a predictive tool to facilitate rational metabolic engineering
of plants for improved biofuel production.