Comparative genomics of fungal mutants provides a systemic view of extreme cadmium tolerance in eukaryotic microbes

Whether eukaryotic organisms can evolve for higher heavy metal resistance in laboratory conditions remains unknown. In this study, we challenged a macrofungi, Pleurotus ostreatus, in a designed microbial evolution and growth arena (MEGA)-plate with an extreme Cd gradient. Within months, the wild-type strain developed 10 mutants, exhibiting a maximum three-fold increase in Cd tolerance and slower growth rates. Genomic sequencing and re-sequencing of the wild-type and ten mutant strains generated about 51 GB data, allowing a comprehensive comparative genomics analysis. As a result, a total of 2512 common single nucleotide polymorphisms, 70 inserts and deletes, 39 copy number variations and 21 structural variations were found in the 10 mutants. The mutant genes were primarily involved in substrate transport. In combination with transcriptome analysis, we discovered that the ten mutants had a distinct Cd-resistant mechanism compared to the wild-type strain. Genes involved in oxidation-reduction, ion transmembrane transport, and metal compartment/efflux are primarily responsible for the extreme Cd tolerance in the P. ostreatus mutants. Our findings contribute to the understanding of eukaryotic Cd resistance at the genome level and establish a foundation for developing bioremediation tools utilizing highly tolerant macrofungi. Macrofungi have gained significant interest in bioremediation due to their impressive benefits, particularly at low metal concentrations. However, enhancing their metal resistance remains a challenge. In this study, we successfully developed Pleurotus ostreatus mutants with extreme metal tolerance using Adaptive Laboratory Evolution (ALE). Additionally, we inferred the potential genetic mechanisms underlying the extreme cadmium tolerance of the evolved cells from a forward genetics perspective, based on a comparative genomics study. This study demonstrates for the first time the possibility of developing macrofungal species with extreme metal tolerance through ALE, and provides insights into the systemic cellular resistance of eukaryotes to cadmium.