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

生物技术，生物化学和生物质工程计划的NSF奖项支持在植物组织中高效，快速和可规模化生产纤维素酶的技术和工艺的开发。为了满足2007年能源独立和安全法案的要求，将纤维素生物质转化为先进的生物燃料将需要大量的低成本纤维素降解酶。目前用于生产纤维素和半纤维素降解酶的方法主要依赖于深层真菌发酵，并且是昂贵的、能量密集的、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。在这个项目中，多种热稳定性细胞壁降解酶将在本塞姆氏烟草（烟草的亲戚）植物和收获的叶子中产生，利用新的瞬时表达系统。瞬时方法相对于在稳定转化的植物中的植物内生产酶的优点在于其快速，可以利用野生型（非转基因）植物生物量，并且植物生长和酶生产阶段是解耦的，并且可以单独优化。该研究项目将提高对瞬时农杆菌渗透过程，植物病毒扩增子表达系统，植物酶的回收和表征、瞬时农杆菌渗透过程中的放大考虑以及纤维素酶生产的替代生物处理策略的技术经济和环境评估。拟议研究对社会的好处是：1）成本效益高，节能环保-合理的技术，不仅有利于生产纤维素酶和半纤维素酶，以满足美国生物燃料的要求，而且还导致“绿色”以及用于工业酶工业的更有效的生物生产技术，2）增强了对在植物中使用瞬时农杆菌渗入的重组蛋白生产的基本工艺的理解，这将加速利用类似技术用于疫苗和治疗剂生产的公司的增长，第三章 * 通过在联合国系统积极征聘和提供跨学科研究和教育培训机会，扩大妇女和代表人数不足的少数群体的参与植物科学，生物技术和生物工艺工程的接口，和4）广泛的课程影响，将研究成果纳入现有的和新的讲座和实验室课程。

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.