SBIR Phase I: Coupling High-throughput Evolution and Expression Screening for Industrial Production of Biofuel Enzymes

这个小型企业创新研究第一阶段项目解决了可持续纤维素生物燃料的成本效益制造的关键挑战——快速开发更有效的生物质转化酶和可扩展的微生物表达系统。纤维素是地球上最丰富的有机聚合物，是陆生植物生物量的主要结构成分，由于其丰富程度，作为生物燃料的可再生原料具有吸引力。用纤维素生物质而不是淀粉生产液体燃料具有令人信服的经济和环境优势。然而，将纤维素原料（如木材和多年生草）转化为葡萄糖存在挑战，因为将其纤维素转化为可发酵糖效率低且成本高。我们将把高通量微生物蛋白表达筛选系统应用于分子酶增强平台，这样就可以生成优化的纤维素转化酶，并筛选与工业酶生产技术的兼容性。第一阶段的工作将是提高纤维素水解酶的效率，这些酶将需要通过微生物发酵以低成本生产。这一创新将减少开发用于纤维素生物燃料的新型高活性酶所需的总时间和成本，从而加快运输燃料的绿色化。在这项工作中开发的酶可以商业化生产，用于纤维素生物燃料的制造和任何其他以葡萄糖为原料的可再生化学。工业酶市场是全球性的，预计将以每年9%的速度增长，而先进的纤维素酶细分市场仍处于起步阶段。目前纤维素酶产品的低性能和高成本（约占生物燃料生产总成本的20%）扩大了这一巨大的市场机会。通过结合酶开发和表达/生产步骤，我们大大减少了从台式到工业生产的酶增强所需的总体时间。这项技术的成功将转化为一项有利可图的投资，重要的是，它将帮助该国向低碳可再生液体燃料来源过渡，为农业技术和农村发展提供复兴。此外，由于该技术适用于任何固体起始材料，因此该技术可用于快速开发酶来降解任何固体材料，从而进一步打开工业和消费市场。最后，对成功的酶的分析可以为酶的作用机制以及在微生物发酵系统中成功生产这些蛋白质的结构或化学基础提供启示。该奖项是根据2009年美国复苏和再投资法案（公法111-5）资助的。

This Small Business Innovation Research Phase I project addresses a key challenge for cost-effective manufacturing of sustainable, cellulosic biofuels - the rapid development of more efficient enzymes for biomass conversion and scalable microbial expression systems for those enzymes. Cellulose is the most abundant organic polymer on earth, the chief structural component of terrestrial plant biomass, and because of its abundance is attractive as a renewable feedstock for biofuels. Producing liquid fuels from cellulosic biomass rather than starches offers compelling economic and environmental advantages. However, converting cellulosic feedstocks, such as wood and perennial grasses, to glucose presents challenges because the conversion of their cellulose to fermentable sugars is inefficient and costly. We will apply a high-throughput microbial protein expression screening system to a molecular enzyme enhancement platform in such a way that optimized cellulose-converting enzymes can be generated and screened for compatibility with industrial enzyme production techniques. The first phase of the work will be to increase the efficiency of cellulolytic enzymes which to be commercialized will need to be produced at low cost through microbial fermentation. This innovation will reduce the total time and costs necessary to develop new, high-activity enzymes for cellulosic biofuels to speed the greening of transportation fuels. Enzymes developed in this work can be commercially produced for cellulosic biofuels manufacture and any other renewable chemistry that uses glucose as a starting material. The industrial enzyme market is global, expected to grow by 9% per year, and the advanced cellulase market segment is still nascent. This large market opportunity is augmented by the low performance and high cost of current cellulase products (approximately 20% of total biofuel production cost). By combining enzyme development and expression/production steps, we drastically reduce the overall time necessary to take enzyme enhancement from the benchtop to industrial manufacturing. Success of this technology will translate into a profitable venture and, importantly, will help the country transition to a low-carbon renewable liquid fuel source, providing a renaissance in agricultural technology and rural development. Further, because the techniques are applicable to any solid starting material, the technology can be used to rapidly develop enzymes to degrade any solid material, opening further industrial and consumer markets. Finally, analysis of successful enzymes may provide illumination to the mechanisms of enzyme action and also to the structural or chemical basis for successful production of these proteins in microbial fermentation systems.This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).