适应性实验室进化和基因工程改善了Pseudomonas putida KT244040中铁甲酸盐的利用
Allison Z Werner1, Young-Saeng C Avina1, Josefin Johnsen2
1Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO, USA; BOTTLE Consortium, Golden, CO, USA.
Metabolic engineering
|December 19, 2024
概括
适应性实验室进化增强了Pseudomonas putida的表现
科学领域:
- 生物技术是生物技术.
- 合成生物学 合成生物学
- 微生物工程 微生物工程
背景情况:
- 聚乙烯二甲 (PET) 是一种广泛使用的塑料,可分解为二甲酸 (TPA) 和乙烯基醇 (EG).
- 这些单体可以回收成新的PET (闭环) 或转化为有价值的产品 (开环).
- 伪菌 KT2440 是一种正在为PET单体转化进行工程的细菌.
研究的目的:
- 提高Pseudomonas putida KT2440使用适应性实验室进化 (ALE) 代谢二甲酸 (TPA) 的能力.
- 为了确定增强细菌生长和TPA利用的基因修饰,以改善PET上循环.
主要方法:
- 在使用TPA,TPA和EG混合物和葡萄糖的P. putida菌株上进行了自动化ALE活动.
- 通过全基因组再测序来分析进化菌株,以确定遗传突变.
- 关键突变进行了逆向工程,并测试了它们对TPA利用的影响.
主要成果:
- 在TPA和TPA-EG混合物中,ALE显著增加了4.1倍和3.5倍的生长率,分别超过约350代.
- 表现最好的进化分离物在TPA和TPA-EG.上显示出0.15-0.20h-1的增长率增加.
- 全基因组测序揭示了全球调节器 (gacS, gacA, turA) 的融合突变以及影响异质PET催化基因 (tphABII) 的重组.
结论:
- 在P. putida. 中,ALE有效增强TPA代谢作用.
- 确定了五种基因干预措施,包括删除全球调节器和途径基因重复,以改善TPA利用率.
- 这些发现有助于开发用于PET上循环的强大的全细胞生物催化剂.
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