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的生物质中存在的抑制性材料（例如木质素片段、糠醛和其他多酚）都提出了挑战，并且用于推高其生物转化的成本。事实上，美国能源部和美国农业部的一项联合研究估计，从木质纤维素生产乙醇的成本大约是从淀粉来源（如玉米）生产乙醇的成本的两倍。本建议中提出的实验计划是在NSERC的主持下，在我们实验室过去24年中进行的纤维素分解和相关酶研究的延续。我们的目标是通过工程改造具有增强性能的纤维素分解酶来降低木质纤维素生物质的预处理成本。为此，我们提出：1）研究用亚磺酸酯官能团取代转化酶的一般碱性催化残基以调节纤维素分解酶的催化电位和pH分布的普遍适用性; 2）继续C. fimi CenA和CbhA基因以产生具有更酸性pH活性谱的纤维素分解酶;和3）筛选通过随机诱变产生的突变体以改变pH活性谱。我们还建议测试的假设，即碳水化合物结合模块的一些纤维素分解酶具有破坏非水解纤维素的结晶度的能力。这些结合模块将独立于它们的亲本酶产生，并且它们破坏结晶纤维素结构的能力将通过原子力显微镜在真实的时间内观察到。增强这种非水解活性的尝试将通过编码基因的定点诱变以产生具有特定氨基酸置换的模块来进行。 最后，我建议继续我们的糖苷水解酶的结构和功能关系的基础研究，试图将转化酶转化为保留在水解产物中的底物的异头构型。

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.