A Bait-and-Switch Strategy Links Phenotypes to Genes Coding for Polymer-Degrading Enzymes in Intact Microbiomes.

Natural microbial communities, with their vast diversity and complexity, are among the richest sources of untapped novel enzymes. Identifying novel enzymes can be challenging because microbiomes often lack clear, measurable phenotypes, unlike laboratory cultures where enzymatic activity can be linked to genetic elements. These constraints have left much of the functional diversity within microbiomes inaccessible to enzyme discovery efforts. Here, we present a genotype/phenotype association framework directly on microbial communities for enzyme discovery. For this, we developed a 'bait-and-switch' treatment strategy that generates measurable dual phenotypes directly within intact microbiomes. Using soil microbiomes as a test system, we applied chitin-rich compost as 'bait' to enrich chitin-degrading organisms, followed by glucose addition to functionally 'switch' the community. This treatment produced a distinct phenotypic signature: prevalence of known chitin degradation genes increases during the bait phase, and their transcripts are rapidly downregulated during the switch phase. By performing hypothesis-free association analysis of protein domains with this dual phenotype, we identified the glycoside hydrolase 18 as the most significantly associated protein domain. Experimental validation confirmed chitinase activity in 63% of tested enzymes, including candidates from unculturable bacteria and those with previously uncharacterized domain architectures. This species-independent, reference-free approach to discover novel enzymes has broad applications in microbiome engineering, biopolymer processing and systems biology, offering a generalizable strategy for functional gene discovery in complex microbial systems.