충남대학교 생체분자공학연구실 | BioMolE Lab at CNU

Background: Methane (CH4) is an attractive carbon feedstock for sustainable biomanufacturing, but poor CH4 solubility and gas-liquid mass-transfer limitations continue to constrain methanotroph-based CH4 valorization. Here, we evaluated biocompatible bionanofluids as additive-based process intensification tools and examined whether two operationally distinct bionanofluid application strategies produce distinct time-dependent physiological responses beyond simple growth enhancement.

Results: Two bionanofluids, chitosan/oleamide (CS/OA) nanoparticles and tannic acid–Fe3+ complex-coated cellulose nanocrystals (TA-Fe3+CNCs), were evaluated in 5-L cultures of Methylosinus trichosporium OB3b using two different application approaches: CS/OA seed adaptation followed by cultivation in conventional medium, and TA-Fe3+CNCs supplementation in the main culture. In both approaches, time-resolved profiling of gas uptake, nitrogen species, extracellular metabolites, and poly(3-hydroxybutyrate) (PHB) showed higher CH4 and O2 utilization, with 1.5–1.6- and 2.0–2.1-fold increases in specific CH4 and O2 uptake rates, respectively, and faster growth, with 1.3–1.5-fold higher µmax, than in the conventional control. These changes were accompanied by consistently lower PHB content, faster nitrate depletion, higher ammonium accumulation, and distinct extracellular metabolite patterns. Notably, CS/OA seed adaptation was associated with transient succinate accumulation during exponential growth, whereas TA-Fe3+CNCs supplementation led to faster methanol accumulation without succinate release. These observations are consistent with a process-level response in which higher CH4/O2 uptake and faster growth coincided with lower PHB content under the tested conditions. As an initial proof-of-concept experiment, a consecutive strategy combining CS/OA seed adaptation with TA-Fe3+CNCs supplementation in Methylomonas sp. DH-1 was associated with greater transient succinate accumulation and a higher peak succinate titer than under the conventional condition.

Conclusions: Comparative time-resolved profiling of two different bionanofluid approaches indicated that bionanofluid application was associated with higher CH4/O2 uptake, faster growth, lower PHB accumulation, and distinct time-dependent metabolite patterns under the tested conditions. These findings support the use of biocompatible bionanofluids as practical media additives that can be applied without major bioreactor redesign and can motivate future component-controlled testing of sequential strategies for growth-linked CH4 bioconversion.

Biomass-derived aromatic compounds, including those obtained from lignin (which is the most abundant aromatic biopolymer on Earth), are valuable for sustainable chemical production. Various lignin-degrading approaches have been developed to cleave recalcitrant bonds. The incorporation of biocatalysts that operate under environmentally friendly and mild conditions with high substrate specificity is considered one of the emerging strategies for lignin valorization. In this study, an archaeal tyrosinase (Tyr-CNK), derived from the marine archaeon Candidatus nitrosopumilus koreensis, is characterized as a versatile biocatalyst for lignin biodegradation and valorization, based on kinetic studies, protein structure determination, and analysis. Notably, the extremely shallow active site pocket and the unique noncanonical caddy domain, which facilitate efficient copper incorporation without obstructing the active site, collectively empower Tyr-CNK with remarkable catalytic efficiency toward various lignin model compounds, such as p-coumaric acid, 4-phenoxyphenol, 4-(benzyloxy)phenol, and guaiacyl glycerol-β-guaiacyl ether. Together with molecular docking simulations, these catalytic and structural features indicate that Tyr-CNK serves as an efficient biocatalyst for the hydroxylation and oxidative degradation of lignin-derived phenolic compounds. Given its versatility, efficiency, and structural uniqueness, Tyr-CNK demonstrates great promise for expanding the catalytic repertoire for biomass conversions and offering new opportunities in sustainable biocatalysis, enzymatic and microbial biodegradation and biomass valorization.

Carbon monoxide (CO) serves as a promising carbon source, but it adversely affects CO-metabolizing microorganisms. To address this issue, fermentation should occur under mass-transfer limitation conditions, where the rate of gaseous CO transfer is lower than its consumption rate. However, maintaining these conditions becomes challenging as microorganism physiology deteriorates during fermentation, requiring a novel strategy to sustain mass-transfer limitation conditions. In this study, cellulose-based nanocrystals coated with a tannic acid-Fe³⁺ complex (TA-Fe³⁺CNC) were developed. These nanocrystals exhibited reversible CO adsorption/desorption characteristics, enabling the transformation from kinetic to mass-transfer limitation conditions. This approach effectively mitigated substrate inhibition from CO with negligibly impacting cell physiology. Kinetic studies demonstrated the effectiveness of TA-Fe³⁺CNCs in mitigating CO inhibition under high CO partial pressures. Furthermore, computational modeling validated the successful shifting from kinetic to mass-transfer limitation conditions at the molecular level. Overall, this study introduces a novel platform for stabilizing CO metabolism by microorganisms.

Numerous biological systems contain vesicle-like biomolecular compartments without membranes, which contribute to diverse functions including gene regulation, stress response, signaling, and skin barrier formation. Coacervation, as a form of liquid–liquid phase separation (LLPS), is recognized as a representative precursor to the formation and assembly of membrane-less vesicle-like structures, although their formation mechanism remains unclear. In this study, a coacervation-driven membrane-less vesicle-like structure is constructed using two proteins, GG1234 (an anionic intrinsically disordered protein) and bhBMP-2 (a bioengineered human bone morphogenetic protein 2). GG1234 formed both simple coacervates by itself and complex coacervates with the relatively cationic bhBMP-2 under acidic conditions. Upon addition of dissolved bhBMP-2 to the simple coacervates of GG1234, a phase transition from spherical simple coacervates to vesicular condensates occurred via the interactions between GG1234 and bhBMP-2 on the surface of the highly viscoelastic GG1234 simple coacervates. Furthermore, the shell structure in the outer region of the GG1234/bhBMP-2 vesicular condensates exhibited gel-like properties, leading to the formation of multiphasic vesicle-like compartments. A potential mechanism is proposed for the formation of the membrane-less GG1234/bhBMP-2 vesicle-like compartments. This study provides a dynamic process underlying the formation of biomolecular multiphasic condensates, thereby enhancing the understanding of these biomolecular structures.