放线菌模块型聚酮合酶的系统发育组学分析及其在聚酮类化合物筛选中的应用

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

【目的】通过分析模块型聚酮合酶(polyketide synthase,PKS)的系统进化关系,阐明酮基合成酶(ketosynthase,KS)和酰基转移酶(acyltransferase,AT)序列与聚酮产物之间的关系,为放线菌天然产物的筛选提供指导。【方法】从PKSDB数据库的20个模块型PKS基因簇中调取所有KS(190个)和AT(195个)氨基酸序列,利用MEGA 4.0软件分别构建KS、AT、KS+AT 3种序列模式的系统发育树,并计算KS序列的簇内和簇间平均进化距离。设计了一对KS结构域的引物,通过PCR方法对20株活性放线菌分离菌株进行了筛选,测定了阳性菌株的KS序列,和已知的相关KS序列构建系统发育树,并对阳性菌株进行了发酵培养和代谢产物分析。【结果】放线菌来源的同一PKS的KS序列倾向于聚成一个进化枝,且按照其产物结构聚类;同一PKS的KS簇内平均进化距离小于0.300,不同PKS的KS簇间平均进化距离一般大于0.300。AT系统发育树按照其底物特异性聚成两个大的分枝;同一PKS的部分AT分别处于两个分枝,其余AT散在分布。KS+AT系统发育树则综合了KS树和AT树的拓扑结构特点。获得13株KS阳性分离菌株,它们的多数KS序列按照菌株分别聚类,其中4株菌的大部分KS各自聚成独特的簇,5株菌的大部分KS分别处在已知PKS进化枝内。从3株阳性菌中分离到预期的聚酮类产物。【结论】放线菌中KS的进化方式以垂直进化为主,而AT则以水平进化为主;KS序列与产物结构相关,且KS簇间平均进化距离可作为不同PKS的判定标准;相对于AT和KS+AT,KS系统发育组学分析更适用于指导放线菌聚酮类产物的筛选。

[ 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 acyhransferase （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 elades, 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.