Towards cellulosic ethanol: bioengineering of cellulases

纤维素生物质转化为乙醇的限速步骤是从原料中有效释放葡萄糖。底物的结晶度、糖苷键的高固有稳定性以及预处理生物质中木质素碎片、糠醛和其他多酚类等抑制物质的存在都构成了挑战，并提高了其生物转化的成本。事实上，美国能源部和美国农业部的一项联合研究估计，从木质纤维素中生产乙醇的成本大约是从淀粉来源（如玉米）生产乙醇的两倍。本提案中提出的实验计划代表了过去24年来在NSERC的支持下，我们实验室对纤维素分解和相关酶的研究的延续。我们的目标是通过增强性能的工程纤维素水解酶来降低木质纤维素生物质预处理的成本。为此，我们建议：1)研究用亚硫酸盐官能团取代转化酶的一般碱催化残基来调节纤维素水解酶的催化电位和pH谱的一般适用性；2)继续对C. fimi的CenA和CbhA基因进行基本原理和随机诱变，以产生更具酸性ph -活性谱的纤维素水解酶；3)筛选随机诱变产生的突变体，以改变pH活性谱。我们还提出测试一些纤维素水解酶的碳水化合物结合模块具有非水解破坏纤维素结晶度的能力的假设。这些结合模块将独立于它们的母体酶而产生，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。为了增强这种非水解活性，将对编码基因进行定点诱变，以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的结构和功能关系的基础研究，尝试将一种转化酶转化为在水解产物中保留底物的端粒构型的酶。

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.