A Computational and Experimental Investigation of Lignin Metabolism in Arabidopsis thaliana.

Predominantly localized in plant secondary cell walls, lignin is a highly crosslinked, aromatic polymer that imparts structural support to plant vasculature, and renders biomass recalcitrant to pretreatment techniques impeding the economical production of biofuels. Lignin is synthesized via the phenylpropanoid pathway where the primary precursor phenylalanine (Phe) undergoes a series of functional modifications catalyzed by 11 enzyme families to produce p-coumaryl, coniferyl, and sinapyl alcohol, which undergo random polymerization into lignin. Several metabolic engineering efforts have aimed to alter lignin content and composition, and make biofuel feedstock more amenable to pretreatment techniques. Despite significant advances, several questions pertaining to carbon flux distribution in the phenylpropanoid network remain unanswered. Furthermore, complexity of the metabolic pathway and a lack of sensitive analytical tools add to the challenges of mechanistically understanding lignin synthesis. In this work, I describe improvements in analytical techniques used to characterize phenylpropanoid metabolism that have been applied to obtain a comprehensive quantitative mass balance of the phenylpropanoid pathway. Finally, machine learning and artificial intelligence were utilized to make predictions about optimal lignin amount and composition for improving saccharification. In summary, the overarching goal of this thesis was to further the understanding of lignin metabolism in the model system, Arabidopis thaliana, employing a combination of experimental and computational strategies. First, we developed comprehensive and sensitive analytical methods based on liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) to quantify intermediates of the phenylpropanoid pathway. Compared to existing targeted profiling techniques, the methods were capable of quantifying a wider range of phenylpropanoid intermediates, at lower concentrations, with minimal sample preparation. The technique was used to generate flux maps for wild type and mutant Arabidopsis stems that were fed exogenously 13C6-Phe. Flux maps computed in this work; (i) suggest the presence of a hitherto uncharacterized alternative route to caffeic acid and lignin synthesis, (ii) shed light on flux splits at key branch points of the network, and (iii) indicate presence of inactive pools for a number of metabolites. Finally, we present a machine learning based model that captures the non-linear relationship between lignin content and composition, and saccharification efficiency. A support vector machine (SVM) based regression technique was developed to predict saccharification efficiency and biomass yields as a function of lignin content, and composition of monomers that make up lignin, namely p-coumaryl (H), coniferyl (G), and sinapyl (S) alcohol derived lignin. The model was trained on data obtained from the literature and validated on Arabidopsis mutants that were excluded from the training data set. Functional forms obtained from SVM regression were further optimized using genetic algorithms (GA) to maximize total sugar yields. Our efforts resulted in two optimal solutions with lower lignin content and interestingly varying H:G:S composition that were conducive to saccharide extractability

Thermodynamic Analysis of Phenylpropanoid Pathway in Arabidopsis Thanliana

Biofuels represent a renewable alternative to traditional fossil fuels. As dependence on fossil fuels rise so does the importance of improving the production of alternative fuels. Lignin poses one obstacle in the development of such alternative fuels. Its presence strengthens cell walls and hinders degradation of polysaccharides into monosaccharides, increasing cost and time while decreasing efficiency of the process. Lignin is composed of three monolignols, each of which is produced through the Phenylpropanoid pathway; a series of chemical reactions. This work aims to determine which reactions in the pathway are least thermodynamically favorable and thus most limiting. From metabolic mapping techniques on the Phenylpropanoid pathway in Arabidopsis Thanliana and thermodynamic data on the Gibbs free energy of formation for the biochemical compounds, the change in Gibbs free energy of the reaction at intracellular conditions is calculated. For compounds which data is unavailable, Group Contribution methods are used to determine the Gibbs free energy of formation. Reactions involving Cinnamoyl-CoA reductase, shikimate O-hydroxycinnamoyltransferase, and 4-coumarate-CoA ligase yielded positive Gibbs free energy values in the pathway. Since reactions involving these enzymes have positive Gibbs free energy values, these reactions require the greatest concentration of enzyme in order to facilitate production of the three monolignols. Knocking out these enzymes should result in a decrease in monolignol and lignin production

Multifaceted plant responses to circumvent Phe hyperaccumulation by downregulation of flux through the shikimate pathway and by vacuolar Phe sequestration

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/139951/1/tpj13730_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/139951/2/tpj13730.pd

A Computational and Experimental Investigation of Lignin Metabolism in Arabidopsis

Predominantly localized in plant secondary cell walls, lignin is a highly cross-linked, aromatic polymer that imparts structural support to plant vasculature, and renders biomass recalcitrant to pretreatment techniques impeding the economical production of biofuels. Lignin is synthesized via the phenylpropanoid pathway where the primary precursor phenylalanine (Phe) undergoes a series of functional modifications catalyzed by 11 enzyme families to produce p-coumaryl, coniferyl, and sinapyl alcohol, which undergo random polymerization into lignin. Several metabolic engineering efforts have aimed to alter lignin content and composition, and make biofuel feedstock more amenable to pretreatment techniques. Despite significant advances, several questions pertaining to carbon flux distribution in the phenylpropanoid network remain unanswered. Furthermore, complexity of the metabolic pathway and a lack of sensitive analytical tools add to the challenges of mechanistically understanding lignin synthesis. In this work, I describe improvements in analytical techniques used to characterize phenylpropanoid metabolism that have been applied to obtain a comprehensive quantitative mass balance of the phenylpropanoid pathway. Finally, machine learning and artificial intelligence were utilized to make predictions about optimal lignin amount and composition for improving saccharification. In summary, the overarching goal of this thesis was to further the understanding of lignin metabolism in the model system, Arabidopis thaliana, employing a combination of experimental and computational strategies. First, we developed comprehensive and sensitive analytical methods based on liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) to quantify intermediates of the phenylpropanoid pathway. Compared to existing targeted profiling techniques, the methods were capable of quantifying a wider range of phenylpropanoid intermediates, at lower concentrations, with minimal sample preparation. The technique was used to generate flux maps for wild type and mutant Arabidopsis stems that were fed exogenously 13C 6-Phe. Flux maps computed in this work; (i) suggest the presence of a hitherto uncharacterized alternative route to caffeic acid and lignin synthesis, (ii) shed light on flux splits at key branch points of the network, and (iii) indicate presence of inactive pools for a number of metabolites. Finally, we present a machine learning based model that captures the non-linear relationship between lignin content and composition, and saccharification efficiency. A support vector machine (SVM) based regression technique was developed to predict saccharification efficiency and biomass yields as a function of lignin content, and composition of monomers that make up lignin, namely p-coumaryl (H), coniferyl (G), and sinapyl (S) alcohol derived lignin. The model was trained on data obtained from the literature and validated on Arabidopsis mutants that were excluded from the training data set. Functional forms obtained from SVM regression were further optimized using genetic algorithms (GA) to maximize total sugar yields. Our efforts resulted in two optimal solutions with lower lignin content and interestingly varying H:G:S composition that were conducive to saccharide extractability

A Computational and Experimental Investigation of Lignin Metabolism in Arabidopsis

Predominantly localized in plant secondary cell walls, lignin is a highly cross-linked, aromatic polymer that imparts structural support to plant vasculature, and renders biomass recalcitrant to pretreatment techniques impeding the economical production of biofuels. Lignin is synthesized via the phenylpropanoid pathway where the primary precursor phenylalanine (Phe) undergoes a series of functional modifications catalyzed by 11 enzyme families to produce p-coumaryl, coniferyl, and sinapyl alcohol, which undergo random polymerization into lignin. Several metabolic engineering efforts have aimed to alter lignin content and composition, and make biofuel feedstock more amenable to pretreatment techniques. Despite significant advances, several questions pertaining to carbon flux distribution in the phenylpropanoid network remain unanswered. Furthermore, complexity of the metabolic pathway and a lack of sensitive analytical tools add to the challenges of mechanistically understanding lignin synthesis. In this work, I describe improvements in analytical techniques used to characterize phenylpropanoid metabolism that have been applied to obtain a comprehensive quantitative mass balance of the phenylpropanoid pathway. Finally, machine learning and artificial intelligence were utilized to make predictions about optimal lignin amount and composition for improving saccharification. In summary, the overarching goal of this thesis was to further the understanding of lignin metabolism in the model system, Arabidopis thaliana, employing a combination of experimental and computational strategies. First, we developed comprehensive and sensitive analytical methods based on liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) to quantify intermediates of the phenylpropanoid pathway. Compared to existing targeted profiling techniques, the methods were capable of quantifying a wider range of phenylpropanoid intermediates, at lower concentrations, with minimal sample preparation. The technique was used to generate flux maps for wild type and mutant Arabidopsis stems that were fed exogenously 13C 6-Phe. Flux maps computed in this work; (i) suggest the presence of a hitherto uncharacterized alternative route to caffeic acid and lignin synthesis, (ii) shed light on flux splits at key branch points of the network, and (iii) indicate presence of inactive pools for a number of metabolites. Finally, we present a machine learning based model that captures the non-linear relationship between lignin content and composition, and saccharification efficiency. A support vector machine (SVM) based regression technique was developed to predict saccharification efficiency and biomass yields as a function of lignin content, and composition of monomers that make up lignin, namely p-coumaryl (H), coniferyl (G), and sinapyl (S) alcohol derived lignin. The model was trained on data obtained from the literature and validated on Arabidopsis mutants that were excluded from the training data set. Functional forms obtained from SVM regression were further optimized using genetic algorithms (GA) to maximize total sugar yields. Our efforts resulted in two optimal solutions with lower lignin content and interestingly varying H:G:S composition that were conducive to saccharide extractability

A 13C isotope labeling method for the measurement of lignin metabolic flux in Arabidopsis stems

Abstract Background Metabolic fluxes represent the functional phenotypes of biochemical pathways and are essential to reveal the distribution of precursors among metabolic networks. Although analysis of metabolic fluxes, facilitated by stable isotope labeling and mass spectrometry detection, has been applied in the studies of plant metabolism, we lack experimental measurements for carbon flux towards lignin, one of the most abundant polymers in nature. Results We developed a feeding strategy of excised Arabidopsis stems with 13C labeled phenylalanine (Phe) for the analysis of lignin biosynthetic flux. We optimized the feeding methods and found the stems continued to grow and lignify. Consistent with lignification profiles along the stems, higher levels of phenylpropanoids and activities of lignin biosynthetic enzymes were detected in the base of the stem. In the feeding experiments, 13C labeled Phe was quickly accumulated and used for the synthesis of phenylpropanoid intermediates and lignin. The intermediates displayed two different patterns of labeling kinetics during the feeding period. Analysis of lignin showed rapid incorporation of label into all three subunits in the polymers. Conclusions Our feeding results demonstrate the effectiveness of the stem feeding system and suggest a potential application for the investigations of other aspects in plant metabolism. The supply of exogenous Phe leading to a higher lignin deposition rate indicates the availability of Phe is a determining factor for lignification rates

MOESM1 of A 13C isotope labeling method for the measurement of lignin metabolic flux in Arabidopsis stems

Additional file 1. Figure S1. A simplified pathway illustrating the enzymes and metabolites involved in lignin biosynthesis. PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate CoA ligase; HCT, hydroxycinnamoyl CoA:shikimatehydroxycinnamoyl transferase; C3′H, p-coumaroyl shikimate 3′-hydroxylase; CSE, caffeoyl shikimate esterase; CCoAOMT, caffeoyl CoA O-methyltransferase; F5H, ferulate5-hydroxylase; COMT, caffeic acid O-methyltransferase; CCR, cinnamoyl CoA reductase; CAD, cinnamyl alcohol dehydrogenase. SALDH, sinapaldehydedehydrogenase. Figure S2. Excised stems incubated in tubes with MS medium in growth chamber. (A) An Arabidopsis stem was excised and placed into a 1.5 mL tube containing liquid MS medium. (B) Arabidopsis stems incubated in MS medium were placed in a rack to perform feeding experiment (picture taken from side). (C) Stems were sitting away from each other to mimic their growth in the soil (picture taken from top). Figure S3. Medium absorbed by the excised stems during the feeding process. The loss of medium from each tube with an excised stem was measured after feeding for 0, 40, 90, 180, and 240 min. Data represented mean ± SD (n = 45). Figure S4. Hierarchical clustering of labeling percentage profiles of soluble phenylpropanoids from the base of Arabidopsis stems supplied with 1 mM [13C6]-Phe over the feeding time course. The averaged labeling percentage data of each metabolite over the time course from Figure 5 were clustered based on squared Euclidian distance. Figure S5. Metabolic profiles of soluble phenylpropanoids from the base of Arabidopsis stems supplied with 1 mM [13C6]-Phe over the feeding time course. Sum of endogenous and 13C6 labeled compounds was quantified with LC/MS-MS and normalized to fresh weight of stem tissue. The plot of each metabolite measured was placed above its name on the pathway. Dashed lines mean multiple steps. Data represent mean ± SD (n = 3)