Substrate acceptance and activity of an alkane monooxygenase from Marinobacter sp.

For a sustainable production of polymers, the controlled functionalization of simple organic precursors is a key aspect, where highly precise enzymes are envisioned to replace intensive chemical reactions. The alkane monooxygenase AlkB from P. putida GPo1¹ is one promising candidate, which hydroxylates terminal methyl groups in a broad range of substrates with high regioselectivity². It was reported to functionalize medium chain fatty acid methyl esters with even higher rates than its natural substrate octane³, which makes AlkB especially interesting for the synthesis of bifunctional hydroxy fatty acids, as precursors of lactones and polymers^4,5. This work focuses on AlkB from Marinobacter sp., a related monooxygenase that was identified in a marine metagenomic sample. AlkB from M. sp. was heterologously expressed in E. coli and its acceptance of FAMEs and alcohol acetates was evaluated in whole-cell biotransformation experiments. To elucidate the role of selected amino acids affecting the substrate scope, single and double mutants of the monooxygenase were created in a rational enzyme engineering approach and compared regarding their relative activities.

van Beilen, J. B. et al. Analysis of Pseudomonas putida alkane-degradation gene clusters and flanking insertion sequences: evolution and regulation of the alk genes. Microbiology 147, 1621–1630 (2001).van Beilen, J. B., Kingma, J. & Witholt, B. Substrate specificity of the alkane hydroxylase system of Pseudomonas oleovorans GPo1. Enzyme Microb. Technol. 16, 904–911 (1994).Schrewe, M., Magnusson, A. O., Willrodt, C., Bühler, B. & Schmid, A. Kinetic Analysis of Terminal and Unactivated C-H Bond Oxyfunctionalization in Fatty Acid Methyl Esters by Monooxygenase-Based Whole-Cell Biocatalysis. Adv. Synth. Catal. 353, 3485–3495 (2011).Schaffer, S. & Haas, T. Biocatalytic and Fermentative Production of α,ω-Bifunctional Polymer Precursors. Org. Process Res. Dev. 18, 752–766 (2014).Bińczak, J., Dziuba, K. & Chrobok, A. Recent Developments in Lactone Monomers and Polymer Synthesis and Application. Mater. (Basel, Switzerland) 14, (2021).