screening and genome mining of polyether-producing strains in actinomycetes
DESCRIPTION
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. - PowerPoint PPT PresentationTRANSCRIPT
Screening and genome mining of polyether-producing strains in actinomycetes
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
INTRODUCTIONINTRODUCTION
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 cancer stem cells has led to a revived interest in the useful biological activity, highly complex structure and intriguing biosynthetic mechanisms of 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-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)
ACKNOWLEDGEMENTSACKNOWLEDGEMENTS
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.
Correspondence: [email protected]
RESULTSRESULTS
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.
DISCUSSIONSDISCUSSIONS
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.
METHODSMETHODS 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.
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.
PKS modulePKS module
PKS module PKS module
Streptomyces lasaliensis JCM 3373
Streptomyces FXJ1.172
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.
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FIG. 4. HPLC comparative metabolic profiling of wild type and epo deletion mutant strain FXJ1.172.
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FIG. 5. High resolution mass spectrometry of NC-1.
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FIG. 6. Reassembled NRPS gene cluster and its domain annotation.
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FIG. 7. HPLC metabolic profiling of strain FXJ1.172 after adding different aromatic amino acids.