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Objective Efficient carbon-fixing microorganisms are a critical functional resource for achieving the “dual carbon” goals. However, the unstable carbon fixation performance makes natural strains difficult to directly meet industrial application needs. The molecular mechanisms underlying the enhancement of carbon fixation performance by atmospheric and room temperature plasma (ARTP) mutagenesis remain unclear. Methods Five carbon-fixing bacterial strains preserved in our laboratory were used as the starting strains. Through ARTP mutagenesis combined with directed screening and carbon-fixing enzyme activity tracking, a genetically stable and efficient carbon-fixing mutant B4-5 was constructed. Whole-genome sequencing, combined analysis of single nucleotide polymorphism (SNP) and insertion/deletion (InDel), and metabolic characterization were employed to systematically elucidate the carbon fixation enhancement mechanism. Results The mutant B4-5 showed increases of 33.16%, 72.54%, and 72.61% in key carbon-fixing enzyme activity, carbon assimilation amount, and carbon assimilation rate, respectively, with the Calvin cycle serving as the core carbon fixation pathway. Whole-genome comparison revealed that the genome of the mutant was highly collinear with that of the parent strain (similarity>98.50%), indicating that there were no large-scale chromosomal structural variations in the genome of the mutant. The combined analysis of SNP and InDel identified four key mutation sites (spoⅡE, nprR, glnQ, and murB) related to carbon fixation performance, and these sites optimized carbon source allocation, coordinated carbon-nitrogen metabolism balance, and reprogrammed carbon flux. Finally, a cascade mechanism of genomic micro-variation-metabolic regulation-phenotype enhancement was established. Conclusion This study clarifies the regulatory mechanism underlying the enhancement of carbon fixation metabolism by ARTP mutagenesis, providing a theoretical basis and engineered strain resources for the development of microbial carbon neutralization technologies.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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