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 (C₁₆), pristane, and glucose. During growth on C₁₆, 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 C₁₂–C₁₆ n-alkanes and enhanced the growth of the pCom8-alkB recombinant on C₂₈ (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.