Aldehyde decarbonylases (ADs), which convert acyl aldehydes into alkanes, supply promising solution for producing alkanes from renewable feedstock. However the instability of ADs impedes their further application. Therefore, the current study aimed to investigate the degradation mechanism of ADs and engineer it towards high stability. Here, we describe the discovery of a degradation tag (degron) in the AD from marine cyanobacterium Prochlorococcus marinus using error-prone PCR-based directed evolution system. Bioinformatic analysis revealed that this C-terminal degron is common in bacterial ADs and identified a conserved C-terminal motif, RMSAYGLAAA, representing the AD degron (ADcon). Furthermore, we demonstrated that the ATP-dependent proteases ClpAP and Lon are involved in the degradation of AD-tagged proteins in E. coli, thereby limiting alkane production. Deletion or modification of the degron motif increased alkane production in vivo. This work revealed the presence of a novel degron in bacterial ADs responsible for its instability. The in vivo experiments proved eliminating or modifying the degron could stabilize AD, thereby producing higher titers of alkanes.

中文翻译：

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使用定向进化在细菌醛脱羰酶中鉴定出一种新的 C 末端 degron。

将酰基醛转化为烷烃的醛脱羰酶 (AD) 为从可再生原料生产烷烃提供了有前景的解决方案。然而，AD的不稳定性阻碍了它们的进一步应用。因此，目前的研究旨在研究 AD 的降解机制并将其设计为高稳定性。在这里，我们描述了使用容易出错的基于 PCR 的定向进化系统从海洋蓝藻 Prochlorococcus marinus 在 AD 中发现的降解标签 (degron)。生物信息学分析表明，这种 C 末端 degron 在细菌 AD 中很常见，并确定了一个保守的 C 末端基序 RMSAYGLAAA，代表 AD degron (ADcon)。此外，我们证明了 ATP 依赖性蛋白酶 ClpAP 和 Lon 参与了大肠杆菌中 AD 标记蛋白的降解，从而限制烷烃的产生。degron 基序的缺失或修饰增加了体内烷烃的产生。这项工作揭示了细菌 AD 中存在一种新的 degron 导致其不稳定性。体内实验证明消除或修饰degron可以稳定AD，从而产生更高滴度的烷烃。