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

该项目主要针对 实现这一目标的障碍 巨大潜力 乙醇嗜热菌 生产：产品选择性低 （乙醇产量/产物产量 除乙醇外）。 三项任务 将被执行。任务1 包括克隆梭菌 热纤目的基因（乙酸 激酶和/或磷酸转乙酰酶， 乳酸脱氢酶） 大肠杆菌，删除一部分 的靶基因，使 相应酶 非功能性的， 质粒构建体， 缺失基因的重新导入 回到C。热纤 任务2 涉及开发一个 电穿孔方案， 将缺失基因导入C. 非复制性的热纤 质粒 任务3利用连续 文化同时验证 菌株稳定性，评价乙醇 耐受性，并检查代谢 和/或路径的动力学影响 操纵 这个项目很新颖 在新兴领域的背景下， 代谢（Metabolic） 因为它：（1）使用 宿主生物体， 遗传学不发达;（2） 利用基因缺失来操纵 在分支路径中流动， （3）过度表达;（4） 不需要复制质粒或 质粒携带基因的表达。

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.