Unearthing the genetic resources of Arabian sea seamount and metagenomic insights into phosphate cycling genes for next generation plant biostimulants

Deep-sea encompasses a wide diversity of microbiomes including bacteria, fungi and viruses which play crucial significant roles in nutrient biogeochemical cycling thereby imparting majorly to functional biodiversity of these hotspots. Sea mounts harboring microbes with extremophilic properties found in deep oceans could be conserved as living repository by functional metagenomics approach which is a potent source to screen bioactive compounds and novel enzymes thereby could address biological question on developing next generation plant biostimulants. This study outlines construction of fosmid metagenome library and adapted combined strategy of functional and nanopore sequence-based metagenomic screening to unveil phosphatase enzymes from Arabian Sea seamount sediment. About 9068 metagenomic clones were generated with an average insert size of 38 kb and stored in pools of 1024 clones, out of which 42 were found to be positive for phosphatase. Five clones with high phosphatase activity were further characterized and NIOT F41 showed the greatest specific activity for phosphatase (41.2 U/mg). Gluconic (1041 mg/L), oxalic (327 mg/L), and succinic acids (610 mg/L) were the predominant organic acids produced by recombinant clones. Fosmid DNA were extracted from five potential clones for nanopore-based metagenomics sequencing which generated an average of 6,00,786 reads. Taxonomic analysis revealed an abundance of Proteobacteria and Firmicutes phyla harboring phosphate-solubilising bacteria Pseudomonas aeruginosa, Bacillus subtilis and Staphylococcus warneri. Furthermore, functional annotation using phosphorus cycling database (PCycDB) predicted variation in relative abundance of phosphatase gene clusters encoding alkaline phosphatase (PhoD, PhoX and PhoA) and acid phosphatase (OlpA, PhoNand PhoC) produced by recombinant clones. In the pot assay, potential metagenomic clones exhibited positive impacts on shoot length (9.1 ± 1.1 cm, p < 0.05), root length (2.05 ± 0.05 cm, p < 0.05), wet biomass (39.3 ± 0.65 mg, p < 0.05), and dry biomass (5.1 ± 1.15 mg, p < 0.05) compared to the negative control indicating significant effect on promoting plant growth. The advanced nanopore sequencing and functional metagenomics methods employed in this study could serve as a marine biodiversity conservation approach for deep-sea microbes hidden in sea mount sediments towards harnessing potential next generation plant biostimulants with promising biotechnological application for sustainable agriculture.