A comprehensive study of the promoting effect of manganese on white rot fungal treatment for enzymatic hydrolysis of woody and grass lignocellulose

Background The efficiency of biological systems as an option for pretreating lignocellulosic biomass has to be improved to make the process practical. Fungal treatment with manganese (Mn) addition for improving lignocellulosic biomass fractionation and enzyme accessibility were investigated in this study. The broad-spectrum effect was tested on two different types of feedstocks with three fungal species. Since the physicochemical and structural properties of biomass were the main changes caused by fungal degradation, detailed characterization of biomass structural features was conducted to understand the mechanism of Mn-enhanced biomass saccharification. Results The glucose yields of fungal-treated poplar and wheat straw increased by 2.97- and 5.71-fold, respectively, after Mn addition. Particularly, over 90% of glucose yield was achieved in Mn-assisted Pleurotus ostreatus-treated wheat straw. A comparison study using pyrolysis gas chromatography mass spectrometry (Py-GC/MS) and two-dimensional 1H–13C heteronuclear single quantum coherence (2D HSQC) nuclear magnetic resonance (NMR) spectroscopy was conducted to elucidate the role of Mn addition on fungal disruption of the cross-linked structure of whole plant cell wall. The increased Cα-oxidized products was consistent with the enhanced cleavage of the major β-O-4 ether linkages in poplar and wheat straw lignin or in the wheat straw lignin–carbohydrate complexes (LCCs), which led to the reduced condensation degree in lignin and decreased lignin content in Mn-assisted fungal-treated biomass. The correlation analysis and principal component analysis (PCA) further demonstrated that Mn addition to fungal treatment enhanced bond cleavage in lignin, especially the β-O-4 ether linkage cleavage played the dominant role in removing the biomass recalcitrance and contributing to the glucose yield enhancement. Meanwhile, enhanced deconstruction of LCCs was important in reducing wheat straw recalcitrance. The findings provided not only mechanistic insights into the Mn-enhanced biomass digestibility by fungus, but also a strategy for improving biological pretreatment efficiency of lignocellulose. Conclusion The mechanism of enhanced saccharification of biomass by Mn-assisted fungal treatment mainly through Cα-oxidative cleavage of β-O-4 ether linkages further led to the decreased condensation degree in lignin, as a result, biomass recalcitrance was significantly reduced by Mn addition. Graphic abstract

Knockout of the lignin pathway gene BnF5H decreases the S/G lignin composition ratio and improves S. sclerotiorum resistance in B. napus

Ferulate-5-hydroxylase (F5H) is a key enzyme involved in the conversion of the guaiacyl monolignol (G-monolignol) to the syringyl monolignol (S-monolignol) in angiosperms. The monolignol ratio has been proposed to affect biomass recalcitrance and the resistance to plant disease. Sclerotinia sclerotiorum (S. sclerotiorum) stem rot of Brassica napus (B. napus) causes heavy damage in oilseed rape production. To date, there is no information about the effect of the lignin monomer ratio on the resistance to S. sclerotiorum in B. napus. Four dominantly expressed BnF5H genes were knocked out by CRISPR/Cas9 simultaneously in B. napus, and the f5h mutant KO-7 was generated. The S/G lignin composition ratio was decreased compared to that of the wild type (WT) based on the results of Mӓule staining and 2D-NMR profiling. The resistance to S. sclerotiorum in stems and leaves increased in KO-7. Furthermore, we found that the stem strength of KO-7 was significantly increased compared to that of the WT. Collectively, for the first time, we demonstrate that knockout of the lignin pathway gene F5H decreases the S/G ratio, improves S. sclerotiorum resistance in B. napus, and increases stem strength.

Two softwood GUX clades are responsible for distinct glucuronic acid patterns on xylan

Wood of coniferous (softwood) trees, is a globally significant carbon sink and an important source of biomass for industrial applications. Despite its importance, very little is known about the genetic basis of softwood biosynthesis. Glucomannan and xylan are the main hemicelluloses in softwood secondary cell walls. Xylan interacts with the cellulose fibrils in a two-fold screw configuration. Moreover, we have shown that xylan GUX (GlucUronic acid substitution of Xylan)-dependent branching with glucuronic acid is critical for biomass recalcitrance. Here, we investigated the decoration patterns of xylan by softwood GUX enzymes. Using in vitro and in planta assays we demonstrate that two distinct clades of conifer GUX enzymes are active glucuronyltransferases. Interestingly, these enzymes have different specific activities, with one adding evenly spaced GlcA branches and the other one being also capable of glucuronidating two consecutive xyloses. Since xylan patterning might modulate xylan-cellulose and xylan-lignin interactions, our result further the understanding of softwood biosynthesis and can contribute to strategies aimed at modifying softwood cell wall properties.

Tailoring poplar lignin without yield penalty by combining a null and haploinsufficient CINNAMOYL-CoA REDUCTASE2 allele

Lignin causes lignocellulosic biomass recalcitrance to enzymatic hydrolysis. Engineered low-lignin plants have reduced recalcitrance but often exhibit yield penalties, offsetting their gains in fermentable sugar yield. Here, CRISPR/Cas9-generated CCR2(−/*) line 12 poplars have one knockout CCR2 allele while the other contains a 3-bp deletion, resulting in a 114I115A-to-114T conversion in the corresponding protein. Despite having 10% less lignin, CCR2(−/*) line 12 grows normally. On a plant basis, the saccharification efficiency of CCR2(−/*) line 12 is increased by 25–41%, depending on the pretreatment. Analysis of monoallelic CCR2 knockout lines shows that the reduced lignin amount in CCR2(−/*) line 12 is due to the combination of a null and the specific haploinsufficient CCR2 allele. Analysis of another CCR2(−/*) line shows that depending on the specific CCR2 amino-acid change, lignin amount and growth can be affected to different extents. Our findings open up new possibilities for stably fine-tuning residual gene function in planta.