Towards cellulosic ethanol: bioengineering of cellulases

纤维素生物质转化为乙醇的速度限制步骤是从原料中有效地释放葡萄糖。底物的结晶度，糖苷键的高固有稳定性，以及预处理后的生物质中存在的抑制物质，如木质素碎片、糠醛和其他多酚类物质，都构成了挑战，并推高了其生物转化成本。事实上，美国能源部和美国农业部的一项联合研究估计，用木质纤维素生产乙醇的成本大约是用玉米等淀粉生产乙醇的成本的两倍。这项建议中提出的实验计划代表了过去24年来在NSERC的主持下在我们实验室进行的关于纤维素分解和相关酶的研究的继续。我们的目标是通过设计具有更高性能的纤维素酶来降低木质纤维生物质的前处理成本。为此，我们建议：1)研究用亚磺酸盐官能团取代转化酶的一般碱基催化残基来调节纤维素酶的催化潜力和pH谱的普遍适用性；2)继续C.FIMI Cena和CbhA基因的基本原理和随机突变，以产生具有更酸性pH活性谱的纤维素酶；以及3)筛选通过随机突变产生的突变株以改变pH活性谱。我们还提出了一个假设，即某些纤维素酶的碳水化合物结合模块具有以非水解性方式破坏纤维素结晶度的能力。这些结合模块将独立于它们的母酶产生，它们破坏结晶纤维素结构的能力将通过原子力显微镜实时观察。增强这种非水解性的尝试将通过对编码基因进行定点突变来产生具有特定氨基酸替代的模块。最后，我建议继续我们对糖苷水解酶结构和功能关系的基础研究，尝试将转化酶转化为保留水解物中底物的异常构型的酶。

The rate limiting step in the conversion of cellulosic biomass to ethanol is the efficient release of glucose from feedstocks. The crystallinity of the substrate, the high inherent stability of the glycosidic bond, and the presence of inhibitory materials, such as lignin fragments, furfurals, and other polyphenolics, in the pre-treated biomass all pose challenges and serve to drive up costs for its bioconversion. Indeed, a joint study by the U.S. Department of Energy and the U.S. Department of Agriculture estimated that the cost of production of ethanol from lignocellulosics is approximately double that for the same production from starch sources, such as corn. The experimental plan presented in this proposal represents a continuation of studies on cellulolytic and related enzymes conducted in our laboratory over the past 24 years under the auspices of NSERC. Our objective is to reduce the cost of pre-treatment of lignocellulosic biomass by engineering cellulolytic enzymes with enhanced properties. To this end, we propose to: 1) investigate the general applicability of replacing the general base catalytic residues of an inverting enzyme with a sulfinate functional group to modulate the catalytic potential and pH profile of cellulolytic enzymes; 2) continue both the rationale and random mutagenesis of C. fimi CenA and CbhA genes to generate cellulolytic enzymes with more acidic pH-activity profiles; and 3) screen mutants generated by random mutagenesis for altered pH activity profiles. We also propose to test the hypothesis that the carbohydrate-binding modules of some cellulolytic enzymes possess the ability to disrupt the crystallinity of cellulose non-hydrolytically. These binding modules will be produced independent of their parent enzyme and their ability to disrupt the structure of crystalline cellulose will be observed in real time by atomic force microscopy. Attempts to enhance this non-hydrolytic activity will be made by site-directed mutagenesis of the encoding genes to produce modules with specific amino acid replacements. Finally, I propose to continue our basic studies on the structure and function relationship of glycoside hydrolases by attempting to convert an inverting enzyme into on that retains the anomeric configuration of the substrate in the hydrolytic product.