Transcript Minghao Liu

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
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 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.
 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 polyether 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.
 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 ionophoreproducing strains from our isolates as well as their
genome mining for other new natural products.
METHODS
PCR-based
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.
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
PKS module
 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) .
PKS module
Streptomyces lasaliensis JCM 3373
Polyether biosynthetic gene clusters were cloned by Fosmid
library screening combined with genome sequencing and then
identified by gene inactivation combined with product analysis.
PKS module PKS module
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：
 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).
Streptomyces FXJ1.172
FIG. 3. Comparison of two lasalocid gene clusters by Mauve.
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-Prediction of physicochemical properties
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-Substrate inducing and temperature shifting
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-Comparative metabolic profiling (Gene inactivation)
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DISCUSSIONS
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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.
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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.
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.
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ACKNOWLEDGEMENTS
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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.
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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.
Correspondence: [email protected]