A novel C-terminal degron identified in bacterial aldehyde decarbonylases using directed evolution

Yilan Liu; Jinjin Chen; Anna N Khusnutdinova; Kevin Correia; Patrick Diep; Khorcheska A Batyrova; Kayla Nemr; Robert Flick; Peter Stogios; Alexander F Yakunin; Radhakrishnan Mahadevan

doi:10.1186/s13068-020-01753-5

A novel C-terminal degron identified in bacterial aldehyde decarbonylases using directed evolution

Biotechnol Biofuels. 2020 Jun 29:13:114. doi: 10.1186/s13068-020-01753-5. eCollection 2020.

Authors

Affiliations

¹ Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, Toronto, ON M5S 3E5 Canada.
² Centre for Environmental Biotechnology, School of Natural Sciences, Bangor University, Bangor, LL57 2UW UK.
³ Institute of Biomedical Engineering, University of Toronto, 200 College Street, Toronto, ON M5S 3E5 Canada.

Abstract

Background: 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.

Results: 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.

Conclusion: 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.

Keywords: Aldehyde decarbonylase; Alkane; Degradation tag; Directed evolution; Protease.