SBIR Phase II: High-yield Fermentation of Sugars to Levulinic Acid

这个小型企业创新研究第二阶段项目的重点是开发一种用于生产乙酰丙酸(LA)的高产发酵路线。LA是最适合生物炼油厂生产的C5积木之一，因为它具有更高的价值、广泛的应用以及可能很快被化学工业采用。在第一阶段，该项目设计并实验验证了生产乳酸的新发酵途径的概念。这项第二阶段工作的重点将是从技术概念验证过渡到实验室规模发酵过程的开发。首先将优化所设计的途径中的限制性酶步骤，以达到与经济生产所需的通量/产率一致的活性水平。含有原始和优化酶的设计途径的变体随后将被克隆到合适的发酵有机体中(S)。利用计算和实验代谢工程工具，将进行敲除和敲除突变，以进一步优化途径中的通量/产量，同时优化宿主细胞的生长。这项工作代表了酶设计的第一个商业应用，合理地设计了没有任何天然对应物的新的代谢途径，使我们更接近设计细胞工厂的梦想。该项目更广泛的影响/商业潜力是推动美国绿色化学工业的发展，并使美国能够在一种新的可再生化学材料的商业生产中处于领先地位。由于缺乏一种高产量的替代昂贵的热化学工艺，阻碍了乙酰丙酸(LA)的广泛采用。由于LA可以通过化学或生物化学方法转化为合成橡胶(通过异戊二烯和丁烯)、生物燃料(如煤油和HMF)、聚合物(如尼龙)和聚合物添加剂(用于改变聚合物特性)，因此潜在市场每年超过200亿美元。当被认为是最终产品时，LA的交易价格比大多数商业生物炼油厂目前的产品乙醇高得多，因此可以帮助他们的产品供应多样化，并显著提高他们的利润率。

This Small Business Innovation Research Phase II project focuses on the development of a high-yield fermentation route for the production of levulinic acid (LA). LA is one of the best-suited C5 building blocks for bio-refinery production due to higher value, broad applications, and likely quick adoption by the chemical industry. During Phase I, this project has designed and experimentally validated the concept of a novel fermentation pathway for the production of LA. The focus of this Phase II work will be to transition from this technical proof-of-concept to the development of a lab-scale fermentation process. The limiting enzymatic steps in the designed pathway will first be optimized to reach levels of activity consistent with the flux/yield required for economical production. Variants of the designed pathway incorporating the original and optimized enzymes will subsequently be cloned into suitable fermentation organism(s). Using computational and experimental metabolic engineering tools, knock-out and knock-down mutations will be performed to further optimize flux/yield in the pathway while optimizing for host cell growth. This work represents the first commercial application of enzyme design to rationally engineer novel metabolic pathway that do not have any natural counterpart, bringing us closer to the dream of designer cell factories. The broader impact/commercial potential of this project is the advancement of a U.S. green chemistry industry and to allow America to take the lead in the commercial production of a new renewable chemical building block. The lack of a high-yield alternative to costly thermo-chemical processes has been preventing widespread adoption of levulinic acid (LA). Because LA can be converted, chemically or biochemically, to synthetic rubber (through isoprene and butenes), bio-fuels (such as kerosene and HMF), polymers (for instance, nylons) and polymer additives (for changing polymer characteristics), the addressable market is in excess of $20B annually. When considered as the end product, LA trades at a considerable higher price than ethanol, the current product of most commercial bio-refineries, and thus can help diversify their product offering and considerably increase their margins.