The Lichen Flavin-Dependent Halogenase, DnHal: Identification, Heterologous Expression and Functional...

Area of Science:

• Biochemistry
• Enzymology
• Fungal Biotechnology

Background:

• Enzymatic halogenation is crucial for chemical diversity.
• Most known flavin-dependent halogenases (F-Hals) are bacterial; none were previously identified in lichenized fungi.
• Fungi are known producers of halogenated compounds.

Purpose of the Study:

• To mine for and characterize a novel flavin-dependent halogenase (F-Hal) from lichenized fungi.
• To investigate the biocatalytic activity of the identified F-Hal.
• To explore the potential of fungal F-Hals in green biocatalysis.

Main Methods:

• Transcriptomic data mining of Dirinaria sp. for putative F-Hal genes.
• Phylogenetic analysis of the F-Hal family.
• Codon optimization, cloning, and expression of the F-Hal gene in Pichia pastoris.
• Enzyme purification and characterization of biocatalytic activity.
• Mass spectrometry analysis of reaction products.

Main Results:

• A putative F-Hal gene, designated dnhal, was identified from Dirinaria sp.
• Phylogenetic analysis indicated dnhal belongs to the non-tryptophan F-Hals group.
• The purified recombinant enzyme exhibited biocatalytic activity, halogenating tryptophan and methyl haematommate.
• Mass spectrometry confirmed the formation of chlorinated products.

Conclusions:

• This study reports the first flavin-dependent halogenase from lichenized fungi.
• The identified enzyme demonstrates the ability to halogenate both tryptophan and aromatic compounds.
• This discovery opens avenues for understanding fungal F-Hals and developing green biocatalysis methods for halogenated compounds.