For half a century, natural products from microorganisms have been the main source of
medicines for treating infectious disease. The most important chemical class of these
antibiotics, apart from the penicillins, is the polyketides. They are made by the stepwise
building of long carbon chains, two atoms at a time, by multifunctional enzymes that
determine the chain length, oxidation state, and pattern of branching, cyclisation, and
stereochemistry of the molecules in a combinatorial fashion to produce an enormous variety
of structures. Recent elucidation of the genetic ‘programming’ of the enzymes has opened a
new field of drug discovery based on rationally engineering the enzymes to produce
‘unnatural natural products’ with novel properties.
Following the development of penicillin for the treatment of septicemia in the early
1940s, numerous antibiotics were discovered and introduced into medicine. While a fungus
makes penicillin, semisynthetic derivatives of which have been a mainstay of antibacterial
therapy for decades, most natural antibacterial antibiotics come from a group of
soil-dwelling, filamentous bacteria called the actinomycetes, of which
Streptomyces is the best-known genus. These organisms make an
amazing array of so-called secondary metabolites that have evolved to give their producers
a competitive advantage in the complex soil environment, where they are exposed to stresses
of all kinds (Challis and Hopwood 2003). The compounds have many functions, but those with
antibiotic activity are the most important from the human perspective. Actinomycete
antibiotics include such antibacterial compounds as tetracycline and erythromycin,
antifungal agents like candicidin and amphotericin, anticancer drugs such as doxorubicin,
and the antiparasitic avermectin (Walsh 2003).
While many different chemical classes are represented amongst actinomycete antibiotics,
one class accounts for an extraordinary proportion of the important compounds, including
all those mentioned above. This chemical family is made up of the polyketides. They are
synthesized by multifunctional enzymes called polyketide synthases (PKSs), which are
related to the fatty acid synthases that make the lipids essential for the integrity of
cell membranes, but they carry out much more complex biosynthetic routines. Repeated rounds
of carbon chain building and modification use a series of independently variable reactions
selected according to a ‘program’ characteristic of each PKS (Reeves 2003). Recent research
has focused on determining this program so as to be able to modify it in rational ways by
genetic engineering and thus generate novel drug candidates. The resulting field of
‘combinatorial biosynthesis’ of ‘unnatural natural products’ has been given added urgency
by the rise of multidrug-resistant pathogens, of which MRSA (methicillin-resistant
Staphylococcus aureus ) is simply the most discussed of a series
of threats (Walsh 2003). How do PKSs work and how can we make new ones?
Molecular Diversity
The heart of PKS function is the synthesis of long chains of carbon atoms by joining
(condensing) together small organic acids, such as acetic and malonic acid, by a so-called
ketosynthase function. This uses the building units in the form of activated derivatives,
called coenzyme A (CoA) esters, so we speak of acetyl-CoA and malonyl-CoA, for example. The
special form of condensation that joins them is driven by loss of carbon dioxide. Thus,
when acetyl-CoA, with two carbon atoms, joins with malonyl-CoA, with three carbons, one of
the latter is lost and a chain of four carbon atoms results (Figure 1A). Further rounds of
condensation extend the chain by two carbons at each step. If the chain-extender unit,
instead of being malonyl-CoA, is methylmalonyl-CoA, which has four carbon atoms, the linear
carbon chain is still extended by two carbons, and the ‘extra’ carbon forms a methyl side
branch. More complex extender units produce more complex side branches.
Choices of the number and type of the building units are variables in determining
polyketide structure. Another concerns the keto groups (C=O) that appear at every alternate
carbon atom in the growing chain as a result of the condensation process (accounting for
the name polyketide). They may remain intact. Alternatively, some may be modified or
removed by a series of three steps (Figure 1B), any of which may be omitted. This results
in keto groups remaining at some points in the chain; hydroxyl groups (–OH), formed by
reduction of a keto group, at others; double bonds between some adjacent carbon atoms,
resulting from removal of the hydroxyl by loss of water (dehydration); or full saturation
with hydrogen atoms elsewhere, arising by ‘enoyl’ reduction of the double bond (Figure 1C).
A further variable concerns the stereochemistry of the hydroxyl groups and methyl or other
carbon branches, each of which can exist in two possible configurations. Finally, the
nascent carbon chain adopts different folding patterns after it leaves the PKS, and
‘tailoring’ enzymes can then add sugars or other chemical groups to it at many alternative
positions, enabled by the pattern of chemical reactivity built into the polyketide by the
PKS. The challenge has been to understand the programming of the PKS that accounts for this
gamut of structural variation.
During the 1990s, the ability to manipulate actinomycete genes, developed over previous
decades, mainly using the model species
Streptomyces coelicolor (Hopwood 1999), was combined with
chemical and biochemical experiments to begin to crack this ‘polyketide code’. The first
studies were on organisms making antibiotics of the ‘aromatic’ family, which includes
tetracycline and doxorubicin, as well as the model compounds actinorhodin (made by
S. coelicolor itself) and tetracenomycin. The main variable in
their structure is carbon chain length, with few choices of different building units or
keto group modification, so the programming would (in principle) be simple. The DNA
sequences responsible for such PKSs revealed sets of genes encoding proteins, including
ketosynthases, ketoreductases, and acyl carrier proteins (ACPs) (the unit of the PKS on
which the growing carbon chain is tethered; see Figure 1A), that would come together to
form a multicomponent PKS resembling a typical bacterial fatty acid synthase. In contrast,
the DNA sequence of the gene set for the complex polyketide erythromycin, made by a
relative of
Streptomyces called
Saccharopolyspora erythraea , which has more involved
programming, revealed multifunctional proteins with the various enzymic functions carried
out by active sites on the same polypeptide chain, as in a mammalian fatty acid
synthase.
The big surprise, though, was the finding of six sets, or modules, of such active sites,
corresponding to the six rounds of condensation needed to build the carbon chain (Cortes et
al. 1990; Donadio et al. 1991). The modules each contain an acyl transferase (to load the
extender unit onto the enzyme), as well as a ketosynthase and an ACP domain, together with
exactly those reductive activities needed to generate the required pattern of modification
of the chain at each step of elongation. Thus was born an ‘assembly line’ model in which
the program for the PKS is hardwired into the DNA and expressed in a linear array of active
sites (domains) along the giant protein. This consists of the six chain-building modules,
preceded by a short module for loading the starter unit and ending in a domain for
releasing the completed carbon chain from the PKS. The carbon chain of the polyketide would
be assembled and modified progressively as the molecule moved along the protein,
interacting with each domain in turn, which would select extender units, make carbon–carbon
bonds, and modify keto groups as appropriate, depending on the presence or absence of
domains for the three steps in the reductive cycle.
The model arose from the gene sequence, but was rapidly tested by mutating individual
domains or adding or deleting whole modules and by observing predicted changes in the
polyketide product. Soon, dozens of engineered compounds had been made, and the field
mushroomed with the isolation of more and more clusters of genes for complex polyketides
that both proved the generality of the model (with minor variations) and filled the need
for spare parts for the engineering of countless new polyketides (Shen 2003). Several
biotech companies were founded to exploit the potential for drug discovery.
Aromatic PKS Programming
Meanwhile, the programming of the aromatic PKSs was harder to understand. They had been
found to contain only a single ketosynthase, which has to operate a specific number of
times to build a carbon chain of the correct length, so how is this determined? How does a
single reductive enzyme know which keto groups to modify? And how is the starter unit for
building the carbon chain selected (the extender units are normally all malonyl-CoA, so no
choice is involved)? Considerable progress had been made in constructing novel compounds by
mixing and matching PKS subunits, but this was largely based on empirical knowledge about
which components to put together (McDaniel et al. 1995). A specific subunit of the PKS,
named the chain length factor (CLF), was deduced to have a major influence on carbon chain
length (McDaniel et al. 1993), but this conclusion was not universally accepted in the
absence of experimental evidence on its mode of action. Two recent publications by the
Khosla laboratory at Stanford University describe significant advances in understanding
aromatic PKS programming and promise to turn the spotlight back onto engineered members of
this class of compounds as potential drug candidates by allowing rational manipulation of
the two key variables: carbon chain length and choice of starter unit.
In the first paper (Tang et al. 2003), the authors explore the hypothesis that the CLF
exerts control over carbon chain length by associating closely with the ketosynthase, a
protein with which it shares considerable amino acid sequence similarity, giving rise to a
channel of a certain size at the interface between the two proteins. By systematically
changing amino acids at four key positions in the CLF, the size of the channel was altered.
Thus, large amino acid residues in the CLF of a PKS that makes a 16-carbon chain were
replaced by less bulky residues found in one that builds a 20-carbon chain, and the chain
length of the product increased as expected. The authors propose that the length of the
channel is the main factor in controlling the number of chain-extension steps that can take
place to fill it. While protein–protein interactions with other PKS subunits may modulate
this chain length control, the work represents a major step in understanding and
manipulating the chain length of aromatic polyketides.
What about the choice of starter unit? Most aromatic polyketides start with acetyl-CoA.
An important earlier publication by Leadlay and colleagues (Bisang et al. 1999) had shown
that this is not loaded directly onto the PKS, as had been assumed, but is derived by loss
of carbon dioxide from a molecule of malonyl-CoA previously loaded onto the enzyme. This
decarboxylation is catalysed by the CLF as an activity independent of its role in
influencing carbon chain length. There are, however, certain aromatic polyketides,
including the anticancer drug doxorubicin, an antiparasitic agent called frenolicin, and
the estrogen receptor agonist R1128, that have different starters.
What Tang et al. (2004) have deduced, as described in this issue of
PloS Biology , is that the PKSs for these compounds consist of two
modules of active sites. The components of each module are not activities carried on the
same protein, as in the PKSs for the complex polyketides, but are all separate proteins.
They form functional modules nevertheless. The newly recognized modules in the producers of
compounds that start with nonacetate units have a dedicated ACP and a special ketosynthase
that carries out a first condensation, joining the unusual starter unit to the first
malonyl-CoA extender unit. The starter module then hands the resulting ‘diketide’ on to the
second module (first reducing it, if appropriate, using reductive enzymes ‘borrowed’ from
fatty acid biosynthesis) for typical extension by successive condensation with malonyl-CoA
units to complete the chain. If the starter module is not present, the second module
defaults to its typical habit of decarboxylating malonyl-CoA to acetyl-CoA and starts the
chain with that.
The excitement of the work for biotechnology is that it offers the prospect of
engineering promising drug candidates by making novel combinations of starter and extender
modules and perhaps of feeding the starter modules with a whole range of unnatural
substrates (Kalaitzis et al. 2003). It is encouraging that already, in the
proof-of-principle studies reported by Tang et al. (2004), some products with improved in
vitro antitumor activity were obtained.
