Novel cytochrome P450s for various hydroxylation of steroids from filamentous fungi.

Hydroxylated steroids are value-added products with diverse biological activities mediated by cytochrome P450 enzymes, however, few has been thoroughly characterized in fungi. This study introduces a rapid identification strategy for filamentous fungi P450 enzymes through transcriptome and bioinformatics analysis. Five novel enzymes (CYP68J5, CYP68L10, CYP68J3, CYP68N1 and CYP68N3) were identified and characterized in Saccharomyces cerevisiae or Aspergillus oryzae. Molecular docking and dynamics simulations were employed to elucidate hydroxylation preferences of CYP68J5 (11α, 7α bihydroxylase) and CYP68N1 (11α hydroxylase). Additionally, redox partners (cytochrome P450 reductase and cytochrome b5) and ABC transporter were co-expressed with CYP68N1 to enhance 11α-OH-androstenedione (11α-OH-4AD) production. The engineered cell factory, co-expressing CPR1 and CYP68N1, achieved a significant increase of 11α-OH-4AD production, reaching 0.845 g·L-1, which increased by 14 times compared to the original strain. This study provides a comprehensive approach for identifying and implementing novel cytochrome P450 enzymes, paving the way for sustainable production of steroidal products.

Effects of arbuscular mycorrhizae and extraradical mycelium of subtropical tree species on soil nitrogen mineralization and enzyme activities.

Through symbiosis with plants, arbuscular mycorrhizal (AM) fungi effectively improve the availability of soil nitrogen (N). However, the mechanism through which AM and associated extraradical mycelium affect soil N mineralization remains unknow. We carried out an in situ soil culture experiment by using in-growth cores in plantations of three subtropical tree species, Cunninghamia lanceolata, Schima superba, and Liquidambar formosana. We measured soil physical and chemical properties, net N mineralization rate, and the activities of four kinds of hydrolase (leucine aminopeptidase (LAP), ß-1,4-N-acetylglucosaminidase (NAG), ß-1,4-glucosidase (ßG), cellobiohydrolase (CB)) and two kinds of oxidases (polyphenol oxidase (POX) and peroxidase (PER)) involved in soil organic matter (SOM) mineralization in treatments of mycorrhiza (with absorbing roots and hyphae), hyphae (hyphae only), and control (mycorrhiza-free). The results showed that mycorrhizal treatments significantly affected soil total carbon and pH but did not affect N mineralization rates and all enzymatic activities. Tree species significantly affected net ammonification rate, net N mineralization rate and activities of NAG, ßG, CB, POX and PER. The net N mineralization rate and enzyme activities in the C. lanceolata stand were significantly higher than that in monoculture broad-leaved stands of either S. superba or L. formosana. There was no interactive effect of mycorrhizal treatment and tree species on any of soil properties, nor on enzymatic activities or net N mineralization rates. Soil pH was negatively and significantly correlated with five kinds of enzymatic activities except for LAP, while net N mineralization rate significantly correlated with ammonium nitrogen content, available phosphorus content, and the activity level of ßG, CB, POX, and PER. In conclusion, there was no difference in enzymatic activities and N mineralization rates between rhizosphere and hyphosphere soils of three subtropical tree species in the whole growing season. The activity of particular carbon cycle-related enzymes was closely related to soil N mineralization rate. It is suggested that differences in litter quality and root functional traits among different tree species affect soil enzyme activities and N mineralization rates through organic matter inputs and shaping soil condition.

Prophage Gene Rv2650c Enhances Intracellular Survival of Mycobacterium smegmatis.

BACKGROUND: Induced by the pathogen Mycobacterium tuberculosis, tuberculosis remains one of the most dangerous infectious diseases in the world. As a special virus, prophage is domesticated by its host and are major contributors to virulence factors for bacterial pathogenicity. The function of prophages and their genes in M. tuberculosis is still unknown. METHODS: Rv2650c is a prophage gene in M. tuberculosis genome. We constructed recombinant Mycobacterium smegmatis (M. smegmatis) to observe bacteria morphology and analyze the resistance to various adverse environments. Recombinant and control strains were used to infect macrophages, respectively. Furthermore, we performed ELISA experiments of infected macrophages. RESULTS: Rv2650c affected the spread of colonies of M. smegmatis and enhanced the resistance of M. smegmatis to macrophages and various stress agents such as acid, oxidative stress, and surfactant. ELISA experiments revealed that the Rv2650c can inhibit the expression of inflammatory factors TNF-α, IL-10, IL-1ß, and IL-6. CONCLUSION: This study demonstrates that the prophage gene Rv2650c can inhibit the spread of colonies and the expression of inflammatory factors and promote intracellular survival of M. smegmatis. These results build the foundation for the discovery of virulence factors of M. tuberculosis, and provide novel insights into the function of the prophage in Mycobacterium.