Novel thermostable antibiotic resistance enzymes from the Atlantis II Deep Red Sea brine pool

The advent of metagenomics has greatly facilitated the discovery of enzymes with useful biochemical characteristics for industrial and biomedical applications, from environmental niches. In this study, we used sequence‐based metagenomics to identify two antibiotic resistance enzymes from the secluded, lower convective layer of Atlantis II Deep Red Sea brine pool (68°C, ~2200 m depth and 250‰ salinity). We assembled > 4 000 000 metagenomic reads, producing 43 555 contigs. Open reading frames (ORFs) called from these contigs were aligned to polypeptides from the Comprehensive Antibiotic Resistance Database using BLASTX. Two ORFs were selected for further analysis. The ORFs putatively coded for 3′‐aminoglycoside phosphotransferase [APH(3′)] and a class A beta‐lactamase (ABL). Both genes were cloned, expressed and characterized for activity and thermal stability. Both enzymes were active in vitro, while only APH(3′) was active in vivo. Interestingly, APH(3′) proved to be thermostable (T (m) = 61.7°C and ~40% residual activity after 30 min of incubation at 65°C). On the other hand, ABL was not as thermostable, with a T (m) = 43.3°C. In conclusion, we have discovered two novel AR enzymes with potential application as thermophilic selection markers.