A fast workflow to explore active enzymes from environmental samples through functional metagenomics

Area of Science:

• Microbiology
• Enzymology
• Bioinformatics

Background:

• Traditional culture-based methods limit discovering novel enzymes.
• Functional metagenomics offers a powerful alternative for enzyme discovery from environmental samples.
• Axis kuhlii, an Indonesian endemic deer, harbors a unique microbiome for enzymatic exploration.

Purpose of the Study:

• To discover active cellulases from Axis kuhlii stool samples using functional metagenomics.
• To develop an efficient workflow for expressing metagenomic sequences directly in Komagatella phaffii.
• To link metagenomic data with activity screening for rapid enzyme identification.

Main Methods:

• Metagenomic sequencing to analyze enzyme diversity.
• Multiplex PCR and rolling circle amplification (RCA) for gene library construction and streamlined cloning.
• Direct expression of metagenomic sequences in Komagatella phaffii, bypassing Escherichia coli.
• Semi-high-throughput screening for identifying active enzymes.

Main Results:

• Successfully established direct expression of metagenomic sequences in K. phaffii.
• Identified and confirmed enzyme activity of eight cellulases, including five endoglucanases and three β-glucosidases.
• Demonstrated the efficiency of the combined metagenomic sequencing, multiplex PCR, RCA, and screening workflow.

Conclusions:

• Functional metagenomics provides a reliable platform for enzyme discovery and characterization from complex microbiomes.
• The developed workflow effectively bridges computational prediction and experimental validation.
• This study highlights the potential of exploring unique environmental niches like the Axis kuhlii microbiome for novel enzyme discovery.