Pathway Engineering to Improve Ethanol Production by Thermo-philic Bacteria (Collaborative Research)

提高嗜热细菌乙醇生产的途径工程（合作研究）

• 批准号: 9314449
• 负责人: Arnold Demain
• 金额: $ 9.95万
• 依托单位: Massachusetts Institute of Technology
• 依托单位国家: 美国
• 项目类别: Continuing grant
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-06-15 至 1995-11-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9314449&HistoricalAwards=false
• 关键词: Pathway; Engineering; Improve; Ethanol; Production

This project addresses the primary obstacle to realizing the significant potential of thermophilic bacteria for ethanol production: low product selectivity (yield of ethanol/yield of products other than ethanol). Three tasks are to be carried out. Task 1 involves cloning Clostridium thermocellum target genes (acetate kinase and/or phosphotransacetylase, lactate dehydrogenase) in Escherichia coli, deleting a portion of the target genes to render the corresponding enzymes non-functional, and preparing plasmid constructs for reintroduction of the deleted genes back into C. thermocellum. Task 2 involves development of an electroporation protocol to introduce deleted genes into C. thermocellum on a non-replicative plasmid. Task 3 utilizes continuous culture to simultaneously verify strain stability, evaluate ethanol tolerance, and examine the metabolic and/or kinetic impact(s) of pathway manipulation. This project is novel in the context of the emerging field of pathway (or metabolic) engineering because it: (1) uses a host organism for which knowledge of genetics is not well developed; (2) uses gene deletion to manipulate flux in a branched pathway rather than overexpression; and (3) does not require a replicative plasmid or expression of plasmid-borne genes.

该项目解决了实现嗜热菌在乙醇生产中的巨大潜力的主要障碍：低产量选择性(乙醇产量/除乙醇以外的产品产量)。要抓好三项工作。任务1涉及克隆热细胞梭菌的靶基因(醋酸激酶和/或磷酸转乙酰酶，乳酸脱氢酶)，删除目标基因的一部分以使相应的酶不起作用，以及准备用于将缺失的基因重新引入高温假单胞菌的质粒构建体。任务2包括开发一种电穿孔方案，将缺失的基因导入到非复制体上的高温假单胞菌中质粒组。任务3利用连续培养来同时验证菌株的稳定性，评估乙醇耐受性，和检查代谢和/或动力学的影响(S)的途径操纵。这个项目在途径(或代谢)工程这一新兴领域的背景下是新颖的，因为它：(1)使用宿主生物这方面的遗传学知识还不发达；(2)利用基因缺失来操纵分支途径中的通量，而不是过度表达；以及(3)不需要复制质粒或表达质粒携带的基因。