重编程epi-Isozizaene合成酶的循环级联,以产生替代的烯产品
Samuel A Eaton1, David W Christianson1
1Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States.
Biochemistry
|July 14, 2023
概括
在epi-isozizaene synthase (EIZS) 中的芳香残留物发生突变会重定向基生物合成. 突变的F95S产生bisabolane前体,一个潜在的生物燃料,证明EIZS作为一个平台的设计者循环.
科学领域:
- 酵素学和合成生物学 合成生物学
- 自然产品生物合成 自然产品生物合成
- 蛋白质工程是指蛋白质工程.
背景情况:
- 来自Streptomyces coelicolor的I类赛斯基特环酸 (sesquiterpene cyclase epi-isozizaene synthase,EIZS) 合成了epi-isozizaene,这是albflavenone抗生素生物合成的一个关键中间体.
- 在EIZS活跃区域中,有四种芳香残留物 (F95,F96,F198,W203) 形成产品轮,指导基板和中间形状.
- 修改活性位点的芳香残留物提供了一种改变EIZS催化活性并产生新型基产品的策略.
研究的目的:
- 在生物化学和结构上表征EIZS突变体,用极性残留物替代芳香残留物.
- 为了研究活性部位修改对塞斯基烯循环路径的影响.
- 探索工程EIZS生产有价值的甲,包括生物燃料前体的潜力.
主要方法:
- EIZS的局部定向突变发生,以极性残留物 (氨酸,氨酸,氨酸) 取代芳香残留物 (F95,F96,F198).
- 突变酶的生物化学表征以识别循环化产物.
- 用X射线晶体学来确定EIZS突变的结构,并分析活性位点的变化.
主要成果:
- 四个EIZS突变 (F95S,F96H,F198S,F198T) 已经成功生成和表征.
- 值得注意的是,F95S EIZS突变产生的混合物是单环六烯前体的比萨博兰,一个潜在的D2柴油替代品.
- X射线结构揭示了微妙的活性部位轮变化,将特定的芳香残留与明显的碳酸介质的控制相关联.
结论:
- 通过芳香到极性残留物的替代,EIZS活动地点工程有效地重定向了四二烯生物合成.
- F95S突变证明了EIZS作为生产用于生物燃料应用的bisabolane等sesquiterpenes的平台的潜力.
- 欧洲工业区 (EIZS) 作为一种多功能支架,用于开发合成生物学的设计循环,产生用于制药和生物燃料的高价值化合物.
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