A novel C-terminal protein degron identified in bacterial aldehyde decarbonylases using directed enzyme evolution
September 15-19, 2019
Metabolic engineers have successfully synthesized alkanes, the bulk component of gasoline, using microbial cell factories as a sustainable alternative to petroleum-based fuels. Aldehyde decarbonylases (AD), enzymes which transform acyl aldehydes into alkanes, have been identified as the bottleneck in these alkane producing pathways. Previous studies demonstrated degradation of AD in E. coli cells via unknown molecular mechanism. Here, we present the discovery of a degradation tag (degron) in AD from Prochlorococcus marinus. AD variants were generated by random mutation using error-prone PCR, transferred into E. coli, and grown in chemostat culture with 2g/L hexanal to select for positive mutations. A short C-terminal sequence of AD from P. marinus was proven to be an intact degron by fusing to fluorescent proteins. Statistical analysis of C-terminal sequences of 371 non-redundant ADs from bacteria revealed a conserved sequence in this region, which was proven to be an effective degron. We also showed that ATP-dependent proteases clpAP and lon are responsible for the degradation of AD degron tagged protein. Furthermore, our results indicate that the AD degron caused 91.4% of green fluorescent protein (GFP) degradation when fused to its C-terminus, whereas its elimination in AD enhanced alkane production in vivo. Thus, our work demonstrated the presence of a protein degron tag in bacterial ADs, thereby facilitating further improvements in AD-based alkane production pathways.
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Yilan Liu, Jinjin Chen, Radhakrishnan Mahadevan, Anna Khusnutdinova, Kevin Correia, and Alexander F. Yakunin, "A novel C-terminal protein degron identified in bacterial aldehyde decarbonylases using directed enzyme evolution" in "Enzyme Engineering XXV", Huimin Zhao, University of Illinois at Urbana-Champaign, USA John Wong, Pfizer, USA Eds, ECI Symposium Series, (2019). https://dc.engconfintl.org/enzyme_xxv/13