Phylogenomic analysis of modular polyketide synthases in actinomycetes and its application in product screening

Abstract:

Abstract: [Objective] To elucidate phylogenetic relationships of modular polyketide synthases (PKS) in actinomycetes, and to provide a guide for screening and discovery of polyketides. [Methods] We retrieved amino acid sequences of 190 ketosynthases (KS) and 195 acyltransferase (AT) of 20 modular polyketide synthases from database PKSDB, and constructed Neighbor-Joining trees based on amino acid sequences of KS, AT and KS+AT respectively using MEGA 4.0. We computed the mean distances within and between groups of KSs. We designed a pair of degenerate primers based on two conserved regions of KS, and screened 20 bioactive isolates by PCR. After sequencing the KS genes of positive strains, we constructed a Neighbor-joining tree of the 89 KSs identified in this study with the 160 known ones. We also fermented 13 KS-positive isolates and carried out chemical analysis of the fermentation extracts. [Results] KSs from the same PKSs of actinomycetes tended to group respectively into clades, and KSs responsible for synthesis of products with similar structures tended to cluster together. The mean distances within groups of KSs from each PKS pathway were less than 0.300, and the mean distances between groups of KSs from different PKS pathways were generally more than 0.300. All the ATs grouped into two big clades according to the substrate specificity. Some ATs from the same pathway were grouped within the two big clades respectively, and the rest were scattered. The tree of KS+AT integrated the topological structures of both KS tree and AT tree. The majority KSs from individual isolates tended to group into clades. Most KSs from four of the isolates grouped into four distinct clades, and most KSs from another five isolates respectively fell into four clades of known pathways. We isolated and identified three predicted compounds as expected. [Conclusion] Vertical evolution is likely to be the dominant evolutionary mode of KS genes in actinomycetes, while AT genes may have evolved mainly through horizontal gene transfer. Considering that KSs cluster according to the corresponding product structures, and that the mean distances 0.300 between groups of KSs could sever as an evaluation criteria for different PKS pathways, the phylogenomic analysis of KS is more suitable than that of AT or KS+AT for guiding the screening of polyketide compouds from actinomycetes.