Cloning, expression, and characterization of Baeyer–Villiger monooxygenases from eukaryotic Exophiala jeanselmei strain KUFI-6N

The fungus Exophiala jeanselmei strain KUFI-6N produces a unique cycloalkanone monooxygenase (ExCAMO) that displays an uncommon substrate spectrum of Baeyer–Villiger oxidation of 4–10-membered ring ketones. In this study, we aimed to identify and sequence the gene encoding ExCAMO from KUFI-6N and overexpress the gene in Escherichia coli. We found that the primary structure of ExCAMO is most closely related to the cycloalkanone monooxygenase from Cylindrocarpon radicicola ATCC 11011, with 54.2% amino acid identity. ExCAMO was functionally expressed in Escherichia coli and its substrate spectrum and kinetic parameters investigated. Substrate profiling indicated that ExCAMO is unusual among known Baeyer–Villiger monooxygenases owing to its ability to accept a variety of substrates, including C4–C12 membered ring ketones. ExCAMO has high affinity and catalytic efficiency toward cycloalkanones, the highest being toward cyclohexanone. Five other genes encoding Baeyer–Villiger monooxygenases were also cloned and expressed in Escherichia coli.

Evaluation of Fungal Activity Through In Silico Analysis of Medicinal Plants Against Exophiala Jeanselmei

Phaeohyphomycosis is a fungal infectious disease commonly called as dermal problem which is caused by dematiaceous fungi, Exophiala jeanselmei. Chitin was the main component of fungal cell and no effective inhibitor was identified still in chitin synthase I. The protein chitin synthase I play a major role in drug metabolism as well as signal processing molecule and therefore have been targeted in the present study. The medicinal plants being a solution for several human ailments, also act as a reservoir for secondary metabolites, has taken its credit as a cure from our ancient times. The biological activity of the Myricetin was analysed using the pass online tool. The value of Probability to be active (Pa) = 0.241 Probability to be inactive (Pi) = 0.021. The several compounds retrieved from the plants Acalypha indica, Achyranthus aspera, Brassica niger, Cassia auriculata, Cleome gynandra, Clitoria ternatea, Ipomoea hederaceae, Leucas aspera, Mimosa pudica, Phyllanthus niruri, Ocimum basilicum, Ocimum sanctum, Tridax procumbens, Vitex negundo and Waltheria indica were analyzed for its possible significant interaction with the target protein using molecular docking studies. The compound Myricetin had Binding energy of -7.32 Kcal/mol and formed hydrogen bonds with the residue HIS 29 showing the bond length of 1.8 Å and residue THR 3 showing the bond length of 1.9 Å. The future perspective of the study is to determine the stability of the protein-compound interaction through docking studies.

Exploiting the Advantages of Molecular Tools for the Monitoring of Fungal Indoor Air Contamination: First Detection of Exophiala jeanselmei in Indoor Air of Air-Conditioned Offices

Today, indoor air pollution is considered a public health issue. Among the impacting pollutants, indoor airborne fungi are increasingly highlighted. Most of the monitoring protocols are culture-based, but these are unable to detect the uncultivable and/or dead fraction or species suppressed by fast-growing fungi, even though this fraction could impact health. Among the contaminants suspected to be part of this fraction, Exophiala jeanselmei is an interesting case study. Known to be pathogenic, this black yeast grows in humid environments such as air-conditioning systems, where it has been previously detected using classical culture-based methods. However, until now, this fungus was never detected in indoor air in contact with these air-conditioning systems. This study shows the first detection of E. jeanselmei in indoor air collected from offices in contact with contaminated air-conditioning reservoirs. While its presence in indoor air could not be demonstrated with culture-based methods, it was found by real-time PCR and massive parallel sequencing. The latter also allowed obtaining a broader view on the fungal diversity in the tested samples. Similar approaches were applied on water samples collected from the conditioning reservoirs to trace the source of contamination. The comparison of results obtained with both methods confirmed that the molecular tools could improve indoor air monitoring, especially of dead and/or uncultivable contaminants or when competition between species could occur.