Simultaneous Overexpression of FERULOYL-CoA 6'-HYDROXYLASE 1 and COUMARIN SYNTHASE Leads to Coumarin-Enriched Lignin and Improved Saccharification in Greenhouse- and Field-Grown Poplar.

The urgent need for renewable resources has increased the interest in woody biomass to manufacture bio-based products. However, lignin recalcitrance limits the enzymatic conversion of wood into fermentable sugars, posing a major challenge for biomass deconstruction. To address this problem, we aimed at incorporating the coumarin scopoletin into the lignin polymer of poplar (Populus tremula × P. alba) by expressing FERULOYL-CoA 6'-HYDROXYLASE 1 (F6'H1) and COUMARIN SYNTHASE (COSY) in lignifying cells. Three constructs were evaluated: two bicistronic constructs, SCOP1 (COSY followed by F6'H1) and SCOP2 (F6'H1 followed by COSY), and one monocistronic, SCOP3 (only F6'H1). SCOP1 poplars produced most free scopoletin without altering overall lignin, cellulose or hemicellulose content. SCOP2 poplars were overall less efficient in scopoletin production and most of these lines showed a severe biomass yield penalty, whereas SCOP3 caused plant lethality. NMR and metabolic analyses confirmed that scopoletin cross-coupled with G and S monomers during lignification in SCOP1 lines. In addition to scopoletin, the detection of benzodioxane structures revealed the incorporation of dihydroxycoumarins. Overall coumarin incorporation in lignin amounted up to 2.3%. After alkaline pretreatment, wood from greenhouse-grown SCOP1 poplars released up to 29% more glucose compared to the wild type upon limited saccharification. Field-testing of three SCOP1 lines showed a 6 to 11% increase in saccharification efficiency, with the line containing the lowest scopoletin levels maintaining normal growth. These results demonstrate that engineering lignin composition in poplar can improve saccharification, and emphasize the importance of construct design, translational research and field validation.