Abstract
Application of genetically engineered bacterial strains for biodegradation of hydrocarbons is a sustainable solution for treating pollutants as well as in industrial applications. However, the process of bioengineering should be carefully carried out to optimize the output. Investigation of regulatory genes for bioengineering is essential for developing synthetic circuits for effective biocatalysts. Here we focus on LcaR, a putative transcriptional regulator affecting the expression of alkB2 and lcaR operon that has a high potential to become a tool in designing such pathways. Four LcaR dimers bind specifically to the upstream regulatory region where divergent promoters of alkB2 and lcaR genes are located with high affinity at a Kd of 0.94 ± 0.17 nM and a Hill coefficient is 1.7 ± 0.3 demonstrating cooperativity in the association. Ligand binding alters the conformation of LcaR, which releases the regulator from its cognate DNA. Tetradecanal and hexadecanal act as natural ligands of LcaR with an IC50 values of 3.96 ± 0.59 µg/ml and 0.68 ± 0.21 µg/ml, respectively. The structure and function of transcription factors homologous to LcaR have not been characterized to date. This study provides insight into regulatory mechanisms of alkane degradation with a direction towards potential applications in bioengineering for bioremediation and industrial applications.
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Abbreviations
- LcaR :
-
Long chain alkane regulator
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Acknowledgements
Financial support from National Science Foundation (NSF/RG/2015/BT/03) and international collaboration grant from Osaka University. Ms. S. Mauran (University of Colombo), Dr. N.V. Chandrasekharan (University of Colombo, Sri Lanka), Dr. W.W.P. Rodrigo (Industrial Technology Institute, Sri Lanka), Dr. Saminda Fernando (Open University, Sri Lanka), Tetsuko Nakaniwa (Osaka University, Japan) and Yuko Misumi (Osaka University, Japan) are gratefully acknowledged for providing technical support and insightful discussions.
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This project was supported by the National Science Foundation, Sri Lanka under Grant NSF/RG/2015/BT/03 and Osaka University, Japan under international collaborative grant.
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Inoka C. Perera designed the experiments and supervised experiments, findings and manuscript writing. Erandika H. Hemamali designed and performed the experiments and manuscript writing. Laksiri P. Weerasinghe synthesized and purified the ligand used in this study. Hideaki Tanaka and Genji Kurisu contributed to design and supervision of the experiments. The first draft of the manuscript was written by Erandika Harshani Hemamali and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
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10532_2021_9970_MOESM1_ESM.tif
Supplementary file1 (TIF 10555 KB). Attenuation of DNA binding by selected ligands. The effect of hexane, tridecane, hexadecane, cetyl alcohol, and meristic acid on LcaR+lcaO complex. LcaR+lcaO complex was challenged with increasing concentration of predicted ligands, a) Hexane (2.5 – 20 %), b) Tridecane (2.5 – 20 %), c) Hexadecane (2.5 – 20 %), d) Cetyl alcohol (1 – 5 mg/ml) and e) myristic acid (1 – 5 mg/ml).
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Hemamali, E.H., Weerasinghe, L.P., Tanaka, H. et al. LcaR: a regulatory switch from Pseudomonas aeruginosa for bioengineering alkane degrading bacteria. Biodegradation 33, 117–133 (2022). https://doi.org/10.1007/s10532-021-09970-x
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DOI: https://doi.org/10.1007/s10532-021-09970-x