Rerouting of the lignin biosynthetic pathway by inhibition of cytosolic shikimate recycling in transgenic hybrid aspen

Abstract

Lignin is a phenolic polymer deposited in the plant cell wall, and is mainly polymerized from three canonical monomers (monolignols), i.e. p-coumaryl, coniferyl and sinapyl alcohols. After polymerization, these alcohols form different lignin substructures. In dicotyledons, monolignols are biosynthesized from phenylalanine, an aromatic amino acid. Shikimate acts at two positions in the route to the lignin building blocks. It is part of the shikimate pathway that provides the precursor for the biosynthesis of phenylalanine, and is involved in the transesterification of p-coumaroyl-CoA to p-coumaroyl shikimate, one of the key steps in the biosynthesis of coniferyl and sinapyl alcohols. The shikimate residue in p-coumaroyl shikimate is released in later steps, and the resulting shikimate becomes available again for the biosynthesis of new p-coumaroyl shikimate molecules. In this study, we inhibited cytosolic shikimate recycling in transgenic hybrid aspen by accelerated phosphorylation of shikimate in the cytosol through expression of a bacterial shikimate kinase (SK). This expression elicited an increase in p-hydroxyphenyl units of lignin and, by contrast, a decrease in guaiacyl and syringyl units. Transgenic plants with high SK activity produced a lignin content comparable to that in wild-type plants, and had an increased processability via enzymatic saccharification. Although expression of many genes was altered in the transgenic plants, elevated SK activity did not exert a significant effect on the expression of the majority of genes responsible for lignin biosynthesis. The present results indicate that cytosolic shikimate recycling is crucial to the monomeric composition of lignin rather than for lignin content.

Preparation and Characterization of Bisphenol A-Based Thermosetting Epoxies Based on Modified Lignin

“…This result indicated that the amount of ECH and its chemical composition influenced the thermal stability of the modified lignin samples. After graft modification by the epoxy group, the initial degradation temperature of the modified lignin was higher than that of unmodified lignin EL0, which could be due to the better thermal stability of EL0 side chains and their inhibitory effects on the thermal degradation of copolymers . The residue chars of EL0, EL10, EL20, and EL30 were 39.5, 37.2, 32.4, and 36.1%, respectively.…”

Lignin engineering in forest trees: From gene discovery to field trials

“…(2022) P. alba × P. glandulosa miR393 suppression via STTM slightly ↑ n.d. n.d. n.d. ↑ Chu et al. (2021) P. tremula × Populus tremuloides HpSK overexpression WT ↑S/G, increased H units ↑ n.d. ↓ Hu et al. (2022) P. alba × P. grandidentata QsuB overexpression ↓33% ↑S/G ↑ n.d. WT Unda et al.…”

“…Besides core genes of the lignin biosynthetic pathway, metabolic flux through the pathway can also be engineered via the depletion of essential co-factors. The expression of a bacterial SHIKIMATE KINASE ( SK ) gene in poplar results in a reduced shikimate pool in the cytoplasm, thereby hindering the HCT reaction for which shikimate is an essential substrate ( Hu et al., 2022 ). The resulting transgenic poplars have a lignin content comparable to that of WT plants, but an increased amount of H units, a reduction in ether-linked G and S units, and a biomass yield penalty ( Hu et al., 2022 ).…”

“…The expression of a bacterial SHIKIMATE KINASE ( SK ) gene in poplar results in a reduced shikimate pool in the cytoplasm, thereby hindering the HCT reaction for which shikimate is an essential substrate ( Hu et al., 2022 ). The resulting transgenic poplars have a lignin content comparable to that of WT plants, but an increased amount of H units, a reduction in ether-linked G and S units, and a biomass yield penalty ( Hu et al., 2022 ). The expression of another bacterial gene, QsuB , which encodes a 3-dehydroshikimate dehydratase, in poplar enables the plants to convert 3-dehydroshikimic acid into 3,4-dihydroxybenzoic acid ( Unda et al., 2022 ).…”

“…Because lignin is the main contributor to the recalcitrance of lignocellulosic biomass toward deconstruction, numerous research efforts have focused on altering lignin amount and/or composition, aiming to increase wood processing efficiency ( Chanoca et al., 2019 ; Bryant et al., 2020 ). However, the in planta effects of pathway perturbations are often unpredictable because the flux through the lignin biosynthesis pathway is regulated at multiple levels ( Figure 2 ): (i) at the transcriptional level ( Zhong et al., 2010 ; Bonawitz et al., 2014 ; Ohtani and Demura, 2019 ); (ii) by post-translational protein modifications ( Wang et al., 2015 ); (iii) by the abundance of enzyme-inhibiting pathway intermediates ( Wang et al., 2014 ; Eudes et al., 2016 ; Yokoyama et al., 2021 ); (iv) by the compartmentalization of substrates ( Widhalm et al., 2015 ; Eudes et al., 2016 ; Guo et al., 2018 ; Vanholme et al., 2019 ); (v) by the different activities and spatial expression patterns of gene family members ( Li et al., 2015 ; Vanholme et al., 2019 ); (vi) by the formation of protein complexes ( Chen et al., 2011 ; Widhalm et al., 2015 ; Gou et al., 2018 ; Yan et al., 2019 ); (vii) by the availability of co-factors and -substrates ( Tang et al., 2014 ; Vanholme et al., 2019 ; Hu et al., 2022 ); (viii) by pathway intermediate-triggered proteolysis of pathway enzymes ( Guan et al., 2022 ); (ix) by branching pathways diverting the flux away from monolignols toward other sinks and products, such as flavonoids, benzenoids, phenylpropanoids, and monomer-glucosides ( Vanholme et al. (2012) ; and (x) by the different parallel routes through which the pathway flux can flow via enzymes that often accept multiple substrates ( Vanholme et al., 2019 ; Tsai et al., 2020 ).…”