Polyketides are a broad family of pharmaceutically and industrially important natural products.1-5 They are produced by bacteria, fungi, plants, and other organisms, and display a plethora of chemical structures and functions. Examples include lasalocid A 1, a coccidiostat biosynthesised from the soil bacterium Streptomyces lasaliensis6; actinorhodin 2, a complex aromatic antibiotic from Streptomyces. coelicolor7; phloroglucinol 3, a small aromatic metabolite from Pseudomonas fluorescens that displays a range of activities including antibacterial action8; resveratrol 4, an antioxidant found in grapes commonly reported as being responsible for the so- called ‘French paradox’9
; and lovastatin 5, a cholesterol lowering compound, and precursor to the drug simvastatin, biosynthesised by the fungus Aspergillus terreus10
(Figure 1). Polyketides also include many anticancer and antitumour agents,
insecticides, antifungals and antiviral compounds.
Figure 1 Examples of polyketides from bacteria (Lasalocid A 1, Actinorhodin 2, and Phloroglucinol 3), plants (Resveratrol 4) and fungi (Lovastatin 5).
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1.1.1. Fundamentals of polyketide biosynthesis
Polyketides are biosynthesised by polyketide synthase (PKS) enzymes. PKSs are roughly categorised into three types per the organisation of their domains and their catalytic activities (Scheme 1). While type I and type III PKSs are single proteins comprising multiple domains/catalytic activities, type II PKSs consist of discrete enzymes or domains that act iteratively to produce complex aromatic compounds.11-
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Type I PKSs can be either ‘modular’ or iterative. Modular PKSs biosynthesise polyketides in a step-wise manner, utilising ‘modules’: these are groups of enzymes or domains which catalyse loading, elongation or termination of the polyketide chain where each domain is used only once. Conversely, iterative type PKSs use each of their domains multiple times. Type III PKSs are always iterative, but differ from type II in that coenzyme A (CoA) is the acyl carrier, whereas an acyl carrier protein (ACP) is used in all other types of PKS.
The common and key step in polyketide biosynthesis is carbon chain formation and elongation by decarboxylative Claisen condensation of malonate units (bound to acyl carrier proteins (ACPs) in type I and II PKSs or to Coenzyme A in type III PSKs) onto acyl groups bound to ketosynthase (KS) domains (Scheme 1). Upon new carbon-carbon bond formation, the extended polyketide chain bound to ACPs or CoAs is transferred onto KSs of the same enzyme/module for iterative systems, or to downstream modules in modular systems for further elaboration. This process is repeated a pre-programmed number of times until an ACP bound n-ketide is generated (Scheme 2). Starter and extender units include methyl, ethyl or propionyl malonyl.
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Scheme 1 Illustration of different polyketide synthases (PKSs): From top to bottom: Type I modular PKSs use modules for single rounds of chain extension and modification, as exemplified by the first two modules of the lasalocid A PKS which are each responsible for one round of polyketide chain extension. Type I iterative PKSs use their domains iteratively, as shown, for the biosynthesis of Terreic acid 614 via the precursor 6-methylsalicylic acid (6- MSA) 50, showing the multiple usage of each domain. Type II PKS such as that responsible for the biosynthesis of actinorhodin 2. Type III PKSs such as that responsible for the biosynthesis of resveratrol 4, use coenzyme A (CoA) as the acyl carrier. KS = ketosynthase, AT = acyl transferase, ACP = acyl carrier protein, DH = dehydratase, KR = ketoreductase, ER = enoylreductase, and CLF = chain length factor.)
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Scheme 2 Polyketide chain extension mechanism for the actinorhodin 2 type II minimal system. ACP = acyl carrier protein, KS = ketosynthase, CLF = chain length factor.
Acyl transferase (AT) domains are present in type I PKSs; they are responsible for selectively loading ACPs with malonyl starter and extender units provided by CoAs. ATs can either be cis- or trans-. The former are paired with a specific ACP, whereas the latter can transfer extender units to one or more ACPs.15 The 6- deoxyerthyronolide B synthase (DEBS) contains a cis-AT, and examples that harbour trans-ATs include PKSs that biosynthesise leinamycin16, bryostatin17 and disorazole15, 18. However, this domain is not necessary in all PKSs as some ACPs can self-malonate, such as in the biosynthesis of actinorhodin 2.19
Further product complexity is achieved by the action of reductive enzymes throughout chain extension. Ketoreductase (KR) domains stereoselectively reduce a β-keto group to a hydroxyl group, using nicotinamide adenine dinucleotide phosphate (NADPH) as a co-factor. Further hydrolysis of the hydroxyl group can be performed by a dehydratase (DH), producing a double bond that can be even further reduced by an enoylreductase (ER).20
6 The acyl carrier protein (ACP) plays a key role in shuttling the growing polyketide chains to different domains, and provides the starter unit to the ketosynthase (KS) for chain extension to begin (See section 1.2.1 for further detail).
Thioesterase (TE) domains catalyse the hydrolysis or cyclisation of advanced thioester intermediates, releasing the fully biosynthesised polyketide from the PKS. However, TEs are not always present in PKSs as spontaneous hydrolysis or cyclisation can occur to release the compound from the PKS.
‘Tailoring’ enzymes perform further polyketide structural modifications; they are often post-PKS enzymes, and include methyltransferases, cyclases, aromatases and glycosyltransferases, amongst many others. Product tailoring modifications are often crucial to their bioactivity. For example, the glycosylation of erythromycin is essential for its antibiotic action.21 The tailoring can also take place on the enzyme bound intermediates such as the epoxidation of the late stage intermediates leading to the polyether rings in lasalocid A 1.22
The biosynthesis of polyketides can be very complex, and include numerous stereoselective reactions and tightly regulated steps that, as yet, are not easily attainable by synthetic chemistry. When polyketide generation is achieved by synthetic chemistry, the number of steps becomes far greater than that required within living organisms, and at far greater cost. For example, the optimised chemical synthesis of the lasalocid A 1 requires 29 chemical steps with approximately 0.55% final yield.23 Therefore, when the compounds are pharmaceutically active it makes economic sense to use microorganism fermentation.
7 Generating analogues of natural products requires a similar long chemical synthetic process: if it were possible to produce these analogues by bioengineering organisms to introduce the favourable modifications this would cut the time and cost of the process, as well as make them environmentally more acceptable by avoiding the use of industrial chemicals. Currently, bioengineering of PKSs is in its early stages and understanding the fundamentals of how these enzymes function is crucial towards achieving this goal.