PATHWAY ENGINEERING IN E COLI FOR THYMIDINE PRODUCTION

大肠杆菌生产胸苷的途径工程

• 批准号: 2069995
• 负责人: DAVID M ANDERSON
• 金额: $ 37.37万
• 依托单位: CHEMGEN CORPORATION
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-09-01 至 1997-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2069995
• 关键词: Escherichia coli; biosynthesis; biotechnology; fermentation; genetic techniques; high performance liquid chromatography; molecular cloning; nuclear magnetic resonance spectroscopy; technology /technique development; thymidine

DESCRIPTION: The objective of this Phase II proposal is to complete the genetic development for a low cost, fermentation based, production method for thymidine. Thymidine, currently produced by chemical synthesis, is an important raw material for several anti-viral pharmaceuticals and other biomedical applications. Previous work at ChemGen to engineer E. coli for thymidine production established the technical feasibility. Several enzymatic steps of the de novo pathway for thymidine production have been engineered resulting in inducible non-inhibited thymidine production by dividing cells. In small scale fermentation, one strain produced 5.2 g/Liter thymidine from glucose. ChemGen proposes to complete the strain development by deregulating the first steps of the biosynthetic pathway utilizing Bacillus genes (cpa and pyrB) that code for enzymes not feed-back inhibited by pyrimidine nucleotides. Once regulated in concert with the rest of the system, this modification should effectively uncouple thymidine production from cell growth to allow commercial production. The technical innovation in this proposal lies in the first demonstration that an essential, long biosynthetic pathway, that is normally highly regulated, can be successfully modified for a commercial production process using a rational design. The final strain will contain at least seven genes foreign to the host. These genes code for enzymes with allosteric properties predicted to be favorable for thymidine production. The design includes genes from five sources, as well as a number of engineered host genes and chromosomal mutations. The investigators believe success will set a precedent for synthesis of worthwhile biochemical pharmaceutical intermediates by use of a rational approach to genetic modification and engineering of complex biosynthetic pathways. PROPOSED COMMERCIAL APPLICATION: AZT (Retrovir) is an important drug treatment for AIDS patients. The deoxyribonucleoside thymidine is the key chemical precursor, and single most expensive component of AZT manufacture. Thymidine currently sells for $500 to $600/Kg, but this new fermentation process is expected eventually to reduce the production cost of thymidine to less that $100/Kg. Other AIDS drugs, d4T and fluorothymidine, currently in trials are also produced from thymidine.

描述：该第二阶段提案的目的是完成 低成本，基于发酵的生产方法的遗传发展 用于胸苷。胸苷目前由化学合成生产，是 几种抗病毒药物的重要原材料和 其他生物医学应用。在Chemgen的工程师E. 胸苷生产的大肠杆菌建立了技术可行性。 胸苷生产的新从头途径的几个酶促步骤 已经设计了导致诱导的非抑制胸苷 通过分裂细胞生产。在小型发酵中，一个菌株 从葡萄糖产生5.2 g/升胸苷。 Chemgen提议 通过放弃受管制的菌株发育 使用芽孢杆菌基因（CPA和PYRB）代码的生物合成途径 对于不被嘧啶核苷酸抑制的酶。一次 与系统的其余部分协同调节，此修改 应有效地将胸苷的产生从细胞生长到 允许商业生产。该提案的技术创新 这是第一个演示，是必不可少的，长生物合成的 通常受到高度调节的途径可以成功修改 用于使用理性设计的商业生产过程。决赛 菌株将至少包含宿主外来的七个基因。这些 具有变构特性的酶的基因代码为 有利于胸苷的产生。该设计包括五个基因 来源以及许多工程的宿主基因和染色体 突变。调查人员认为，成功将为 合成有价值的生化药品中间体的合成 一种合理的遗传修饰和工程方法的方法 复杂的生物合成途径。 拟议的商业应用：AZT（Retrovir）是重要的药物 艾滋病患者的治疗。脱氧核苷胸苷是 关键的化学前体和AZT最昂贵的组成部分 生产。胸苷目前的售价为500至600美元/公斤，但此新 预计发酵过程最终会减少生产 胸苷的成本低至$ 100/kg。其他艾滋病药物，D4T和 目前正在试验中的氟氨酰胺也是由胸苷产生的。