A Rapid, Scalable Method for Plant-Made Production of Cellulases

该奖项由美国国家科学基金会颁发，由生物技术、生化和生物质工程项目颁发，支持在植物组织中高效、快速和规模化生产纤维素酶的技术和工艺的开发。为了满足2007年《能源独立与安全法》的要求，将纤维素生物质转化为先进的生物燃料将需要大量低成本的纤维素降解酶。目前生产纤维素和半纤维素降解酶的方法主要依赖于浸没真菌发酵，并且价格昂贵，能源密集型，产生二氧化碳，并且消耗用于生产微生物生长的纤维素原料。在本项目中，多种耐热细胞壁降解酶将利用新的瞬态表达系统在本烟（Nicotiana benthamiana）植物和收获的叶片中产生。在稳定转化的植物中，瞬态方法相对于在植物内生产酶的优点是速度快，可以利用野生型（非转基因）植物的生物量，并且植物生长和酶生产阶段是解耦的，可以单独优化。智力优势：该研究项目将提高对瞬时农业渗透过程、植物病毒扩增子表达系统、植物酶的恢复和特性、瞬时农业渗透过程的规模考虑以及生产纤维素酶的替代生物处理策略的技术经济和环境评估的基本生物过程工程理解。更广泛的影响:拟议的研究对社会的好处是：1)具有成本效益，节能和环保的技术，不仅对纤维素酶和半纤维素酶的生产有用，以满足美国生物燃料的要求，而且还会为工业酶行业带来“更环保”和更有效的生物生产技术；2)加强对利用植物短暂农业渗透生产重组蛋白的基本过程的了解，这将加速利用类似技术生产疫苗和治疗药物的公司的发展；3)通过积极招聘和跨学科研究以及在植物科学、生物技术和生物过程工程方面的教育培训机会，扩大妇女和代表性不足的少数群体的参与；4)通过将研究成果纳入现有的和新的讲座和实验课程，广泛的课程影响。

This NSF award by the Biotechnology, Biochemical and Biomass Engineering program supports the development of technologies and processes for efficient, rapid and scalable production of cellulase enzymes in plant tissues. To meet the requirements of the Energy Independence and Security Act of 2007, conversion of cellulosic biomass into advanced biofuels will require a significant amount of cellulose degrading enzymes at a low cost. Current methods for production of cellulose and hemicellulose degrading enzymes rely primarily on submerged fungal fermentation and are expensive, energy intensive, generate carbon dioxide, and consume cellulosic feedstock for growth of the production microorganisms. In this project, multiple thermostable cell wall degrading enzymes will be produced in Nicotiana benthamiana (a relative of tobacco) plants and harvested leaves, utilizing novel transient expression systems. Advantages of the transient approach over in-planta production of enzymes in stably transformed plants are that it is rapid, can utilize wild type (non-transgenic) plant biomass, and that the plant growth and enzyme production stages are decoupled and can be separately optimized.Intellectual merit: This research project will improve fundamental bioprocess engineering understanding of transient agroinfiltration processes, plant viral amplicon expression systems, recovery and characterization of plant-made enzymes, scale-up considerations in transient agroinfiltration processes, and techno-economic and environmental assessment of alternative bioprocessing strategies for production of cellulase enzymes.Broader impact: The benefits of the proposed research to society are 1) cost-effective, energyefficient and environmentally-sound technologies that will not only be useful for the production of cellulase and hemicellulase enzymes to meet the U.S biofuels mandates but also lead to "greener" and more efficient bioproduction technologies for the industrial enzyme industry, 2)enhanced fundamental process understanding of recombinant protein production using transient agroinfiltration in plants which will accelerate growth of companies utilizing similar technologies for vaccine and therapeutics production, 3) broadening participation of women and underrepresented minorities through proactive recruitment and interdisciplinary research and educational training opportunities at the interface of plant science, biotechnology and bioprocess engineering, and 4) broad curricular impact by incorporating research findings into existing and new lecture and laboratory courses.