TY - JOUR

T1 - A novel C-terminal degron identified in bacterial aldehyde decarbonylases using directed evolution

AU - Liu, Yilan

AU - Chen, Jinjin

AU - Khusnutdinova, Anna N

AU - Correia, Kevin

AU - Diep, Patrick

AU - Batyrova, Khorcheska A

AU - Nemr, Kayla

AU - Flick, Robert

AU - Stogios, Peter

AU - Yakunin, Alexander F

AU - Mahadevan, Radhakrishnan

N1 - © The Author(s) 2020.

PY - 2020/6/29

Y1 - 2020/6/29

N2 - 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.

AB - 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.

U2 - 10.1186/s13068-020-01753-5

DO - 10.1186/s13068-020-01753-5

M3 - Article

C2 - 32612677

VL - 13

SP - 114

JO - Biotechnology for Biofuels

JF - Biotechnology for Biofuels

SN - 1754-6834

ER -