Presentation Information
[P01-133]Integrated omics analysis revealed metabolic alterations of a toluene-degrading Acinetobacter in the gas phase
○Shori Inoue1, Taisei Naobayashi2, Kanako Tokiyoshi2, Shogo Yoshimoto1, Maiko Hattori1, Teppei Niide3, Hiroshi Shimizu3, Yoshihiro Toya3, Hiroshi Tsugawa2, Katsutoshi Hori1 (1. Nagoya university (Japan), 2. Tokyo University of Agriculture and Technology (Japan), 3. University of Osaka (Japan))
Keywords:
Bacteria,Bioproduction,Omics analysis,Metabolism,Toluene
[Purpose]
Gas-phase bioprocesses that immobilize microbial cells on solid carriers enable the efficient conversion of poorly water-soluble gaseous substrates, thereby offering significant potential to advance bioremediation and bioproduction (1). However, microorganisms in the gas phase are exposed to various environmental stresses, mainly due to the absence of bulk water. While the survival strategies of microorganisms in gaseous environments have been studied in environmental microbiology, the metabolic adaptations that sustain active cellular metabolism remain poorly understood. In this study, we aimed to elucidate the comprehensive metabolic alterations of the toluene-degrading bacterium Acinetobacter sp. Tol 5 during toluene assimilation in both gas- and aqueous-phase conditions. This strain serves as a promising microbial chassis for gas-phase bioreactions due to its high adhesiveness and robust tolerance to desiccation (2).
[Method]
Tol 5 cells immobilized on porous polyurethane carriers were incubated in either aqueous conditions (nitrogen-depleted medium or PBS) or a gas-phase condition supplied with toluene vapor. After 24 hours of toluene degradation, cellular metabolites and mRNA were extracted for integrated metabolomic and transcriptomic analyses. Furthermore, to clarify the contribution of the detected metabolic pathways, we constructed and evaluated gene deletion mutants targeting the primary toluene assimilation route.
[Results]
Principal component analysis demonstrated that the metabolomic and transcriptomic profiles clearly differed depending on the incubation conditions. Metabolite set enrichment analysis revealed that the most prominent metabolic alterations occurred in nitrogen metabolism and storage lipid utilization. Under gas-phase conditions, the degradation of amino acids and nucleic acids was significantly promoted, and the intracellular glutamate pool was maintained at high levels. Notably, citrulline specifically accumulated in the gas phase, representing a stress response similar to that reported in Cucurbitaceae plants during drought. Furthermore, the degradation of intracellular storage lipids was significantly enhanced. We also discovered that an alternative toluene metabolic pathway was activated in the gas phase, and mutants lacking the major toluene dioxygenase route successfully degraded toluene exclusively under gas-phase conditions.
[Consideration]
Our results reveal a metabolic adaptation of Tol 5 in the gas phase. The accumulation of glutamate and citrulline likely protects the cells from severe osmotic and oxidative stress, while the enhanced utilization of storage lipids provides essential energy and metabolic water for survival in this environment. Concurrently, an alternative catabolic pathway for toluene assimilation was specifically activated in the gas phase, which is likely linked to these central metabolic reorganizations.
[Conclusion]
These findings provide critical insights into the sophisticated adaptation strategies of bacteria in gaseous environments. This knowledge offers fundamental information for the rational design of robust gas-phase bioprocesses.
(1) Cordero-Soto IN, et al., Biochem. Eng. J. 164, 107767 (2020).
(2) Usami A, Ishikawa M, and Hori K, Green Chem. 22, 1258-1268 (2020).
Gas-phase bioprocesses that immobilize microbial cells on solid carriers enable the efficient conversion of poorly water-soluble gaseous substrates, thereby offering significant potential to advance bioremediation and bioproduction (1). However, microorganisms in the gas phase are exposed to various environmental stresses, mainly due to the absence of bulk water. While the survival strategies of microorganisms in gaseous environments have been studied in environmental microbiology, the metabolic adaptations that sustain active cellular metabolism remain poorly understood. In this study, we aimed to elucidate the comprehensive metabolic alterations of the toluene-degrading bacterium Acinetobacter sp. Tol 5 during toluene assimilation in both gas- and aqueous-phase conditions. This strain serves as a promising microbial chassis for gas-phase bioreactions due to its high adhesiveness and robust tolerance to desiccation (2).
[Method]
Tol 5 cells immobilized on porous polyurethane carriers were incubated in either aqueous conditions (nitrogen-depleted medium or PBS) or a gas-phase condition supplied with toluene vapor. After 24 hours of toluene degradation, cellular metabolites and mRNA were extracted for integrated metabolomic and transcriptomic analyses. Furthermore, to clarify the contribution of the detected metabolic pathways, we constructed and evaluated gene deletion mutants targeting the primary toluene assimilation route.
[Results]
Principal component analysis demonstrated that the metabolomic and transcriptomic profiles clearly differed depending on the incubation conditions. Metabolite set enrichment analysis revealed that the most prominent metabolic alterations occurred in nitrogen metabolism and storage lipid utilization. Under gas-phase conditions, the degradation of amino acids and nucleic acids was significantly promoted, and the intracellular glutamate pool was maintained at high levels. Notably, citrulline specifically accumulated in the gas phase, representing a stress response similar to that reported in Cucurbitaceae plants during drought. Furthermore, the degradation of intracellular storage lipids was significantly enhanced. We also discovered that an alternative toluene metabolic pathway was activated in the gas phase, and mutants lacking the major toluene dioxygenase route successfully degraded toluene exclusively under gas-phase conditions.
[Consideration]
Our results reveal a metabolic adaptation of Tol 5 in the gas phase. The accumulation of glutamate and citrulline likely protects the cells from severe osmotic and oxidative stress, while the enhanced utilization of storage lipids provides essential energy and metabolic water for survival in this environment. Concurrently, an alternative catabolic pathway for toluene assimilation was specifically activated in the gas phase, which is likely linked to these central metabolic reorganizations.
[Conclusion]
These findings provide critical insights into the sophisticated adaptation strategies of bacteria in gaseous environments. This knowledge offers fundamental information for the rational design of robust gas-phase bioprocesses.
(1) Cordero-Soto IN, et al., Biochem. Eng. J. 164, 107767 (2020).
(2) Usami A, Ishikawa M, and Hori K, Green Chem. 22, 1258-1268 (2020).
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