Abstract
The entanglement of lignin polymers with cellulose and hemicellulose in plant cell walls is a major biological barrier to the economically viable production of biofuels from woody biomass. Recent efforts of reducing this recalcitrance with transgenic techniques have been showing promise for ameliorating or even obviating the need for costly pretreatments that are otherwise required to remove lignin from cellulose and hemicelluloses. At the same time, genetic manipulations of lignin biosynthetic enzymes have sometimes yielded unforeseen consequences on lignin composition, thus raising the question of whether the current understanding of the pathway is indeed correct. To address this question systemically, we developed and applied a novel modeling approach that, instead of analyzing the pathway within a single target context, permits a comprehensive, simultaneous investigation of different datasets in wild type and transgenic plants. Specifically, the proposed approach combines static flux-based analysis with a Monte Carlo simulation in which very many randomly chosen sets of parameter values are evaluated against kinetic models of lignin biosynthesis in different stem internodes of wild type and lignin-modified alfalfa plants. In addition to four new postulates that address the reversibility of some key reactions, the modeling effort led to two novel postulates regarding the control of the lignin biosynthetic pathway. The first posits functionally independent pathways toward the synthesis of different lignin monomers, while the second postulate proposes a novel feedforward regulatory mechanism. Subsequent laboratory experiments have identified the signaling molecule salicylic acid as a potential mediator of the postulated control mechanism. Overall, the results demonstrate that mathematical modeling can be a valuable complement to conventional transgenic approaches and that it can provide biological insights that are otherwise difficult to obtain.
Highlights
The complex, interwoven structure of lignin, cellulose, and hemicellulose polymers in plant cell walls is the main cause for the recalcitrance of lignocellulosic feedstocks to microbial and enzymatic deconstruction towards fermentable sugars
flux balance analysis (FBA)-guided elucidation of three principal branch points Accounting for recent experimental observations, we adopted a revised pathway structure of monolignol biosynthesis in alfalfa stems that includes the CCR2-catalyzed reduction of caffeoyl-CoA to caffeyl aldehyde and the subsequent synthesis of coniferyl aldehyde by caffeic acid 3-O-methyltransferase (COMT) (Figure 1: black and red colored reactions), as explained earlier
The FBA-derived steady-state flux analysis for wild-type plants supports this argument. It suggests that variation in lignin composition from young to mature internodes is accomplished by modulating the flux partitioning at three principal branch points: p-coumaroyl-CoA, coniferyl aldehyde, and coniferyl alcohol
Summary
The complex, interwoven structure of lignin, cellulose, and hemicellulose polymers in plant cell walls is the main cause for the recalcitrance of lignocellulosic feedstocks to microbial and enzymatic deconstruction towards fermentable sugars. This recalcitrance, in turn, accounts for the high cost of biofuel production from renewable sources [1]. Most pertinent genes have been identified in model species with complete sequence information, and knowledge from these genomes is currently being used for homology searches in species where sequencing efforts are ongoing Such homology investigations are often effective, but caution is necessary, because multiple genes with similar sequences and annotations pointing to the same enzyme may possess distinct expression patterns and substrate preferences [5]. As a case in point, alfalfa (Medicago sativa L.), the organism used for our analysis, exhibits substantial differences in cell wall composition among the different internodes of young plants
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