Wout Boerjan - Academia.edu (original) (raw)

Papers by Wout Boerjan

Modification of lignin quality in transgenic poplars

info:eu-repo/semantics/nonPublishe

Holzforschung, 2021

Lignin content and its molecular structure influence various wood characteristics. In this study,... more Lignin content and its molecular structure influence various wood characteristics. In this study, the anatomical and physicochemical properties of wood derived from a naturally occurring mulberry mutant deficient in cinnamyl alcohol dehydrogenase (CAD), a key enzyme in lignin biosynthesis, were analyzed using conventional staining assays on stem sections, length and width measurements of xylem fiber cells, wood pulping and saccharification assays, and sugar compositional analysis of extractive-free wood powder. The present data indicate that the mutation in the CAD gene leads to improved wood delignification efficiency, increased pulp yield under alkaline pulping conditions, and enhanced saccharification efficiency following alkaline pretreatment. This study opens up new avenues for the multipurpose use of the mulberry CAD-deficient mutant as a raw material for biorefinery processes, in addition to its traditional use as a favored feed for silkworms.

ABSTRACTLignin is a complex polymer precisely deposited in the cell wall of specialised plant cel... more ABSTRACTLignin is a complex polymer precisely deposited in the cell wall of specialised plant cells, where it provides essential cellular functions. Plants coordinate timing, location, abundance and composition of lignin deposition in response to endogenous and exogenous cues. In roots, a fine band of lignin, the Casparian strip encircles endodermal cells. This forms an extracellular barrier to solutes and water and plays a critical role in maintaining nutrient homeostasis. A signalling pathway senses the integrity of this diffusion barrier and can induce over-lignification to compensate for barrier defects. Here, we report that activation of this endodermal sensing mechanism triggers a transcriptional reprogramming strongly inducing the phenylpropanoid pathway and immune signaling. This leads to deposition of compensatory lignin that is chemically distinct from Casparian strip lignin. We also report that a complete loss of endodermal lignification drastically impacts mineral nutrie...

Suppression of the Arabidopsis cinnamoyl-CoA reductase 1-6 intronic T-DNA mutation by epigenetic modification

Plant Physiology

Arabidopsis (Arabidopsis thaliana) transfer DNA (T-DNA) insertion collections are popular resourc... more Arabidopsis (Arabidopsis thaliana) transfer DNA (T-DNA) insertion collections are popular resources for fundamental plant research. Cinnamoyl-CoA reductase 1 (CCR1) catalyzes an essential step in the biosynthesis of the cell wall polymer lignin. Accordingly, the intronic T-DNA insertion mutant ccr1-6 has reduced lignin levels and shows a stunted growth phenotype. Here, we report restoration of the ccr1-6 mutant phenotype and CCR1 expression levels after a genetic cross with a UDP-glucosyltransferase 72e1 (ugt72e1),-e2,-e3 T-DNA mutant. We discovered that the phenotypic recovery was not dependent on the UGT72E family loss of function but due to an epigenetic phenomenon called trans T-DNA suppression. Via trans T-DNA suppression, the gene function of an intronic T-DNA mutant was restored after the introduction of an additional T-DNA sharing identical sequences, leading to heterochromatinization and splicing out of the T-DNA–containing intron. Consequently, the suppressed ccr1-6 allele...

Frontiers in Plant Science

The potential of whole genome duplication to increase plant biomass yield is well-known. In Arabi... more The potential of whole genome duplication to increase plant biomass yield is well-known. In Arabidopsis tetraploids, an increase in biomass yield was accompanied by a reduction in lignin content and, as a result, a higher saccharification efficiency was achieved compared with diploid controls. Here, we evaluated whether the results obtained in Arabidopsis could be translated into poplar and whether the enhanced saccharification yield upon alkaline pretreatment of hairpin-downregulated CINNAMYL ALCOHOL DEHYDROGENASE1 (hpCAD) transgenic poplar could be further improved upon a whole genome duplication. Using a colchicine treatment, wild-type (WT) Populus tremula x P. alba cv. INRA 717-1B4, a commonly used model clone in tree biotechnology research, and hpCAD tetraploids were generated and grown in the greenhouse. In parallel, WT tetraploid poplars were grown in the field. In contrast to Arabidopsis, a whole genome duplication of poplar had a negative impact on the biomass yield of both...

Frontiers in Plant Science

Lignocellulosic biomass is recalcitrant toward deconstruction into simple sugars mainly due to th... more Lignocellulosic biomass is recalcitrant toward deconstruction into simple sugars mainly due to the presence of lignin. By engineering plants to partially replace traditional lignin monomers with alternative ones, lignin degradability and extractability can be enhanced. Previously, the alternative monomer curcumin has been successfully produced and incorporated into lignified cell walls of Arabidopsis by the heterologous expression of DIKETIDE-CoA SYNTHASE (DCS) and CURCUMIN SYNTHASE2 (CURS2). The resulting transgenic plants did not suffer from yield penalties and had an increased saccharification yield after alkaline pretreatment. Here, we translated this strategy into the bio-energy crop poplar. Via the heterologous expression of DCS and CURS2 under the control of the secondary cell wall CELLULOSE SYNTHASE A8-B promoter (ProCesA8-B), curcumin was also produced and incorporated into the lignified cell walls of poplar. ProCesA8-B:DCS_CURS2 transgenic poplars, however, suffered from s...

BackgroundGiven its tolerance to stress and its richness in particular secondary metabolites, the... more BackgroundGiven its tolerance to stress and its richness in particular secondary metabolites, the tobacco tree, Nicotiana glauca, has been considered a promising biorefinery feedstock that would not be competitive with food and fodder crops.ResultsHere we present a 3.5 Gbp draft sequence and annotation of the genome of N. glauca spanning 731,465 scaffold sequences, with an N50 size of approximately 92 kbases. Furthermore, we supply a comprehensive transcriptome and metabolome analysis of leaf development comprising multiple techniques and platforms.The genome sequence is predicted to cover nearly 80% of the estimated total genome size of N. glauca. With 73,799 genes predicted and a BUSCO score of 94.9%, we have assembled the majority of gene-rich regions successfully. RNA-Seq data revealed stage-and/or tissue-specific expression of genes, and we determined a general trend of a decrease of tricarboxylic acid cycle metabolites and an increase of terpenoids as well as some of their cor...

Journal of Experimental Botany, 2019

Wood is extensively used as a construction material. Despite increasing knowledge of its mechanic... more Wood is extensively used as a construction material. Despite increasing knowledge of its mechanical properties, the contribution of the cell-wall matrix polymers to wood mechanics is still not well understood. Previous studies have shown that axial stiffness correlates with lignin content only for cellulose microfibril angles larger than around 20°, while no influence is found for smaller angles. Here, by analysing the wood of poplar with reduced lignin content due to down-regulation of CAFFEOYL SHIKIMATE ESTERASE, we show that lignin content also influences axial stiffness at smaller angles. Micro-tensile tests of the xylem revealed that axial stiffness was strongly reduced in the low-lignin transgenic lines. Strikingly, microfibril angles were around 15° for both wild-type and transgenic poplars, suggesting that cellulose orientation is not responsible for the observed changes in mechanical behavior. Multiple linear regression analysis showed that the decrease in stiffness was alm...

Plant Physiology, 2018

S.C. designed the experiments, performed most of the experiments, analyzed the data and wrote the... more S.C. designed the experiments, performed most of the experiments, analyzed the data and wrote the article; N.D.S. created and selected the set of polyploids and complemented the writing; R.V.A. did the experiments on the developmental series and complemented the writing; J.U.F. performed the carbohydrate micro-array and complemented the writing; R.D.R. and M.D.B. prepared the sections and did the transmission electron microscopy; D.G. and W.T.W. complemented the writing; B.V. and W.B. conceived the project, assisted in designing the experiments, supervised the experiments, and assisted in writing the article.

Plant Physiology, 2016

Auxin steers numerous physiological processes in plants, making the tight control of its endogeno... more Auxin steers numerous physiological processes in plants, making the tight control of its endogenous levels and spatiotemporal distribution a necessity. This regulation is achieved by different mechanisms, including auxin biosynthesis, metabolic conversions, degradation, and transport. Here, we introduce cis-cinnamic acid (c-CA) as a novel and unique addition to a small group of endogenous molecules affecting in planta auxin concentrations. c-CA is the photo-isomerization product of the phenylpropanoid pathway intermediate trans-CA (t-CA). When grown on c-CA-containing medium, an evolutionary diverse set of plant species were shown to exhibit phenotypes characteristic for high auxin levels, including inhibition of primary root growth, induction of root hairs, and promotion of adventitious and lateral rooting. By molecular docking and receptor binding assays, we showed that c-CA itself is neither an auxin nor an anti-auxin, and auxin profiling data revealed that c-CA does not significantly interfere with auxin biosynthesis. Single cell-based auxin accumulation assays showed that c-CA, and not t-CA, is a potent inhibitor of auxin efflux. Auxin signaling reporters detected changes in spatiotemporal distribution of the auxin response along the root of c-CA-treated plants, and long-distance auxin transport assays showed no inhibition of rootward auxin transport. Overall, these results suggest that the phenotypes of c-CA-treated plants are the consequence of a local change in auxin accumulation, induced by the inhibition of auxin efflux. This work reveals a novel mechanism how plants may regulate auxin levels and adds a novel, naturally occurring molecule to the chemical toolbox for the studies of auxin homeostasis.

Phenylcoumaran benzylic ether reductase (PCBER) is one of the most abundant proteins in poplar (P... more Phenylcoumaran benzylic ether reductase (PCBER) is one of the most abundant proteins in poplar (Populus spp) xylem, but its biological role has remained obscure. In this work, metabolite profiling of transgenic poplar trees downregulated in PCBER revealed both the in vivo substrate and product of PCBER. Based on mass spectrometry and NMR data, the substrate was identified as a hexosylated 8-5-coupling product between sinapyl alcohol and guaiacylglycerol, and the product was identified as its benzyl-reduced form. This activity was confirmed in vitro using a purified recombinant PCBER expressed in Escherichia coli. Assays performed on 20 synthetic substrate analogs revealed the enzyme specificity. In addition, the xylem of PCBER-downregulated trees accumulated over 2000-fold higher levels of cysteine adducts of monolignol dimers. These compounds could be generated in vitro by simple oxidative coupling assays involving monolignols and cysteine. Altogether, our data suggest that the function of PCBER is to reduce phenylpropanoid dimers in planta to form antioxidants that protect the plant against oxidative damage. In addition to describing the catalytic activity of one of the most abundant enzymes in wood, we provide experimental evidence for the antioxidant role of a phenylpropanoid coupling product in planta.

Holzforschung, 2021

Hydroxycinnamoyl shikimates were reported in 2005 to be intermediates in monolignol biosynthesis.... more Hydroxycinnamoyl shikimates were reported in 2005 to be intermediates in monolignol biosynthesis. 3-Hydroxylation of p-coumarate, originally thought to occur via coumarate 3-hydroxylase (C3H) from p-coumaric acid or its CoA thioester, was revealed to be via the action of coumaroyl shikimate 3′-hydroxylase (C3′H) utilizing p-coumaroyl shikimate as the substrate, itself derived from p-coumaroyl-CoA via hydroxycinnamoyl-CoA: shikimate hydroxycinnamoyltransferase (HCT). The same HCT was conjectured to convert the product, caffeoyl shikimate, to caffeoyl-CoA to continue on the pathway starting with its 3-O-methylation. At least in some plants, however, a more recently discovered caffeoyl shikimate esterase (CSE) enzyme hydrolyzes caffeoyl shikimate to caffeic acid from which it must again produce its CoA thioester to continue on the monolignol biosynthetic pathway. HCT and CSE are therefore monolignol biosynthetic pathway enzymes that have provided new opportunities to misregulate lignif...

The Plant Journal, 2021

The intricate architecture of cell walls and the complex cross-linking of their components hinder... more The intricate architecture of cell walls and the complex cross-linking of their components hinders some industrial and agricultural applications of plant biomass. Xylan is a key structural element of grass cell walls, closely interacting with other cell wall components such as cellulose and lignin. The main branching points of grass xylan, 3-linked L-arabinosyl substitutions, can be modified by ferulic acid (a hydroxycinnamic acid), which cross-links xylan to other xylan chains and lignin. XAX1 (Xylosyl arabinosyl substitution of xylan 1), a rice (Oryza sativa) member of the glycosyltransferase family GT61, has been described to add xylosyl residues to arabinosyl substitutions modified by ferulic acid. In this study, we characterize hydroxycinnamic acid-decorated arabinosyl substitutions present on rice xylan and their cross-linking, in order to decipher the role of XAX1 in xylan synthesis. Our results show a general reduction of hydroxycinnamic acidmodified 3-linked arabinosyl substitutions in xax1 mutant rice regardless of their modification with a xylosyl residue. Moreover, structures resembling the direct cross-link between xylan and lignin (ferulated arabinosyl substitutions bound to lignin monomers and dimers), together with diferulates known to cross-link xylan, are strongly reduced in xax1. Interestingly, apart from feruloyl and p-coumaroyl modifications on arabinose, putative caffeoyl and oxalyl modifications were characterized, which were also reduced in xax1. Our results suggest an alternative function of XAX1 in the transfer of hydroxycinnamic acid-modified arabinosyl substitutions to xylan, rather than xylosyl transfer to arabinosyl substitutions. Ultimately, XAX1 plays a fundamental role in cross-linking, providing a potential target for the improvement of use of grass biomass.

Genetic engineering of lignin composition in Populus trees

Plant Biotechnology Journal, 2021

Lignins are cell-wall-located aromatic polymers that provide strength and hydrophobicity to woody... more Lignins are cell-wall-located aromatic polymers that provide strength and hydrophobicity to woody tissues. Lignin monomers are synthesized via the phenylpropanoid pathway, wherein CAFFEOYL SHIKIMATE ESTERASE (CSE) converts caffeoyl shikimate into caffeic acid. Here, we explored the role of the two CSE homologs in poplar (Populus tremula x P. alba). Reporter lines showed that the expression conferred by both CSE1 and CSE2 promoters is similar. CRISPR-Cas9-generated cse1 and cse2 single mutants had a wild-type lignin level. Nevertheless, CSE1 and CSE2 are not completely redundant, as both single mutants accumulated caffeoyl shikimate. In contrast, the cse1 cse2 double mutants had a 35% reduction in lignin and associated growth penalty. The reduced lignin content translated into a four-fold increase in cellulose-to-glucose conversion upon limited saccharification. Phenolic profiling of the double mutants revealed large metabolic shifts, including an accumulation of p-coumaroyl, 5hydroxyferuloyl, feruloyl and sinapoyl shikimate, in addition to caffeoyl shikimate. This indicates that the CSEs have a broad substrate specificity, which was confirmed by in vitro enzyme kinetics. Taken together, our results suggest an alternative path within the phenylpropanoid pathway at the level of the hydroxycinnamoyl shikimates, and show that CSE is a promising target to improve plants for the biorefinery.

Computational and Structural Biotechnology Journal, 2021

Despite the scientific and economic importance of maize, little is known about its specialized me... more Despite the scientific and economic importance of maize, little is known about its specialized metabolism. Here, five maize organs were profiled using different reversed-phase liquid chromatography-mass spectrometry methods. The resulting spectral metadata, combined with candidate substrate-product pair (CSPP) networks, allowed the structural characterization of 427 of the 5,420 profiled compounds, including phenylpropanoids, flavonoids, benzoxazinoids, and auxin-related compounds, among others. Only 75 of the 427 compounds were already described in maize. Analysis of the CSPP networks showed that phenylpropanoids are present in all organs, whereas other metabolic classes are rather organ-enriched. Frequently occurring CSPP mass differences often corresponded with glycosyl-and acyltransferase reactions. The interplay of glycosylations and acylations yields a wide variety of mixed glycosides, bearing substructures corresponding to the different biochemical classes. For example, in the tassel, many phenylpropanoid and flavonoid-bearing glycosides also contain auxinderived moieties. The characterized compounds and mass differences are an important step forward in metabolic pathway discovery and systems biology research. The spectral metadata of the 5,420 compounds is publicly available (DynLib spectral database, ).

Proceedings of the National Academy of Sciences, 2019

Lignin is an abundant aromatic polymer found in plant secondary cell walls. In recent years, lign... more Lignin is an abundant aromatic polymer found in plant secondary cell walls. In recent years, lignin has attracted renewed interest as a feedstock for bio-based chemicals via catalytic and biological approaches and has emerged as a target for genetic engineering to improve lignocellulose digestibility by altering its composition. In lignin biosynthesis and microbial conversion, small phenolic lignin precursors or degradation products cross membrane bilayers through an unidentified translocation mechanism prior to incorporation into lignin polymers (synthesis) or catabolism (bioconversion), with both passive and transporter-assisted mechanisms postulated. To test the passive permeation potential of these phenolics, we performed molecular dynamics simulations for 69 monomeric and dimeric lignin-related phenolics with 3 model membranes to determine the membrane partitioning and permeability coefficients for each compound. The results support an accessible passive permeation mechanism fo...

Editorial overview: Plant biotechnology – lignin engineering

Current Opinion in Biotechnology, 2019

It is particularly timely to publish a special issue on lignin biosynthesis and its engineering g... more It is particularly timely to publish a special issue on lignin biosynthesis and its engineering giving the depth and breadth of amazing breakthroughs in fundamental understanding and applications that have occurred in recent years. Lignins are polymers that are mainly deposited in secondary-thickened plant cell walls. There, they are produced in a polymerization step (lignifica-tion) from their component monomers by a process of simple combinatorial radical coupling that was once considered an enigma. The monomers that have been known for decades are the p-coumaryl, coniferyl, and sinapyl alcohols. However, the development of new analytical tools along with analyses of lignin structures from a broader range of plant species has led to the discovery of many additional monomers, giving rise to a rich diversity of polymers with different physicochemical properties. As a consequence, the very definition of lignin continues to evolve as new species of plants are characterized in greater detail. The fact that lignin polymerization is a combinatorial radical coupling process, and that so many monomers naturally couple into the lignin polymer, has given rise to the concept that lignin structure is malleable and that plants can be engineered to make lignin structures that are tailored for various end-use applications. Indeed, in industrial operations producing pulp or fermentable sugars from wood, lignin needs to be extracted by expensive chemical treatments, and the efficiency of this process heavily depends on lignin's amount and composition. On the other hand, lignin is increasingly considered as a valuable source of aromatic building blocks for the chemical industry. As is now becoming evident, particular lignin structures can be designed and engineered into plants to maximize lignin-derived aromatic monomers streams. The acceleration in the interest in lignin and its engineering, caused by the urgent need for a transition from a fossil-based to a biobased economy, is illustrated by the steadily increasing number of papers covering 'lignin' with 'gene' terms, from 725 in 2010 to 1516 in 2017, with citations escalating at a far greater rate. When we conceived this issue, our objective was to cover lignin research extending from biosynthetic pathway revelations up to its valorization, but always with a focus on biotechnology, and with minimal overlap among the individual reviews. Given that several research groups work on the very same topic, it was, unfortunately, not possible to invite all of the main players in the lignin field and we apologize for this. The following are just a few of the highlights covered in the various contributions to this special issue as a testament to the state-of-the-art advances and practical implications of lignin bioengineering. Vanholme et al. [1] review the biosynthetic pathways towards the 35 metabolites from over 10 metabolic classes that have now been identified as lignin building blocks. They examine (Belgium). He specializes in lignin biosynthesis (gene and pathway discovery) and in lignin engineering in model plants (Arabidopsis), and crops (maize and poplar) to speed up the transition from a fossil-based to a bio-based economy.

Lignin biosynthesis and its integration into metabolism

Current Opinion in Biotechnology, 2019

Lignin is a principal structural component of cell walls in higher terrestrial plants. It reinfor... more Lignin is a principal structural component of cell walls in higher terrestrial plants. It reinforces the cell walls, facilitates water transport, and acts as a physical barrier to pathogens. Lignin is typically described as being composed of p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units that derive from the polymerization of the hydroxycinnamyl alcohols, p-coumaryl, coniferyl, and sinapyl alcohol, respectively. However, lignin also derives from various other aromatic monomers. Here, we review the biosynthetic pathway to the lignin monomers, and how flux through the pathway is regulated. Upon perturbation of the phenylpropanoid pathway, pathway intermediates may successfully incorporate into the lignin polymer, thereby affecting its physicochemical properties, or may remain soluble as such or as derivatized molecules that might interfere with physiological processes.

Modification of lignin quality in transgenic poplars

info:eu-repo/semantics/nonPublishe

Holzforschung, 2021

Lignin content and its molecular structure influence various wood characteristics. In this study,... more Lignin content and its molecular structure influence various wood characteristics. In this study, the anatomical and physicochemical properties of wood derived from a naturally occurring mulberry mutant deficient in cinnamyl alcohol dehydrogenase (CAD), a key enzyme in lignin biosynthesis, were analyzed using conventional staining assays on stem sections, length and width measurements of xylem fiber cells, wood pulping and saccharification assays, and sugar compositional analysis of extractive-free wood powder. The present data indicate that the mutation in the CAD gene leads to improved wood delignification efficiency, increased pulp yield under alkaline pulping conditions, and enhanced saccharification efficiency following alkaline pretreatment. This study opens up new avenues for the multipurpose use of the mulberry CAD-deficient mutant as a raw material for biorefinery processes, in addition to its traditional use as a favored feed for silkworms.

ABSTRACTLignin is a complex polymer precisely deposited in the cell wall of specialised plant cel... more ABSTRACTLignin is a complex polymer precisely deposited in the cell wall of specialised plant cells, where it provides essential cellular functions. Plants coordinate timing, location, abundance and composition of lignin deposition in response to endogenous and exogenous cues. In roots, a fine band of lignin, the Casparian strip encircles endodermal cells. This forms an extracellular barrier to solutes and water and plays a critical role in maintaining nutrient homeostasis. A signalling pathway senses the integrity of this diffusion barrier and can induce over-lignification to compensate for barrier defects. Here, we report that activation of this endodermal sensing mechanism triggers a transcriptional reprogramming strongly inducing the phenylpropanoid pathway and immune signaling. This leads to deposition of compensatory lignin that is chemically distinct from Casparian strip lignin. We also report that a complete loss of endodermal lignification drastically impacts mineral nutrie...

Suppression of the Arabidopsis cinnamoyl-CoA reductase 1-6 intronic T-DNA mutation by epigenetic modification

Plant Physiology

Arabidopsis (Arabidopsis thaliana) transfer DNA (T-DNA) insertion collections are popular resourc... more Arabidopsis (Arabidopsis thaliana) transfer DNA (T-DNA) insertion collections are popular resources for fundamental plant research. Cinnamoyl-CoA reductase 1 (CCR1) catalyzes an essential step in the biosynthesis of the cell wall polymer lignin. Accordingly, the intronic T-DNA insertion mutant ccr1-6 has reduced lignin levels and shows a stunted growth phenotype. Here, we report restoration of the ccr1-6 mutant phenotype and CCR1 expression levels after a genetic cross with a UDP-glucosyltransferase 72e1 (ugt72e1),-e2,-e3 T-DNA mutant. We discovered that the phenotypic recovery was not dependent on the UGT72E family loss of function but due to an epigenetic phenomenon called trans T-DNA suppression. Via trans T-DNA suppression, the gene function of an intronic T-DNA mutant was restored after the introduction of an additional T-DNA sharing identical sequences, leading to heterochromatinization and splicing out of the T-DNA–containing intron. Consequently, the suppressed ccr1-6 allele...

Frontiers in Plant Science

The potential of whole genome duplication to increase plant biomass yield is well-known. In Arabi... more The potential of whole genome duplication to increase plant biomass yield is well-known. In Arabidopsis tetraploids, an increase in biomass yield was accompanied by a reduction in lignin content and, as a result, a higher saccharification efficiency was achieved compared with diploid controls. Here, we evaluated whether the results obtained in Arabidopsis could be translated into poplar and whether the enhanced saccharification yield upon alkaline pretreatment of hairpin-downregulated CINNAMYL ALCOHOL DEHYDROGENASE1 (hpCAD) transgenic poplar could be further improved upon a whole genome duplication. Using a colchicine treatment, wild-type (WT) Populus tremula x P. alba cv. INRA 717-1B4, a commonly used model clone in tree biotechnology research, and hpCAD tetraploids were generated and grown in the greenhouse. In parallel, WT tetraploid poplars were grown in the field. In contrast to Arabidopsis, a whole genome duplication of poplar had a negative impact on the biomass yield of both...

Frontiers in Plant Science

Lignocellulosic biomass is recalcitrant toward deconstruction into simple sugars mainly due to th... more Lignocellulosic biomass is recalcitrant toward deconstruction into simple sugars mainly due to the presence of lignin. By engineering plants to partially replace traditional lignin monomers with alternative ones, lignin degradability and extractability can be enhanced. Previously, the alternative monomer curcumin has been successfully produced and incorporated into lignified cell walls of Arabidopsis by the heterologous expression of DIKETIDE-CoA SYNTHASE (DCS) and CURCUMIN SYNTHASE2 (CURS2). The resulting transgenic plants did not suffer from yield penalties and had an increased saccharification yield after alkaline pretreatment. Here, we translated this strategy into the bio-energy crop poplar. Via the heterologous expression of DCS and CURS2 under the control of the secondary cell wall CELLULOSE SYNTHASE A8-B promoter (ProCesA8-B), curcumin was also produced and incorporated into the lignified cell walls of poplar. ProCesA8-B:DCS_CURS2 transgenic poplars, however, suffered from s...

BackgroundGiven its tolerance to stress and its richness in particular secondary metabolites, the... more BackgroundGiven its tolerance to stress and its richness in particular secondary metabolites, the tobacco tree, Nicotiana glauca, has been considered a promising biorefinery feedstock that would not be competitive with food and fodder crops.ResultsHere we present a 3.5 Gbp draft sequence and annotation of the genome of N. glauca spanning 731,465 scaffold sequences, with an N50 size of approximately 92 kbases. Furthermore, we supply a comprehensive transcriptome and metabolome analysis of leaf development comprising multiple techniques and platforms.The genome sequence is predicted to cover nearly 80% of the estimated total genome size of N. glauca. With 73,799 genes predicted and a BUSCO score of 94.9%, we have assembled the majority of gene-rich regions successfully. RNA-Seq data revealed stage-and/or tissue-specific expression of genes, and we determined a general trend of a decrease of tricarboxylic acid cycle metabolites and an increase of terpenoids as well as some of their cor...

Journal of Experimental Botany, 2019

Wood is extensively used as a construction material. Despite increasing knowledge of its mechanic... more Wood is extensively used as a construction material. Despite increasing knowledge of its mechanical properties, the contribution of the cell-wall matrix polymers to wood mechanics is still not well understood. Previous studies have shown that axial stiffness correlates with lignin content only for cellulose microfibril angles larger than around 20°, while no influence is found for smaller angles. Here, by analysing the wood of poplar with reduced lignin content due to down-regulation of CAFFEOYL SHIKIMATE ESTERASE, we show that lignin content also influences axial stiffness at smaller angles. Micro-tensile tests of the xylem revealed that axial stiffness was strongly reduced in the low-lignin transgenic lines. Strikingly, microfibril angles were around 15° for both wild-type and transgenic poplars, suggesting that cellulose orientation is not responsible for the observed changes in mechanical behavior. Multiple linear regression analysis showed that the decrease in stiffness was alm...

Plant Physiology, 2018

S.C. designed the experiments, performed most of the experiments, analyzed the data and wrote the... more S.C. designed the experiments, performed most of the experiments, analyzed the data and wrote the article; N.D.S. created and selected the set of polyploids and complemented the writing; R.V.A. did the experiments on the developmental series and complemented the writing; J.U.F. performed the carbohydrate micro-array and complemented the writing; R.D.R. and M.D.B. prepared the sections and did the transmission electron microscopy; D.G. and W.T.W. complemented the writing; B.V. and W.B. conceived the project, assisted in designing the experiments, supervised the experiments, and assisted in writing the article.

Plant Physiology, 2016

Auxin steers numerous physiological processes in plants, making the tight control of its endogeno... more Auxin steers numerous physiological processes in plants, making the tight control of its endogenous levels and spatiotemporal distribution a necessity. This regulation is achieved by different mechanisms, including auxin biosynthesis, metabolic conversions, degradation, and transport. Here, we introduce cis-cinnamic acid (c-CA) as a novel and unique addition to a small group of endogenous molecules affecting in planta auxin concentrations. c-CA is the photo-isomerization product of the phenylpropanoid pathway intermediate trans-CA (t-CA). When grown on c-CA-containing medium, an evolutionary diverse set of plant species were shown to exhibit phenotypes characteristic for high auxin levels, including inhibition of primary root growth, induction of root hairs, and promotion of adventitious and lateral rooting. By molecular docking and receptor binding assays, we showed that c-CA itself is neither an auxin nor an anti-auxin, and auxin profiling data revealed that c-CA does not significantly interfere with auxin biosynthesis. Single cell-based auxin accumulation assays showed that c-CA, and not t-CA, is a potent inhibitor of auxin efflux. Auxin signaling reporters detected changes in spatiotemporal distribution of the auxin response along the root of c-CA-treated plants, and long-distance auxin transport assays showed no inhibition of rootward auxin transport. Overall, these results suggest that the phenotypes of c-CA-treated plants are the consequence of a local change in auxin accumulation, induced by the inhibition of auxin efflux. This work reveals a novel mechanism how plants may regulate auxin levels and adds a novel, naturally occurring molecule to the chemical toolbox for the studies of auxin homeostasis.

Phenylcoumaran benzylic ether reductase (PCBER) is one of the most abundant proteins in poplar (P... more Phenylcoumaran benzylic ether reductase (PCBER) is one of the most abundant proteins in poplar (Populus spp) xylem, but its biological role has remained obscure. In this work, metabolite profiling of transgenic poplar trees downregulated in PCBER revealed both the in vivo substrate and product of PCBER. Based on mass spectrometry and NMR data, the substrate was identified as a hexosylated 8-5-coupling product between sinapyl alcohol and guaiacylglycerol, and the product was identified as its benzyl-reduced form. This activity was confirmed in vitro using a purified recombinant PCBER expressed in Escherichia coli. Assays performed on 20 synthetic substrate analogs revealed the enzyme specificity. In addition, the xylem of PCBER-downregulated trees accumulated over 2000-fold higher levels of cysteine adducts of monolignol dimers. These compounds could be generated in vitro by simple oxidative coupling assays involving monolignols and cysteine. Altogether, our data suggest that the function of PCBER is to reduce phenylpropanoid dimers in planta to form antioxidants that protect the plant against oxidative damage. In addition to describing the catalytic activity of one of the most abundant enzymes in wood, we provide experimental evidence for the antioxidant role of a phenylpropanoid coupling product in planta.

Holzforschung, 2021

Hydroxycinnamoyl shikimates were reported in 2005 to be intermediates in monolignol biosynthesis.... more Hydroxycinnamoyl shikimates were reported in 2005 to be intermediates in monolignol biosynthesis. 3-Hydroxylation of p-coumarate, originally thought to occur via coumarate 3-hydroxylase (C3H) from p-coumaric acid or its CoA thioester, was revealed to be via the action of coumaroyl shikimate 3′-hydroxylase (C3′H) utilizing p-coumaroyl shikimate as the substrate, itself derived from p-coumaroyl-CoA via hydroxycinnamoyl-CoA: shikimate hydroxycinnamoyltransferase (HCT). The same HCT was conjectured to convert the product, caffeoyl shikimate, to caffeoyl-CoA to continue on the pathway starting with its 3-O-methylation. At least in some plants, however, a more recently discovered caffeoyl shikimate esterase (CSE) enzyme hydrolyzes caffeoyl shikimate to caffeic acid from which it must again produce its CoA thioester to continue on the monolignol biosynthetic pathway. HCT and CSE are therefore monolignol biosynthetic pathway enzymes that have provided new opportunities to misregulate lignif...

The Plant Journal, 2021

The intricate architecture of cell walls and the complex cross-linking of their components hinder... more The intricate architecture of cell walls and the complex cross-linking of their components hinders some industrial and agricultural applications of plant biomass. Xylan is a key structural element of grass cell walls, closely interacting with other cell wall components such as cellulose and lignin. The main branching points of grass xylan, 3-linked L-arabinosyl substitutions, can be modified by ferulic acid (a hydroxycinnamic acid), which cross-links xylan to other xylan chains and lignin. XAX1 (Xylosyl arabinosyl substitution of xylan 1), a rice (Oryza sativa) member of the glycosyltransferase family GT61, has been described to add xylosyl residues to arabinosyl substitutions modified by ferulic acid. In this study, we characterize hydroxycinnamic acid-decorated arabinosyl substitutions present on rice xylan and their cross-linking, in order to decipher the role of XAX1 in xylan synthesis. Our results show a general reduction of hydroxycinnamic acidmodified 3-linked arabinosyl substitutions in xax1 mutant rice regardless of their modification with a xylosyl residue. Moreover, structures resembling the direct cross-link between xylan and lignin (ferulated arabinosyl substitutions bound to lignin monomers and dimers), together with diferulates known to cross-link xylan, are strongly reduced in xax1. Interestingly, apart from feruloyl and p-coumaroyl modifications on arabinose, putative caffeoyl and oxalyl modifications were characterized, which were also reduced in xax1. Our results suggest an alternative function of XAX1 in the transfer of hydroxycinnamic acid-modified arabinosyl substitutions to xylan, rather than xylosyl transfer to arabinosyl substitutions. Ultimately, XAX1 plays a fundamental role in cross-linking, providing a potential target for the improvement of use of grass biomass.

Genetic engineering of lignin composition in Populus trees

Plant Biotechnology Journal, 2021

Lignins are cell-wall-located aromatic polymers that provide strength and hydrophobicity to woody... more Lignins are cell-wall-located aromatic polymers that provide strength and hydrophobicity to woody tissues. Lignin monomers are synthesized via the phenylpropanoid pathway, wherein CAFFEOYL SHIKIMATE ESTERASE (CSE) converts caffeoyl shikimate into caffeic acid. Here, we explored the role of the two CSE homologs in poplar (Populus tremula x P. alba). Reporter lines showed that the expression conferred by both CSE1 and CSE2 promoters is similar. CRISPR-Cas9-generated cse1 and cse2 single mutants had a wild-type lignin level. Nevertheless, CSE1 and CSE2 are not completely redundant, as both single mutants accumulated caffeoyl shikimate. In contrast, the cse1 cse2 double mutants had a 35% reduction in lignin and associated growth penalty. The reduced lignin content translated into a four-fold increase in cellulose-to-glucose conversion upon limited saccharification. Phenolic profiling of the double mutants revealed large metabolic shifts, including an accumulation of p-coumaroyl, 5hydroxyferuloyl, feruloyl and sinapoyl shikimate, in addition to caffeoyl shikimate. This indicates that the CSEs have a broad substrate specificity, which was confirmed by in vitro enzyme kinetics. Taken together, our results suggest an alternative path within the phenylpropanoid pathway at the level of the hydroxycinnamoyl shikimates, and show that CSE is a promising target to improve plants for the biorefinery.

Computational and Structural Biotechnology Journal, 2021

Despite the scientific and economic importance of maize, little is known about its specialized me... more Despite the scientific and economic importance of maize, little is known about its specialized metabolism. Here, five maize organs were profiled using different reversed-phase liquid chromatography-mass spectrometry methods. The resulting spectral metadata, combined with candidate substrate-product pair (CSPP) networks, allowed the structural characterization of 427 of the 5,420 profiled compounds, including phenylpropanoids, flavonoids, benzoxazinoids, and auxin-related compounds, among others. Only 75 of the 427 compounds were already described in maize. Analysis of the CSPP networks showed that phenylpropanoids are present in all organs, whereas other metabolic classes are rather organ-enriched. Frequently occurring CSPP mass differences often corresponded with glycosyl-and acyltransferase reactions. The interplay of glycosylations and acylations yields a wide variety of mixed glycosides, bearing substructures corresponding to the different biochemical classes. For example, in the tassel, many phenylpropanoid and flavonoid-bearing glycosides also contain auxinderived moieties. The characterized compounds and mass differences are an important step forward in metabolic pathway discovery and systems biology research. The spectral metadata of the 5,420 compounds is publicly available (DynLib spectral database, ).

Proceedings of the National Academy of Sciences, 2019

Lignin is an abundant aromatic polymer found in plant secondary cell walls. In recent years, lign... more Lignin is an abundant aromatic polymer found in plant secondary cell walls. In recent years, lignin has attracted renewed interest as a feedstock for bio-based chemicals via catalytic and biological approaches and has emerged as a target for genetic engineering to improve lignocellulose digestibility by altering its composition. In lignin biosynthesis and microbial conversion, small phenolic lignin precursors or degradation products cross membrane bilayers through an unidentified translocation mechanism prior to incorporation into lignin polymers (synthesis) or catabolism (bioconversion), with both passive and transporter-assisted mechanisms postulated. To test the passive permeation potential of these phenolics, we performed molecular dynamics simulations for 69 monomeric and dimeric lignin-related phenolics with 3 model membranes to determine the membrane partitioning and permeability coefficients for each compound. The results support an accessible passive permeation mechanism fo...

Editorial overview: Plant biotechnology – lignin engineering

Current Opinion in Biotechnology, 2019

It is particularly timely to publish a special issue on lignin biosynthesis and its engineering g... more It is particularly timely to publish a special issue on lignin biosynthesis and its engineering giving the depth and breadth of amazing breakthroughs in fundamental understanding and applications that have occurred in recent years. Lignins are polymers that are mainly deposited in secondary-thickened plant cell walls. There, they are produced in a polymerization step (lignifica-tion) from their component monomers by a process of simple combinatorial radical coupling that was once considered an enigma. The monomers that have been known for decades are the p-coumaryl, coniferyl, and sinapyl alcohols. However, the development of new analytical tools along with analyses of lignin structures from a broader range of plant species has led to the discovery of many additional monomers, giving rise to a rich diversity of polymers with different physicochemical properties. As a consequence, the very definition of lignin continues to evolve as new species of plants are characterized in greater detail. The fact that lignin polymerization is a combinatorial radical coupling process, and that so many monomers naturally couple into the lignin polymer, has given rise to the concept that lignin structure is malleable and that plants can be engineered to make lignin structures that are tailored for various end-use applications. Indeed, in industrial operations producing pulp or fermentable sugars from wood, lignin needs to be extracted by expensive chemical treatments, and the efficiency of this process heavily depends on lignin's amount and composition. On the other hand, lignin is increasingly considered as a valuable source of aromatic building blocks for the chemical industry. As is now becoming evident, particular lignin structures can be designed and engineered into plants to maximize lignin-derived aromatic monomers streams. The acceleration in the interest in lignin and its engineering, caused by the urgent need for a transition from a fossil-based to a biobased economy, is illustrated by the steadily increasing number of papers covering 'lignin' with 'gene' terms, from 725 in 2010 to 1516 in 2017, with citations escalating at a far greater rate. When we conceived this issue, our objective was to cover lignin research extending from biosynthetic pathway revelations up to its valorization, but always with a focus on biotechnology, and with minimal overlap among the individual reviews. Given that several research groups work on the very same topic, it was, unfortunately, not possible to invite all of the main players in the lignin field and we apologize for this. The following are just a few of the highlights covered in the various contributions to this special issue as a testament to the state-of-the-art advances and practical implications of lignin bioengineering. Vanholme et al. [1] review the biosynthetic pathways towards the 35 metabolites from over 10 metabolic classes that have now been identified as lignin building blocks. They examine (Belgium). He specializes in lignin biosynthesis (gene and pathway discovery) and in lignin engineering in model plants (Arabidopsis), and crops (maize and poplar) to speed up the transition from a fossil-based to a bio-based economy.

Lignin biosynthesis and its integration into metabolism

Current Opinion in Biotechnology, 2019

Lignin is a principal structural component of cell walls in higher terrestrial plants. It reinfor... more Lignin is a principal structural component of cell walls in higher terrestrial plants. It reinforces the cell walls, facilitates water transport, and acts as a physical barrier to pathogens. Lignin is typically described as being composed of p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units that derive from the polymerization of the hydroxycinnamyl alcohols, p-coumaryl, coniferyl, and sinapyl alcohol, respectively. However, lignin also derives from various other aromatic monomers. Here, we review the biosynthetic pathway to the lignin monomers, and how flux through the pathway is regulated. Upon perturbation of the phenylpropanoid pathway, pathway intermediates may successfully incorporate into the lignin polymer, thereby affecting its physicochemical properties, or may remain soluble as such or as derivatized molecules that might interfere with physiological processes.

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Lignin is a phenolic heteropolymer that is deposited in secondary-thickened cell walls, where it ... more Lignin is a phenolic heteropolymer that is deposited in secondary-thickened cell walls, where it provides mechanical strength. A recent structural characterization of cell walls from monocot species showed that the flavone tricin is part of the native lignin polymer, where it is hypothesized to initiate lignin chains. In this study, we investigated the consequences of altered tricin levels on lignin structure and cell wall recalcitrance by phenolic profiling, nuclear magnetic resonance, and saccharification assays of the naturally silenced maize (Zea mays) C2-Idf (inhibitor diffuse) mutant, defective in the CHALCONE SYNTHASE Colorless2 (C2) gene. We show that the C2-Idf mutant produces highly reduced levels of apigenin-and tricin-related flavonoids, resulting in a strongly reduced incorporation of tricin into the lignin polymer. Moreover, the lignin was enriched in b-b and b-5 units, lending support to the contention that tricin acts to initiate lignin chains and that, in the absence of tricin, more monolignol dimerization reactions occur. In addition, the C2-Idf mutation resulted in strikingly higher Klason lignin levels in the leaves. As a consequence, the leaves of C2-Idf mutants had significantly reduced saccharification efficiencies compared with those of control plants. These findings are instructive for lignin engineering strategies to improve biomass processing and biochemical production.

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Caffeoyl shikimate esterase (CSE) was recently shown to play an essential role in lignin biosynth... more Caffeoyl shikimate esterase (CSE) was recently shown to play an essential role in lignin biosynthesis in Arabidopsis (Arabidopsis thaliana) and later in Medicago truncatula. However, the general function of this enzyme was recently questioned by the apparent lack of CSE activity in lignifying tissues of different plant species. Here, we show that down-regulation of CSE in hybrid poplar (Populus tremula 3 Populus alba) resulted in up to 25% reduced lignin deposition, increased levels of p-hydroxyphenyl units in the lignin polymer, and a relatively higher cellulose content. The transgenic trees were morphologically indistinguishable from the wild type. Ultra-high-performance liquid chromatography-mass spectrometry-based phenolic profiling revealed a reduced abundance of several oligolignols containing guaiacyl and syringyl units and their corresponding hydroxycinnamaldehyde units, in agreement with the reduced flux toward coniferyl and sinapyl alcohol. These trees accumulated the CSE substrate caffeoyl shikimate along with other compounds belonging to the metabolic classes of benzenoids and hydroxycinnamates. Furthermore, the reduced lignin amount combined with the relative increase in cellulose content in the CSE down-regulated lines resulted in up to 62% more glucose released per plant upon limited saccharification when no pretreatment was applied and by up to 86% and 91% when acid and alkaline pretreatments were used. Our results show that CSE is not only important for the lignification process in poplar but is also a promising target for the development of improved lignocellulosic biomass crops for sugar platform biorefineries.

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In the search for renewable energy sources, genetic engineering is a promising strategy to improv... more In the search for renewable energy sources, genetic engineering is a promising strategy to improve plant cell wall composition for biofuel and bioproducts generation. Lignin is a major factor determining saccharification efficiency and, therefore, is a prime target to engineer. Here, lignin content and composition were modified in poplar (Populus tremula 3 Populus alba) by specifically down-regulating CINNAMYL ALCOHOL DEHYDROGENASE1 (CAD1) by a hairpin-RNA-mediated silencing approach, which resulted in only 5% residual CAD1 transcript abundance. These transgenic lines showed no biomass penalty despite a 10% reduction in Klason lignin content and severe shifts in lignin composition. Nuclear magnetic resonance spectroscopy and thioacidolysis revealed a strong increase (up to 20-fold) in sinapaldehyde incorporation into lignin, whereas coniferaldehyde was not increased markedly. Accordingly, ultra-high-performance liquid chromatography-mass spectrometry-based phenolic profiling revealed a more than 24,000-fold accumulation of a newly identified compound made from 8-8 coupling of two sinapaldehyde radicals. However, no additional cinnamaldehyde coupling products could be detected in the CAD1-deficient poplars. Instead, the transgenic lines accumulated a range of hydroxycinnamate-derived metabolites, of which the most prominent accumulation (over 8,500-fold) was observed for a compound that was identified by purification and nuclear magnetic resonance as syringyl lactic acid hexoside. Our data suggest that, upon down-regulation of CAD1, coniferaldehyde is converted into ferulic acid and derivatives, whereas sinapaldehyde is either oxidatively coupled into S9(8-8)S9 and lignin or converted to sinapic acid and derivatives. The most prominent sink of the increased flux to hydroxycinnamates is syringyl lactic acid hexoside. Furthermore, low-extent saccharification assays, under different pretreatment conditions, showed strongly increased glucose (up to +81%) and xylose (up to +153%) release, suggesting that down-regulating CAD1 is a promising strategy for improving lignocellulosic biomass for the sugar platform industry.

Lignin is a major polymer in the secondary plant cell wall and composed of hydrophobic interlinke... more Lignin is a major polymer in the secondary plant cell wall and composed of hydrophobic interlinked hydroxyphenylpropanoid units. The presence of lignin hampers conversion of plant biomass into biofuels; plants with modified lignin are therefore being investigated for increased digestibility. The bacterium Sphingomonas paucimobilis produces lignin-degrading enzymes including LigD, LigF and LigG involved in cleaving the most abundant lignin interunit linkage, the b-aryl ether bond. In this study, we expressed the LigD, LigF and LigG (LigDFG) genes in Arabidopsis thaliana to introduce postlignification modifications into the lignin structure. The three enzymes were targeted to the secretory pathway. Phenolic metabolite profiling and 2D HSQC NMR of the transgenic lines showed an increase in oxidized guaiacyl and syringyl units without concomitant increase in oxidized b-aryl ether units, showing lignin bond cleavage. Saccharification yield increased significantly in transgenic lines expressing LigDFG, showing the applicability of our approach. Additional new information on substrate specificity of the LigDFG enzymes is also provided.

Lignin is an abundant aromatic polymer found in plant secondary cell walls. In recent years, lign... more Lignin is an abundant aromatic polymer found in plant secondary cell walls. In recent years, lignin has attracted renewed interest as a feedstock for bio-based chemicals via catalytic and biological approaches and has emerged as a target for genetic engineering to improve lignocellulose digestibility by altering its composition. In lignin biosynthesis and microbial conversion, small phenolic lignin precursors or degradation products cross membrane bilayers through an unidentified translocation mechanism prior to incorporation into lignin polymers (synthesis) or catabolism (bioconversion), with both passive and transporter-assisted mechanisms postulated. To test the passive permeation potential of these phenolics, we performed molecular dynamics simulations for 69 monomeric and dimeric lignin-related phe-nolics with 3 model membranes to determine the membrane partitioning and permeability coefficients for each compound. The results support an accessible passive permeation mechanism for most compounds, including monolignols, dimeric phenolics, and the flavonoid, tricin. Computed lignin partition coefficients are consistent with concentration enrichment near lipid car-bonyl groups, and permeability coefficients are sufficient to keep pace with cellular metabolism. Interactions between methoxy and hydroxy groups are found to reduce membrane partitioning and improve permeability. Only carboxylate-modified or glyco-sylated lignin phenolics are predicted to require transporters for membrane translocation. Overall, the results suggest that most lignin-related compounds can passively traverse plant and microbial membranes on timescales commensurate with required biological activities, with any potential transport regulation mechanism in lignin synthesis, catabolism, or bioconversion requiring compound functionalization. molecular dynamics | lignin permeability | lignin biosynthesis | biological funneling | free energy calculation

It is particularly timely to publish a special issue on lignin biosynthesis and its engineering g... more It is particularly timely to publish a special issue on lignin biosynthesis and its engineering giving the depth and breadth of amazing breakthroughs in fundamental understanding and applications that have occurred in recent years. Lignins are polymers that are mainly deposited in secondary-thickened plant cell walls. There, they are produced in a polymerization step (lignifica-tion) from their component monomers by a process of simple combinatorial radical coupling that was once considered an enigma. The monomers that have been known for decades are the p-coumaryl, coniferyl, and sinapyl alcohols. However, the development of new analytical tools along with analyses of lignin structures from a broader range of plant species has led to the discovery of many additional monomers, giving rise to a rich diversity of polymers with different physicochemical properties. As a consequence, the very definition of lignin continues to evolve as new species of plants are characterized in greater detail. The fact that lignin polymerization is a combinatorial radical coupling process, and that so many monomers naturally couple into the lignin polymer, has given rise to the concept that lignin structure is malleable and that plants can be engineered to make lignin structures that are tailored for various end-use applications. Indeed, in industrial operations producing pulp or fermentable sugars from wood, lignin needs to be extracted by expensive chemical treatments, and the efficiency of this process heavily depends on lignin's amount and composition. On the other hand, lignin is increasingly considered as a valuable source of aromatic building blocks for the chemical industry. As is now becoming evident, particular lignin structures can be designed and engineered into plants to maximize lignin-derived aromatic monomers streams. The acceleration in the interest in lignin and its engineering, caused by the urgent need for a transition from a fossil-based to a biobased economy, is illustrated by the steadily increasing number of papers covering 'lignin' with 'gene' terms, from 725 in 2010 to 1516 in 2017, with citations escalating at a far greater rate. When we conceived this issue, our objective was to cover lignin research extending from biosynthetic pathway revelations up to its valorization, but always with a focus on biotechnology, and with minimal overlap among the individual reviews. Given that several research groups work on the very same topic, it was, unfortunately, not possible to invite all of the main players in the lignin field and we apologize for this. The following are just a few of the highlights covered in the various contributions to this special issue as a testament to the state-of-the-art advances and practical implications of lignin bioengineering. Vanholme et al. [1] review the biosynthetic pathways towards the 35 metabolites from over 10 metabolic classes that have now been identified as lignin building blocks. They examine (Belgium). He specializes in lignin biosynthesis (gene and pathway discovery) and in lignin engineering in model plants (Arabidopsis), and crops (maize and poplar) to speed up the transition from a fossil-based to a bio-based economy.