Selection and enrichment of microbial species with an increased lignocellulolytic phenotype from a native soil microbiome by activity-based probing

Abstract

Multi-omic analyses can provide information on the potential for activity within a microbial community but often lack specificity to link functions to cell, primarily offer potential for function or rely on annotated databases. Functional assays are necessary for understanding in situ microbial activity to better describe and improve microbiome biology. Targeting enzyme activity through activity-based protein profiling enhances the accuracy of functional studies. Here, we introduce a pipeline of coupling activity-based probing with fluorescence-activated cell sorting, culturing, and downstream activity assays to isolate and examine viable populations of cells expressing a function of interest. We applied our approach to a soil microbiome using two activity-based probes to enrich for communities with elevated activity for lignocellulose-degradation phenotypes as determined by four fluorogenic kinetic assays. Our approach efficiently separated and identified microbial members with heightened activity for glycosyl hydrolases, and by expanding this workflow to various probes for other function, this process can be applied to unique phenotype targets of interest.

Fig. 1. Workflow diagram for the application of ABP-FACS for kinetic MUB assay.

A Microbes are extracted from an environmental soil sample and incubated in media overnight. B Activity-based probes are developed with a warhead to target specific enzymes, a linker as a bridge to the functional tag, and a fluorophore tag to allow for downstream analyses. C The designed activity-based probes are applied to the cell community to target enzymatic reactions for the phenotype of interest. D Fluorescence-activated cell sorting is used to sort populations of cells depending on the presence or absence of probe labeling. E Sorted microbial populations are plated and incubated with fluorogenic compounds to track enzymatic activity over time as a proxy for phenotypic activity. Image generated using BioRender.

Fig. 2. Kinetic assay of four biological replicates showing the averages of three technical replicates for each line.

Values shown for time in hours of assay and substrate consumed as measured by fluorescence intensity converted to MUB concentration in µM. A GH4a sorted populations with glucose-MUB. B GH4a sorted populations with cellobiose-MUB. C SYF161 sorted populations with xylose-MUB. D SYF161 sorted populations with xylobiose-MUB.

Fig. 3. Shannon Diversity of probe sorted populations at four time during the kinetics incubation.

A GH4a sorted populations incubated with glucose-MUB with time points occurring at 15, 25, 35, and 45 h. B GH4a sorted populations incubated with cellobiose-MUB with time points occurring at 15, 25, 35, and 45 h. C SYF161 sorted populations incubated with xylose-MUB with time points occurring at 28, 38, 48, and 64 h. D SYF161 sorted populations incubated with xylobiose-MUB with time points occurring at 28, 38, 48, and 64 h. Pairwise Wilcoxon test performed to determine significance for bulk or depleted Shannon Diversity compared to enriched samples. Significance (p < 0.05) is denoted with a star (*).

Fig. 4. Ordination for GH4a probe.

Nonmetric multidimensional scaling (NMDS) for Bray Curtis Dissimilarity of community relative abundance with SIMPER analysis with ASVs representing 70% of the sample differences for comparisons between enriched vs bulk and enriched vs depleted. A–D Sorted populations for glucose-MUB incubations. A Time point 1–15 h. B Time point 2–25 h. C Time point 3–35 h. D Time point 4–45 h. E–H Sorted populations for cellobiose-MUB incubations. E Time point 1–15 h. F Time point 2–25 h. G Time point 3–35 h. H Time point 4–45 h.

Fig. 5. Ordination for SYF161 probe.

Nonmetric multidimensional scaling (NMDS) for Bray Curtis Dissimilarity of community relative abundance with SIMPER analysis with ASVs representing 70% of the sample differences for comparisons between enriched vs bulk and enriched vs depleted. A–D Sorted populations for xylose-MUB incubations. A Time point 1–28 h. B Time point 2–38 h. C Time point 3–48 h. D Time point 4–64 h. E–H Sorted populations for xylobiose-MUB incubations. E Time point 1–28 h. F Time point 2–38 h. G Time point 3–48 h. H Time point 4–64 h.