使用工程化酸盐合成酶和Escherichia coli的酸果酸酶变体增加了酸盐生产
Jeffrey K Dodelin1, Abigail E Rose1, Hemshikha Rajpurohit2
1Department of Microbiology, University of Georgia, Athens, Georgia, USA.
Biotechnology and bioengineering
|December 10, 2024
概括
在大肠杆菌中修改酸盐合成酶 (GltA) 通过优化乙-CoA的利用来增强酸盐的产生. GltA [K167A] 变种实现了在美酸盐合成中产量和生产率的最佳平衡.
科学领域:
- 代谢工程是代谢工程.
- 合成生物学 合成生物学
- 生物化学工程 生物化学工程
背景情况:
- 梅瓦诺酸是异oprenoid生物合成的关键前体.
- 大肠杆菌中的本源代谢途径与乙-CoA竞争,乙-CoA是美酸盐合成的关键基质.
- 基甲基谷氨基-CoA减少酶是美酸盐形成的最后一种酶,它需要NADPH,这可能会限制产量.
研究的目的:
- 通过工程化染色体基因参与乙-CoA代谢和NADPH形成,增强大肠杆菌中美酸盐产量和生产率.
- 调查修改酸盐合成酶 (GltA) 和酸果酶 (PfkA) 对美酸盐生产的影响.
主要方法:
- 在大肠杆菌菌株中设计了9种酸盐合成酶 (GltA) 和4种酸果酸酶 (PfkA) 变异,这些变异过度表达异构甲酸盐路径基因.
- 工程菌株与野生型菌株和淘汰菌株在摇瓶和1升控制生物反应器实验中进行了基因工程菌株的 mevalonate 生产的比较.
- 分析了胺酸和限制对美酸产量和生产力的影响.
主要成果:
- 与野生类型相比,GltA变异通常提高了美酸盐产量,特别是在没有胺酸的情况下.
- 而PfkA变种的产量比野生型PfkA菌株的产量更低.
- GltA[K167A]变种显示出产量 (0.20 g/g) 和生产率 (0.87 g/L·h) 之间的最佳平衡.
- 使用GltA[K167A]的制过程在31小时内实现了最终度为36.9g/L的美瓦酸盐,产量为0.31g/g.
结论:
- 染色体修饰GltA是一种有效的策略,用于调节细胞内乙-CoA池,以增强乙-CoA衍生化合物的产生,如美瓦隆酸盐.
- 在这个系统中,乙-CoA的可用性,而不是NADPH,被确定为甲酸盐生产的主要限制因素.
- GltA[K167A]变种代表了对高产量和高生产率美酸盐制造的有希望的菌株.
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