Catalase plays a significant role in improving animal intestinal health and growth performance; however, its industrial application in feed is largely limited by poor thermostability and low acid tolerance. We identified an acid-resistant catalase from Talaromyces pinophilus and improved its thermal stability. We integrated multiple thermal stability design strategies, constructed a mutation library using algorithms from different design platforms, and further refined the library with computational tools. This approach successfully generated the combinatorial mutant M3 (E38Q/S69P/S187A). Its half-life at 80 °C increased by 2.64 times, and two fold increase in half life at 90 ℃, while the specific activity remained unchanged. Molecular dynamics simulations show that this mutational strategy can significantly enhance the thermal stability of catalase through a cascading effect of ‘local rigidity enhancement—global conformational compaction—hydrophobic core stabilization’. Additionally, under simulated high-temperature and gastric acid conditions, the mutant strain demonstrated a high capacity for decomposing H2O2, significantly enhancing its practical value for use in animal feed.
Stabilizing enzyme functionality during downstream processing remains a central challenge in scalable biomanufacturing. This study presents a process-integrated evaluation of microencapsulation strategies to preserve the activity of a fibrinolytic protease derived from Bacillus tequilensis HSFI-5, with an emphasis on the interplay among the encapsulant matrix, drying method, and enzymatic accessibility. Encapsulation systems based on maltodextrin, Arabic gum, chitosan, carrageenan, and alginate (5%–15% w/v) were processed by freeze-drying and spray-drying and assessed for activity retention and microencapsulation yield. Results demonstrated that both formulation and processing conditions significantly influenced functional performance (P < 0.05). Lower polymer concentrations favored higher apparent enzymatic activity, whereas higher concentrations improved powder recovery, revealing a trade-off between catalytic accessibility and process efficiency. A moderate negative correlation between activity retention and yield (r = − 0.62, P = 0.018) supports a matrix-dependent diffusion constraint. A comparative analysis of crude, diluted, and microencapsulated systems showed that the reduced apparent activity in encapsulated formulations may be associated with matrix-related accessibility constraints. This interpretation is supported by the increased activity observed after mechanical disruption, suggesting that the decrease was not solely due to complete enzyme inactivation. Among the evaluated systems, maltodextrin-based formulations exhibited a favorable balance between activity retention and yield, particularly under spray-drying conditions, indicating compatibility with scalable processing. Qualitative clot degradation assays confirmed preservation of fibrinolytic functionality after encapsulation, although with a delayed response consistent with controlled enzyme release. Morphological analysis revealed spherical, relatively smooth microcapsules that may facilitate improved hydration and substrate diffusion. Collectively, these findings establish a process-level framework that links matrix composition, drying strategy, and mass-transfer behavior to enzymatic performance. This work highlights the importance of designing encapsulation systems that balance structural protection with functional accessibility, thereby providing a rational basis for developing stable, scalable enzyme formulations for biomanufacturing applications.
Thermotolerant microbial hosts offer advantages for industrial bioprocesses, yet direct oil-based bioconversion requires efficient coupling of extracellular lipid hydrolysis with intracellular carbon assimilation. In the thermotolerant bacterium Cupriavidus cauae PHS1, direct utilization of triacylglycerol-rich substrates is constrained by the lack of extracellular lipolytic activity and limited glycerol metabolism. Here, the underexplored non-model bacterium C. cauae PHS1 was engineered as a thermotolerant chassis for direct conversion of palm oil (PO) and waste frying oil (WFO) into polyhydroxybutyrate (PHB) at 42 °C. Heterologous expression of a thermostable secretory lipase from C. necator H16 conferred extracellular oil-hydrolysis capability, and adaptive laboratory evolution was applied to improve glycerol metabolism. This combined strategy enabled direct utilization of triacylglycerol-derived carbon and enhanced PHB accumulation from both a model oil feedstock and a complex waste oil feedstock. Under non-optimized culture conditions, the engineered strain produced PHB from 10 g/L PO, reaching 5.11 g/L cell dry weight (CDW) with 64.18% PHB content, and from 10 g/L WFO, achieving 4.03 g/L CDW with 52.61% PHB content. These results support the potential of C. cauae PHS1 as a promising thermotolerant chassis for sustainable PHB biomanufacturing from complex waste oil feedstocks.
Decomposed wheat straw is a promising seedling substrate but is vulnerable to contamination by filamentous fungi, notably Aspergillus flavus. Multifunctional microbes that combine strong cellulolytic capacity with antifungal activity are needed to enable cleaner bioprocessing and reduce mycotoxin risk. This study sought to identify such a strain and verify its performance from screening to crop-relevant application. From 2,102 Bacillus isolates collected between 2018 and 2023, we selected Bacillus amyloliquefaciens SQ1 exhibiting both cellulolytic activity and antifungal efficacy against A. flavus. During straw decomposition, SQ1 accelerated cellulose and hemicellulose loss, decreased C/N and lignin/N ratios, and rapidly shifted pH toward neutrality. SQ1 suppressed fungal proliferation; Cryo-SEM revealed fractured and collapsed A. flavus hyphae with markedly reduced spore adhesion. Transcriptomic profiling of A. flavus under SQ1 exposure revealed coordinated repression, spanning cell-envelope biogenesis and sterol synthesis (down-regulation of fks1, aflY, erg2, erg11), diminished developmental competence (ligA), and attenuated toxin regulation (aflR). SQ1-amended decomposed straw reduced the relative abundance of Aspergillus and lowered aflatoxin B1 (AFB1, C17H12O6) content. Using the SQ1-amended decomposed straw as a substrate improved peanut seedling survival, plant height, and leaf chlorophyll. SQ1 functions as a dual-purpose biocatalyst, breaking down cellulose while inhibiting fungal growth to enhance straw biotransformation, mitigate mycotoxin risk, and support early crop growth. These findings highlight a sustainable and value-added approach to wheat straw utilization, with practical implications for improved substrate management and cleaner production in crop systems.
Clostridium acetobutylicum ATCC 824 (pCD07239), designated CD07239 in this study, was previously constructed by introducing the Clostridioides difficile CD630_0723–CD630_0729 gene cluster into the parental ATCC 824 strain. The CD07239 strain initiates butanol production during the early growth phase. To investigate the transcriptional basis of this phenotype, we performed RNA-seq-based transcriptome analysis using phenotype-defined RNA samples collected during batch fermentation. At 3 h, CD07239 had already produced approximately 0.1 g/L butanol, whereas butanol was not detected in ATCC 824. At 9 h, both strains produced butanol, but CD07239 accumulated a substantially higher level than ATCC 824. Principal component analysis revealed that the transcriptomes were separated according to fermentation stage and strain background. In ATCC 824, major solventogenic genes, including adhE1, ctfA, ctfB, and adc, were strongly induced at 9 h compared with 3 h. In contrast, early butanol production in CD07239 at 3 h was not accompanied by premature induction of these genes. Instead, adhE2 showed markedly high expression in CD07239, with log2 fold changes of 6.4 at 3 h and 6.6 at 9 h compared with ATCC 824. Additional adhE2 expression in ATCC 824 did not reproduce early butanol production, suggesting that adhE2 alone is insufficient to induce this phenotype. These results indicate that early butanol production in CD07239 is associated with an adhE2-dominant transcriptional state distinct from the conventional solventogenic transition of ATCC 824.
Grapevine trunk diseases threaten sustainable viticulture, while wine lees remain an underexploited biocontrol-relevant by-product within the circular economy framework. An integrated bioprocess was developed to upgrade lees into high-molecular-weight biomolecule mixtures and low-molecular-weight peptide-enriched fractions through a defined sequence of unit operations: fermentation design by using Saccharomyces monocultures and sequential inoculations with non-Saccharomyces strains, ultrasound-assisted autolysis, enzymatic hydrolysis, and 3 kDa ultrafiltration. The antifungal activity of the products was tested in vitro and in vivo against Phaeomoniella chlamydospora and Phaeoacremonium minimum, the two main pathogens that cause Petri disease in grapevine. The results demonstrated that high-molecular-weight autolysates showed no inhibition. The post-hydrolysis fractions reduced mycelial growth of Phaeoacremonium minimum by 45%–75% and Phaeomoniella chlamydospora by 40%–57%, and decreased conidiation by up to 92% and 61%, respectively. They also reduced symptoms (wood discoloration) by 47%–52% and 35%–73% and endophytic fungal growth by 63%–84% and 80%–91% respectively. Those results indicate an association between hydrolysis and the appearance of antifungal activity. Untargeted metabolomics employing liquid chromatography–mass spectrometry (UHPLC–ESI–TIMS–QTOF) highlighted strain- and fermentation-dependent secondary metabolite signatures, with 448 features differentiating across fermentation conditions. Activity-based correlation fingerprinting identified 49 putative biomarkers negatively associated with pathogen growth. Overall, fermentation design functions as a controllable upstream quality parameter, offering a scalable circular economy pathway for the production of antifungal bioproducts derived from winery waste.
Lacto-N-neotetraose (LNnT), a pivotal oligosaccharide in human milk, plays a significant role in modulating the intestinal microbiota of infants, and exhibits potential applications in antiviral therapy. Corynebacterium glutamicum is widely used in industrial fermentation, but it is incapable of synthesizing LNnT under natural conditions. Previously, we constructed a recombinant C. glutamicum strain CL014 for LNnT production, but the yield was limited. In this study, the key enzyme β-1,4-galactosyltransferase LgtB was engineered to improve LNnT biosynthesis in CL014. Fourteen mutant strains were constructed by replacing the critical residue Arg24 of LgtB to different amino acids and screened for the optimal LNnT production. The highest LNnT production was obtained in the mutant CL014-T. CL014-T could produce 1.40 g/L LNnT in shake flask cultivation, which is a 33.3% increase compared with the control strain CL014. Molecular docking simulations demonstrated that the mutation in CL014-T promotes the formation of additional hydrogen bonds in the flexible loop region, enhancing conformational flexibility and optimizing substrate interactions. Further mechanistic insights obtained through molecular dynamics simulations uncovered that the increased dynamic flexibility in two functional regions and the formation of more core hydrogen bonds collectively contribute to enhanced binding stability and improved product yield in the mutant. After 96 h feed-batch fermentation, CL014-T produced 2.29 g/L LNnT. This work demonstrates that C. glutamicum has the potential to be engineered as an efficient cell factory for human milk oligosaccharide production.