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Genome-based bioprospecting of microbes for new therapeutics
Sergey B Zotchev1, Olga N Sekurova1 and Leonard Katz2
Bioprospecting of natural sources for new medicines has a long
and successful history, exemplified by the fact that over 50% of
all drugs currently on the market are either derived from or
inspired by natural products. However, development of new
natural product-based therapeutics has been on the decline
over the past 20 years, mainly owing to frequent re-discovery of
already known compounds coupled with high costs for
screening, characterization and development. With the onset of
the genomic era allowing rapid sequencing and analysis of
bacterial and fungal genomes, it became evident that these
organisms possess ‘hidden treasures’ in the form of gene
clusters potentially governing biosynthesis of novel biologically
active compounds. This review highlights current progress in
mining for and expression of these gene clusters, which may
revolutionize the drug discovery pipelines in the near future.
Addresses
1
Department of Biotechnology, Norwegian University of Science and
Technology, Trondeim, Norway
2
Synthetic Biology Engineering Research Center, University of
California-Berkeley, Berkeley, USA
Corresponding author: Zotchev, Sergey B ([email protected])
Current Opinion in Biotechnology 2012, 23:941–947
This review comes from a themed issue on Pharmaceutical
biotechnology
Edited by Francis E Nano and José F Rodrı́guez
For a complete overview see the Issue and the Editorial
Available online 3rd May 2012
0958-1669/$ – see front matter, # 2012 Elsevier Ltd. All rights
reserved.
http://dx.doi.org/10.1016/j.copbio.2012.04.002
Microbial genomes and secondary metabolite
biosynthesis gene clusters
Drug discovery from natural products has for a long time
been dependent on the high-throughput screening of
extracts prepared from microorganisms, plants and higher
organisms. The former, in particular bacteria of the order
Actinomycetales, and filamentous fungi, proved to be extremely rich sources of new antibiotics, anti-cancer therapeutics, cholesterol-lowering drugs, and so on. Over the
years, however, the discovery of novel compounds from
these sources dropped dramatically, mostly because of the
frequent isolation of the already known compounds. This
tendency made some to believe that microorganisms as
sources for new therapeutics have been exhausted. This
view has been challenged with the onset of genomics era,
when rapid sequencing of genes and genomes became a
routine. Genome sequencing and analysis of actinomycete
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bacterium Streptomyces coelicolor A3(2) published in 2002
[1] revealed unprecedented potential of this microbe to
synthesize compounds previously undetected by conventional methods of cultivation, extraction, and bioactivity
testing. Apparently, genes encoding biosynthesis of these
compounds were expressed at a very low level (or not at all)
in laboratory conditions, making the detection extremely
difficult. Soon after, scientists from Ecopia (now part of
Thallion Pharmaceuticals Inc.) published their work on
genome-guided discovery of novel compounds based on
genomic snapshots allowing detection of antibiotic biosynthesis genes. This has been followed by manipulation
of media and growth conditions leading to expression of
cryptic gene clusters, and production of potentially novel
enediynes [2]. Recent advances in DNA sequencing
made the ‘genomic snapshot’ approach obsolete, providing
almost complete genome sequences available for mining
of genes of interest in a matter of weeks. The task of
mining remained, however, quite laborious, until the development of a software antiSMASH [3], that allows to
efficiently detect secondary metabolite gene clusters in
the genomes of bacteria and fungi. antiSMASH is also able
to partially predict types of compounds that can be produced if the gene cluster is fully functional. Since the
secondary metabolite gene clusters are abundant, especially in the genomes of actinomycete bacteria, some kind of
prioritization will be required in order to minimize rediscovery, and to focus on the potentially novel and
promising compounds. The latter can be assisted by an
array of the pathway analysis and prediction tools recently
reviewed by Medema et al. [4]. Gene cluster and pathway
analyses also provide important information that can guide
researchers in the identification and isolation of compounds specified by particular gene clusters. For example,
Kersten et al. [5] have recently described a method of
peptidogenomics, where structures of peptides predicted
to be synthesized via both ribosomal and non-ribosomal
pathways are correlated with mass spectrometry of cell
extracts. This method allows rapid identification of new
peptide-based natural products, simultaneously linking
them to corresponding gene clusters that can be used
for boosting the compounds’ yields, as described in the
section below.
Activation and heterologous expression of
secondary metabolite biosynthesis genes
As mentioned above, low abundance of natural products
often prevents them from being discovered during conventional screening. Furthermore, even if a compound is
detected, low yield and unstable production may render
attempts at purification, structure elucidation and comprehensive biological testing extremely inefficient and
Current Opinion in Biotechnology 2012, 23:941–947
942 Pharmaceutical biotechnology
time consuming. A number of genomic-based techniques
are currently being employed to increase titers. One is
based on the fact that secondary metabolite gene clusters
almost always contain regulatory genes, which control
expression of structural biosynthetic genes. These pathway-specific regulators can be manipulated in order to
increase the production of cluster-specified compound
through, for example, inactivation of a repressor, or overexpression of a positive regulator. Using the repressor
deletion approach, Gottelt et al. [6] succeeded in expression of a silent gene cluster in S. coelicolor governing
biosynthesis of a previously undetected compound with
antibacterial activity. Overexpression of a transcriptional
activator from an orphan gene cluster in Streptomyces
ambofaciens resulted in production of a novel macrolide
with antitumor activity [7]. Tandem amplification of a
biosynthesis gene cluster also led to increased production
levels of an antibiotic. Murakami et al. [8] introduced
the oriT-like recombination sites RsA and RsB from
Streptomyces kanamyceticus into the genome of S. coelicolor
to flank the gene cluster responsible for biosynthesis of
the polyketide actinorhodin, as well as the gene zouA that
encodes a TraA-like relaxase and demonstrated a 20-fold
increase in the titers of actinorhodin resulting from tandem amplification of the act genes.
These approaches can only be attempted if the producing
host is genetically tractable. Although methods for
genetic manipulation have been developed for several
strains, many actinomycetes have proven to be refractory
to the introduction of heterologous DNA, or have exhibited resistance to all usable drugs for genetic selection.
Hence, an alternative approach to detection of potentially
novel products is through the heterologous expression of
the corresponding gene clusters. While many different
microbial hosts have been used for expression of heterologous genes, E. coli, the yeast Saccharomyces cerevisiae,
and several Streptomyces species have been shown to
Figure 1
Microbial
isolate
DNA
Bioinformatics
Tools development
Genome reduction
Host
Precursors pathways
engineering
Superhost
Product
Gene clusters
No product
BstXI
BamHI
EcoRI
BstXI
Nhel
AmR
Promoter exchange
Regulators
BamHI
attP
int
Testing
BstXI
EcoRI
Apol
Re-factoring
Re-design and
complete synthesis
BstXI
BamHI
pCL9-Phz2
BamHI
BstXI
Recombinant
plasmids
Current Opinion in Biotechnology
Overall tentative scheme for genome-based bioprospecting.
Current Opinion in Biotechnology 2012, 23:941–947
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Genome-based bioprospecting Zotchev, Sekurova and Katz 943
express large clusters leading to the production of a small
molecule secondary metabolite at reasonable initial titers.
E. coli has the advantage of the availability of numerous
vectors (plasmids, cosmids, BACs) and promoters for
heterologous expression, and can express genes with
G + C codon bias as high as 73% [9], but does not
recognize promoters from Streptomyces. In addition, some
precursors required for the biosynthesis of secondary
metabolites (e.g. methylmalonyl-CoA for the biosynthesis of many polyketides) are not produced in E. coli.
Finally, it is known that type I polyketide synthase
proteins from Streptomyces do not always fold correctly
in E. coli [10]. S. cerevisiae has been used to express a
heterologous polyketide synthase gene from a fungus to
produce the polyketide 6-methylsalicylic acid [11] and is
also being developed for expression of other heterologous
genes. Employing a series of E. coli vectors carrying yeast
promoters for the initial cloning of selected genes from
random sources and a YAC system for the subsequent
assembly and expression of the genes in yeast, Naesby
et al. [12] demonstrated production of several flavonoids
in S. cerevisiae (Figure 1).
Streptomyces have a long history of use as hosts for the
production of secondary metabolites from heterologous
sources, dating to 1984 when a strain of Streptomyces
parvulus produced the polyketide actinorhodin after
the cloning of the corresponding gene cluster from the
producing host S. coelicolor [13]. In 2010 Baltz reviewed
the heterologous production of 17 secondary metabolites
of differing classes (polyketides, aminoglycosides, glycopeptides, etc.) from different actinomycete sources in 14
Streptomyces hosts, representing variants of 7 different
species [14]. Additional reports have appeared describing
heterologous gene expression in Streptomyces since then.
Recent efforts have focused on improving streptomycete
hosts for production of heterologous products by deleting
genes encoding the biosynthesis of native secondary
metabolites. Gomez-Escribano and Bibb deleted four
gene clusters encoding the biosynthesis of actinorhodin,
prodiginine, CPK and CDA from the S. coelicolor M145
chromosome and introduced mutations into rpoB, and
demonstrated that the ‘improved’ host exhibited significant increase in the titer of heterologously produced
chloramphenicol and congocidine compared to the
parental strain after the respective biosynthesis clusters
were introduced into the two hosts [15]. Komatsu et al.
deleted 1.4 MB of non-essential DNA, representing ca.
18% of the genome, from the ends of the Streptomyces
avermitilis linear chromosome and demonstrated an
absence of secondary metabolite production from this
strain [16]. When used as a host for heterologous gene
expression, they showed substantial improvement in the
titers of cephamycin and streptomycin over those produced in the parental strain. In general, Streptomyces
vectors and promoters exist for the expression of heterologous DNA on autonomously replicating high and low
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copy number plasmids or in the chromosome after sitespecific integration in a variety of phage or plasmid
attachment sites in many Streptomyces hosts. In addition,
a number of selective markers as well as methods to
introduce DNA, including electroporation or conjugation
from E. coli have been developed for a large number of
Streptomyces hosts.
Synthetic biology approaches to heterologous
expression of new clusters
Gene clusters identified that are thought to produce novel
compounds can be cloned directly into any of the hosts
mentioned above, but it is not likely that they will be
expressed in E. coli or S. cerevisiae without major genetic
engineering efforts. If the source of the cluster is Streptomyces, it is possible that the incoming genes will be
expressed and compound will be detected, particularly
if an improved or minimized host is employed. There is
also a greater likelihood that expression (and compound
detection) will be achieved if the host producing strain is a
streptomycete, in particular one of the strains described in
the preceding section. However, in cases when gene
clusters originate from bacteria phylogenetically distinct
from streptomycetes, a considerable effort may be
required to achieve their expression and production of
compounds they specify. In a recent review, Medema
et al. suggested a way how such clusters can be rebuilt, or
‘re-factored’, to achieve optimal performance [17].
Definitely, synthetic biology can and will be employed
to address the potential problems with codon usage,
promoter recognition by new hosts’ sigma factors, as well
as difficulties with translation that may arise from suboptimal binding of hosts’ ribosomes to heterologous
Shine–Dalgarno (SD) sequences on mRNAs [18]. As
for any other synthetic biology application, generation of
well-characterized ‘parts’ (i.e. promoters, SD sequences,
genes, terminators) and ‘devices’ (i.e. regulatory circuits
and defined gene sets, for example, for precursor biosynthesis) to be used for cluster re-factoring will be of pivotal
importance. Most of the currently available standardized
synthetic biology parts are designed for E. coli, and will
most probably not function properly in streptomycetes.
Another problem for the expression of heterologous gene
cluster in Streptomyces is the diversity among members of
this genus of bacteria. Different species of Streptomyces
will possess a variety of secondary metabolite gene clusters and co-evolved precursor biosynthetic pathways. As
well, the different species can have different sigma factors
and 16S rRNA sequences that affect transcription and
translation of foreign genes. All these factors are important for heterologous gene expression, leading to successful functional expression of a particular gene cluster in
one host but not the other [14]. Ideally, a single streptomycete host shall be developed for synthetic biologydriven and genome-based drug discovery, which would
have following features: (i) genetically tractable, with a
variety of vectors/markers available; (ii) all endogenous
Current Opinion in Biotechnology 2012, 23:941–947
Modern methods for DNA assembly.
Method
Principle
Advantages
Disadvantages
Applications examples
Reference
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SLIC (Sequence and
Ligase Independent
Cloning)
30 exonuclease and
polymerase activities of
T4 DNA polymerase
and ca. 25-bp homology
at the ends of DNA parts
- standardized
- scarless
- mostly sequence and
ligase-independent
- limitation in size of final
DNA molecule (max. 20 kb)
- strong secondary structures
at the ends of parts can interfere
with DNA assembly
Rapid method for creating of
recombinant plasmids, generating
multiprotein complex production
[21,22]
[21]
‘Gibson’ ligation
Isothermal one-pot reaction
based on activities of
T5 exonuclease, Phusion
DNA polymerase and
Taq ligase;
All parts contain ca.
25-bp homologous
flanking sequences
-
standardized
scarless
mostly sequence-independent
multi-part
one-pot isothermal reaction
big assemblies up to 300 kb
reported
- expensive enzyme mix
- parts smaller than 250bp
should be avoided
- strong secondary structures
at the ends of parts can
interfere with DNA assembly
Creation of combinatorial library of
promoter and gene cassettes for
pathway engineering [24]
[23]
CPEC (Circular
Polymerase
Extension Cloning)
Single PCR cycle without
primers, DNA parts contain
25-bp homologous ends,
reaction is catalyzed
by Phusion DNA
polymerase
-
standardized
scarless
largely sequence-independent
multi-part assembly is possible
single enzyme is needed
can be useful in cases of DNA
parts with strong secondary
structures owing to high
temperature of the reaction
- PCR-introduced mutations are
possible, especially for DNA
assemblies larger then 10-kb
- the method is still not ideal
for sequences with many repeats
or GC-rich DNA
Cloning of complex combinatorial
libraries and pathways [25]
[25]
Golden Gate
assembly
and variants
Use of restriction
enzymes (type IIS
endonucleases,
e.g. BsaI)
-
standardized
quasi-scarless
one-step restriction-ligation reaction
homology of DNA parts is not
necessary
- multi-part assembly is possible
- only partly sequence-independent,
as internal sites for restriction
enzymes should be avoided in
all parts for assembly
Generating libraries of
recombinant genes [27]
[26,27]
Bio BricksTM
assembly
and variants
Simultaneously
performed restriction
digest and
ligation reactions
- standardized
- cheap and simple
- kits are available
- scars in DNA sequence are
unavoidable
- laborious for multi-parts
assemblies
For large-scale assemblies by
mixing and matching parts from
Registry of Standard Biological
parts [29]
[28]
SLiCE (Seamless
Ligation
Cloning Extract)
Assembly of DNA
molecules in single
in vitro recombination
reaction using bacterial
RecA-deficient cell extracts,
l Red recombination
system enhances the
efficiency of cloning
-
Not tested for multi-part assembly
Not tested for high-GC DNA
assembly
Not tested for assembly of
large (>4 kb) parts
General cloning method for
creating recombinant plasmids
[30]
[30]
fast one-tube reaction
inexpensive
mostly sequence-independent
seamless
high fidelity
944 Pharmaceutical biotechnology
Current Opinion in Biotechnology 2012, 23:941–947
Table 1
[34]
Complete assembly of
chromosomes [35]
- useful for assembly of large
DNA molecules
DNA assembly in
Bacillus (domino
method)
In vivo DNA assembly
in B. subtilis using
specific integrative vectors
-
laborious
sequential
time-consuming
long homologous
overlaps for recombination
are required
[31]
- very accurate and efficient
- parallel multi-part assembly
- very large (bacterial
chromosome-size)
assemblies possible
TAR (TransformationAssisted
Recombination) and
modifications
In vivo assembly via
homologous recombination
in S. cerevisae
DNA parts must contain
homologous overlaps
of 25-40 bp
- laborious compared to the
in vitro methods
Assembly of entire synthetic
microbial genomes [32]
Pathway assembly [15,33]
Genome-based bioprospecting Zotchev, Sekurova and Katz 945
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(active) secondary metabolite biosynthesis gene clusters
deleted; (iii) engineered and controllable pathways for
biosynthesis of a variety of secondary metabolite precursors; (iv) engineered catabolic pathways allowing utilization of a variety of cheap nutrients; (v) re-designed
developmental program that is de-coupled from secondary metabolism and allowing rapid and significant
accumulation of biomass without the loss of productivity.
Once parts, devices and optimized hosts for heterologous
cluster expression become available, there still remains a
problem of efficient cloning of the clusters, which usually
span 20–150 kb of DNA. Construction of gene libraries,
screening them for specific clusters, and stitching clusters
together from parts scattered between different clones
has become obsolete. Methods for assembly of DNA –
genes, biosynthetic pathways and even entire genomes –
from small parts have been developed recently, and are
now important new tools for synthetic biology. These
techniques enable not only the reconstruction of natural
pathways but also allow the creation of new pathways
with predictable properties from individual parts and
devices. Modern techniques for DNA assembly could
be conditionally divided into methods using restriction
enzymes, such as Golden Gate, Bio Brick or more recently
BglBricks, as well as sequence-independent protocols,
such as Gibson isothermal assembly, SLIC and SPEC
(see Table 1 for references). In vivo DNA assembly in
yeast and Bacillus proved to be useful for bigger and more
complex (e.g. chromosome) assemblies. Successful
assembly of antibiotic biosynthesis gene clusters in yeast
with subsequent transfer to Streptomyces host and heterologous expression was recently demonstrated by Shao
et al. [15]. An overview of these methods is given in
Table 1. It can be envisaged, that using the aforementioned technologies, gene clusters may be both assembled
in a suitable vector, and re-assembled by combining the
original genes amplified from bacterial genomes with predefined parts that will ensure their functionality. DNA
synthesis represents an attractive alternative to cluster reassembly. Although still rather expensive, this approach
allows unprecedented freedom in terms of choosing hostoptimized codon usage and regulatory elements to provide for optimal performance. Bioinformatics tools for
gene design become increasingly available (e.g. GeneDesigner, https://www.dna20.com/genedesigner2/), and
will greatly assist in cluster re-factoring, ultimately leading to the discovery of novel bioactive compounds and
thus creating new opportunities for drug discovery.
Conclusion
New generation sequencing and innovative bioinformatics allowing rapid access to and analyses of microbial
genomes have provided a glimpse of ‘hidden treasures’,
manifested in biosynthetic genes for hereto undetected
and potentially valuable natural products. Most of these
genes, however, remain silent in laboratory conditions,
Current Opinion in Biotechnology 2012, 23:941–947
946 Pharmaceutical biotechnology
and must be heterologously expressed and/or activated in
order to ensure production of respective secondary
metabolites in quantities sufficient for structure elucidation and biological testing. Although several success
stories have been reported recently [6,7], reliably harnessing the ‘silent’ biosynthetic genes on a large scale
remains a challenging task, especially in bacteria that are
difficult to manipulate genetically. Heterologous expression in a defined genetically tractable bacterial host
remains the best option, but in many cases both transcriptional and translational signals in the gene clusters
foreign to the host must be replaced. Here, synthetic
biology will definitely play a major role in re-factoring of
the clusters in order to gain control over their functioning.
Smart cluster re-design [4,18,19], molecular genetic tools,
characterized ‘parts’ and ‘devices’ that are being developed for bacteria [15,20], coupled to the plummeting
costs of DNA synthesis will soon open new possibilities
for drug discovery based on realization of bacterial genetic
potential to synthesize bioactive compounds.
Acknowledgements
The authors would like to thank the Research Council of Norway and the
Synthetic Biology Engineering Research Center (UC Berkeley) for financial
support.
References and recommended reading
Papers of particular interest, published within the period of review,
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of special interest
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