High Potential for Biomass-Degrading Enzymes Revealed by Hot Spring Metagenomics

Abstract

Enzyme stability and activity at elevated temperatures are important aspects in biotechnological industries, such as the conversion of plant biomass into biofuels. In order to reduce the costs and increase the efficiency of biomass conversion, better enzymatic processing must be developed. Hot springs represent a treasure trove of underexplored microbiological and protein chemistry diversity. Herein, we conduct an exploratory study into the diversity of hot spring biomass-degrading potential. We describe the taxonomic diversity and carbohydrate active enzyme (CAZyme) coding potential in 71 publicly available metagenomic datasets from 58 globally distributed terrestrial geothermal features. Through taxonomic profiling, we detected a wide diversity of microbes unique to varying temperature and pH ranges. Biomass-degrading enzyme potential included all five classes of CAZymes and we described the presence or absence of genes encoding 19 glycosyl hydrolases hypothesized to be involved with cellulose, hemicellulose, and oligosaccharide degradation. Our results highlight hot springs as a promising system for the further discovery and development of thermo-stable biomass-degrading enzymes that can be applied toward generation of renewable biofuels. This study lays a foundation for future research to further investigate the functional diversity of hot spring biomass-degrading enzymes and their potential utility in biotechnological processing.

Keywords: CAZyme; bioprospecting; biotechnology; cellulases; thermophilic enzymes.

FIGURE 1

(A) Temperature and pH scatter plot of all 71 metagenomes used in this study. Points are colored by country of sample origin. Dashed gray lines denote the temperature and pH categories used. Temperature categories: mesophilic (<45.0°C), thermophilic (45.0–79.9°C), and hyperthermophilic (>80.0°C). pH categories: highly acidic (<4.0), mildly acidic (4.0–5.9), neutral (6.0–7.9), and alkaline (>8.0). (B–F) Violin plots of sequencing statistics for all 71 metagenomes. The four metagenomes of particular interest to this study are highlighted. Black square denotes median value.

FIGURE 2

Observed taxa and Shannon Diversity metrics for the taxonomic profile assignment of metagenomic contigs at genus level classification. Taxonomy was assigned to metagenomic contigs using the Kraken2 standard database and contig abundances were calculated using Bracken. (A) α-diversity split by temperature categories, which were: mesophilic (<45.0°C), thermophilic (45.0–79.9°C), and hyperthermophilic (>80.0°C). (B) α-diversity split by pH categories, which were: highly acidic (<4.0), mildly acidic (4.0–5.9), neutral (6.0–7.9), and alkaline (>8.0). * denotes significance p < 0.05. Statistics calculated with pairwise comparisons and p-values adjusted by the Holm method. (C,D) Canonical correspondence analysis (CCA) of β-diversity calculated by Bray Curtis dissimilarity with points colored by sample temperature or pH, respectively.

FIGURE 3

Heatmap showing presence-absence of specific CAZymes with predicted cellulase, hemicellulase, and oligosaccharide-degrading enzyme activities in the 71 metagenomes. Dark blue color represents the presence of a CAZyme gene sequence. Sample clustering was based on hierarchical cluster analysis of dissimilarity of the CAZyme presence-absence profile. Two metagenomes from non-hot spring lignocellulolytic environments are shown on the left and indicated with a gray square (2030936001—Nasutitermes corniger P3 gut compartment) and triangle (2209111003—São Paulo Zoo compost). Four hot spring metagenomes of particular interest to this study are indicated with colored circles. 3300029977—LCB-024—blue, 3300006865—Larsen N4—yellow, 3300007072—Dewar Creek DC9—green, and 3300029625—SJ3—orange.

FIGURE 4

Sankey diagram illustrating taxonomic distribution of CAZyme-containing contigs and the hot spring metagenomes they originated from. Indicated on the left is the enzyme type determined by the most abundant Pfam domain for the most abundant glycosyl hydrolase family for contigs identified by dbCAN2 using the CAZy database (Pfam00150—GH5—Cellulase, Pfam00331—GH10—Hemicellulase, Pfam00933—GH3—Oligosaccharide-degrading enzymes). In the center is the taxonomic affiliation of the contigs determined by IMG. Taxonomically unclassified contigs were removed prior to plotting. Shown on the right is the hot spring metagenome location for the taxa. The height of the Sankey bars are assigned proportionately to the relative abundances of CAZyme-containing contigs and taxa.