Screening and genome mining of polyether-producing strains in actinomycetes

1. FIG. 1. Neighbour-joining tree of 44 putative polyether epoxidases and five known polyether epoxidases. FIG. 2. Neighbour-joining tree of 16S rRNA genes from polyether epoxidase gene-positive strains. Phe, aqueous phase Tyr, aqueous phase lasalocid aqueous phase NC-1 PKS module PKS module Streptomyceslasaliensis JCM 3373 Screening and genome mining of polyether-producing strains in actinomycetes Trp, aqueous phase Trp, lipid phase PKS module PKS module 6 Minghao Liu, Hao Wang, Ning Liu, Jisheng Ruan and Ying Huang* State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China none 2 Streptomyces FXJ1.172 1 5 3 4 RESULTS INTRODUCTION • Polyether ionophores are a unique class of polyketides with broad-spectrum activity which have been successfully used in veterinary medicine and in animal husbandry. Recent research on their outstanding potency for the control of drug-resistant bacteria and cancerstem cells has led to a revived interest in the useful biological activity, highlycomplex structure and intriguing biosynthetic mechanismsof these compounds for further application purposes. • Interestingly, we found polyether producers may have great secondary metabolism potentials to be unravelled based on genome sequencing. Here we described our recent progress on screening for polyether ionophore-producing strains from our isolates as well as their genome mining for other new natural products. • PCR screening of 1068 actinomycetes revealed that isolates from acidic soils collected in Jiangxi Province could be a good source of polyether producers, for their higher occurrences of putative poly-ether epoxidase genes with greater sequence diversity and novelty than those from other habitats (Figs. 1 and 2). • These results spur genome sequencing of some representative strains from the acidic soils, e.g. FXJ1.172 which produces lasalocid and FXJ1.264 which produces a new etheromycin analogue. METHODS • PCR-based screening methods • Primers were designed for the epoxidase gene which is conserved and critical in all of the five so far published complete gene clusters of polyether ionophores. • Cloning of polyether biosynthetic gene clusters • Polyether biosynthetic gene clusters were cloned by Fosmid library screening combined with genome sequencing and then identified by gene inactivation combined with product analysis. • Genome mining for natural product • Genome was sequenced using Roche 454 GS FLX and then accessed its secondary metabolism potential by antiSMASH (http://antismash.secondarymetabolites.org/). • Multiple methods were applied to activate these cryptic gene clusters for corresponding natural products： • -Prediction of physicochemical properties • -Substrate inducing and temperature shifting • -Comparative metabolic profiling (Gene inactivation) • The size of FXJ.172’s genome was approximately 9Mb with 37 predicted biosynthetic gene clusters including one for lasalocid. Comparing the lasalocid biosynthetic gene cluster in FXJ1.172 and the reported one in S. lasaliensis JCM 3373, a great rearrangement of PKS modules was discovered while no significant difference was detected between their corresponding products (Fig. 3) . • Production of lasalocid in FXJ1.172 was successfully abolished by deletion of the epoxidase-encoding gene and intriguingly a entirely distinct compound named NC-1 (structure under elucidation) was isolated instead (Figs. 4 and 5). FIG. 3. Comparison of two lasalocid gene clusters by Mauve. lipid phase ACKNOWLEDGEMENTS lipid phase aqueous phase This study was supported by the Natural Science Foundation of China (NSFC; no. 31170010) and by the Specialized Research Fund for the State Key Laboratories of China. FIG. 4. HPLC comparative metabolic profiling of wild type and epo deletion mutant strain FXJ1.172. FIG. 5. High resolution mass spectrometry of NC-1. • A potential NRPS gene cluster was reassembled in FXJ1.172 during genome comparison and annotation, and it shows a demand of aromatic amino acid for its biosynthesis by substrate specificity prediction of its A domains (Fig. 6). Different aromatic amino acids were added to a basic medium for FXJ1.172 fermentation, and the metabolic profile was greatly changed when adding tryptophan (Fig. 7). Several new peaks were screened through HPLC and were under elucidation. DISCUSSIONS • Our study indicates that a strong correlation does exist between polyether epoxidases and polyether ionophores and thus establishes a feasible genetic screening strategy that is useful for the rapid identification of known and the discovery of unknown polyether products in actinomycetes. • Genome sequencing reveals that the polyether producing strains harbor many cryptic gene clusters that are of great potential productivity for new natural products. And a combination of different genome mining methods can efficiently help us to exploit these natural treasures. FIG. 6. Reassembled NRPS gene cluster and its domain annotation. FIG. 7. HPLC metabolic profiling of strain FXJ1.172 after adding different aromatic amino acids. Correspondence: huangy@im.ac.cn