创建一个shikimate路径的变体
Ningqing Ran1, K M Draths, J W Frost
1Department of Chemistry, Michigan State University, East Lansing, Michigan 48824, USA.
Journal of the American Chemical Society
|June 4, 2004
概括
工程酶克服了对关键代谢物的竞争,增加了微生物生产的天然产品的产量. 石基胺通路的这种进步增强了必需化合物的生物合成.
科学领域:
- 代谢工程是代谢工程.
- 合成生物学 合成生物学
- 微生物生物技术 微生物生物技术
背景情况:
- 石基胺通路对于微生物中产生芳香化合物至关重要.
- 碳水化合物转移酶系统对烯酸酸 (PEP) 的竞争限制了微生物生产产量.
- 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) 合成酶是消耗PEP的石基胺途径中的一个关键酶.
研究的目的:
- 为了设计一种新的shikimate路径变体来规避PEP竞争.
- 加强DAHP和下游天然产品的生产.
- 评估工程路径在微生物生长和新陈代谢中的功能.
主要方法:
- 来自大肠杆菌和K. pneumoniae. 的2-Keto-3-deoxy-6-phosphogalactonate (KDPGal) 代酶的定向演变.
- 进化KDPGal阿尔多酶同酶的表征,以改善DAHP合成活性.
- 构建和分析缺乏本地DAHP合成酶的大肠杆菌菌株,但依赖于进化的KDPGal阿尔多酶.
主要成果:
- 进化的KDPGal阿尔多酶异酶在催化DAHP形成方面表现出4-8倍高的特异性活性.
- 该工程途径成功地从酸盐和d-erythrose 4-phosphate中产生DAHP.
- 大肠杆菌构造利用进化的KDPGal阿尔多酶支持微生物生长和3-脱水基胺合成.
结论:
- 工程化shikimate通路变体有效绕过了PEP竞争.
- 定向进化KDPGal基酶提供了一种可行的策略,用于增强微生物生产的shikimate通路产品.
- 这种方法有可能改善各种自然产品的工业生物合成.
相关概念视频
Export of Mitochondrial and Chloroplast Genes
A eukaryotic cell can have up to three different types of genetic systems: nuclear, mitochondrial, and chloroplast. During evolution, organelles have exported many genes to the nucleus; this transfer is still ongoing in some plant species. Approximately 18% of the Arabidopsis thaliana nuclear genome is thought to be derived from the chloroplast’s cyanobacterial ancestor, and around 75% of the yeast genome derived from the mitochondria’s bacterial ancestor. This export has occurred irrespective...
Aldehydes and Ketones with Alcohols: Hemiacetal Formation
Similar to water, alcohols can add to the carbonyl carbon of the aldehydes and ketones. The addition of one molecule of alcohol to the carbonyl compound forms the hemiacetal or half acetal. As depicted below, in a hemiacetal, the carbon is directly linked to an OH and OR group.
Aldehydes and Ketones with HCN: Cyanohydrin Formation Overview
Cyanohydrins are compounds that contain –CN and –OH groups on the same carbon atom. They are formed by the nucleophilic addition of the cyanide ions to the carbonyl group. Cyanide ions are highly basic and nucleophilic and can be generated from HCN under aqueous conditions. However, since HCN is a weak acid, the number of cyanide ions generated is very small. Hence, a small amount of base or KCN/NaCN is added to HCN to increase the concentration of the cyanide ions in the reaction mixture.
Aldehydes and Ketones with Amines: Imine and Enamine Formation Overview
Primary amines react with carbonyl compounds—aldehydes and ketones—to generate imines. Imines consist of a C=N double bond and are named Schiff bases after its discoverer—the German chemist Hugo Schiff. On the other hand, secondary amines react with carbonyl compounds to give enamines. In enamines, the presence of a C=C double bond adjacent to the nitrogen atom leads to the delocalization of the lone pair.
[3,3] Sigmatropic Rearrangement of 1,5-Dienes: Cope Rearrangement
The Cope rearrangement is classified as a [3,3] sigmatropic shift in 1,5-dienes, leading to a more stable, isomeric 1,5-diene. The reaction involves a concerted movement of six electrons, four from two π bonds and two from a σ bond, via an energetically favorable chair-like transition state.
Chemical Shift: Internal References and Solvent Effects
In an NMR sample, precise measurement of the absolute absorption frequencies of nuclei is difficult. A standard internal reference compound is added, and the frequency difference between the reference signal and sample signals is measured.
The internal reference compound generally used in NMR spectroscopy is tetramethylsilane (TMS). TMS is preferred because it is chemically inert, soluble in NMR solvents, and easily removable. Also, the highly shielded methyl protons in TMS yield an intense...
The internal reference compound generally used in NMR spectroscopy is tetramethylsilane (TMS). TMS is preferred because it is chemically inert, soluble in NMR solvents, and easily removable. Also, the highly shielded methyl protons in TMS yield an intense...


