Latha Gandla Madhavi, Derba-Maceluch Marta, Liu Xiaokun, Gerber Lorenz, Master Emma R, Mellerowicz Ewa J, Jönsson Leif J
Department of Chemistry, Umeå University, SE-901 87 Umeå, Sweden.
Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, SE-901 83 Umeå, Sweden.
Phytochemistry. 2015 Apr;112:210-20. doi: 10.1016/j.phytochem.2014.06.002. Epub 2014 Jul 2.
The secondary walls of angiosperms contain large amounts of glucuronoxylan that is thought to be covalently linked to lignin via ester bonds between 4-O-methyl-α-D-glucuronic acid (4-O-Me-GlcA) moieties in glucuronoxylan and alcohol groups in lignin. This linkage is proposed to be hydrolysed by glucuronoyl esterases (GCEs) secreted by wood-degrading fungi. We report effects of overexpression of a GCE from the white-rot basidiomycete Phanerochaete carnosa, PcGCE, in hybrid aspen (Populus tremula L. x tremuloides Michx.) on the wood composition and the saccharification efficiency. The recombinant enzyme, which was targeted to the plant cell wall using the signal peptide from hybrid aspen cellulase PttCel9B3, was constitutively expressed resulting in the appearance of GCE activity in protein extracts from developing wood. Diffuse reflectance FT-IR spectroscopy and pyrolysis-GC/MS analyses showed significant alternation in wood chemistry of transgenic plants including an increase in lignin content and S/G ratio, and a decrease in carbohydrate content. Sequential wood extractions confirmed a massive (+43%) increase of Klason lignin, which was accompanied by a ca. 5% decrease in cellulose, and ca. 20% decrease in wood extractives. Analysis of the monosaccharide composition using methanolysis showed a reduction of 4-O-Me-GlcA content without a change in Xyl contents in transgenic lines, suggesting that the covalent links between 4-O-Me-GlcA moieties and lignin protect these moieties from degradation. Enzymatic saccharification without pretreatment resulted in significant decreases of the yields of Gal, Glc, Xyl and Man in transgenic lines, consistent with their increased recalcitrance caused by the increased lignin content. In contrast, the enzymatic saccharification after acid pretreatment resulted in Glc yields similar to wild-type despite of their lower cellulose content. These data indicate that whereas PcGCE expression in hybrid aspen increases lignin deposition, the inhibitory effects of lignin are efficiently removed during acid pretreatment, and the extent of wood cellulose conversion during hydrolysis after acid pretreatment is improved in the transgenic lines possible due to reduced cell wall cross-links between cell wall biopolymers by PcGCE.
被子植物的次生壁含有大量的葡糖醛酸木聚糖，据认为其通过葡糖醛酸木聚糖中的4-O-甲基-α-D-葡糖醛酸（4-O-Me-GlcA）部分与木质素中的醇基团之间的酯键与木质素共价连接。这种连接被认为可被木材降解真菌分泌的葡糖醛酸酯酶（GCEs）水解。我们报道了来自白腐担子菌肉色显革菌（Phanerochaete carnosa）的一种GCE（PcGCE）在杂种杨（Populus tremula L. x tremuloides Michx.）中过表达对木材成分和糖化效率的影响。使用杂种杨纤维素酶PttCel9B3的信号肽将重组酶靶向到植物细胞壁，其组成型表达导致在发育木材的蛋白质提取物中出现GCE活性。漫反射傅里叶变换红外光谱和热解-气相色谱/质谱分析表明转基因植物的木材化学有显著变化，包括木质素含量和S/G比增加，碳水化合物含量降低。连续木材提取证实克拉森木质素大量增加（+43%），同时纤维素含量约降低5%，木材提取物约降低20%。使用甲醇解分析单糖组成表明转基因株系中4-O-Me-GlcA含量降低而木糖含量不变，这表明4-O-Me-GlcA部分与木质素之间的共价连接保护这些部分不被降解。未经预处理的酶促糖化导致转基因株系中半乳糖、葡萄糖、木糖和甘露糖的产量显著降低，这与其因木质素含量增加而导致的更高抗性一致。相反，酸预处理后的酶促糖化导致葡萄糖产量与野生型相似，尽管其纤维素含量较低。这些数据表明，虽然杂种杨中PcGCE的表达增加了木质素沉积，但在酸预处理过程中木质素的抑制作用被有效去除，并且由于PcGCE减少了细胞壁生物聚合物之间的细胞壁交联，转基因株系中酸预处理后水解过程中木材纤维素的转化程度得到了改善。
