A new approach to zip‐lignin: 3,4‐dihydroxybenzoate is compatible with lignification

Abstract

Renewed interests in the development of bioenergy, biochemicals, and biomaterials have elicited new strategies for engineering the lignin of biomass feedstock plants. This study shows, for the first time, that 3,4-dihydroxybenzoate (DHB) is compatible with the radical coupling reactions that assemble polymeric lignin in plants.We introduced a bacterial 3-dehydroshikimate dehydratase into hybrid poplar (Populus alba 9 grandidentata) to divert carbon flux away from the shikimate pathway, which lies upstream of lignin biosynthesis.Transgenic poplar wood had up to 33% less lignin with p-hydroxyphenyl units comprising as much as 10% of the lignin. Mild alkaline hydrolysis of transgenic wood released fewer ester-linked p-hydroxybenzoate groups than control trees, and revealed the novel incorporation of cell-wall-bound DHB, as well as glycosides of 3,4-dihydroxybenzoic acid (DHBA). Two-dimensional nuclear magnetic resonance (2D-NMR) analysis uncovered DHBA-derived benzodioxane structures suggesting that DHB moieties were integrated into the lignin polymer backbone. In addition, up to 40% more glucose was released from transgenic wood following ionic liquid pretreatment and enzymatic hydrolysis.This work highlights the potential of diverting carbon flux from the shikimate pathway for lignin engineering and describes a new type of 'zip-lignin' derived from the incorporation of DHB into poplar lignin.

Expression of Dehydroshikimate Dehydratase in Sorghum Improves Biomass Yield, Accumulation of Protocatechuate, and Biorefinery Economics

“…These DHBA titers are comparable to those obtained from engineered plants harvested at the later senesced mature stage in the greenhouse experiment (Figure ), suggesting that DHBA remains stable within biomass during plant development. These are important observations considering that sorghum cultivated for the manufacturing of advanced biofuels and bioproducts can be harvested either at full maturity or at an earlier stage that is compatible with the ensiling process. , Overall, the DHBA titers measured in QsuB sorghum biomass are approximately 10-fold higher than those measured previously in switchgrass modified with the qsuB gene, but lower than those from QsuB poplar in which DHBA ranged between 0.7–1.2% on a DW basis in woody biomass. , Besides differences in crop types, the use of different promoters to drive qsuB expression in these crops can explain the variability observed for the DHBA content.…”

“…Unlike in Arabidopsis, switchgrass, and poplar in which expression of qsuB resulted in reductions of lignin content in biomass, − engineered QsuB sorghum lines produced the same quantity of lignin compared to controls in both greenhouse and field conditions (Tables S5 and S7). In Arabidopsis, switchgrass, and poplar QsuB lines, the reduction of lignin has been attributed to a reduction of the shikimate pool that is used by hydroxycinnamoyl-coenzyme A:shikimate hydroxycinnamoyl transferase (HCT) during lignin biosynthesis because 3-dehydroshikimate is both the substrate of QsuB and the direct precursor of shikimate in plastids. − However, recent studies highlighted that HCT may not have a major role for lignin biosynthesis in certain plants including sorghum, which may explain our data on the lignin content in QsuB modified sorghum. Nevertheless, lowering biomass recalcitrance via reducing lignin is an important trait for bioenergy crops to limit the costs associated with pretreatment and enzymatic hydrolysis.…”

“…DHBA is found in several plant species, but the exact metabolic steps and genes involved in its biosynthesis remain unknown . Overproduction of DHBA in planta can be achieved by the heterologous expression of plastid-targeted bacterial 3-dehydroshikimate dehydratase (QsuB) to convert endogenous 3-dehydroshikimate into DHBA (Figure a). − …”

Biological funneling of phenolics from transgenic plants engineered to express the bacterial 3-dehydroshikimate dehydratase (qsuB) gene

“…The QsuB sorghum line pSbUbi::qsuB #22 described in Tian et al (2022) was used for this study. It contains a codonoptimized sequence encoding a plastid-targeted version of QsuB under the control of the promoter of a sorghum polyubiquitin gene for constitutive qsuB expression.…”

Engineering Curcumin Biosynthesis in Poplar Affects Lignification and Biomass Yield

“…Similarly to the engineered rice plants, biomass from the transgenic trees had increased saccharification yields from plants that grew normally. Finally, heterologous expression of a gene encoding a bacterial 3-DEHYDROSHIKIMATE DEHYDRATASE in hybrid poplar resulted in reduced lignin amounts, the incorporation of monolignol-3,4-dihydroxybenzoate conjugates in the lignin, and improved saccharification without affecting growth ( Unda et al, 2022 ).…”