STTR Phase I: Anaerobic fermentation system for isoprene production from lignocellulose

这个小型企业技术转移（STTR）第一阶段项目将开发一种兼性厌氧细菌，该细菌将从纤维素中获得的糖发酵成异戊二烯，异戊二烯是一种用于生产乳胶橡胶、塑料和药品的有价值的原料。异戊二烯也用于生产化学品，或聚合成石油衍生航空燃料的替代品。这项工作将建立在大肠杆菌原型的基础上，通过重新设计基因结构并在兼性厌氧宿主凝结芽孢杆菌中表达它们。本研究的第一阶段将合成并克隆一组异戊二烯合成酶（IspS）基因到凝固芽孢杆菌中；这项工作将首先评估10个独立的潜在IPS分子，以确定在细菌中产生异戊二烯的最佳分子。将通过气相色谱-质谱顶空分析评估工程B.凝固菌株的异戊二烯产量。在I期结束时，在大肠杆菌中以至少一个数量级的速率实现木质纤维素源厌氧异戊二烯生产将证明可行性。第二阶段将专注于通过该酶的途径改善代谢物的通量。该项目的更广泛的影响/商业潜力将是开发一种高效的凝结芽孢杆菌菌株，将纤维素材料（如纸浆和纸浆厂污泥）中的糖发酵成异戊二烯。造纸工业每年产生约200万吨纤维污泥。这种材料要么被丢弃在垃圾填埋场，要么被焚烧，从而增加了数百万美元的造纸成本。这种污泥的组成是约50%的纤维素和半纤维素在干重的基础上，使其成为一个很好的发酵原料时，转化为其组成的五碳和六碳糖。异戊二烯是一种平台化学品，能够在未来的生物经济中发挥核心作用。异戊二烯的全球年消费量为每年17亿磅，其中95%以上用于生产异戊二烯橡胶、苯乙烯-异戊二烯-苯乙烯嵌段共聚物（SIS）和丁基橡胶，估计价格为每磅1.30美元。天平用于生产高价值的类异戊二烯衍生物，如维生素、保健品、农药、香料、香精和药品。该项目还将提高对异戊二烯和其他高价值类异戊二烯化合物的生物合成途径的了解。

This Small Business Technology Transfer (STTR) Phase I project will develop a facultative anaerobic bacterium that ferments sugars obtained from cellulosic sources into isoprene, a valuable feedstock used in the production of latex rubber, plastics, and pharmaceuticals. Isoprene also is used for the production of chemicals, or polymerized into replacements for petroleum-derived jet fuel. This work will build on prototype E. coli work by redesigning gene constructs and expressing them in the facultative anaerobic host Bacillus coagulans. Phase I of this research will entail synthesizing and cloning a set of isoprene synthase (IspS) genes into B. coagulans; this work will first evaluate ten separate potential IPS molecules to determine the best one for isoprene production in bacteria. The engineered B. coagulans strains will be evaluated for isoprene production by GC-MS headspace analysis. Achieving anaerobic isoprene production from lignocellulosic sources within at least an order of magnitude of the rates achieved in E. coli at the end of Phase I will demonstrate feasibility. Phase II will then focus on improving the flux of metabolites through the pathway to this enzyme. The broader impact/commercial potential of this project will be to develop a high efficiency bacterial strain of Bacillus coagulans to ferment sugars derived from cellulosic materials, such as pulp and pulp mill sludge into isoprene. The paper industry produces an estimated 2 million tons of fiber sludge annually. This material is either discarded in a landfill or burned, adding millions of dollars to the cost of paper production. The composition of this sludge is ~50% cellulose and hemicellulose on a dry weight basis, making it an excellent fermentation feedstock when converted into its constituent five and six carbon sugars. Isoprene is a platform chemical that is capable of playing a central role in the future bio-economy. Annual global consumption of isoprene is 1.7 billion pounds per year, of which more than 95% is used in the production of isoprene rubber, styrene-isoprene-styrene block copolymer (SIS), and butyl rubber, with an estimated price of $1.30 per pound. The balance is used in the production of high-value isoprenoid derivatives such as vitamins, nutraceuticals, pesticides, fragrances, flavors and pharmaceuticals. The project also will provide an improved understanding of the biosynthetic pathway of isoprene and other high-value isoprenoid compounds.