Synergetic effect of fungal pretreatment and lignin modification on delignification and saccharification: a case study of a natural lignin mutant in mulberry.

Background: Fungal pretreatment for partial separation of lignocellulosic components may reduce lignocellulose recalcitrance during the production of biofuels and biochemicals. Quantitative and qualitative modification of plant lignin through genetic engineering or traditional breeding may also reduce the recalcitrance. This study was conducted to examine the effects of combining these two approaches using three white rot fungi and mulberry wood with an altered lignin structure.

Results: Mulberry wood prepared from homozygotes or heterozygotes with a loss-of-function in the cinnamyl alcohol dehydrogenase gene (CAD) was pretreated with three fungal species. Both heterozygous (CAD/cad) and homozygous (cad/cad, null mutant) mulberry plants were derived from the same parents via backcrossing between Sekizaisou (cad/cad, seed parent), a natural lignin mutant, and its F1 progeny (CAD/cad, pollen parent). Homozygote wood and the isolated lignin exhibited an abnormal color. Lignin in homozygotes without fungal pretreatment exhibited a lower syringyl/guaiacyl ratio, molar mass, and thioacidolysis product yield than those in heterozygotes. Pretreatment with Phanerochaete chrysosporium achieved the highest delignification efficiency with a significant reduction in the cellulose content in both mulberry genotypes. In contrast, Ceriporiopsis subvermispora selectively removed lignin, with a weaker reduction in the cellulose content. The degree of delignification by C. subvermispora was significantly higher in homozygotes than in heterozygotes. Trametes versicolor tended to have a lower delignification capacity and smaller effect of subsequent enzymatic sugar release toward the wood from both genotypes than the other two fungi, making it less suitable for fungal pretreatment. Thioacidolysis assays indicated that cinnamaldehyde β-O-4, a typical subunit in the homozygote lignin, did not contribute to the high degradability of the lignin. The saccharification efficiency tended to be higher in homozygote wood than in heterozygote wood under all fungal pretreatment conditions.

Conclusions: Although further optimization of various system conditions is required, our findings suggest that CAD deficiency promotes delignification and subsequent enzymatic saccharification and may improve the biorefining efficiency of wood when combined with fungal pretreatment.