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

## TL;DR

A new method was developed to quickly find active enzymes from environmental samples using metagenomics, avoiding traditional culture methods.

## Contribution

An efficient workflow was developed for enzyme discovery using metagenomic expression in Komagatella phaffii without intermediate E. coli steps.

## Key findings

- Five endoglucanases and three β-glucosidases with confirmed activity were discovered.
- A semi-high-throughput screening method enabled rapid enzyme identification.
- The workflow combines metagenomic sequencing, multiplex PCR, and rolling circle amplification.

## Abstract

Functional metagenomics has emerged as an effective tool for discovering novel enzymes directly from environmental samples, overcoming the limitations of traditional culture-based methods. In this study, we used a functional metagenomic approach on stool samples from Axis kuhlii, an endemic deer species from Indonesia, to identify active cellulases. We created an efficient workflow for expression of metagenomic sequences directly in Komagatella phaffii by combining metagenomic sequencing to investigate enzyme diversity, multiplex PCR to build a genes library, and rolling circle amplification (RCA) to streamline the cloning process, eliminating the need for intermediate Escherichia coli transformation and propagation steps. Furthermore, a semi-high-throughput screening method was used to evaluate multiple samples at once, allowing for the rapid identification of active enzymes. Using this approach, we discovered five endoglucanases and three β-glucosidases with confirmed enzyme activity. This study shows that functional metagenomics can bridge the gap between computational predictions and experimental validation, providing a reliable platform for enzyme discovery and characterization from complex environmental microbiomes.

• We established K. phaffii expression of metagenomic sequences via multiplex PCR and RCA.

• This approach links metagenomic and activity screening to enable enzyme discovery.

• Eight active cellulases were obtained from environmental samples through this approach.

The online version contains supplementary material available at 10.1007/s00253-026-13805-1.