Flux modeling for monolignol biosynthesis.

The pathway of monolignol biosynthesis involves many components interacting in a metabolic grid to regulate the supply and ratios of monolignols for lignification. The complexity of the pathway challenges any intuitive prediction of the output without mathematical modeling. Several models have been presented to quantify the metabolic flux for monolignol biosynthesis and the regulation of lignin content, composition, and structure in plant cell walls. Constraint-based models using data from transgenic plants were formulated to describe steady-state flux distribution in the pathway. Kinetic-based models using enzyme reaction and inhibition constants were developed to predict flux dynamics for monolignol biosynthesis in wood-forming cells. This review summarizes the recent progress in flux modeling and its application to lignin engineering for improved plant development and utilization.

Improving wood properties for wood utilization through multi-omics integration in lignin biosynthesis.

A multi-omics quantitative integrative analysis of lignin biosynthesis can advance the strategic engineering of wood for timber, pulp, and biofuels. Lignin is polymerized from three monomers (monolignols) produced by a grid-like pathway. The pathway in wood formation of Populus trichocarpa has at least 21 genes, encoding enzymes that mediate 37 reactions on 24 metabolites, leading to lignin and affecting wood properties. We perturb these 21 pathway genes and integrate transcriptomic, proteomic, fluxomic and phenomic data from 221 lines selected from ~2000 transgenics (6-month-old). The integrative analysis estimates how changing expression of pathway gene or gene combination affects protein abundance, metabolic-flux, metabolite concentrations, and 25 wood traits, including lignin, tree-growth, density, strength, and saccharification. The analysis then predicts improvements in any of these 25 traits individually or in combinations, through engineering expression of specific monolignol genes. The analysis may lead to greater understanding of other pathways for improved growth and adaptation.

Assessing the impact of the 4CL enzyme complex on the robustness of monolignol biosynthesis using metabolic pathway analysis.

Lignin is a polymer present in the secondary cell walls of all vascular plants. It is a known barrier to pulping and the extraction of high-energy sugars from cellulosic biomass. The challenge faced with predicting outcomes of transgenic plants with reduced lignin is due in part to the presence of unique protein-protein interactions that influence the regulation and metabolic flux in the pathway. Yet, it is unclear why certain plants have evolved to create these protein complexes. In this study, we use mathematical models to investigate the role that the protein complex, formed specifically between Ptr4CL3 and Ptr4CL5 enzymes, have on the monolignol biosynthesis pathway. The role of this Ptr4CL3-Ptr4CL5 enzyme complex on the steady state flux distribution was quantified by performing Monte Carlo simulations. The effect of this complex on the robustness and the homeostatic properties of the pathway were identified by performing sensitivity and stability analyses, respectively. Results from these robustness and stability analyses suggest that the monolignol biosynthetic pathway is resilient to mild perturbations in the presence of the Ptr4CL3-Ptr4CL5 complex. Specifically, the presence of Ptr4CL3-Ptr4CL5 complex increased the stability of the pathway by 22%. The robustness in the pathway is maintained due to the presence of multiple enzyme isoforms as well as the presence of alternative pathways resulting from the presence of the Ptr4CL3-Ptr4CL5 complex.

Complete proteomic-based enzyme reaction and inhibition kinetics reveal how monolignol biosynthetic enzyme families affect metabolic flux and lignin in Populus trichocarpa.

We established a predictive kinetic metabolic-flux model for the 21 enzymes and 24 metabolites of the monolignol biosynthetic pathway using Populus trichocarpa secondary differentiating xylem. To establish this model, a comprehensive study was performed to obtain the reaction and inhibition kinetic parameters of all 21 enzymes based on functional recombinant proteins. A total of 104 Michaelis-Menten kinetic parameters and 85 inhibition kinetic parameters were derived from these enzymes. Through mass spectrometry, we obtained the absolute quantities of all 21 pathway enzymes in the secondary differentiating xylem. This extensive experimental data set, generated from a single tissue specialized in wood formation, was used to construct the predictive kinetic metabolic-flux model to provide a comprehensive mathematical description of the monolignol biosynthetic pathway. The model was validated using experimental data from transgenic P. trichocarpa plants. The model predicts how pathway enzymes affect lignin content and composition, explains a long-standing paradox regarding the regulation of monolignol subunit ratios in lignin, and reveals novel mechanisms involved in the regulation of lignin biosynthesis. This model provides an explanation of the effects of genetic and transgenic perturbations of the monolignol biosynthetic pathway in flowering plants.