Revealing the Native Multi-Enzyme Synergy of Acinetobacter calcoaceticus SDH15 for Ultra-Long-Chain Alkane Biodegradation

• Acinetobacter calcoaceticus SDH15 broadens the alkane biodegradation range to C56. • Four-module enzymatic system revealed via integrated genomics and transcriptomics. • AlkMa monooxygenase upregulated 327-fold, driving ultra-long-chain alkane oxidation. Polyethylene (PE) biodegradation is critical, yet microbial degradation of its ultra-long-chain alkane (ULCA, > C22) components remains limited and poorly understood beyond C40. This study identified Acinetobacter calcoaceticus ( A. calcoaceticus ) SDH15, a novel strain capable of degrading solid n -alkanes up to C56, thereby significantly extending the known biological range of alkane catabolism. Integrated genomic and transcriptomic analyses revealed a four-module enzymatic system driving this process. The first module, chemotaxis and adsorption, employs an upregulated PilH-Chp chemosensory system and type IV fimbriae (T4P), with fimF/G expression increased up to 332-fold, for substrate sensing and attachment. The second module, transmembrane transport, involves upregulated genes encoding outer membrane proteins ( fadL , ompW , and tonB ) and inner membrane transporters ( mfs / emrAB-tolC ; up to 18.9-fold) that facilitate alkane uptake. The third module, oxidative degradation, is initiated by the AlkB-type alkane monooxygenase AlkMa, which is encoded by the dramatically upregulated alkMa gene (327.7-fold for C22) and is essential for ULCAs oxidation, followed by alcohol and aldehyde dehydrogenases (ALDHs). The fourth module, energy metabolism, couples β-oxidation with an adaptive glyoxylate cycle to conserve carbon. Furthermore, electrospray ionization mass spectrometry enabled direct detection of C56 fatty acid intermediates, providing molecular evidence of the pathway’s broad substrate capacity. Functional validation through alkMa gene deletion confirmed its indispensable role in the initial oxidation of C22–C30 alkanes. This study presents a comprehensive model of native multi-enzyme synergy underlying ULCAs biodegradation, offering a framework for engineering microbial systems for PE remediation.