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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 × 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.

Our ability to study and valorize the lignin fraction of biomass is hampered by the fundamental and still unmet challenge of precisely quantifying native lignin's structural features. Here, we developed a rapid elevated‐temperature 1H−13C Heteronuclear Single‐Quantum Coherence Zero (HSQC0) NMR method that enables this precise quantification of native lignin structural characteristics even with whole plant cell wall (WPCW) NMR spectroscopy, overcoming fast spin relaxation in the gel phase. We also formulated a Gaussian fitting algorithm to perform automatic and reliable spectral integration. By combining HSQC0 measurements with yield measurements following depolymerisation, we can confirm the combinatorial nature of radical coupling reactions during biosynthesis leading to a random sequential organization of linkages within a largely linear lignin chain. Such analyses illustrate how this analytical method can greatly facilitate the study of native lignin structure, which can then be used for fundamental studies or to understand lignin depolymerization methods like reductive catalytic fractionation or aldehyde‐assisted fractionation. [ABSTRACT FROM AUTHOR]

Enhancing the production of biochemicals from lignocellulosic biomass is one potential way to decrease society's dependence on fossil fuels. Aromatic compounds obtained from plant biomass can be used as substrates for microbial production of dicarboxylic acids such as 2-pyrone-4,6-dicarboxylic acid (PDC) and cis,cis-muconic acid, which are building blocks for the manufacturing of polymer-based fibers and materials. In this study, we used an engineered strain of the bacterium Novosphingobium aromaticivorans to investigate how to increase PDC productivity in flow-through bioreactors receiving aqueous solutions of aromatics. At the best operational conditions tested, we achieved stable PDC production rates of 0.77 gPDC L−1 h−1 with p-hydroxybenzoic acid, 1.93 gPDC L−1 h−1 with syringic acid, and 1.53 gPDC L−1 h−1 with the products from alkaline pretreated poplar biomass. PDC titers in these reactors ranged from 7.7 to 15 g L−1 (42 to 80 mM) and were limited by aromatic solubility in the case of syringic acid, or by accumulation of protocatechuic acid from p-hydroxybenzoic acid when high aromatic loading rates were used. The use of high aromatic loading rates, hollow-fiber membranes to concentrate the microbial cells, and NH4OH for pH control were factors that contributed to this study achieving the highest PDC productivities reported to date. Overall, our findings demonstrate strategies that can be used to increase bioreactor productivity when aromatic substrates are delivered in aqueous form. These findings may also provide useful insight for production of other biochemicals from aromatic streams using N. aromaticivorans or other microbial chassis. [ABSTRACT FROM AUTHOR]

Utility vectors with promoters that confer desired spatial and temporal expression patterns are useful tools for studying gene and cellular function and for industrial applications. To target the expression of DNA sequences of interest to cells forming plant secondary cell walls, which generate most of the vegetative biomass, upstream regulatory sequences of the Brachypodium distachyon lignin biosynthetic gene BdPMT and the cellulose synthase genes BdCESA7 and BdCESA8 were isolated and cloned into binary vectors designed for Agrobacterium-mediated transformation of monocots. Expression patterns were assessed using the β-glucuronidase gene GUSPlus and X-glucuronide staining. All three promoters showed strong expression levels in stem tissue at the base of internodes where cell wall deposition is most active, in both vascular bundle xylem vessels and tracheids, and in interfascicular tissues, with expression less pronounced in developmentally older tissues. In leaves, BdCESA7 and BdCESA8 promoter-driven expression was strongest in leaf veins, leaf margins, and trichomes; relatively weaker and patchy expression was observed in the epidermis. BdPMT promoter-driven expression was similar to the BdCESA promoters expression patterns, including strong expression in trichomes. The intensity and extent of GUS staining varied considerably between transgenic lines, suggesting that positional effects influenced promoter activity. Introducing the BdPMT and BdCESA8 Open Reading Frames into BdPMT and BdCESA8 utility promoter binary vectors, respectively, and transforming those constructs into Brachypodium pmt and cesa8 loss-of-function mutants resulted in rescue of the corresponding mutant phenotypes. This work therefore validates the functionality of these utility promoter binary vectors for use in Brachypodium and likely other grass species. The identification, in Bdcesa8-1 T-DNA mutant stems, of an 80% reduction in crystalline cellulose levels confirms that the BdCESA8 gene is a secondary-cell-wall-forming cellulose synthase.

Litter chemistry is one of the most studied controls on decomposition in terrestrial ecosystems. Solar radiation has been shown to increase litter decomposition rates in arid ecosystems through the process of photodegradation. However, it remains unclear how photodegradation affects litter chemistry, especially the abundance and composition of lignin, which is thought to play a key role in photodegradation. Using two-dimensional nuclear magnetic resonance (2D NMR) spectroscopic methods, we quantified the molecular-level changes in litter chemistry associated with photodegradation. Litter of Bromus diandrus was exposed in the field to two levels of radiation [with and without ultraviolet (UV) wavelengths] and two durations of exposure (2.5 months during summer, and 1 year). Through fiber analysis by sequential digestion, we found that the litter hemicellulose fraction decreased significantly from 31.6 to 24.9 % after 1 year of decomposition. In litter exposed for 1 year, the hemicellulose fraction was significantly lower in litter with UV exposure compared to litter without UV exposure (23.8 vs. 25.9 %). These results indicate that UV photodegradation has a small but significant effect on litter chemistry compared to other decomposition processes. Even though fiber analysis showed no loss of total lignin, 2D NMR analysis demonstrated that UV exposure reduced the major lignin structural units containing β-aryl ether inter-unit linkages by 9 % and decreased the relative abundance of lignin p-hydroxyphenyl units by 20 %. The 2D NMR analysis also revealed that lignin guaiacyl units were preferentially lost after 1 year of decomposition relative to the reference material, but no effects of UV exposure on guaiacyl units were observed. These results suggest that photodegradation causes partial degradation, not necessarily complete breakdown, of lignin structures. Our data also demonstrate that applications of 2D NMR methods are valuable for acquiring detailed information on lignin and polysaccharide chemistry during both biotic and abiotic decomposition processes.

Summary: Ester‐linked p‐hydroxybenzoate occurs naturally in poplar lignin as pendent groups that can be released by mild alkaline hydrolysis. These 'clip‐off' phenolics can be separated from biomass and upgraded into diverse high‐value bioproducts. We introduced a bacterial chorismate pyruvate lyase gene into transgenic poplar trees with the aim of producing more p‐hydroxybenzoate from chorismate, itself a metabolic precursor to lignin. By driving heterologous expression specifically in the plastids of cells undergoing secondary wall formation, this strategy achieved a 50% increase in cell‐wall‐bound p‐hydroxybenzoate in mature wood and nearly 10 times more in developing xylem relative to control trees. Comparable amounts also remained as soluble p‐hydroxybenzoate‐containing xylem metabolites, pointing to even greater engineering potential. Mass spectrometry imaging showed that the elevated p‐hydroxybenzoylation was largely restricted to the cell walls of fibres. Finally, transgenic lines outperformed control trees in assays of saccharification potential. This study highlights the biotech potential of cell‐wall‐bound phenolate esters and demonstrates the importance of substrate supply in lignin engineering. [ABSTRACT FROM AUTHOR]

Summary: Ester‐linked p‐coumarate (pCA) is a hallmark feature of the secondary cell walls in commelinid monocot plants. It has been shown that pCA groups arise during lignin polymerisation from the participation of monolignol conjugates assembled by p‐coumaroyl‐CoA:monolignol transferase (PMT) enzymes, members of the BAHD superfamily of acyltransferases.Herein, we report that a eudicot species, kenaf (Hibiscus cannabinus), naturally contains p‐coumaroylated lignin in the core tissues of the stems but not in the bast fibres. Moreover, we identified a novel acyltransferase, HcPMT, that shares <30% amino acid identity with known monocot PMT sequences.Recombinant HcPMT showed a preference in enzyme assays for p‐coumaroyl‐CoA and benzoyl‐CoA as acyl donor substrates and sinapyl alcohol as an acyl acceptor. Heterologous expression of HcPMT in hybrid poplar trees led to the incorporation of pCA in lignin, but no improvement in the saccharification potential of the wood.This work illustrates the value in mining diverse plant taxa for new monolignol acyltransferases. Furthermore, the occurrence of pCA outside monocot lineages may represent another example of convergent evolution in lignin structure. This discovery expands textbook views on cell wall biochemistry and provides a new molecular tool for engineering the lignin of biomass feedstock plants. [ABSTRACT FROM AUTHOR]