Sequence-based screening for self-sufficient P450 monooxygenase from a metagenome library

Area of Science:

• Biocatalysis and Enzyme Engineering
• Environmental Microbiology

Background:

• Cytochrome P450 monooxygenases (CYPs) are versatile oxidation catalysts.
• Self-sufficient CYPs, with fused reductive and P450 domains, offer robust biocatalytic potential.
• Expanding the genetic diversity of self-sufficient CYPs is crucial for novel applications.

Purpose of the Study:

• To develop a sequence-based screening system for identifying self-sufficient CYP genes within a soil metagenome.
• To discover and characterize novel self-sufficient CYP enzymes from environmental DNA.

Main Methods:

• Construction of a soil metagenome library.
• Sequence-based screening for self-sufficient CYP genes.
• Phylogenetic analysis of identified CYP genes.
• Heterologous expression and biochemical characterization of the novel CYP enzyme.

Main Results:

• A novel self-sufficient CYP gene, syk181, was successfully identified from the soil metagenome.
• Phylogenetic analysis placed SYK181 distinctly, with 46% identity to CYP102A1.
• Heterologously expressed SYK181 demonstrated significant hydroxylase activity on fatty acids, naphthalene, and phenanthrene.

Conclusions:

• Sequence-based screening of metagenomes is an effective strategy for discovering self-sufficient CYP genes.
• The novel enzyme SYK181 possesses self-sufficient hydroxylase activity towards diverse substrates, including fatty acids and aromatic compounds.
• SYK181 represents the first self-sufficient CYP isolated directly from a metagenome, offering a valuable resource for engineering biocatalysts for bioconversion and biodegradation.