外源嗜热FAE酶表达促进芒草生物质降解转化的过程与机制

The formation of lignin-ferulate-arabinoxylan complexes by ferulic acids (FA) covalently cross-linked with polymers of hemicellulose and lignin provides a molecular structure basis for rigid cell wall recalcitrance to biodegradation in grass plants. It was suggested that the ferulic acid esterase (FAE) that was exogenously expressed in plant could hydrolyze the FA esters in cell walls and break the covalent cross-links between hemicellulose and lignin polymers, resulting in cell wall depolymerization and consequent easy pretreatment and enzymatic conversion of lignocellulose. However, the mesophilic FAE activity was proved to disrupt host plant cell growth and development and as a result reduce the biomass yield and stress resistances of transgenic plants. Our previous study confirmed that the activity of thermophilic lignocellulose degradation enzymes was activated by high temperature when their coding genes were introduced into model plant and expressed, which ensures the normal growth and development of transgenic plants. In this project, a strategy of genic transformation with heat-resistance enzyme gene will be implemented in which several recombinant genes encoding a bacterial thermophilic FAE will be introduced into Miscanthus sinensis, an elite biomass crop. Then, the dynamic accumulation and activity of the thermophilic FAE protein under different subcellular targeting strategy in transgenic plants, the effects of enzyme deposition on performance of agronomic characters as well as on cell wall ultrastructure and chemical component contents of plants, the activating and regulating mechanisms of thermophilic enzyme in the pretreatment of transgenic biomass, and the contribution and function of the FAE enzyme in biomass pretreatment will be estimated. This study will bring forth a novel theory, method and practice for developing novel crops with an characteristic of easy degradation to promote the biomass industry.

阿魏酸与半纤维素、木质素共价交联形成木质素—阿魏酸—阿拉伯木聚糖复合物，是禾本科植物细胞壁形成坚固抗降解屏障的重要分子基础。在植物中异源表达阿魏酸酯酶(FAE)，可水解细胞壁中的阿魏酸酯键，促进细胞壁解聚，有效提高生物质预处理能力，降低酶解转化成本。但目前仍面临转基因的常温FAE酶活性干扰宿主植物生长发育、降低抗逆性等技术挑战。我们的前期研究表明，将嗜热木质纤维素水解酶基因在模式植物中异源表达，其酶活性在高温条件下受控激活，可以确保宿主植物生长发育正常。本项目拟采用高温酶基因转化策略，将细菌源嗜热FAE酶重组基因导入芒草中，研究酶在不同亚细胞分区定位表达对其积累量及酶活性的效应，阐明目标重组蛋白积累对转基因植株生长发育安全性的影响，提出转基因嗜热FAE酶活性的调控机制，评估该酶对生物质预处理效率的贡献与作用，为培育在可控条件下生物质高效自降解转化的作物品种创新理论与方法和提供实践基础。

植物细胞中异源表达阿魏酸酯酶（FAE）可水解细胞壁木质纤维素大分子中的阿魏酸酯键，促进细胞壁解聚，但常温FAE酶活性会干扰宿主植物正常生长发育和降低抗逆性。本研究首先对一个热纤梭菌FAE酶的酶学性质进行了表征，发现重组嗜热FAE酶具有较好的温度和pH值稳定性，发挥催化活性的适宜pH值为5.0~9.0，适宜温度为50℃~70℃。为了增强微生物源基因在植物细胞中的表达效率，我们对FAE-CBM6编码基因的密码子进行了优化，优化后的基因序列长度1,188 bp，共改变297个碱基，占碱基总数的25.0%，改变258个密码子，占密码子总数的65.15%。解决了芒草胚性愈伤组织长期继代保存的难题，并建立了农杆菌介导转化芒草的转基因方法，阳性转化频率达到62.22%。在此基础上，成功将嗜热FAE编码基因导入芒草和拟南芥中使之在不同亚细胞空间异源表达。结果显示，嗜热FAE酶对转基因植株的生长发育无明显不利影响，在不同植物亚细胞空间表达的嗜热FAE酶在70℃下的酶活性远高于25℃的，且在胞质中表达该酶的活性最高可达1152.23 U/mg。转基因植株生物质在不同自降解温度及不同NaOH预处理强度下，释放的还原性单糖产率均显著高于野生型，而生物质经70℃自降解处理后释放的还原单糖产率显著高于25℃自降解处理的，其中，胞质中表达嗜热FAE酶的转基因生物质在不同预处理条件下均可以获得最高的还原性单糖产量，介于49.55%~79.53%，是野生型的2.55~2.75倍，由此说明嗜热FAE酶在植物细胞中的表达导致生物质发生了自降解反应，且高温下的降解效率更显著。本研究有力地证明了将植物细胞作为生物反应器异源表达微生物源的木质纤维素降解酶，实现确保植物生长发育安全与促进生物质降解转化的双重目标是完全可行的。本研究的结果可以为能源植物、牧草和农作物秸秆的高效资源化利用提供新的思路与方法。