Dual-functional Bacillus cereus X1 drives hydrocarbon biodegradation and carbon sequestration via glnQ-mediated...

Petroleum hydrocarbon contamination poses dual environmental challenges: direct ecosystem pollution and indirect exacerbation of greenhouse gas emissions via microbial mineralization, while natural carbon fixation pathways (e.g., the RuBisCO-mediated Calvin cycle) are insufficient at offsetting CO₂ release due to RuBisCO's inherent catalytic limitations. To address this, we engineered a dual-functional Bacillus cereus X1 strain capable of coupled hydrocarbon degradation and enhanced CO₂ fixation through optimization of a glnQ-centered metabolic network. Starting from oil-contaminated sludge, 40 carbon-fixing strains were isolated, with B. cereus X1 as the top performer, exhibiting the highest RuBisCO level (74.4 ± 2.8 U/g) together with confirmed n-alkane-degrading activity. Using Atmospheric Room-Temperature Plasma (ARTP) mutagenesis, we generated mutant derivatives (X1-Mut13/29) displaying a 1.29-1.34-fold elevation in RuBisCO level to the wild type that was stably inherited over ten serial passages. Whole-genome resequencing identified a shared nonsynonymous variant in glnQ (c.836 C>T; p.Ala279Val), encoding the ATP-binding subunit of a glutamine ABC transporter. Expression of the mutant glnQ^A279V allele in wild-type X1 generated X1-glnQ, which exhibited 2.13 times the RuBisCO level of the empty-vector control and the lowest mean residual TPH concentration among the inoculated treatments in oily-residue microcosms (690 ± 62.23 mg kg^-1 dry residue). Transcriptomics profiling revealed that glnQ overexpression upregulated key enzymes of the Calvin cycle (e.g., fbaA, glpX, tktA), glycolysis (gapA, pgk), and tricarboxylic acid (TCA) cycle, thereby enhancing NADPH/ATP supply to support carbon fixation particularly for RuBP regeneration. Mechanistically, glnQ overexpression influenced cellular C/N status, redirecting glutamine-derived α-ketoglutarate to replenish TCA cycle intermediates and fuel anaplerotic CO₂ assimilation. Collectively, this study establishes a novel "degrade-and-sequester" biocatalyst that enables synergistic pollution remediation and carbon fixation, providing a blueprint for engineering microbial carbon loops in oil-contaminated ecosystems. SYNOPSIS: This study engineers a hydrocarbon-degrading Bacillus cereus X1 strain with enhanced CO₂ fixation capacity via glnQ-mediated metabolic reprogramming, offering a novel bioremediation strategy for petroleum-contaminated environments. By leveraging glnQ-a key regulator of nitrogen and carbon metabolism-as a metabolic hub, the strain is designed to couple hydrocarbon catabolism with CO₂ sequestration, addressing the dual challenges of environmental pollution and greenhouse gas emissions. These findings support partial CO₂ reassimilation as a potential route to improve carbon-use efficiency during bioremediation, advancing sustainable solutions for contaminated ecosystem restoration.