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The Impact of Bacterial Genomics on Natural Product Research.

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Reviews
R. Mller and H. B. Bode
Metabolic Pathway Mining
DOI: 10.1002/anie.200501080
The Impact of Bacterial Genomics on Natural Product
Research
Helge B. Bode and Rolf Mller*
Keywords:
combinatorial biosynthesis · genomics ·
heterologous expession ·
natural products
Dedicated to Prof. Dr. Eckhard Leistner
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Chemie
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2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Angewandte
Chemie
Natural Product Discovery
T
“ heres life in the old dog yet!” This adage also holds true for natural
product research. After the era of natural products was declared to be
over, because of the introduction of combinatorial synthesis techniques, natural product research has taken a surprising turn back
towards a major field of pharmaceutical research. Current challenges,
such as emerging multidrug-resistant bacteria, might be overcome by
developments which combine genomic knowledge with applied biology and chemistry to identify, produce, and alter the structure of new
lead compounds. Significant biological activity is reported much less
frequently for synthetic compounds, a fact reflected in the large
proportion of natural products and their derivatives in clinical use.
This Review describes the impact of microbial genomics on natural
products research, in particularly the search for new lead structures
and their optimization. The limitations of this research are also
discussed, thus allowing a look into future developments.
1. Introduction
Without medically applied natural products[+] human life
would be less comfortable and definitely much shorter: More
than 75 % of all antibacterial and approximately 50 % of all
anticancer compounds currently in clinical use are either
natural products themselves or derivatives thereof.[1] Clinically useful antibacterial natural products are often the drugs
of last resort for the treatment of multidrug-resistant and,
without treatment, often deadly microorganisms. However,
resistance against new antibiotics, including those representing our last line of defence, is emerging. Even with the
adoption of antibiotics with novel mechanisms of action, we
do not have to ask the question if resistance will develop but
only when it will occur. This situation is mainly a consequence
of the enormous speed at which microorganisms exchange
and mutate their genes. The more often antibiotics are used,
the more rapidly resistance is spread.[2] It is predictable that
the winner in this game will always be the microbe, simply
because of its huge numbers, short generation times, and
mutation rates. Nevertheless, pharmaceutical research must
try to fight back to maintain the status quo. However, to find a
promising new natural product for the treatment of any kind
of disease is an expensive and difficult challenge,[3, 4] which has
led major pharmaceutical companies to rely solely on
synthetic chemicals.[5, 6]
The reason for this can be seen in several disadvantages
that natural products appear to have compared to synthetic
chemicals: 1) The isolation and characterization is often
laborious and, therefore; 2) it is not possible to obtain as
many natural products as synthetic chemicals in the same
time; 3) by definition there is a biological source involved in
the production of any natural product, which requires
experienced handling plus specialized and expensive equipment.
However, these disadvantages are more than balanced by
the positive aspects of natural products, and some of the
arguments against them can in fact be turned into strong
Angew. Chem. Int. Ed. 2005, 44, 6828 – 6846
From the Contents
1. Introduction
6829
2. Approaches to Explore the
Genetic Potential
6833
3. Be Prepared! Natural Product
Research in the Postgenomic
Era
6843
advantages:
1) Natural
products
occupy a complementary region of
chemical space compared with synthetic compounds as discussed extensively in recent reviews.[7–12] These
differences range from simple features such as elemental
composition and molecular weight to specific structural
elements such as ring size and type, and overall complexity
and stereochemistry in general. 2) Natural products have
evolved in a biological context and the percentage of
biologically active natural products is much higher than that
of synthetic compounds (although not all of them have an
identified biological activity).[13] These two findings result in a
new trend of “natural productlike” synthetic strategies: new
lead compounds can be identified from natural product
scaffolds or by creating analogues to natural products.[14–16]
These approaches have also led to a cooperation between
natural product research and combinatorial chemistry, the
latter of which was earlier thought to completely replace the
need for novel natural products. In general, it is now accepted
that both disciplines support, fertilize, and rely on one
another.[10, 17] 3) The biological source of the compound is
often renewable and thus opens up the possibility to scale-up
production. This is especially true if the producing organism is
easy to cultivate, and is a major reason why bacteria and fungi
are the dominant source of the natural products in clinical use.
Furthermore, because of our increased understanding of
genetics and the principles of biological regulation the
production of these compounds can be optimized (see
Section 2.5). The biosynthesis of the compounds themselves
can also be manipulated to yield new derivatives with possibly
superior qualities (see Section 2.6). Possible ways to obtain
[*] Jr. Prof. Dr. H. B. Bode, Prof. Dr. R. M+ller
Institut f+r Pharmazeutische Biotechnologie
Universit/t des Saarlandes
Postfach 151150, 66041 Saarbr+cken (Germany)
Fax: (+ 49) 681-3025473
E-mail: rom@mx.uni-saarland.de
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
[+] The expressions natural product and secondary metabolite are used
interchangeably throughout this Review.
2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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R. Mller and H. B. Bode
these goals for pharmacologically relevant natural products
are the focus of this Review and will be described in later
sections. Most of the work cited was performed with
polyketides and nonribosomally biosynthesized peptides.
Since these natural products are derived from enzymatic
systems called polyketide synthases (PKSs) and nonribosomal
peptide synthetases (NRPSs), these enzymes and the resulting secondary metabolites are introduced briefly (Scheme 1).
It should be noted that many bacterial natural products are
derived from other pathways, such as the isoprenoid or
shikimate pathways.[18–20]
Bacterial PKSs (type I) are multifunctional enzymes that
are organized into modules, each of which harbors a set of
distinct, non-iteratively acting activities responsible for several biosynthetic steps during the catalysis of one cycle of the
polyketide chain elongation.[21] Type I PKSs are involved in
the biosynthesis of macrolides, polyethers, and polyenes.
Type II PKSs are multienzyme complexes that carry a single
set of iteratively acting proteins and are involved in the
biosynthesis of aromatic and often polycyclic polyketides.[22, 23]
Type III PKSs, also known as chalcone synthase like PKSs, are
homodimeric enzymes which iteratively act as condensing
enzymes, as exemplified by the formation of flaviolin.[23]
NRPSs are similar to the type I PKSs in that they are
multifunctional enzymes that are organized into modules with
sets of non-iteratively acting activities for the incorporation
and processing of one amino acid per module.[24, 25]
Figure 1. Currently finished genomes ordered according to their intermediate taxonomic rank. Classes and orders that harbor experimentally
verified secondary metabolite producing members are shown in gray.
1.1. Increase in Sequence Information
Since the publication of the first complete microbial
genome sequence of Haemophilus influenzae in 1995,[31] more
than 250 fully sequenced genomes have been reported
(Figure 1) and more than 600 prokaryotes and 460 eukaryotes
are currently being sequenced.[32] Although there was a clear
focus on pathogenic bacteria in the early days of genome
sequencing, biotechnology is catching up rapidly: currently
47 % of all bacterial-sequencing projects deal with organisms
having industrial applications, including secondary metabolism (52 % of the projects remain focused on pathogens and
about 1 % involve the sequence of exotic organisms).[32] The
value of the exponentially growing sequence information is
Helge B. Bode was born in 1973 and studied
chemistry and biology in Gttingen and
Stockholm. After his Diploma and PhD
(2000) studies in natural product chemistry
with Prof. A. Zeeck, he received a Diploma
in molecular microbiology with Prof. G.
Braus in 2001. After postdoctoral research
with R. M/ller in Braunschweig and Prof. D
Kaiser in Stanford, he was appointed Junior
professor for Natural Product Biotechnology
at Saarland University in April 2004. His
research focuses on the elucidation of the
biochemical basis of the complex life cycle of
myxobacteria.
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immeasurable because bioinformatic methods, along with
large-scale approaches such as transcriptomics and proteomics, are only just beginning to reveal insights into numerous
important research topics such as virulence genes, host–
pathogen interactions, new molecular targets, and novel ways
to produce secondary metabolites. Sequence data from
multiple isolates or different strains of a single pathogen are
accumulating. This type of information has proven invaluable
for providing new insights into the genetic variability present
in a particular species as well as facilitating correlations
between genotype and phenotype.[33]
Unexpectedly, genome sequencing of known producers of
secondary metabolites (Streptomyces sp.[34, 35] and ArabidopRolf M/ller studied pharmacy at Bonn University and received his PhD with Prof. E.
Leistner in 1994. He worked for two years as
a research fellow of the DFG with Prof. H.
Floss in Seattle, and then studied myxobacteria at the German Research Centre for
Biotechnology (GBF) for his Habilitation
(TU Braunschweig, 2001). In 2003 he
received the BioFuture prize of the BMBF
and in the same year was appointed head of
the Department of Pharmaceutical Biotechnology at Saarland University. His recent
work focuses on the biotechnological exploitation of myxobacterial genome projects and the heterologous expression
and modification of complex megasynthetases in pseudomonads and fastgrowing myxobacteria.
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sis[36]) revealed the capacity
to synthesize many more
secondary metabolites than
were actually known from
these organisms. Even more
surprising, many organisms
that were not known as
secondary metabolite producers, habor the typical
genes and often huge biosynthesis gene clusters for
the production of several
hypothetical or unidentified
natural products. A possible
explanation for this might
be that only a few taxa
(plants, fungi, and some
bacterial taxa) have been
investigated with respect to
secondary
metabolism.
Almost all bacteria, fungi,
and plants sequenced to
date show at least some
biosynthesis genes typical
for secondary metabolite
production, thus indicating
the old evolutionary origin
of these genes.
1.2. Plants—Potential and
Problems
Scheme 1. Mechanisms and structures of bacterial PKSs and NRPSs. a) Generalized example of a type I PKS
consisting of non-iteratively acting domains; acyltransferase (AT), acyl carrier protein (ACP), ketosynthase
(KS), ketoreductase (KR), dehydratase (DH), enoylreductase (ER); a and p indicate the specificity of the AT
domain for malonyl-CoA or methylmalonyl-CoA.[21] b) Biosynthesis of erythromycin A is a well-studied example
of the action of a type I PKS; 6-deoxyerythronolide B synthase (DEBS). c) Type II PKS consisting of iteratively
acting subunits as exemplified for actinorhodin biosynthesis; chain length factor (KSb).[21, 26] d) Type III PKS
consisting of an iteratively acting enzyme as exemplified by flaviolin biosynthesis.[23, 27] e) Generalized example
of NRPS biosynthesis; adenylation (A), peptidyl carrier protein (T), condensation (C), epimerization (E),
heterocyclization (HC), oxidation (Ox).[28, 29] f) Formation of tyrocidine A is a well-documented example for
NRPS biosynthesis.[29, 30]
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Plants have the greatest
potential to produce any
type of natural product.
This situation is exemplified
by the enormous variety of
low- molecular-weight compounds present in most
plant materials.[37] The complexity of plant metabolomes is far beyond those
from
other
organisms.
However, the organization
of the genes in a typical
plant
genome
makes
research dealing with plant
secondary metabolism a
major challenge: 1) The
genes for the production of
secondary metabolites are
not found in one genomic
region (that is, as a biosynthetic gene cluster, such as
those usually present in
bacteria or fungi) but scattered throughout the entire
genome. Therefore, the
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R. Mller and H. B. Bode
identification of a complete biosynthetic pathway in plants is
extremely tedious and time-consuming. 2) The exon/intron
structure of the genes leads to a number of problems.
Therefore, it is necessary to work with cDNA which is
generated from enriched mRNA pools to identify the genes of
interest.
A good example of these challenges is the potent
anticancer agent paclitaxel (taxol), which was isolated from
the bark of the pacific yew as a pure compound in 1971. This
source is environmentally unfriendly and gave rise to serious
supply issues, and today it is produced by a semisynthetic
approach starting from 10-deacetylbaccatin III, which can be
isolated from yew tree needles (and thus a renewable source)
in large quantities.[38] The broad use of paclitaxel in breast
cancer treatment has triggered several efforts to produce this
valuable compound more efficiently, either in plant cell
cultures[39] or alternatively in fast-growing bacterial hosts
after heterologous expression of the required biosynthetic
machinery. Unfortunately, the complex biosynthesis of taxol
requires approximately 19 biosynthetic steps, which explains
that despite groundbreaking efforts, especially by the Croteau
research group, several genes for the biosynthesis are still
unknown (Scheme 2).[40–43] Interestingly, endophytic fungi
have been isolated from various yew trees that produce
paclitaxel. Despite their small production titers, these fungi
open up the possibility to study paclitaxel biosynthesis in
comparison to the plant system and lead to potential
biotechnological processes.[39]
1.3. Fungi—The Promised Land?
Fungi have been a major source of natural products used
in food processing and medicine as well as for religious rituals
since the very early days of human culture. The modern era of
fungal natural product research started with the well-known
story of a contaminated agar plate in Alexander FlemingEs
laboratory in 1928, which eventually led to the clinical use of
penicillin to treat bacterial infections.[44] Since then, fungi
have been an impressive source of biologically active lead
structures for pharmaceutical development, including the
immunosuppressant cyclosporin[45] and the cholesterol-lowering agent lovastatin.[46]
Fungal genomics started with the availability of the
complete sequence of the model yeast Saccharomyces cerevisiae in 1997.[47] Since then genome sequences of additional
model strains used to study fungal molecular biology and/or
fermentative biotechnology have been reported (Schizosaccharomyces pombe (2002),[48] Neurospora crassa (2003),[49]
Phanerochaete chrysosporium (2004),[50] Yarrowia lipolytica
(2004), and Kluyveromyces lactis (2004)[51]).
The first complete sequence of a fungus that produced
secondary metabolites was the white-rot fungus Phanerochaete chrysosporium in 2004.[50] More than 50 genes involved
in secondary metabolite biosynthesis including several genes
encoding PKS and NRPS have been identified in the 30 Mbp
genome. It is noteworthy that the biosynthesis of polyketides
often involves post-PKS assembly steps, including the action
of cytochrome P450 like enzymes, and an impressive number
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Scheme 2. Biosynthesis of paclitaxel (taxol). Enzymatic activities
encoded by genes that have not been cloned yet are shown in
bold.[40–43]
of corresponding genes (more than 100) have been identified
in this genome. Fortunately, most genes for secondary
metabolite biosynthesis in P. chrysosporium and in fungi in
general are clustered on the chromosome so that the
identification and study of complete secondary metabolite
biosynthetic pathways is much easier than in plants.
Currently 113 fungal genome projects are in progress
worldwide.[32] The majority of these involve classical secondary metabolite producers (for example, Penicillium chrysogenum and 13 different Aspergillus sp.) or plant pathogenic
strains which are also known for the production of natural
products (for example, Fusarium sp., Botrytis sp., and Magnaporthe sp.). These projects will undoubtedly add valuable
insights into fungal secondary metabolite biosynthesis, which
is currently little studied relative to related bacterial systems.
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One forseeable problem with fungal systems is the lack of
genetic methods for the manipulation of many strains and the
fact that in some cases the biosynthetic genes are not
clustered on the chromosome. Nevertheless, there is enormous potential in fungal systems, and most of the advantages
discussed below with respect to bacteria also apply for fungi.
1.4. Bacteria—The Bigger the Better?
Research focussing on bacteria and in particular on
bacterial secondary metabolism has a number of advantages
in comparison to plants and fungi: 1) Genes for the biosynthesis of the desired compounds are mostly clustered; 2) the
coding density of bacterial genomes is high and thus sequence
costs can be reduced; 3) no introns are present, thus enabling
expression methods starting directly from chromosomal
DNA; 4) the compact genetic composition allows the fast
identification of bacterial gene clusters and their transfer into
different expression hosts; and 5) fermentation processes can
be developed, thus enabling large-scale production (which is
also true for fungi).
Many more bacteria have been sequenced than eukaryotes because of their relatively small genome sizes. Early
sequencing efforts focused mostly on pathogenic bacteria,
while 47 % of all current bacterial sequencing projects deal
with biotechnologically relevant strains. Up to now only a few
species have been sequenced that were known in advance to
be secondary metabolite producers. However, the sequencing
of these strains revealed a plethora of biosynthetic gene
clusters for the production of additional unknown secondary
metabolites (Figure 2). The best studied examples are Strep-
Figure 2. Selected fully sequenced prokaryotic secondary metabolite
producers with numbers of isolated polyketides and nonribosomal
peptides at the time of the genome sequence (black bars). Identified
gene clusters encoding PKS and NRPS in the genome are shown as
gray bars.[32] The data was taken from the original publications or
generated by BLAST searches of KS, KR, TE, ACP, T, and A domains.
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tomyces coelicolor and S. avermitilis, which were known to
produce 4 and 3 secondary metabolites, respectively, but
actually possess 20 and 25 clusters for secondary metabolite
biosynthesis, respectively.[34, 35] A detailed post-genomic
examination of the production profile of S. coelicolor has
already led to the isolation of two additional compounds, the
existence of which had been postulated after genome
annotation.[52, 53]
Statistical analysis of all bacterial genomes sequenced to
date revealed that there is a positive correlation between the
genome size and the number of genes involved in the
biosynthesis of secondary metabolites.[54] Large genomes
found in most producers of secondary metabolites might be
a consequence of the complex ecological niches they
occupy—requiring adaptation to several microenvironments
and/or competition between species. Further ecologically
relevant species need to be sequenced to learn more about
this dependency.[55] However, two questions arise from the
tremendous discrepancy between isolated compounds and
genomic potential: How can we identify and obtain the
hypothetical products that have not yet been isolated? How
can we use the enormous genetic potential in the most
efficient way? We will try to address these questions in the
following sections by using selected examples from the recent
literature.
2. Approaches to Explore the Genetic Potential
2.1. Random, but Simple and Successful
The easiest way to obtain more secondary metabolites
from a single strain bearing the genetic potential to produce
several compounds is to vary the culture conditions. The
rationale behind this approach is that the production of
secondary metabolites might be a specific response of the
producing organisms to a changing environment.[13] For
example, soil bacteria living in the rhizosphere have to
compete with fungi for nutrients. Thus, the production of
antifungal compounds would be advantageous in such an
environment. In contrast, the artificial media composition,
cultivation conditions, and high cell densities used under
laboratory conditions might cause regulatory cascades in the
cells thus enabling the production of some compounds but
disabling the production of others that might be produced
under different and perhaps more “natural” conditions. Since
our knowledge of any ecological network is very poor,
random approaches which vary the easily accessible cultivation parameters are chosen (media composition, aeration,
addition of chemicals, etc.) to trigger the expression of latent
biosynthetic genes. This approach might influence directly or
indirectly the transcription, translation, and enzyme activity/
specificity in the whole organism (Figure 3). Very similar
techniques have been used widely for the optimization of the
production of a desired compound, and their use in screening
for novel natural products has led to some impressive
examples of microbial chemical diversity. These methods
are regularly used today by pharmaceutical and biotech
companies.[15]
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Figure 3. Possible ways to influence the production of secondary metabolites. Isolation of new producing strains and metagenome approaches
prior to methods dealing with secondary metabolites are shown in gray. Genetic modifications and/or addition of genes leading to modified
compounds are shown in red.
After the cultivation of the aspinonene producer Aspergillus ochraceus DSM7428 in different media and cultivation
vessels (resulting in different aeration rates), 15 additional
secondary metabolites belonging to unrelated structural and
biosynthetic families were isolated. Of particular note was the
static cultivation for several days which led to a completely
different metabolite profile (Scheme 3).[56, 57] Similar results
were also obtained with another fungus which produced up to
2.6 g L 1 of a reduced precursor of the main metabolite
(120 mg L 1 in shaking culture) under similar conditions.[58]
Since variation of the growth conditions very often results in
the production of several compounds by a single organism,
this approach was termed “one strain—many compounds”
(OSMAC).[56] Recently, the addition of organic solvents and
signaling compounds to growth media was even used to
increase the chemical diversity of selected strains.[59, 60]
2.2. Possible Ways to Use the Endless Resources
The microbial world cultured in the laboratory represents
less than one percent of the microbial diversity in different
natural environments.[61] The biosynthetic potential of this
unexplored resource is nearly endless and raises the question:
Why do we only work with this limited number of bacteria?
The answer to this is that these “lab” strains behave well in
artificial conditions and can be cultivated easily. Microorganisms currently in use grow fast in pure culture and reach high
cell densities. However, fast growth is not advantageous when
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nutrients are not available. Too much food might even kill
cells if they require a specialized diet. In nature, poor nutrient
conditions represent the rule and not the exception, and areas
completely devoid of nutrients occur.
New techniques are currently being developed to cultivate
and enrich formerly “not-yet-culturable” organisms.[62–65] The
work required is very laborious and time-consuming. Therefore, additional techniques have been developed that circumvent the cultivation of organisms but still enable the exploration of chemical diversity. DNA libraries from environmental
samples are generated bearing the genetic information of all
organisms present at a specific location at the sampling
time.[66–68] After sequencing such so called “metagenomes”,
the genetic information of single organisms can still be
assembled and analyzed by using established bioinformatic
tools, as was most impressively shown recently for the
microbial community of the Sargasso Sea.[69] Furthermore,
the DNA libraries generated in this way can be transferred
into suitable host organisms that can then be tested for the
production of the desired compounds and enzymes (see also
Section 2.5). This methodology is especially important for
those potent biologically active compounds which have been
isolated from organisms not amenable to large-scale cultivation (for example, bryozoae, sponges, and molluscs).[70]
Intriguingly, structural similarities exist between several
marine natural products of unknown origin and compounds
isolated from soil bacteria (Scheme 4). On the basis of genetic
evidence, it is now generally accepted that several of the
“marine” natural products from higher organisms are in fact
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Scheme 3. Secondary metabolites isolated from Aspergillus ochraceus DSM7428 after variation of the cultivation parameters; Shaking culture (SK).[56]
Scheme 4. Examples of structurally related natural products isolated from bacteria and marine organisms (a)[73–77] and of natural products
produced by noncultivated bacterial symbionts of higher organisms (b).[71, 72, 78, 79]
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marine microbial products (for example, onnamide[71] and
bryostatin[72]). Therefore, the isolation of the corresponding
biosynthetic gene clusters and their heterologous production
should become possible in the future, thus enabling the largescale production of the corresponding natural products (see
also Section 2.5).
2.3. Screening of Genomic DNA Leads to the Production of Novel
Secondary Metabolites
The enormous amount of sequence data becoming
available from all types of microbial sources has led to an
increase in bioinformatic approaches to find novel secondary
metabolites. Although this methodology is limited to the
interpretation of biosynthetic genes typical for secondary
metabolism, many novel secondary metabolic gene clusters
can be found because they are frequently related to PKS and/
or NRPS pathways. Farnet and co-workers sequenced numerous biosynthetic gene clusters that were identified by their
similarity to known genes by using a genome scanning
approach. These researches have significantly increased the
small number of known enediyne antibiotic producing
strains[80] by growing the strains identified by genome scanning under different growth conditions and subsequent
product analysis. Therefore, this approach resembles a
combination of DNA sequencing with the OSMAC methodology described above (see Section 2.1).
In addition to the complete sequencing of novel biosynthetic gene clusters, small fragments of these genomic regions
can be used to identify novel metabolites if the host strain can
be genetically manipulated. Numerous strategies for the
amplification of fragments from PKS and/or NRPS genes by
using degenerate PCR have been described and nonsequenced genomic libraries can be probed and screened for
the presence of PKS and/or NRPS gene fragments in single or
multiple biosynthetic gene clusters. Once the genomic regions
involved in natural product biosynthesis have been identified,
the fragments can be used for gene inactivation studies and
the resulting mutants and their wild-type strains can be
compared for their production profile. At least three novel
compounds were found in Stigmatella aurantiaca[81] by using
this approach (myxochelin,[82] myxochromid,[83] and aurafuron[84] ; see Figure 4).
It could be argued that these compounds could have been
found in the strain by an intense variation of the culture
conditions. However, the application of genetic knowledge
has tremendously increased the chances of identifying novel
compounds whose production is dependent on the corresponding genes. In the case of metabolites produced at low
levels, it is difficult to define whether the “novel compounds”
are indeed such or just components of the medium. The latter
can be ruled out if production is dependent on an identified
gene locus.
While many research groups focus on identifying and then
sequencing novel biosynthetic pathways, bioinformaticians
preferentially use the numerous new microbial genome
sequences to predict new pathways by in silico methods. On
the basis of PKS and NRPS enzymology logic,[21, 28, 85] even
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Figure 4. HPLC traces with UV detection (200–400 nm) showing the
correlation of biosynthesis gene clusters and the corresponding natural
products in S. aurantiaca DW4/3-1 by comparison of wild types and
mutants.[81]
structures of unknown compounds can theoretically be
predicted depending on bioinformatic gene cluster analyses.[86–89] A biosynthetic gene cluster of unknown function was
identified from S. aurantiaca that was predicted to be involved
in iron chelator biosynthesis and was indeed found subsequently to be responsible for myxochelin formation in this
strain.[82, 90] Bacillibactin was found in Bacillus subtilis on the
basis of genome analysis,[91] and coelichelin has been predicted to be an iron chelator from S. coelicolor.[92] The latter
compound was indeed found three years later.[53] Although
this bioinformatics methodology for predicting structures
seems very attractive and convincing at first, to date there are
only very few examples for the subsequent identification of
these “theoretical compounds”. This situation is presumably a
consequence of the limitations discussed below.
All predictions were based on the nonribosomal code and/
or in silico analyses of the corresponding biosynthetic PKS
and NRPS gene clusters. It turns out that there are numerous
exceptions to the commonly accepted “rules” for polyketide
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and nonribosomal peptide biosynthesis.[93, 94] One of these
rules is the biosynthetic logic of “colinearity”, which means
that a correlation exists between the number and type of
modules and the number and type of extender units
incorporated by the megasynthetase. Nevertheless, module
skipping[95] as well as iterative use of modules have been
described in PKS systems. Prediction of substrates (for
example, malonyl-CoA and methylmalonyl-CoA) is difficult,
and modules or domains can be inactive or “missing”.[94, 96]
Similarily, NRPS domains have been found to be inactive[83, 97]
and the nonribosomal code, although exceptionally useful in
many cases, turns out to be not applicable in certain
examples.[98, 99] To make the situation even more complicated,
nonclustering of bacterial biosynthetic genes also occurs,
which would make a structure prediction impossible.[99–101]
In general, if biosynthetic systems closely related to those
already described in the literature are found, predictions of
similar structures using bioinformatics seems feasible. For
more complicated cases, predictions will be very difficult
using existing tools alone. For example, neither the mupirocin[102] nor the disorazol biosynthetic genes[103] could have
been used to predict the structure of the compounds
produced. A combined approach involving more experimental biochemistry coupled to advanced bioinformatics should
improve the situation dramatically in the future.
2.4. Activation of “Silent” Biosynthetic Gene Clusters
As a viable alternative to the OSMAC and the genome
scanning approaches[80] described above, the insertion of
inducible promotors into the chromosome capable of driving
the expression of so far uncharacterized “silent” biosynthetic
gene clusters seems practicable. Although a random activation approach of gene expression has been described,[104] we
are not aware of any proof of principle for this technology in
natural product producing microorganisms. Therefore, this
idea awaits practical exploitation, although problems with
biosynthetic gene clusters requiring the insertion of numerous
promotor regions for gene activation have to be expected.
Alternatively, influencing pleiotropic or direct regulatory
mechanisms within the host cell should enable the artificial
expression of uncharacterized genes and might lead to the
(over)production of natural products. Whereas negative
regulators might be inactivated, positive regulators can be
overproduced. A recent example describes the partial activation of a silent angucycline-type gene cluster from a
streptomycete.[105] A putative repressor gene was inactivated
which resulted in the production of angucycline metabolites.
The ferric uptake regulator (Fur) controls numerous regulatory genes, such as pvdS in Pseudomonas aeruginosa, which
encodes an alternative sigma factor. A hierarchical cascade of
direct and indirect iron regulation has been analyzed by
transcriptome analysis in response to iron.[106] Strong iron
regulation was observed for previously identified genes
involved in biosynthesis or uptake of the siderophores
pyoverdine and pyochelin. Iron-controlled regulatory genes
include seven alternative sigma factors and five other transcriptional regulators.
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Additionally, xenobiotics and pheromones can influence
the expression of biosynthetic pathways.[107] Although the role
of S-adenosyl methionine (SAM) in antibiotic production is
far from understood, a correlation between actinorhodin
production and the level of SAM in the cell has been
reported.[108, 109] Another example of defined chemicals that
exhibit secondary metabolite regulatory functions in streptomycetes are the g-butyrolactones.[110] Davies and co-workers
have shown convincingly that even antibiotics themselves,
when present at subinhibitory concentrations, influence the
expression of numerous genes.[111] Secondary metabolites
might very well be involved in the regulation of their own
biosynthesis or of the biosynthesis of similar or totally
unrelated compounds in different microorganisms and thus
might be used to induce the production of additional
compounds. Recently, Uchiyama et al.[112] have shown in an
elegant approach that novel metagenomic catabolic gene
clusters can be identified. They measured the expression of a
metagenome reporter gene library after induction with a
variety of chemicals. Next, they used a fluorescence-activated
cell-sorting system (FACS) to identify clones in which
expression was induced, and then characterized the activated
geneEs product(s). One can envisage cloning fragments of
“silent” biosynthetic gene clusters in a similar approach and
finding low-molecular-weight inducers that enable the expression of secondary metabolite genes. Once inducers of the
respective promotors are identified, they could be used to
trigger expression of the silent genes in the natural host.
However, since the size of many biosynthetic gene clusters
makes complete cloning difficult, this approach does not seem
feasible for complete heterologous expression of a whole
gene cluster (see Section 2.5).
The study of the regulatory mechanisms underlying
secondary metabolism is still tedious and time-consuming
for examples where genomic sequences are not available. This
situation is changing rapidly because of a variety of ongoing
functional genome projects of natural product producers (for
example, S. coelicolor, S. avermitilis, B. subtilis, and Sorangium cellulosum). The availability of genomic information
sets the stage to study complex regulatory mechanisms by
using transcriptomic and proteomic tools. Moreover, the
genome sequence from one species enables the identification
of regulatory and metabolic proteins required for the
biosynthesis of the desired compound from closely related
strains.[113]
2.5. Heterologous Expression of Biosynthetic Gene Clusters
Numerous PKS and NRPS biosynthetic gene clusters have
been cloned over the past decade, and the increasing
biochemical knowledge from studies dealing with the corresponding enzymes has led to the emergence of combinatorial
biosynthesis technologies that aim at generating novel compounds through genetic manipulation.[114, 115] In principle, the
availability of the genetic information makes heterologous
expression possible in improved production hosts. This
technology can also be used to express biosynthetic pathways
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from currently not-yet-culturable microorganisms (see Section 2.2).
Genetic manipulation is straightforward in some model
strains (for example, S. coelicolor and B. subtilis), but the
genetic tools are insufficient or not developed for many
microbial producers of natural products. A number of them
are slow-growing or currently not-yet-culturable and thus not
suited for genetic modification of biosynthetic pathways (for
example, myxobacteria, cyanobacteria, and presumed bacterial symbionts as producers of “marine natural products”; see
Section 2.2).
These circumstances and the rapidly increasing number of
microbial genome sequences have led to a considerable
demand in heterologous expression techniques for complete
secondary metabolite pathways in natural product research
and drug discovery. It is a viable alternative to the improvement of the strain and the fermentation process as well as to
the genetic modification of the native producer strain.[116]
A number of strategies for the heterologous expression of
secondary metabolite pathways have been described, which
range from the targeted expression of specific natural
products to the expression of large unknown DNA fragments
(also termed the expression of the metagenome; see Section 2.2).[117] Bacterial artificial chromosome (BAC) shuttle
vectors were used for constructing environmental libraries,
followed by expression in multiple expression hosts (for
example, pseudomonads, streptomycetes, and Escherichia
coli).[67, 118] However, only products from relatively small
biosynthetic gene clusters have been produced with this
method to date, and the producing heterologous host has
mostly been from the same genus as the natural producer
(Scheme 5).[118]
Similarly, there are a number of reports on the heterologous production of biosynthetic pathways from bacteria in
closely related genera. As an example, Marahiel and coworkers have expressed and engineered the bacitracin
biosynthetic pathway from Bacillus licheniformis in B. subtilis
which produces almost twice as much of bacitracin as the
natural producer.[125] Salas and co-workers have recently
generated more than 30 derivatives of the antitumor compound indolocarbazole in yields comparable to those from the
original producer after heterologous expression of varying
combinations of biosynthetic genes in Streptomyces albus.[126]
Most of the examples in the literature relate to small type II
PKS systems,[127–132] whereas the heterologous expression of
large type I PKS and/or NRPS gene clusters has been
achieved by coexpression from several plasmids, each harboring parts of the biosynthetic pathway.[133, 134] Expression of the
epothilone biosynthetic gene cluster from Sorangium cellulosum in Myxococcus xanthus[135] is another example. The
methodology employed relied on several rounds of cloning
and heterologous host modification because no autonomously
replicating units are known for myxobacteria. Whereas the
yields in this case were only 1 % of that produced in
S. cellulosum, optimization of the medium resulted in a
respectable increase in epothilone titer up to the level of
the original natural producer.[136] Nevertheless, it has to be
considered that the original strain has in the meantime been
significantly improved for production by classical methods,
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Scheme 5. Low-molecular-weight compounds produced from
fragments of different metagenomes.[119–124]
similarly to the soraphen-producing strain S. cellulosum
So ce26.[137]
Only within the last few years has it been shown that the
production of complex natural products in unrelated bacteria
is feasible. In these elegant studies a genetically engineered
E. coli host was used to produce the aglycone precursor of
erythromycin.[116] Subsequently, precursors of ansamycin
macrolactam were produced in E. coli after expression of
part of the biosynthetic operon.[138] Epothilone as well as
soraphen derived from myxobacteria that belong to the genus
Sorangium have been produced in streptomycetes, although
in very low yields.[139, 140] Despite increasing experience with
the various hosts, the product yields have typically been much
lower than those in the original producer. This might be a
consequence of weaknesses in the natural promotor, problems with expression levels of correctly folded proteins,[141]
difficulties with the essential posttranslational activation of
carrier proteins,[142] differences in mRNA stability, or precursor supply, to name just a few possible drawbacks of the
technique. Numerous ongoing studies in many laboratories
are addressing these questions in various ways, including the
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application of novel genetic engineering systems and the use
of a variety of heterologous hosts.
In all of the studies mentioned above, classical and timeconsuming cloning procedures were employed, and the
expression of large secondary metabolite clusters was only
achieved by coexpression from several genetic units.
Recently, the application of recombinogenic engineering[143]
in E. coli for cloning and manipulating large natural product
assembly lines has been used successfully by several research
groups. This technique is particularly suitable for large DNA
molecules such as BACs and cosmids, as it does not rely on
classical cloning procedures. After overexpression of proteins
facilitating efficient homologous recombination (Red/ET) in
E. coli, PKS and NRPS pathways can be altered and
redesigned based on short homology arms.
Researchers at Cubist have applied this methodology to
engineer the daptomycin biosynthetic pathway from Streptomyces roseosporus in E. coli on a BAC; they then expressed it
in a daptomycin-negative mutant of S. roseosporus to produce
novel and active lipopetides.[144] In this example, a closely
related (here in fact the same) expression host was used,
which is feasible and sensible when well-developed hosts are
available. As this prerequisite has not been achieved in many
areas of research, efficient heterologous expression of biosynthetic gene clusters from microbial producers is still a
challenging task, especially for the slow-growing or not-yetculturable sources. In a recent example, Red/ET recombineering[143] has also been used to rebuild the complete assembly
line from the myxobacterial myxochromid PKS/NRPS hybrid
gene cluster in one genetic unit and to introduce the elements
required for conjugation, stable integration, and regulated
expression in the heterologous host Pseudomonas putida.[97]
One of the important advantages of this method is that the
whole secondary metabolite pathway is located on one
construct, which can be accurately manipulated in E. coli
and transferred into the heterologous host strain. Surprisingly,
the fermentation time is reduced threefold when producing
myxochromide S in P. putida while the product yield is
fivefold higher relative to that of the natural producer
S. aurantiaca. P. putida is able to produce all the required
activated precursors for myxochromide S biosynthesis
(acetyl-CoA, malonyl-CoA, and propionyl-CoA) and
expresses a broad spectrum phosphopantetheinyl transferase
which is necessary for activation of the carrier protein.[145] As
genetic manipulation in P. putida is straightforward, it should
be possible to engineer the strain to produce further
precursors that are essential for the biosynthesis of other
secondary metabolites (for example, unusual extension units
and amino acids) from various sources. Therefore, this
strategy, which combines the power of advanced DNA
engineering and mutagenesis in E. coli with the utility of
pseudomonads as the heterologous host, provides a new
alternative for the analysis and mutagenesis of known and
unknown secondary metabolite pathways.
Another alternative approach focuses on artificial DNA
synthesis of complete biosynthetic gene clusters. This
approach depends on the ability to synthesize long, accurate
DNA sequences efficiently. The idea was validated by
building a synthetic PKS gene cluster whose functionality
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was demonstrated by its ability to produce the megaenzyme
and its polyketide product in Escherichia coli by synthesizing
approximately 5-kb segments of DNA, followed by their
assembly into longer sequences by conventional cloning
methods.[146]
It should be pointed out that it is unlikely that there will
be a single solution to the immense variety of heterologous
expression problems in the future. Closely related and wellestablished production hosts which can be manipulated
genetically may be regarded as first-choice organisms. However, since these are often unavailable, as many production
hosts as possible should be further developed to provide a
battery of possible expression technologies, the strength and
weaknesses of which will become evident in the future.
2.6. Combinatorial Biosynthesis
2.6.1. Genetic Engineering of the Natural Host
The modularity and versatility of PKS and NRPS as well
as challenges and opportunities in combinatorial biosynthesis
have been discussed in excellent reviews.[24, 147–153] To illustrate
our growing knowledge related to the genetic engineering and
the production of novel compounds with altered structures,
we refer here to some studies that have been published in
recent years. Further examples of combinatorial biosynthesis
approaches are presented in Section 2.5 (heterologous
expression techniques).
The Leadlay and Staunton research groups have extended
their pioneering work in PKS research by engineering starter
unit specificity in the erythromycin-producing PKS in vivo.[154]
They were even able to show in the same biosynthetic system
that a longer polyketide chain can be generated after insertion
of an extra module into the PKS.[155] The same research group
was also able to produce ivermectin-like drugs after domain
exchange in the avermectin PKS of S. avermitilis although in a
reduced overall yield.[156] In yet another example they
employed the loading domain of the soraphen PKS, which
was speculated to load benzoyl-CoA onto the megasynthase
from S. cellulosum.[157] To prove this hypothesis, this domain
was used to make a DEBS1-TE hybrid (fusing the thioesterase domain of DEBS3 to DEBS1 and exchanging the
erythromycin-loading module against the soraphen loading
module), which resulted in the production of 5-phenyltriketide lactone with a yield of approximately 1 % of the regular
DEBS1-TE triketide.[158] Subsequent studies showed that
additional phenyl-substituted polyketides could also be
made.[159]
Researchers at Kosan Biosciences and Stanford University have performed equally impressive work in PKS engineering.[160, 161] They were able to reengineer the genes that
encode the erythromycin formation to produce the antiparasitic agent megalomicin in Saccharopolyspora erythraea in
better yields than could be obtained from the natural
producer.[162] They also performed studies to understand the
substrate specificity of PKS modules by generating hybrid
multimodular synthases.[163] Katz and co-workers produced a
new epothilone derivative in good yields by engineering the
epothilone megasynthetase (albeit not in the natural pro-
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ducer, see above).[164] The combinatorial biosynthesis of
pikromycin-related macrolides in Streptomyces venezuelae
has also been studied[165] and led to the generation of multiple
bioactive macrolides by hybrid modular PKSs in this streptomycete.[166]
Combinatorial biosynthesis using type II PKSs is the focus
of several research groups.[114, 115, 131, 167] Chromomycin analogues differing in the methylation, acylation, and glycosylation pattern were recently made by manipulating the respective post-PKS genes.[168, 169] Other examples of the engineering
of biosynthetic pathways using such post-PKS enzymes are
derived from the characterization of glycosyl transferases,
which can be employed to generate novel structures in vivo
and in vitro (see Section 2.6.2).[170, 171] It can be expected that
genomic information from natural product producers will
provide increasing knowledge about natureEs secondary
metabolite assembly tool box which can be applied in various
ways to rational pathway engineering.
All the examples reported in this section have resulted in
the production of new compounds, however, there may be
many more examples that did not work at all and will
presumably never appear in the literature. A failed example
of pathway engineering is related to various manipulations of
the myxothiazol megasynthetase in S. aurantiaca. Although
relatively simple genetic manipulations were performed,
neither intermediates nor expected derivatives of the biosyn-
thetic pathway could be observed, no matter what part of the
biosynthetic gene cluster was manipulated (Scheme 6).[172]
2.6.2. In Vitro Studies Using Biosynthetic Enzymes
Biosynthetic genes identified in the search for new
secondary metabolic gene loci are widely used for the
expression of catalytic modules and domains for in vitro
studies with purified enzymes. These recombinant proteins
were initially employed to decipher the biochemistry of
biosynthetic pathways[28, 173, 174] and in vitro reconstitutions of
complex natural product biosyntheses were reported.[90, 175, 176]
In vitro combinatorial biosynthesis techniques have mostly
relied on genetic engineering and mutation in E. coli because
this technique is much faster than the engineering of most, or
probably all, typical secondary metabolite producers. Consequently, most functional biochemical and also combinatorial studies have been performed with proteins that could be
actively expressed in E. coli. Pioneering work by the Marahiel
and Walsh research groups has shown that recombinant
NRPS and PKS proteins can be engineered to produce novel
and altered products.[30, 174, 177] The feasibility of combinatorial
biosynthesis to make commercially useful compounds has
been shown by creating a recombinant NRPS capable of
synthesizing in vitro a-l-aspartyl-l-phenylalanine, the precursor of the artificial sweetener aspartame.[178] New inter-
Scheme 6. Selected variations of erythromycin (a) and its aglycone 6-deoxyerythronolide B (b) obtained by combinatorial biosynthesis and mutasynthesis (italics). Overall, more than 100 derivatives have been generated by using combinatorial biosynthesis and mutasynthesis.
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mediates of epothilone[179] and rifamycin[180, 181] biosynthesis,
but with alternative starter units, were made using purified
proteins from the respective gene clusters. In another study,
the modification of the acyl carrier protein with alternative
starter units enabled the biosynthesis of novel polyketides
with 16 (instead of 14) carbon atoms by employing the
minimal actinorhodin type II PKS.[182] An alternative way to
generate chemical diversity using purified bacterial biosynthetic proteins is exemplified by recent in vitro studies using
the iterative and relatively small type III PKSs. These “plantlike PKSs” were first described by the Horinouchi research
group.[27, 183] It was realized that these enzymes are commonly
found in bacteria[23, 184–186] and that they are versatile systems
for generating novel products.[187–190] Up to 14 compounds of
different structural types (pyrones and phenoles varying in
side-chains length) have been produced in vitro by using
RppA (type III PKS involved in red-brown pigment production) to elongate five different starting units with malonylCoA.[187] Type III PKS are also involved in the formation of
precursors of glycopeptides by NRPS; such enzymes from the
balhimycin and the vancomycin biosynthetic gene clusters
were identified as synthases of 3,5-dihydroxyphenylacetic
acid, which serves as a precursor for the unusual amino acid
3,5-dihydroxyphenylglycine[191, 192] that is part of the glycopeptide backbone.
Recently, proteins or excised catalytic domains from
natural product biosynthetic pathways have also been used
in chemoenzymatic syntheses. PKSs and NRPSs responsible
for the production of macrocyclic compounds often employ
their C-terminal thioesterase (TE) domain to catalyze
enzymatic cyclization of a linear precursor.[193] Walsh and
co-workers showed that excised TE domains retain their
autonomous ability to catalyze the macrocyclization of linear
peptide thioesters with native peptide sequences. Additionally, they can cyclize peptide analogues with alterations in the
peptide sequence and peptide/polyketide-like hybrids with
longer chain lengths.[194] Thus, these macrocyclization catalysts raise the prospect of using TE catalysis for the
generation of macrocyclic peptide libraries.[195] Isolated TEs
can also catalyze the cyclization of linear peptides immobilized on a solid-phase support, which offers the possibility to
merge natural product biosynthesis with combinatorial solidphase chemistry.[196] In an extension of this approach, even
glycosylated glycopeptides could be cyclized in good
yields.[197]
Glycosyltransferases are frequently found as tailoring
enzymes associated with products derived from PKS and
NRPS. They play a key role in the decoration of natural
product core structures with sugar moieties that are often
essential for biological activity. The corresponding proteins
utilize activated sugars and can be altered genetically to
accept and glycosylate a variety of substrates. Thorson and coworkers pioneered this approach and called it “glycorandomization” (Scheme 7).[198, 199] Progress in the understanding of
deoxyhexose and aminosugar biosynthesis will facilitate the
future use of new sugars or those which are difficult to
prepare chemically.[200, 201]
In general, it can be expected that any type of tailoring
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tase (for example, glycosyl transferases, methyl transferases,
hydrolases, P450-dependent enzymes, or prenylating
enzymes) can be used for similar in vitro studies in the
future. As genome sequencing efforts reveal more and more
of the genetic basis of microbial biochemical diversity, the
corresponding biosynthetic genes await exploitation in combinatorial approaches.
2.7. Mutasynthesis
The extraordinary possibilities of the combination of
genomic techniques with “old-fashioned” mutasynthesis[204]
has been excellently reviewed.[205, 206] We will thus only
provide some recent examples which best exemplify the
potential of this approach.
Biosynthesis of synthetic precursors has been used
extensively in polyketide and nonbribosomal peptide producing organisms using precursors that are activated as Nacetylcysteamine (NAC) thioesters which mimic the carrierprotein-bound biosynthetic intermediates.[207–212] In mutasynthesis approaches, the efficiency of the incorporation of an
intermediate which is fed to a culture can be significantly
increased if the biosynthesis of the natural precursor is
blocked by mutagenesis. Hertweck and co-workers generated
the novel cytostatic compound aureonitrile using p-cyanobenzoyl-SNAC as an unnatural starter unit of the aureothin
biosynthesis in a mutant unable to produce the natural primer
p-nitrobenzoyl-CoA.[213] The authors showed that the addition of p-cyanobenzoate is sufficient for the production of
aureonitrile, presumably because the endogenous p-nitrobenzoate CoA ligase can activate both precursors (Scheme 8).
The biosynthesis and the mode of attachment of a wide
range of PKS starter units in bacteria are covered in a recent
review by Moore and Hertweck.[215] A prominent example of
the practical application of a combined mutasynthesis/combinatorial biosynthesis approach is the production of the
unnatural natural compound doramectin, a derivative of the
anthelmintic type I polyketide avermectin that differs in the
starting unit.[216]
Avermectin biosynthesis is usually primed with thioesters
of activated short branched-chain carboxylic acids which are
derived from the degradation of branched-chain amino acids.
Once this degradation pathway is inactivated, avermectin
production is stalled. However, the lack of precursors can be
overcome by the addition of cyclohexylcarboxylic acid (or
other related precursors), which leads to the production of
doramectin, a broad-spectrum antiparasitic compound with
activity superior to avermectin. After a pathway involved in
the biosynthesis of cyclohexylcarboxyl-CoA has been identified in Streptomyces collinus,[217] Reynolds and co-workers
transferred these genes into the S. avermitilis mutant impaired
in the production of the natural starter unit. This approach led
to the direct production of doramectin in excellent yields that
were comparable to those found after the exogenous addition
of cyclohexylcarboxylic acid to the S. avermitilis mutant.[214]
The experiment represents metabolic engineering rather than
mutasynthesis, because no precursor or intermediate is added
to the fermentation broth.
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Scheme 7. In vitro derivatization and synthesis of natural products. a) General outline of the glycorandomization approach.[198] b) Glycorandomization applied to vancomycin.[202] c) Generation of tyrocidine analogues by using a combination of chemical synthesis and thioesterase-catalyzed cyclization.[194, 197, 203]
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Scheme 8. Generation of aureonitrile by mutasynthesis (a) and doramectin by metabolic engineering (b).[213, 214]
Combinations of mutational and chemoenzymatic
approaches have been exploited to generate new rapamycins[218] and aminocoumarins.[219, 220] Novel glycopeptides were
made using the biosynthetic potential of halogenases.[221]
The potential of mutasynthesis can be evaluated prior to
in vivo experiments in studies employing recombinant biosynthetic enzymes, in an analogous way to the work described
in Section 2.6.2. Walsh and co-workers could show in vitro
that the first modules of the epothilone synthetase can utilize
alternative starter units and substrates to generate novel
intermediates.[179, 222] It will be interesting to see whether these
experiments will lead to the large-scale production of novel
epothilones in vivo as well.
As can be deduced from the examples given above, there
often appears to be no clear borderline between the types of
approaches described. Several commonly used phrases such
as “combinatorial biosynthesis” and “mutasynthesis” are not
well defined and might be summarized as different forms of
“metabolic (or biosynthetic) engineering” techniques. The
strengths of the experimental ideas lie in the application of
combined techniques for the particular problem to be solved.
ular basis of natural product formation have been described in
this Review, and technologies exploiting this knowledge for
the generation of new natural products with biological activity
have been summarized. These techniques have already led to
the production of several novel compounds. The prerequisite
of rational pathway engineering is a comprehensive understanding of complex natural product biosynthesis at the
molecular level, but since this has not been achieved in many
cases, numerous manipulations of biosynthetic pathways are
ineffective or lead to low production yields; thus, further in
depth biochemical studies are needed. Furthermore, exponentially increasing sequencing information, novel cultivation
techniques, metagenomic approaches, and progress in heterologous expression and molecular biology in general are
expected to give natural products research a promising future.
The rapid progress in all areas of metabolic/biosynthetic
engineering offers opportunities to alter and/or derivatize
auspicious natural product leads in vivo and in vitro. Considering the structural complexity of natural products, this
approach might well become a cornerstone of pharmaceutical
lead development in the future.
3. Be Prepared! Natural Product Research in the
Postgenomic Era
Research in the authors laboratory was supported by the DFG
and the BMBF. The authors would like to acknowledge Mark
Zabriskie, Barrie Wilkinson, Christian Hertweck, and an
unknown reviewer for providing helpful comments.
The impact of increasing genomic information as well as
biosynthetic and regulatory studies that decipher the molecAngew. Chem. Int. Ed. 2005, 44, 6828 – 6846
Received: March 25, 2005
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