Evolution and enrichment of CYP5035 in Polyporales: functionality of an understudied P450 family

Abstract Bioprospecting for innovative basidiomycete cytochrome P450 enzymes (P450s) is highly desirable due to the fungi’s enormous enzymatic repertoire and outstanding ability to degrade lignin and detoxify various xenobiotics. While fungal metagenomics is progressing rapidly, the biocatalytic potential of the majority of these annotated P450 sequences usually remains concealed, although functional profiling identified several P450 families with versatile substrate scopes towards various natural products. Functional knowledge about the CYP5035 family, for example, is largely insufficient. In this study, the families of the putative P450 sequences of the four white-rot fungi Polyporus arcularius, Polyporus brumalis, Polyporus squamosus and Lentinus tigrinus were assigned, and the CYPomes revealed an unusual enrichment of CYP5035, CYP5136 and CYP5150. By computational analysis of the phylogeny of the former two P450 families, the evolution of their enrichment could be traced back to the Ganoderma macrofungus, indicating their evolutionary benefit. In order to address the knowledge gap on CYP5035 functionality, a representative subgroup of this P450 family of P. arcularius was expressed and screened against a test set of substrates. Thereby, the multifunctional enzyme CYP5035S7 converting several plant natural product classes was discovered. Aligning CYP5035S7 to 102,000 putative P450 sequences of 36 fungal species from Joint Genome Institute-provided genomes located hundreds of further CYP5035 family members, which subfamilies were classified if possible. Exemplified by these specific enzyme analyses, this study gives valuable hints for future bioprospecting of such xenobiotic-detoxifying P450s and for the identification of their biocatalytic potential. Graphical abstract Key points • The P450 families CYP5035 and CYP5136 are unusually enriched in P. arcularius. • Functional screening shows CYP5035 assisting in the fungal detoxification mechanism. • Some Polyporales encompass an unusually large repertoire of detoxification P450s. Supplementary Information The online version contains supplementary material available at 10.1007/s00253-021-11444-2.

CYP512 (purple), CYP5035 (dark yellow), CYP5136 (violet), CYP5141 (brown), CYP5144 (orange), CYP5150 (green) and CYP5359 (blue) have been coloured. The P450 nomenclature of the CYP5035 selected and expressed for a functional screening is shown in red. The tree was constructed using the close-neighbour-interchange algorithm in MEGA X.

Fig. S12:
A minimum evolution tree of the P450ome of P. squamosus involving 184 amino acid sequences. CYP512 (purple), CYP5035 (dark yellow), CYP5136 (violet), CYP5141 (brown), CYP5144 (orange), CYP5150 (green) and CYP5359 (blue) have been coloured. The P450 nomenclature of the CYP5035 selected and expressed for a functional screening is shown in red. The tree was constructed using the close-neighbour-interchange algorithm in MEGA X.

Fig. S13:
A minimum evolution tree of the P450ome of L. tigrinus involving 184 amino acid sequences. CYP512 (purple), CYP5035 (dark yellow), CYP5136 (violet), CYP5141 (brown), CYP5144 (orange), CYP5150 (green) and CYP5359 (blue) have been coloured. The P450 nomenclature of the CYP5035 selected and expressed for a functional screening is shown in red. The tree was constructed using the close-neighbour-interchange algorithm in MEGA X.

Fig. S14:
Displayed is a minimum evolution tree of the CYP5035 and CYP5136 families of P. arcularius (Parc; red) and P. brumalis (Pbru; blue). Evidence for their close phylogeny is an alternating pattern of red and blue sequences almost throughout the tree. This analysis involved 67 amino acid sequences. The tree was constructed using the close-neighbour-interchange algorithm in MEGA X.

Fig. S15:
Displayed is a minimum evolution tree of the CYP5035 family involving 174 amino acid sequences. Phylogeny of CYP5035 enzymes of the fungus P. arcularius and P. brumalis (Parc and Pbru; red) compared to related species L. tigrinus and P. squamosus (Ltig and Psqu; violet), and the other model white-rot fungi Ganoderma sp. and G. sinense (Gsp and Gsin; blue), T. versicolor (Tver; green), P. chrysosporium and P. carnosa (Pchr and Pcar; black), B. adusta and P. brevispora and Heterobasidion irregulare (Badu and Pbre and Hirr; orange) as well as brown-rot fungi P. placenta and Serpula lacrymans (Ppla and Slac; brown) in order to get an insight into the evolution of this P450 family. The tree was constructed using the close-neighbour-interchange algorithm in MEGA X.

Fig. S16:
Displayed is a minimum evolution tree of the CYP5136 family involving 92 amino acid sequences. Phylogeny of CYP5136 enzymes of the fungus P. arcularius and P. brumalis (Parc and Pbru; red) compared to related species L. tigrinus and P. squamosus (Ltig and Psqu; violet), and the other model white-rot fungi Ganoderma sp. and G. sinense (Gsp and Gsin; blue), T. versicolor (Tver; green), P. chrysosporium and P. carnosa (Pchr and Pcar; black), B. adusta and P. brevispora and H. irregulare (Badu and Pbre and Hirr; orange) as well as brown-rot fungus Serpula lacrymans (Slac; brown) in order to get an insight into the evolution of this P450 family. The tree was constructed using the close-neighbourinterchange algorithm in MEGA X.   TCTATCCGCGCGCACACCGCGCTGCTCCTCGGCCCTCCTAGTCTTGCATTGGGATTCCTCGGCTCCACCGGTTCGAG  TTCACGAAGTGTCCTCCATACGCTGCCCCTCGCGTATGCTGCATACGTCGGCGCTTTAACAGTCTACACTATTCTC  TACCGCATATCGCCCTTTCACCCACTCGCTCAGTATCCCGGTCCGCTGGGATGCAAGGTATCGCAGTGGTGGATGG  CATGCAAATCCTGGTCTGGATACGAACACCTGTACATCAGCGAGCTGCACCGCAAGTACGGGGACGTTGTCCGCAT  CGGCCCAAATGAGCTCTCGATCCGGGACGCGTCTGCGATCAGTCCTATCATGAGCATTGCAAAAGGCCCGCAGTAT  GTCGGACGCATGCTCAGCGATGGGATTCACTTGCCTATAATTGGGATTCAAGACCCTGCCGAACATCTGCGCCGCC  GCCGTCCATGGAACCGCGCGTTTACAGTGCCGGCGTTGAGAGGGTACGAGGAGACGATAGCGCGGAGAGCGCGGC  AGCTGGTCGATGCACTCGAGCGTCATAACGGAGGACAGGAAGAGGTCATACTTGGGAAATGGTTCAATGACTTTG  CCTATGACTTCATGTGTGACATGGCGTTTGGCGGTGGTTCTGAGCTACTGCAGGAACGAGACGACAGTAACGTTT  GGCGGGTCCTGGATGAGGGCATGAAAGTTGGCACGCTTCTCGCACATGTCCCCTGGCTAGGGGTGTATCTGAGCC  ACGTCCCGCTTGCCACCGGGGCACTGGACGTTCTCATCTCGCACTGCCGCATGCTCACCACCCAGCGGGTCCAGCG  GGGCGCTACGCAGAAAGACTTGTTCCATTACCTGAACGACGAAGACCTCGCAAATTCCTCCGAGAAGCCTCGGGC  ACAGCCTCCACTGCGTCAACTCACGGACGACGGCTGCCTCATCGTTGTCGCCGGCGCGGACACCACGTCAAGCGCA  CTTACTAGCCTGTTCTACTGCCTGTTGACGAACCCGGAGACATACAGACGGCTGCAAGACGAGGTGGACAAGTTC  TATCCGCGTGGAGAAGACGCTTGCGACACGAGATACCATCGCGAGATGCGCTGGCTGAACGCGGTAATATGCGAG