Discovery of hyperstable carbohydrate‐active enzymes through metagenomics of extreme environments

Abstract

The enzymes from hyperthermophilic microorganisms populating volcanic sites represent interesting cases of protein adaptation and biotransformations under conditions where conventional enzymes quickly denature. The difficulties in cultivating extremophiles severely limit access to this class of biocatalysts. To circumvent this problem, we embarked on the exploration of the biodiversity of the solfatara Pisciarelli, Agnano (Naples, Italy), to discover hyperthermophilic carbohydrate‐active enzymes (CAZymes) and to characterize the entire set of such enzymes in this environment (CAZome). Here, we report the results of the metagenomic analysis of two mud/water pools that greatly differ in both temperature and pH (T = 85 °C and pH 5.5; T = 92 °C and pH 1.5, for Pool1 and Pool2, respectively). DNA deep sequencing and following in silico analysis led to 14 934 and 17 652 complete ORFs in Pool1 and Pool2, respectively. They exclusively belonged to archaeal cells and viruses with great genera variance within the phylum Crenarchaeota, which reflected the difference in temperature and pH of the two Pools. Surprisingly, 30% and 62% of all of the reads obtained from Pool1 and 2, respectively, had no match in nucleotide databanks. Genes associated with carbohydrate metabolism were 15% and 16% of the total in the two Pools, with 278 and 308 putative CAZymes in Pool1 and 2, corresponding to ~ 2.0% of all ORFs. Biochemical characterization of two CAZymes of a previously unknown archaeon revealed a novel subfamily GH5_19 β‐mannanase/β‐1,3‐glucanase whose hemicellulose specificity correlates with the vegetation surrounding the sampling site, and a novel NAD+‐dependent GH109 with a previously unreported β‐N‐acetylglucosaminide/β‐glucoside specificity. Databases The sequencing reads are available in the NCBI Sequence Read Archive (SRA) database under the accession numbers SRR7545549 (Pool1) and SRR7545550 (Pool2). The sequences of GH5_Pool2 and GH109_Pool2 are available in GenBank database under the accession numbers MK869723 and MK86972, respectively. The environmental data relative to Pool1 and Pool2 (NCBI BioProject PRJNA481947) are available in the Biosamples database under the accession numbers SAMN09692669 (Pool1) and SAMN09692670 (Pool2).

Spatial Metagenomics of Three Geothermal Sites in Pisciarelli Hot Spring Focusing on the Biochemical Resources of the Microbial Consortia

“…Reads assigned to the genus Acidianus were also present in the sample from Site C. These, however, represented only 18% of the total reads, while the dominant genus was represented by Metallosphaera (33%) ( Figure 3 ), mainly attributable to the species M. prunae , which is known to grow at temperatures between 55 and 80 °C and pH range 1.0–4.5 [ 25 ]. This result is in line with what was previously observed in another extremely acidic site of the Pisciarelli hot spring, named Pool 2 (92 °C, pH 1.5) (Pool 2 Biosample ID: SAMN09692670), where Metallosphaera sedula was the dominant species [ 20 ]. Nonetheless, the most abundant component (41.5%) of Site C consisted of reads that did not match any known sequence.…”

“…Regarding the unassigned Site A reads, most of them were aligned with clusters 9, 22, and 23. The first two clusters were assigned to the genus Acidianus ( Table S1 ) and presumably represented the result of HGT, as previously reported [ 20 ].…”

“…A dramatic morphological change in the area of interest, which occurred at the end of July 2019, led to the expansion of the area previously known as Pool 1, which merged to the proximal Pool 2 pool (92 °C, pH 1.5), generating a completely new site (Site A). This new pool, if compared to Pool 1, changed both in terms of extension of the pool boundaries and in terms of temperature, showing, in particular, an increase of about 9 °C compared to the conditions previously observed [ 20 ].…”

“…Recently, our group reported on the metagenomic analysis of the microbial community populating the Pisciarelli hot springs (Naples, Italy), identifying the entire carbohydrate active enzymes portfolio (CAZome) that has been cloned and partially characterized [ 20 ].…”

“…In contrast, the majority of the obtained reads from Pool 2 found no match with the NT NCBI database (62%). Among the assigned reads, Crenarchaeota (37%) dominate the site ( Metallosphaera sedula, 31%; Saccharolobus solfataricus, 3%; Acidianus hospitalis , 3%) followed by archaeal viruses (0.36%) [ 20 ]. In that study, we demonstrated that even sites that have been consistently sampled for decades are still largely unexplored in terms of microbial diversity and of their extremozymes.…”