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Metagenomics-Guided Discovery of Potential Bacterial Metallothionein Genes from the Soil Microbiome That Confer Cu and/or Cd Resistance.

ABSTRACT: Metallothionein (MT) genes are valuable genetic materials for developing metal bioremediation tools. Currently, a limited number of prokaryotic MTs have been experimentally identified, which necessitates the expansion of bacterial MT diversity. In this study, we conducted a metagenomics-guided analysis for the discovery of potential bacterial MT genes from the soil microbiome. More specifically, we combined resistance gene enrichment through diversity loss, metagenomic mining with a dedicated MT database, evolutionary trace analysis, DNA chemical synthesis, and functional genomic validation to identify novel MTs. Results showed that Cu stress induced a compositional change in the soil microbiome, with an enrichment of metal-resistant bacteria in soils with higher Cu concentrations. Shotgun metagenomic sequencing was performed to obtain the gene pool of environmental DNA (eDNA), which was subjected to a local BLAST search against an MT database for detecting putative MT genes. Evolutional trace analysis led to the identification of 27 potential MTs with conserved cysteine/histidine motifs different from those of known prokaryotic MTs. Following chemical synthesis of these 27 potential MT genes and heterologous expression in Escherichia coli, six of them were found to improve the hosts' growth substantially and enhanced the hosts' sorption of Cu, Cd, and Zn, among which MT5 led to a 13.7-fold increase in Cd accumulation. Furthermore, four of them restored Cu and/or Cd resistance in two metal-sensitive E. coli strains.IMPORTANCE The metagenomics-guided procedure developed here bypasses the difficulties encountered in classic PCR-based approaches and led to the discovery of novel MT genes, which may be useful in developing bioremediation tools. The procedure used here expands our knowledge on the diversity of bacterial MTs in the environment and may also be applicable to identify other functional genes from eDNA.

SUBMITTER: Li X

PROVIDER: S-EPMC7170490 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundEnergy shortage has become a global problem. Production of biofuels from renewable biomass resources is an inevitable trend of sustainable development. Cellulose is the most abundant and renewable resource in nature. Lack of new cellulases with unique properties has become the bottleneck of the efficient utilization of cellulose. Environmental metagenomes are regarded as huge reservoirs for a variety of cellulases. However, new cellulases cannot be obtained easily by functional screening of metagenomic libraries.ResultsIn this work, a metagenomics-guided strategy for obtaining new cellulases from the metagenome was proposed. Metagenomic sequences of DNA extracted from the anaerobic beer lees converting consortium enriched at thermophilic conditions were assembled, and 23 glycoside hydrolase (GH) sequences affiliated with the GH family 5 were identified. Among the 23 GH sequences, three target sequences (designated as cel7482, cel3623 and cel36) showing low identity with those known GHs were chosen as the putative cellulase genes to be functionally expressed in Escherichia coli after PCR cloning. The three cellulases were classified into endo-β-1,4-glucanases by product pattern analysis. The recombinant cellulases were more active at pH 5.5 and within a temperature range of 60-70 °C. Computer-assisted 3D structure modeling indicated that the active residues in the active site of the recombinant cellulases were more similar to each other compared with non-active site residues. The recombinant cel7482 was extremely tolerant to 2 M NaCl, suggesting that cel7482 may be a halotolerant cellulase. Moreover, the recombinant cel7482 was shown to have an ability to resist three ionic liquids (ILs), which are widely used for cellulose pretreatment. Furthermore, active cel7482 was secreted by the twin-arginine translocation (Tat) pathway of Bacillus subtilis 168 into the culture medium, which facilitates the subsequent purification and reduces the formation of inclusion body in the context of overexpression.ConclusionsThis study demonstrated a simple and efficient method for direct cloning of new cellulase genes from environmental metagenomes. In the future, the metagenomics-guided strategy may be applied to the high-throughput screening of new cellulases from environmental metagenomes.

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Metagenomics-Guided Discovery of Potential Bacterial Metallothionein Genes from the Soil Microbiome That Confer Cu and/or Cd Resistance.

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