BRIGE: Metabolic Cell-Process Engineering (MCPE) for High Biobutanol Production by Clostridium tyrobutyricum

提出的研究涉及工程策略，以提高生物丁醇生产低成本，细菌为基础的生物质技术。技术描述：本项目旨在利用酪酸丁酸梭菌实现高滴度、高产丁醇生产。特别是，一种创新的工程方法，即代谢细胞过程工程（MCPE），将通过了解宿主细胞及其发酵过程之间的相互作用来大幅增加生物丁醇的产量。具体来说，计划是：1)对最近代谢工程的C. tyrobutyricum突变细胞对各种化学物质（例如，电子载体和无机盐）的反应有一个基本的了解，并确定成本有效的化学物质来促进NADH（还原性烟酰胺腺嘌呤二核苷酸）池并从生物质水解物中解毒抑制剂。实验设计（DOE）方法将用于筛选和确定氧化还原平衡和解毒所需的化学物质。2) MCPE通过增加胞内NADH和降低生物质水解液毒性生产双丁醇。一种新型氧化还原工程突变体（MCE）的构建和发酵工艺的开发与定时添加鉴定的化学物质（MPE）将整合并利用在丁醇生产中。更广泛的意义和重要性：丁醇是一种重要的工业溶剂，也是一种安全的替代运输燃料，可以通过现有的管道和加油站进行分散。在过去的几十年里，人们一直在努力利用低价值的生物质生产低成本的生物丁醇。然而，由于与现有工艺相关的各种化学问题，生物丁醇生产仍然存在低产量和有效浓度的问题。拟议的MCPE工程策略有可能使生物丁醇产量翻一番，预计成本低于每加仑2.5美元。因此，成功开发所提出的技术可以在保护自然资源和环境的同时提供安全的可再生能源，从而服务于公众利益。许多生物能源产业和其他学术研究领域将受益于利用MCPE开发新的工程菌株。扩大未被充分代表群体在工程领域的参与：本项目还旨在扩大未被充分代表群体的参与并增加多样性。化学与生物工程系（ChBE）有600多名本科生，历史上吸引了大约40%的女性，超过60%的学生对生物技术行业和研究生课程感兴趣。因此，利用PI独特的6年生物技术产业经验来加强ChBE课程非常重要。具体来说，一系列生物生产单元操作（如新型生物反应器设计和生物丁醇发酵演示运行）将被设计并引入到传统的ChBE 319单元操作实验室课程中。这些新功能可以扩展学生对工业生物生产的认识，有利于他们的职业目标发展。通过Alabama Science in Motion （ASIM）向K-12学生介绍合适的生化反应和滴定实验以及客座讲座，该项目覆盖阿拉巴马州西部9个县，包括贫困的黑带地区。还计划开展外联活动，例如通过SWE和REU招募女性研究人员并在PI实验室指导她们。这项研究是由工程教育和中心部的工程项目扩大参与计划的一部分，即工程项目扩大参与研究启动基金资助的。

The proposed research deals with engineering strategies to improve biobutanol production from low-cost, bacteria-based biomass technologies. Technical Description: This project seeks to achieve high-titer and high-yield butanol production by the bacterium Clostridium tyrobutyricum. Particularly, an innovative engineering approach, i.e., Metabolic Cell-Process Engineering (MCPE), will be developed to substantially increase biobutanol production through understanding the interaction between a host cell and its fermentation process. Specifically, it is planned to: 1) Develop a fundamental understanding of the response of a recently metabolically engineered C. tyrobutyricum mutant cell to various chemicals (e.g., electron carriers and inorganic salts), and identify cost effective chemicals to boost the NADH (Reduced Nicotinamide Adenine Dinucleotide) pool and detoxify the inhibitors from biomass hydrolysate. A Design of Experiment (DOE) approach will be used to screen and identify the chemicals needed for redox balance and detoxification. 2) Double butanol production via MCPE by increasing intracellular NADH and reducing the toxicity of biomass hydrolysate. The construction of a novel redox engineered mutant (MCE) and development of fermentation process with timed addition of the identified chemicals (MPE) will be integrated and utilized in butanol production.Broader Significance and Importance: Butanol is an important industrial solvent and a safe alternative transportation fuel that can be dispersed through existing pipelines and filling stations. Over the past decades, intensive efforts have been made to produce low-cost biobutanol using low-value biomass. However, biobutanol production still suffers from low yields and effective concentrations due to a variety of chemical issues associated with existing processes. The proposed MCPE engineering strategy has the potential to double biobutanol production, at a projected cost under $2.5 per gallon. Therefore, successfully developing the proposed technology can serve the public interest by providing a safe, renewable energy source while protecting natural resources and the environment. Many bioenergy industries and other areas of academic research would benefit from the development of novel engineering strain using MCPE.Broadening Participation of Underrepresented Groups in Engineering: This project also aims to broaden the participation of underrepresented groups and increase diversity. The Department of Chemical and Biological Engineering (ChBE) with over 600 undergraduates has historically attracted about 40 % women and more than 60 % of the students are interested in Biotech industry and graduate programs. Therefore, it is very important to enhance the ChBE curriculum by taking advantage of the unique 6-year Biotech industrial experiences of the PI. Specifically, a serial of bioproduction unit operations (e.g. novel bioreactor design and biobutanol fermentation demo run) will be designed and introduced to the traditional ChBE 319 Unit Operation Lab course. These new features can expand the students' knowledge of industry bioproduction and benefit their career goal development. Suitable biochemical reaction and titration experiments and guest lectures will be introduced to K-12 students through Alabama Science in Motion (ASIM) that covers 9 counties in west Alabama, including the impoverish Black Belt area. Outreach activities such as recruiting women researchers through SWE and REU and mentoring them in PI lab are also planned. This research has been funded through the Broadening Participation Research Initiation Grants in Engineering solicitation, which is part of the Broadening Participation in Engineering Program of the Engineering Education and Centers Division.