STTR Phase I: Engineering Clostritrial Fermentation for Biobutanol Production

智力优势：这个STTR项目将开发新的工程梭菌发酵菌株，以经济地从淀粉和木质纤维素生物质中提取糖，生产丁醇作为生物燃料。丁醇是一种重要的工业溶剂，也是一种比乙醇更好的运输燃料。近年来石油价格的上涨和有限的石油资源引起了人们对厌氧梭状菌发酵生产生物丁醇的浓厚兴趣。然而，传统的丙酮-丁醇-乙醇（ABE）发酵由于丁醇抑制作用强，丁醇产率（20%）、丁醇浓度（16 g/L）和反应器生产率（0.5 g/L*h）较低，且生产微生物（主要是乙酰丁酸梭菌）代谢途径和基因调控复杂，发酵过程难以改善。为了开发一种新的高丁醇生产菌株，将在I期克隆具有灭活的ack（醋酸激酶）和pta（磷酸转乙酰酶）的梭状芽孢杆菌突变菌株，并将其与酒精脱氢酶基因结合，然后在纤维床生物反应器中进一步适应以获得高丁醇耐受性。突变体的功能基因组研究和进一步的代谢工程和工艺开发将在II期进行，以评估从葡萄糖和木糖生产丁醇的可行性和优势。新的发酵工艺可以使丁醇产量和浓度加倍，从而将产品成本降低到具有经济竞争力的燃料应用水平。更广泛的影响：目前，丁醇几乎完全通过石化路线生产。它的用途包括溶剂、橡胶单体和制动液的工业应用。丁醇也被证明是一种很好的替代运输燃料。如果利用先进的发酵技术（如代谢工程的丁醇耐受性突变体）降低生产成本，生物丁醇将有很大的潜力与乙醇竞争，成为一种运输燃料。通过将葡萄糖和木糖的丁醇产率从目前较低的20% （w/w）提高到40%，可以大大提高生物丁醇的经济性。通过工程突变体，生产效率和丁醇产品浓度也可以提高至少100%。总的来说，生物丁醇产品的成本可以降低到每加仑2美元以下。因此，这项技术可以提供比乙醇更经济、更好的生物燃料。该项目将侧重于从工业废物流和低成本生物质原料中生产增值产品，以提高生物炼制工业的经济可行性。成功开发所提出的丁醇发酵技术将满足公众利益，特别是在提供安全的可再生能源，保护自然资源和环境，增加经济机会和生活质量方面。在整个商业开发和制造阶段都将创造就业机会。本项目将培养至少1名博士后学者和1名博士生。

Intellectual Merit:This STTR project will develop novel engineered Clostridia strains for fermentation to economically produce butanol as a biofuel from sugars derived from starchy and lignocellulosic biomass. Butanol is an important industrial solvent and potentially a better transportation fuel than ethanol. Recent rising oil prices and limited petroleum resources have generated high interest in the production of biobutanol by anaerobic Clostridial fermentation. However, the conventional acetone-butanol-ethanol (ABE) fermentation has low butanol yield (20%), butanol concentration (16 g/L) and reactor productivity (0.5 g/L*h) due to a strong butanol inhibition, and the fermentation process is difficult to improve due to the complicated metabolic pathways and gene regulation involved in the production microorganisms, mainly Clostridium acetobutylicum. To develop a novel high-butanol producer, Clostridia mutant strains with inactivated ack (acetate kinase) and pta (phosphotransacetylase) will be cloned with an alcohol dehydrogenase gene in Phase I and the mutants will be further adapted in a fibrous bed bioreactor to attain a high butanol tolerance. Functional genomic studies of the mutants and further metabolic engineering and process development will be carried out in Phase II to evaluate the feasibility and advantages of producing butanol from glucose and xylose. The new fermentation process can double the butanol yield and concentration, thus reducing the product cost to an economically competitive level for fuel application.Broader Impact:Currently, butanol is almost exclusively produced via petrochemical routes. Its uses include industrial applications in solvent, rubber monomers and brake fluids. Butanol has also been shown to be a good alternative transportation fuel. Biobutanol will have a great potential to compete with ethanol as a transportation fuel when its production cost is reduced by using advanced fermentation technologies such as metabolically engineered butanol-tolerant mutants. By increasing the butanol yield from glucose and xylose from the current low of 20 % (w/w) to ~40%, the economics of biobutanol can be greatly improved. With the engineered mutants, the productivity and butanol product concentration can also be improved by at least 100%. Overall, the biobutanol product cost can be reduced to less than $2 per gallon. This technology thus can provide an economical and better biofuel than ethanol. This project will focus on generation of value-added products from industrial waste streams and low-cost biomass feedstocks to enhance the economic viability of the biorefinery industry. Successfully developing the proposed butanol fermentation technology will satisfy the public interest, especially in providing a safe, renewable energy, protecting natural resources and the environment, and enhancing economic opportunity and quality of life. There will be job creation throughout the commercial development and manufacturing phases. At least one postdoctoral scholar and one Ph.D. student will be trained in this project.