Different Routes for Conifer- and Sinapaldehyde and Higher Saccharification upon Deficiency in the Dehydrogenase CAD1

Abstract

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.

Integration of renewable deep eutectic solvents with engineered biomass to achieve a closed-loop biorefinery

“…There have been reports on enhanced saccharification from CAD mutant biomasses (11,(39)(40)(41). It is challenging to directly compare the results from these different studies with the present study because the biomass is derived from different plant species that have various levels of both lignin and CAD deficiency.…”

“…It is challenging to directly compare the results from these different studies with the present study because the biomass is derived from different plant species that have various levels of both lignin and CAD deficiency. However, it would be interesting to test and scale-up DES-based pretreatment process with biomass from CAD-deficient bioenergy crops such as poplar (11), bm1 maize (47), bm6 sorghum (48), and switchgrass (49). It is worth noting that, unlike certain mutants affected in other steps of the lignin biosynthetic pathway, several CAD mutants do not show any visible phenotype or biomass yield penalty.…”

“…HSQC experiments were carried out with a Bruker pulse sequence (hsqcetgpspsi2.2). Assignment of the HSQC spectra is described elsewhere (11,30).…”

“…It has been reported that lignins from CAD-deficient biomass are more readily solubilized and extracted in alkaline conditions due to their higher content of free-phenolic end groups (11,35,36). Based on the principles of hydrogen bond interaction, DES could provide a mild acid-base catalysis mechanism that initiates the cleavage of aryl-ether linkages, leading to lignin separation from the biomass (20).…”

Covalent interactions between lignin and hemicelluloses in plant secondary cell walls

“…In vivo this results in the presence of an unsaturated bond between carbons 7 and 8 in the 8-O-4 and 8-8 linkages [52]. Multiple studies have shown that CAD mutation or downregulation reduces recalcitrance or increases extractability of hemicelluloses or lignin, but any reduction in hemicellulose:lignin crosslinking remains to be investigated [60][61][62][63][64][65]. The recalcitrance differences observed in CAD and COMT mutants may be partially explained by reductions in lignin content.…”