Lyczakowski Jan J, Wicher Krzysztof B, Terrett Oliver M, Faria-Blanc Nuno, Yu Xiaolan, Brown David, Krogh Kristian B R M, Dupree Paul, Busse-Wicher Marta
Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QW UK.
Natural Material Innovation Centre, University of Cambridge, 1 Scroope Terrace, Cambridge, CB2 1PX UK.
Biotechnol Biofuels. 2017 Sep 19;10:224. doi: 10.1186/s13068-017-0902-1. eCollection 2017.
Plant lignocellulosic biomass can be a source of fermentable sugars for the production of second generation biofuels and biochemicals. The recalcitrance of this plant material is one of the major obstacles in its conversion into sugars. Biomass is primarily composed of secondary cell walls, which is made of cellulose, hemicelluloses and lignin. Xylan, a hemicellulose, binds to the cellulose microfibril and is hypothesised to form an interface between lignin and cellulose. Both softwood and hardwood xylan carry glucuronic acid side branches. As xylan branching may be important for biomass recalcitrance and softwood is an abundant, non-food competing, source of biomass it is important to investigate how conifer xylan is synthesised.
Here, we show using Arabidopsis mutant biomass that removal of glucuronosyl substitutions of xylan can allow 30% more glucose and over 700% more xylose to be released during saccharification. Ethanol yields obtained through enzymatic saccharification and fermentation of biomass were double those obtained for non-mutant material. Our analysis of additional xylan branching mutants demonstrates that absence of GlcA is unique in conferring the reduced recalcitrance phenotype. As in hardwoods, conifer xylan is branched with GlcA. We use transcriptomic analysis to identify conifer enzymes that might be responsible for addition of GlcA branches onto xylan in industrially important softwood. Using a combination of in vitro and in vivo activity assays, we demonstrate that a white spruce () gene, , encodes an active glucuronosyl transferase. Glucuronic acid introduced by PgGUX reduces the sugar release of Arabidopsis mutant biomass to wild-type levels indicating that it can fulfil the same biological function as native glucuronosylation.
Removal of glucuronic acid from xylan results in the largest increase in release of fermentable sugars from Arabidopsis plants that grow to the wild-type size. Additionally, plant material used in this work did not undergo any chemical pretreatment, and thus increased monosaccharide release from biomass can be achieved without the use of environmentally hazardous chemical pretreatment procedures. Therefore, the identification of a gymnosperm enzyme, likely to be responsible for softwood xylan glucuronosylation, provides a mutagenesis target for genetically improved forestry trees.
植物木质纤维素生物质可作为生产第二代生物燃料和生物化学品的可发酵糖来源。这种植物材料的顽固性是其转化为糖的主要障碍之一。生物质主要由次生细胞壁组成，次生细胞壁由纤维素、半纤维素和木质素构成。木聚糖是一种半纤维素，它与纤维素微纤丝结合，并被推测在木质素和纤维素之间形成一个界面。软木和硬木的木聚糖都带有葡萄糖醛酸侧链。由于木聚糖分支可能对生物质顽固性很重要，且软木是一种丰富的、不与粮食竞争的生物质来源，因此研究针叶树木聚糖的合成方式很重要。
在这里，我们利用拟南芥突变体生物质表明，去除木聚糖的葡萄糖醛酸取代基可使糖化过程中释放的葡萄糖多30%，木糖多700%以上。通过对生物质进行酶促糖化和发酵获得的乙醇产量是未突变材料的两倍。我们对其他木聚糖分支突变体的分析表明，缺乏葡萄糖醛酸是赋予降低顽固性表型的独特因素。与硬木一样，针叶树木聚糖也带有葡萄糖醛酸分支。我们利用转录组分析来鉴定可能负责在具有工业重要性的软木中将葡萄糖醛酸分支添加到木聚糖上的针叶树酶。通过结合体外和体内活性测定，我们证明白云杉（）基因编码一种活性葡萄糖醛酸转移酶。PgGUX引入的葡萄糖醛酸将拟南芥突变体生物质的糖释放量降低到野生型水平，表明它可以履行与天然葡萄糖醛酸化相同的生物学功能。
从木聚糖中去除葡萄糖醛酸会使生长到野生型大小的拟南芥植物中可发酵糖的释放量增加最多。此外，这项工作中使用的植物材料未经过任何化学预处理，因此无需使用对环境有害的化学预处理程序就能实现生物质中单糖释放量的增加。因此，鉴定出一种可能负责软木木聚糖葡萄糖醛酸化的裸子植物酶，为遗传改良林木提供了一个诱变靶点。
