The recalcitrance of lignocellulosic biomass provided technical and economic challenges in the current biomass conversion processes. Lignin is considered as a crucial recalcitrance component in biomass utilization. An in-depth understanding of lignin biosynthesis can provide clues to overcoming the recalcitrance. Laccases are believed to play a role in the oxidation of lignin monomers, leading to the formation of higher-order lignin. In plants, functions of only a few laccases have been evaluated, so little is known about the effect of laccases on cell wall structure and biomass saccharification. In this study, we screened a gain-of-function mutant with a significant increase in lignin content from Arabidopsis mutant lines overexpressing a full-length poplar cDNA library. Further analysis confirmed that a Chinese white poplar (Populus tomentosa) laccase gene PtoLAC14 was inserted into the mutant, and PtoLAC14 could functionally complement the Arabidopsis lac4 mutant. Overexpression of PtoLAC14 promoted the lignification of poplar and reduced the proportion of syringyl/guaiacyl. In contrast, the CRISPR/Cas9-generated mutation of PtLAC14 results in increased the syringyl/guaiacyl ratios, which led to integrated enhancement on biomass enzymatic saccharification. Notably, the recombinant PtoLAC14 protein showed higher oxidized efficiency to coniferyl alcohol (precursor of guaiacyl unit) in vitro. This study shows that PtoLAC14 plays an important role in the oxidation of guaiacyl deposition on cell wall. The reduced recalcitrance of the PtoLAC14-KO lines suggests that PtoLAC14 is an elite target for cell wall engineering, and genetic manipulation of this gene will facilitate the utilization of lignocellulose.

中文翻译：

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LACCASE14 是愈创木基木质素沉积所必需的，并影响杨树细胞壁的消化率


木质纤维素生物质的顽固性给当前生物质转化过程带来了技术和经济挑战。木质素被认为是生物质利用中关键的顽固成分。对木质素生物合成的深入了解可以为克服这一顽抗提供线索。据信漆酶在木质素单体的氧化中发挥作用，导致高阶木质素的形成。在植物中，仅评估了少数漆酶的功能，因此关于漆酶对细胞壁结构和生物质糖化的影响知之甚少。在本研究中，我们从过度表达全长杨树 cDNA 文库的拟南芥突变株系中筛选出木质素含量显着增加的功能获得突变体。进一步分析证实，该突变体中插入了白杨（Populus tomentosa）漆酶基因PtoLAC14，并且PtoLAC14可以在功能上与拟南芥lac4突变体互补。 PtoLAC14的过表达促进了杨木的木质化，降低了紫丁香基/愈创木基的比例。相比之下，CRISPR/Cas9生成的PtLAC14突变导致紫丁香基/愈创木基比率增加，从而导致生物质酶糖化的综合增强。值得注意的是，重组PtoLAC14蛋白在体外对松柏醇（愈创木基单元的前体）表现出更高的氧化效率。本研究表明PtoLAC14在细胞壁愈创木基沉积的氧化中发挥重要作用。 PtoLAC14-KO 系的顽抗性降低表明 PtoLAC14 是细胞壁工程的精英靶标，对该基因的遗传操作将促进木质纤维素的利用。