Quantitative Proteomics Reveals Substrate-Specific Metabolic Adaptations for n-Alkane and Branched Alkane Degradation in Dietzia sp. CN-3

Although hydrocarbon-degrading bacteria play a critical role in petroleum bioremediation, the substrate-specific metabolic pathways and adaptive mechanisms that underlie their competitive dominance remain poorly understood. Dietzia sp. CN-3, a salt-tolerant bacterium capable of utilizing both linear and branched alkanes, is an ideal model for investigating these regulatory networks. Here, we performed data-independent acquisition (DIA)-based quantitative proteomics to compare the proteomic landscapes of strain CN-3 grown on n -hexadecane (C 16 ), pristane, and glucose. During growth on C 16 , strain CN-3 expressed a proposed terminal and subterminal oxidation pathway converting n -alkanes to acyl-CoA derivatives, involving AlkB and CYP153 hydroxylases, alcohol/aldehyde dehydrogenases, Baeyer-Villiger monooxygenases, and esterases. In contrast, pristane induced an alternative terminal oxidation pathway along with a substantially expanded repertoire of fatty acid β-oxidation enzymes to overcome steric hindrance. Functional heterologous expression of alkB in Pseudomonas fluorescens KOB2Δ1 restored growth on C 12 -C 16 n -alkanes and enhanced the growth of the pCom8-alkB recombinant on C 28 (μ max = 0.065 d -1 , OD max = 0.333), confirming its role in medium- to long-chain alkane utilization. Our findings provide a systematic proteomic framework for understanding alkane oxidation in Dietzia , and offer mechanistic insights into the metabolic strategies that potentially drive strain CN-3's adaptability in hydrocarbon-contaminated environments.