Functional analysis of environmental DNA-derived
type II polyketide synthases reveals structurally
diverse secondary metabolites
Zhiyang Fenga, Dimitris Kallifidasa, and Sean F. Bradya,b,1
aLaboratory of Genetically Encoded Small Molecules, and
bHoward Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York,
Edited by Jerrold Meinwald, Cornell University, Ithaca, NY, and approved June 14, 2011 (received for review March 14, 2011)
A single gram of soil is predicted to contain thousands of unique
bacterial species. The majority of these species remain recalcitrant
to standard culture methods, prohibiting their use as sources of un-
ique bioactive small molecules. The cloning and analysis of DNA
extracted directly from environmental samples (environmental
DNA, eDNA) provides a means of exploring the biosynthetic capa-
city of natural bacterial populations. Environmental DNA libraries
contain large reservoirs of bacterial genetic diversity from which
new secondary metabolite gene clusters can be systematically re-
covered and studied. The identification and heterologous expres-
sion of type II polyketide synthase-containing eDNA clones is
reported here. Functional analysis of three soil DNA-derived poly-
ketide synthase systems in Streptomyces albus revealed diverse
metabolites belonging to well-known, rare, and previously unchar-
acterized structural families. The first of these systems is predicted
to encode the production of the known antibiotic landomycin E.
The second was found to encode the production of a metabolite
with a previously uncharacterized pentacyclic ring system. The
third was found to encode the production of unique KB-3346-5
derivatives, which show activity against methicillin-resistant
Staphylococcus aureus and vancomycin-resistant Enterococcus fae-
calis. These results, together with those of other small-molecule-
directed metagenomic studies, suggest that culture-independent
approaches are capable of accessing biosynthetic diversity that has
not yet been extensively explored using culture-based methods.
The large-scale functional screening of eDNA clones should be a
productive strategy for generating structurally previously unchar-
acterized chemical entities for use in future drug development
therapeutics and the continued need for new antimicrobials
and chemotherapeutics, large screening programs have deem-
phasized the use of microbial extracts over the past two decades.
The reason most frequently cited for this decline is the persistent
rediscovery of known metabolites (1–3). Most environmental
bacteria remain recalcitrant to standard culture methods (4–6),
and the difficulties associated with growing these organisms
prohibit their use as new sources of bioactive small molecules.
Although it is not yet possible to easily culture the majority of
environmental bacteria, it is possible to extract microbial DNA
directly from environmental samples (environmental DNA,
eDNA) and to clone this DNA into cultured bacteria where it
can be functionally characterized. This general approach has
been termed metagenomics (7). The application of metage-
nomics to the study of bacterial secondary metabolism is particu-
larly appealing in light of the fact that the genes required for the
biosynthesis of a natural product are typically clustered on a bac-
terial chromosome. The heterologous expression of natural pro-
duct gene clusters captured on individual clones or on small
numbers of overlapping clones should provide a means of obtain-
ing previously unidentified bioactive small molecules.
espite the historical success of bacterial natural products
as lead structures for the development of small molecule
A structurally diverse collection of aromatic metabolites,
including many important antimicrobials and anticancer agents,
arise from iterative (type II, aromatic) polyketide synthases
(PKSs) (8). Although the gene clusters that encode the biosynth-
esis of these diverse metabolites can differ substantially in gene
content, they all encode a conserved minimal PKS composed of
three proteins: ketosynthase alpha, KSα; ketosynthase beta/chain
length factor, KSβ; and acyl carrier protein, ACP. The minimal
PKS is responsible for the iterative condensation of malonyl-
CoAs into a nascent polyketide chain that is then cyclized,
aromatized, reduced, oxidized, rearranged, and functionalized
in pathway-specific ways to generate the extraordinary structural
diversity that is known to arise from these systems (8, 9). PCR-
based studies as well as shotgun-sequencing efforts indicate that
eDNA samples are rich in unique minimal PKS genes (10–13).
Through the functional characterization of eDNA-derived type
II PKS containing clones, we have identified PKS systems that
encode structurally diverse metabolites including compounds
with unique and rare carbon skeletons. Among the metabolites
we identified are compounds that show activity against both
methicillin-resistant Staphylococcus aureus (MRSA) and vanco-
mycin-resistant (vanA) Enterococcus faecalis (VRE). These stu-
dies suggest that large-scale heterologous expression of eDNA
clones containing diverse KSβgenes will be a productive strategy
for producing previously unidentified bioactive metabolites that
can be used in future drug discovery efforts.
Results and Discussion
Two previously archived eDNA cosmid-based libraries, con-
structed using DNA isolated from arid soils collected in Utah
(UT) and Arizona (AZ), were screened for clones containing
type II PKS systems. To identify type II PKS gene clusters in these
libraries, cosmid DNA isolated from each library was used as the
template in PCR reactions with degenerate primers designed to
amplify full-length KSβgenes (10, 14–16). Amplicons of the cor-
rect predicted size (1.5 kb) were gel purified, sequenced, and
compared to deposited KSβgenes from cultured bacteria (Fig. 1).
Unique KSβgenes were used as probes to recover type II PKS
containing clones from the archived libraries. Recovered cosmids
were then retrofitted with the genetic elements required for con-
jugation and site-specific integration into Streptomyces spp. and
Author contributions: Z.F., D.K., and S.F.B. designed research, performed research,
analyzed data, and wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The X-ray data and structure tables for compound 2 have been deposited
in the Cambridge Structural Database (CSD), Cambridge Crystallographic Data Centre,
Cambridge CB2 1EZ, United Kingdom (CSD reference no. 805477). The sequences reported
in this paper have been deposited in the GenBank database [accession nos. HQ828985
(cosAZ154), HQ828986 (BAC:UT-X26/F129), and HQ828984 (cosAZ97)].
1To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/
www.pnas.org/cgi/doi/10.1073/pnas.1103921108PNAS ∣ August 2, 2011 ∣ vol. 108 ∣ no. 31 ∣ 12629–12634
ACKNOWLEDGMENTS. We thank Emil Lobkovsky from the Cornell University
X-ray crystallography facility. We thank Dr. John D. Bauer for his assistance
with library construction and clone recovery. We thank Ken, Beth, and
Mikaela Morris for their assistance with sample collection. S.F.B. is a Howard
Hughes Medical Institute Early Career Scientist. This work was supported by
National Institutes of Health Grants GM077516 and U54-AI057158.
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