Microbial genomics for the improvement of natural
product discovery
Steven G Van Lanen and Ben Shen
The quest for the discovery of novel natural products has
entered a new chapter with the enormous wealth of genetic
data that is now available. This information has been exploited
by using whole-genome sequence mining to uncover cryptic
pathways, or biosynthetic pathways for previously undetected
metabolites. Alternatively, using known paradigms for
secondary metabolite biosynthesis, genetic information has
been ‘fished out’ of DNA libraries resulting in the discovery of
new natural products and isolation of gene clusters for known
metabolites. Novel natural products have been discovered by
expressing genetic data from uncultured organisms or difficultto-manipulate strains in heterologous hosts. Furthermore,
improvements in heterologous expression have not only helped
to identify gene clusters but have also made it easier to
manipulate these genes in order to generate new compounds.
Finally, and perhaps the most crucial aspect of the efficient and
prosperous use of the abundance of genetic information, novel
enzyme chemistry continues to be discovered, which has aided
our understanding of how natural products are biosynthesized
de novo, and enabled us to rework the current paradigms for
natural product biosynthesis.
Addresses
School of Pharmacy, University of Wisconsin-Madison, 777 Highland
Avenue, Madison, WI 53705, USA
Corresponding author: Shen, Ben (bshen@pharmacy.wisc.edu)
Current Opinion in Microbiology 2006, 9:252–260
This review comes from a themed issue on
Ecology and industrial microbiology
Edited by Arnold Demain and Lubbert Dijkhuizen
Available online 2nd May 2006
1369-5274/$ – see front matter
# 2006 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.mib.2006.04.002
Introduction
Natural products remain a consistent source of drug leads
with more than 40% of new chemical entities reported
since 1981 being derived from microbial natural products
[1–3]. Perhaps more astonishing is that more than 60% of
the anticancer and 70% of the anti-infective antibiotics
currently in clinical use are natural products or natural
product-based. Despite this impressive track record,
most of the big pharmaceutical companies have recently
de-emphasized, downsized or even abandoned their
natural-product discovery efforts, partly because of the
perception that the ‘tank’ of natural products has run dry
Current Opinion in Microbiology 2006, 9:252–260
and that finding new natural-product drug leads is not a
profitable endeavor.
Recent progress in several aspects of natural-product
research and microbial genomics, however, suggests that
the potential of natural-product diversity and discovery is
vastly underestimated, offering several promising alternatives to existing methods for the discovery of new
natural products (Figure 1). First, the exponential growth
in cloning and characterization of natural-product biosynthetic machinery in the past two decades has unveiled
unprecedented molecular insights into natural-product
biosynthesis, including the observation that genes for
natural product biosynthesis are clustered in the microbial
genome and that variations of a few common biosynthetic
machineries can account for the vast structural diversity
observed for natural products. These findings have fundamentally changed the landscape of natural-product
research and discovery by enabling the revision of known
natural-product structures, the prediction of yet-to-be
isolated novel products on the basis of gene sequences,
and the systematic generation of ‘unnatural’ natural products by manipulating genes that govern the naturalproducts biosynthesis (this process is also known as
combinatorial biosynthesis). Second, whole-genome
sequencing has revealed that there are far more biosynthetic gene clusters than there are currently known
metabolites for a given organism, suggesting that the
biosynthetic potential for natural products in microorganisms has been greatly under-explored by traditional methods of natural-product discovery. Third, only 1% of the
microbial community is estimated to have been cultivated in the lab, implying that there is a vast biodiversity
of natural products in microorganisms that remains to be
exploited. Newly emerging cultivating techniques, culture-independent methods, which involve expressing
gene clusters in model heterologous hosts, and chemoenzymatic bioconversion strategies have enabled access to
these previously inaccessible natural-product resources.
Finally, biochemical studies of natural-product biosynthetic enzymes have been extremely successful in the
discovery of new enzyme pathways and unusual chemical
conversions, and to date most known gene clusters
include genes whose deduced products have no homolog
or only have homology to proteins in the database with
unknown function, which is indicative of proteins with
novel functions and possibly the presence of undescribed natural products. Continued discovery and
characterization of these novel enzymes should ultimately enable realization of the full potential of microbial
genomics-based natural-product discovery.
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Microbial genomics for the improvement of natural product discovery Van Lanen and Shen 253
Glossary
Colinearity rule: Chain extensions during polyketide and
polypeptide formation depend on the number, type and organization
of modules such that the genetic architecture is directly reflected in
the final product.
Genome sequence tags (GSTs): Short (700 base pair [bp]) random
DNA fragments from a genomic library that can be used to generate
probes to screen entire DNA libraries.
Synthon: DNA fragments of approximately 500 bp that are amplified
by PCR and used to ultimately create a contiguous synthetic gene
cluster.
Red/ET recombineering: Genetic engineering to reassemble a
biosynthetic gene cluster based on recombination that is directed
by l-phage-derived proteins Reda, Redb, and Redg.
This review, therefore, highlights the interplay between
microbial genomics and natural-product biosynthesis and
illustrates the impact of this relationship on natural product discovery by using a few of the numerous examples
that have been reported in the past a couple of years
(Figure 1). Readers are referred to several other recent
reviews for a more comprehensive coverage on this and
related topics [4–8].
Whole-genome sequence mining
It is possible to estimate the biosynthetic potential for a
given organism by mining the whole-genome sequence,
because natural-product biosynthetic genes are present in
clusters in microbial genomes; however, it is the characteristics of the various biosynthetic machineries that
have enabled the prediction of the types and sometimes
the exact structures of the final natural products. This is
best exemplified by the polyketide and non-ribosomal
peptide biosynthetic gene clusters, featuring the polyketide synthases and nonribosomal peptide synthetases
(NRPSs), which catalyze the biosynthesis of members
of two of the largest families of natural products. Archetypical polyketide synthases are classified into type I, II or
III enzymes according to their structural and catalyticdomain organization, and NRPSs have likewise been
grouped on the basis of their catalytic architecture
[9,10]. Typically, the nascent polyketide or peptide backbone is further modified by tailoring enzymes in postpolyketide synthase or post-NRPS steps of the pathway,
most commonly oxygenases, oxidoreductases and glycosyltransferases or other transferases, in order to imbue
additional structural functionalities to the final natural
product. The conserved features within this machinery
have been cornerstones of the genomics-guided discovery
of natural products, and discovery methods are continually being altered and optimized to reflect the current
paradigms of natural-product biosynthesis [11,12,13].
The genomes of 294 microorganisms have been
sequenced and annotated in the National Centre for
Biotechnology Information genome project, and a
remarkable aspect of this wealth of information is that
the number of genes that are expected to be involved in
secondary metabolite production dramatically outnumbers the amount of known secondary metabolites [14]. An
extreme example of this is in the cyanobacterium Nostoc
punctiforme, which has 22 genes that encode probable
polyketide synthases or NRPSs, although only one of
these has been related to a secondary metabolite
Figure 1
Microbial genomics and natural product biosynthesis and their impact on natural product discovery. See Figures 2 and 3 for structures of
individual natural products (1–18). Highlighted in red boxes are techniques that have been introduced or advanced to utilize the wealth of genomic
data available. Likewise, compounds shown in red have been discovered or more closely examined by the respective methodologies. Related
to microbial genomics is the deciphering of new enzyme pathways (blue box) that has enabled the reworking of current paradigms for natural
product biosynthesis (green) in order to optimize the available techniques to continue the cycle for novel natural product and drug discovery.
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Current Opinion in Microbiology 2006, 9:252–260
254 Ecology and industrial microbiology
Figure 2
Current Opinion in Microbiology 2006, 9:252–260
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Microbial genomics for the improvement of natural product discovery Van Lanen and Shen 255
(nostopeptolide). The complete genomes of 13 Actinobacteria, unequivocally the largest group of antibiotic
producers, also follow this trend. A case in point is that
of the genomes of three members of the genus Corynebacterium that have been sequenced: each contains a
modular type I polyketide synthase, but these have not
been correlated to any secondary metabolite. A last
example is that in a survey of 11 secondary metabolite
producers — including Streptomyces coelicolor, which is the
best characterized model-strain for Streptomyces, and Streptomyces avermitilis, which is an indispensable industrial
Streptomyces strain — unveiled a total of 118 NRPS and
polyketide synthase clusters, only 14 of which had been
assigned to known non-ribosomal peptides or polyketides
at the time the genome sequences were reported (this
represents less than 12% of the biosynthetic potential
available to these organisms) [15].
The use of sequence data to search for novel metabolites
has been exemplified by the discovery of the iron chelator
coelichelin (1, Figure 2) from S. coelicolor M145
(Figure 2a) [16]. The gene cluster for 1 contains three
NRPS modules and, from the colinearity rule (see Glossary) and the authors’ NRPS code, was predicted to
contain a tripeptide containing the synthetic building
blocks of L-d-N-formyl-d-N-hydroxyornithine, L-threonine and L-d-N-hydroxyornithine. By accurately predicting the adenylation domain substrate-specificity and
understanding the role of hydroxamic acid as a divalent
metal chelator, the appropriate condition for production
was identified and a new tris-hydroxamate tetrapeptide
iron chelator, 1, was produced. These results, along with
others not mentioned here [17,18], unquestionably
demonstrate the utility of genome mining for the identification of new secondary metabolites.
Genome scanning
As an alternative approach to whole-genome sequence
mining, genome scanning provides an efficient way to
discover natural-product biosynthetic gene clusters without having the complete genome sequence. This
approach takes advantage of the fact that the genes for
natural-product biosynthesis form clusters in a microbial
genome, the size of which range from 20–200 kilobases
(kb). By shotgun-sequencing a small number of random
genome sequence-tags (GSTs; see Glossary) from a
library of genomic DNA, it is expected that, when analyzed, any given gene cluster will be represented by
multiple GSTs. GSTs derived from genes that are likely
to be involved in the biosynthesis of natural products are
identified and used as probes in order to localize entire
biosynthetic gene clusters. The effectiveness of this
method was elegantly demonstrated initially by isolating
the dynemicin (2) and macromomycin biosynthetic gene
clusters from strains known to produce these enediyne
antitumor antibiotics, and subsequently by identifying 11
cryptic enediyne biosynthetic loci from 70 actinomycete
strains that were previously not known as enediyne
producers (Figure 2b). Armed with this genomic information, enediyne antibiotic production in the strains was
verified by optimizing medium and fermentation conditions [19]. As a consequence of this type of genome
scanning, the enediyne family was revealed to be more
dispersed among actinomycetes than originally
anticipated.
The success of the aforementioned genome-scanning
approach using GSTs depended crucially on the recognition of the enediyne polyketide synthase, a novel iterative
type I enzyme unique to the enediyne family of natural
products [20,21]. Taking advantage of the high sequenceconservation among the enediyne polyketide synthases, a
PCR method for the rapid amplification of the minimal
enediyne polyketide synthase genes was also developed
and successfully applied to the cloning and localization of
the esperamicin (3) and maduropeptin (4) biosynthetic
gene clusters, providing an alternative method to directly
access the enediyne biosynthetic machinery (Figure 2b)
[22].
An approach for obtaining ‘perfect probes’ to identify all
polyketide synthase and NRPS gene clusters in a genome, an approach which in principle is analogous to the
GST-based genome scanning method, was independently developed. The utility of this method was first
verified in silico by scanning the Bacillus subtilis genome to
localize the known polyketide synthase and NRPS clusters, and subsequently used to identify the epothilone (5)
gene cluster from Sorangium cellulosum along with several
other NRPS and polyketide synthase gene clusters
(Figure 2b) [23].
Cultivation and metagenomics
Random sequencing of soil isolates suggests that less than
1% of microorganisms have been cultivated in the lab
using most common cultivation conditions [24,25].
Recent investigations have added support to the idea
that this phenomenon is as a result of the media, incubation times and inoculum size traditionally used, with the
first two of these factors being the most important in the
isolation and culture of rarely isolated soil bacteria. [26].
Using specific enrichment techniques, primarily by varying the media, hundreds of different organisms from the
family of Streptosporangiacae were isolated, and many
were hypothesized to produce novel antibiotics on
the basis of observations of other members of this
family [27]. In a separate example, by changing the media
in combination with using selective agents for motile
(Figure 2 Legend) Natural products, the discovery and isolation of which have benefited from microbial genomics as exemplified by (a) whole
genome mining, (b) genome scanning, (c) metagenomics and (d) heterologous expression.
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Current Opinion in Microbiology 2006, 9:252–260
256 Ecology and industrial microbiology
microorganisms, two new antibiotic-producing actinomycete species were discovered, demonstrating the usefulness of a guided-culturing approach for the isolation of
rare strains [28]. Finally, the culturing of rare organisms
has been improved by the development of new techniques. This is illustrated by the use of microcapsules,
derived from a single encapsulated cell, for high-throughput screening [29]. These culturing techniques will
undoubtedly improve the access to natural products.
A valuable alternative to cultivating rare or slow-growing
organisms is to extract community DNA and produce
clone libraries in a cultivation-independent approach
termed metagenomics [30]. Typically, large genomic
DNA isolates are inserted into suitable carriers, such as
bacterial artificial chromosomes (BACs) or cosmids, to be
evaluated in fast-growing streptomycetes or other heterologous hosts. This methodology has been especially useful for the evaluation of both marine and terrestrial
bacterial symbionts for their potential production of secondary metabolites.
Putative biosynthetic genes for the pederin and byrostatin family of antitumor compounds were identified using a
metagenomics approach. Pederin (6), isolated from
extracts of Paederus species rove beetles, was determined
to have originated from a microbial source that is that has
the highest sequence homology to Pseudomonas aeruginosa
[31,32]. The resulting polyketide synthase predicted for 6
biosynthesis was shown to be evolutionarily distinct from
the archetypical polyketide synthase [33], and this
acyltransferase (AT)-less polyketide synthase phylogenetic grouping has proved useful for the discovery of
putative biosynthetic genes for the synthesis of onnamide
A (7) from the marine sponge Theonella swinhoei [34].
Similarly, a putative biosynthetic gene coding for prebryostatin 1 (8), an antitumor compound isolated from the
marine protozoan Bugula neritina, was also identified
using metagenomics and, similarly to 6, the bryostatin
polyketide synthase belongs to the AT-less polyketide
synthase family [33,35]. These are just a few illustrations of the utility of metagenomics in the search for
natural products (Figure 2c), and they also highlight
examples of an emerging family of polyketide synthases
that use discrete, iterative ATs for polyketide biosynthesis [36–38]. It should be noted that whereas metagenomics has been effective in identifying novel
biosynthetic gene clusters, experimental verification of
the predicted products of these biosynthetic gene clusters
remains a great challenge.
Heterologous expression
With the difficulties associated with obtaining a functional genetic system or detectable production conditions
for a particular secondary metabolite, efforts to heterologously express gene clusters have increased with undeniable success [39]. Escherichia coli, initially perceived as
Current Opinion in Microbiology 2006, 9:252–260
an unsuitable host for heterologous expression, has been
engineered to produce 6-deoxyerythronolide B (9) at
levels comparable to the model Streptomyces host,
S. coelicolor [40]. To further establish E. coli as a suitable
heterologous host, 9 was successfully produced by introducing a synthetic 31.7 kb DNA polyketide gene cluster
that was prepared using a ‘synthon’ approach (see Glossary) [41]. A chemoenzymatic approach was used to
synthesize epothilone using E. coli as a host, by introducing the genes responsible for the second half of the
biosynthetic pathway using a three-plasmid system and
then by feeding the bacteria with a chemically synthesized substrate as an N-acetylcysteamine (SNAC) thioester (Figure 2d) [42].
E. coli has also been used to confirm the locus for the
biosynthetic gene cluster of patellamide A and patellamide
C, two cyclic peptides that are produced by the symbiont
Prochloron didemni [43,44]. This investigation, similar to
that of 6 and 8, represents a cultivation-independent
approach to identify natural products, but also provides
the first conclusive evidence of a gene cluster which has
been isolated from an obligate symbiont by expressing the
marine natural-product pathway in E. coli [44].
Other hosts, besides E. coli, that are easy to genetically
manipulate are amenable to heterologous expression of
various natural products. The biosynthetic pathway for
rebeccamycin (10) was dissected and reconstituted in the
strain Streptomyces albus, which provided an environment
capable of supplying precursors without the need for
further genetic manipulations [45]. Furthermore, a variety of indolocarbazoles [such as the congener staurosporine (11)] were generated by swapping and/or inserting
genes from different loci; this illustrates the utility of
heterologous expression in combinatorial biosynthesis.
Intriguingly, whereas direct introduction of the fredericamycin (FDM) biosynthetic gene cluster from Streptomyces griseus into Streptomyces lividan resulted in little
FDM production unless fdmR, which encodes a pathway-specific activator, is overexpressed under the control
of a strong constitutive promoter; introduction of the
FDM cluster alone into S. albus yielded FDM in amounts
comparable to that from the S. griesus wild type strain [46].
This result not only duplicated the success of S. albus as a
heterologous host, but also underscored how little is
currently known about global and pathway-specific regulation of natural-product biosynthetic gene cluster
expression in heterologous hosts.
Daptomycin (12; Cubicin1), which has been recently
approved for the treatment of Gram-positive infections
of skin and skin structure, represents one of only two
novel antibiotics to reach the market in 30 years. The
daptomycin gene cluster has been cloned from Streptomyces roseosporus and characterized, and a BAC clone
containing the entire 12-gene cluster on a 128 kb DNA
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Microbial genomics for the improvement of natural product discovery Van Lanen and Shen 257
fragment was successfully introduced into S. lividans for
heterologous expression, resulting in production of
18 mg l1 of 12 (Figure 2d) [47]. Although the yield is
still very poor in comparison with the wild type S.
roseosporus strain under the optimized fermentation conditions, to our knowledge this represents the largest gene
cluster that has ever been successfully expressed in a
heterologous host.
Another method combines the ease of genetic manipulation in E. coli with the selection of a separate, suitable host
for natural-product production. The biosynthetic gene
cluster for myxochromide S1 (13), a hybrid polyketide
synthase-NRPS system, was rebuilt and engineered in E.
coli to contain the entire locus and to include a toluic-acid
inducible promoter and a homologous recombination
region (Figure 2d). The assembly was performed using
Red/ET recombineering (see Glossary) in E. coli; the final
DNA was transferred into Pseudomonas putida and integrated into the chromosome [48]. The recombinant P.
putida strain produced the desired product, 13, and
remarkably at a fivefold greater level and in significantly
less incubation time than the wild type.
The above examples not only exemplify the utility of
using heterologous hosts, but this strategy sets the stage
to probe the functional roles of individual open reading
frames or domains and will be useful for combinatorial
biosynthesis to create unnatural natural products with
desired properties. It also illustrates the many challenges
to which we have yet to find solutions. These issues have
to be addressed before the production of natural products
by expressing their biosynthetic gene clusters in model
heterologous hosts becomes a realistic alternative.
Discovering novel chemistry
The discovery of novel natural products using genomics
and the improvement of industrial applications using
bioinformatics depend on understanding the biochemistry of secondary metabolite production. The polyketide
synthase responsible for 9 synthesis has historically been
the archetype for the structure and function of polyketide
synthases [9], and this modular type I system continues to
be the most-studied polyketide synthase [49,50]. However, increasing numbers of polyketide synthases and
NRPSs are being discovered that do not fit into the
existing paradigms; this has been reviewed elsewhere
[37,38]. In addition to polyketide synthase and NRPS,
the analysis of other recombinant enzymes, such as tailoring enzymes, has recently resulted in several discoveries
that were unexpected from sequence predictions.
Coronatine (14, Figure 3) and pyoluteorin (15), two
hybrid polyketide-peptides found in different Pseudomonas species, have recently been shown to be biosynthesized with unexpected halogenation events (Figure 3a).
During 14 biosynthesis, CmaB, a non-heme Fe2+
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a-ketoglutarate-dependent enzyme, was shown to carry
out g-chlorination of a thioester-linked substrate, L-alloisoleucine, followed by cyclopropyl ring formation by a
previously unknown protein, CmaC [51]. With respect
to 15 biosynthesis, PltA (a predicted FADH-dependent
halogenase) was found to not only regiospecifically insert
chlorine at position 5 of thioester-linked proline but also
to further modify the monohalogenated proline intermediate by chlorination at position 4 [52]. The first
transformation, using cryptic halogenation, represents a
unique strategy for biochemical conversions, and the
enzymatic steps of CmaB and PltA were impossible to
predict from DNA sequence alone.
The difficulties associated with making functional predictions solely on the basis of DNA sequence have also
been prominent in the development of a variety of
Streptomyces-derived natural products (Figure 3b). In
the biosynthesis of jadomycin (16), a polyketide isolated
from S. venezuelae ISP 5230, the enzyme JadH was functionally assigned to have both oxygenase and dehydrase
activities, although the former function was only predicted before biochemical analysis [53]. For nystatin
(17) production by S. noursei, nysF was originally proposed
to encode a 40 -phosphopantetheinyl transferase, but
unexpectedly was shown to be a negative regulator of
17 biosynthesis, and therefore deletion of the gene did
not abolish production but actually improved yields by
60% [54]. Finally, although OtcC was correctly predicted
to be an oxygenase involved in oxytetracycline (18)
biosynthesis by S. rimosus, the deletion of otcC resulted
in a novel 17 carbon polyketide (19) instead of the native
19 carbon polyketide, showing that, although the activity
of OtcC was correctly predicted, this deletion not only
resulted in a loss of oxygenase activity but also resulted in
an incorrect chain length being produced [55].
Conclusions and perspectives
Discovery of novel antibiotics and clinically useful natural
products has been in modest decline over the past few
decades, despite estimates that more than one million
cultures were screened by pharmaceutical companies
during this period [2]. Increased antibiotic resistance in
combination with the lengthy process of marketing new
drugs only compounds this decline [56]. Consequently,
new approaches to drug discovery need to be developed
and applied.
Genome sequencing of actinomycetes and other microorganisms has revealed a number of biosynthetic enzymes
that cannot be related to known metabolites, suggesting
that the occurrence of natural products has been underestimated by using classical techniques such as screening
fermentation broths or target-based high-throughput
screening [2,3]. Therefore, ways to take advantage of
the wealth of genomic information have been brought
to the forefront, including directed cultivation methods,
Current Opinion in Microbiology 2006, 9:252–260
258 Ecology and industrial microbiology
Figure 3
Novel chemistry, the discovery of which has influenced by microbial genomics as exemplified by (a) the halogenation steps during coronatine
(14) and pyoluteorin (15) biosynthesis and (b) unusual enzymatic steps during other natural product biosynthesis in Streptomyces. Abbreviation:
PCP, peptidyl carrier protein.
using heterologous hosts to express gene clusters from
metagenomes or organisms with silent phenotypes, or
using different high-throughput techniques that are not
discussed here, such as mass spectroscopy and near-infrared spectroscopy [57–59].
The vast amount of DNA sequence in the public database represents only the beginning of the new genomics
era. Recent advances in DNA sequencing have led to the
statement that we are on the verge of having ‘‘a genome
sequencing center in every lab’’, implying that obtaining
genomic data will no longer be the bottleneck for naturalproduct science [60,61]. Furthermore, when genome
Current Opinion in Microbiology 2006, 9:252–260
sequencing of the ‘first tier’ organisms is completed,
whole genome sequencing will almost certainly expand
and diversify to include other relevant natural-product
producers and other more rare organisms. Moreover, if the
process of genome scanning and mining guides the discovery of a clinically useful compound in a similar fashion
to that shown with coelichelin, whole genome sequencing
will only gain momentum.
It is apparent from the above examples that, although
advancing rapidly, knowledge of the DNA sequence
cannot be simply translated to enzyme function, and
ultimately the structure, of a natural product.
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Microbial genomics for the improvement of natural product discovery Van Lanen and Shen 259
Nevertheless, as has been hinted to throughout this
review, ingenuity, dedication and, if necessary, brute
force can overcome any shortcomings encountered during
the process of drug discovery, and a sense of optimism
should prevail as long as we remain cautious of sequence
annotations until biochemical assignments are made. A
continued and concerted progression in genomics, microbiology and biochemistry will be invaluable in the next
generation of drug discovery, whether this occurs by the
identification of new or rare organisms, by cultivation or
metagenomics techniques, or through rational engineering of the biosynthetic machinery.
14. Jenke-Kodama H, Sandmann A, Müller R, Dittmann E:
Evolutionary implications of bacterial polyketide synthases.
Mol Biol Evol 2005, 22:2027-2039.
Acknowledgements
18. May JJ, Wendrich TM, Marahiel MA: The dhb operon of Bacillus
subtilis encodes the biosynthetic template for the catecholic
siderophore 2,3-dihydroxybenzoate-glycine-threonine
trimeric ester bacillibactin. J Biol Chem 2001, 276:7209-7217.
Current studies on natural product biosynthesis described from the Shen
laboratory were supported in part by National Institutes of Health (NIH)
grants CA94426, CA78747, CA106150 and CA113297. SVL is the recipient
of NIH postdoctoral fellowship CA1059845, and BS is the recipient of an
NIH Independent Scientist Award AI51689.
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