The Quest to Create Fluorinated Medicines Using Engineered Polyketide Synthase Pathways
Imagine a chemical element so rare in living organisms that it appears in only a handful of natural molecules, yet so crucial to modern medicine that it features in 20-30% of all pharmaceutical drugs. This is the story of fluorine—a tiny atom with enormous power to transform medicines, making them more stable, more targeted, and more effective.
For decades, chemists have painstakingly added fluorine to drug molecules through synthetic processes. But what if we could persuade nature's own molecular factories to do this work for us?
Recent research has achieved a remarkable feat: rewiring bacterial biosynthesis pathways to incorporate fluorine into complex natural products. This breakthrough represents a new chapter in our relationship with this elusive element, potentially enabling scientists to produce fluorinated drugs more efficiently and create entirely new therapeutic compounds.
The key to this advancement lies in hijacking some of nature's most sophisticated molecular machinery: polyketide synthases—the cellular assembly lines that build many of our most important antibiotics and cancer treatments 1 .
Fluorine is the petite powerhouse of the pharmaceutical world. As one of the smallest atoms, it can often replace hydrogen in drug molecules without taking up much extra space. But despite its modest size, fluorine exerts an outsized influence on molecular behavior due to its extreme electronegativity—its powerful ability to attract electrons 4 .
Despite fluorine's abundance in the Earth's crust, natural organisms have been remarkably hesitant to incorporate it into their molecular structures. Of the millions of natural compounds discovered to date, fewer than twenty contain fluorine 4 .
Fluorine blocks common metabolic degradation pathways, allowing drugs to remain active in the body longer.
The "polar hydrophobic" nature of fluorine-containing compounds helps drugs cross cellular membranes more efficiently.
Fluorine forms specific interactions with protein binding sites, potentially reducing side effects.
These advantages explain why fluorination has become one of the most valuable strategies in drug design. From blockbuster antidepressants to life-saving antibiotics, fluorine-containing drugs have revolutionized modern medicine 3 7 .
If cells had industrial factories, they would look remarkably like polyketide synthases (PKSs). These massive enzyme complexes operate as molecular assembly lines, systematically building complex organic compounds through a series of coordinated steps 1 .
Like automotive assembly lines where a basic chassis is progressively transformed into a finished vehicle, PKSs start with simple molecular building blocks and gradually assemble them into structurally sophisticated products.
These biological nanofactories produce an astonishing array of therapeutically valuable compounds, including antibiotics (erythromycin), immunosuppressants (rapamycin), and anticancer agents (epothilone) 6 .
| Domain | Function | Role in Assembly Line |
|---|---|---|
| Ketosynthase (KS) | Catalyzes carbon-carbon bond formation | Extends the growing polyketide chain |
| Acyltransferase (AT) | Selects and loads extender units | Feedstock supplier for the assembly line |
| Acyl Carrier Protein (ACP) | Carries the growing chain | Molecular conveyor belt |
| Ketoreductase (KR) | Reduces ketone groups | Controls oxidation state of added units |
| Dehydratase (DH) | Removes water molecules | Introduces double bonds |
| Enoylreductase (ER) | Reduces double bonds | Controls saturation level |
For synthetic biologists, PKSs represent both an opportunity and a challenge. Their modular logic suggests they might be retooled to produce novel compounds, much like reprogramming a physical assembly line to manufacture different products. However, these molecular machines have proven remarkably resistant to engineering efforts 6 .
In a landmark study published in Science, researchers tackled the fluorine incorporation problem with a multi-pronged strategy 1 . Their approach involved engineering both the fluorinated building blocks and the PKS machinery that would incorporate them into polyketide chains.
The research team recognized that fluoroacetate—one of the few naturally occurring fluorinated compounds—could serve as a potential starting point. However, while fluoroacetate had been observed to act as a starter unit in some biological systems, it had never been successfully used as an extender unit for building polyketide backbones 1 .
With a reliable source of fluorinated building blocks established, the team next examined whether PKS enzymes could utilize fluoromalonyl-CoA in chain-extension reactions 1 . They began with a simplified system—the standalone ketosynthase NphT7 from Streptomyces sp. CL190.
The results were encouraging: NphT7 successfully catalyzed the formation of acetofluoroacetyl-CoA using acetyl-CoA as a starter and fluoromalonyl-CoA as an extender 1 . Though the catalytic efficiency was five-fold lower with the fluorinated substrate, the overall yield was comparable to the natural reaction.
| Enzyme/System | Natural Substrate Efficiency | Fluorinated Substrate Efficiency | Key Finding |
|---|---|---|---|
| AckA-Pta System | Nearly quantitative conversion | Similar extent of reaction | Fluorine does not prevent activation |
| ACCase | Rapid conversion | 4.5-fold slower but similar extent | No significant irreversible inhibition |
| MatB | High efficiency | 1000-fold less efficient but functional | Alternative route available |
| NphT7 (KS) | High catalytic efficiency | 5-fold reduction in kcat/KM | Successful C-C bond formation |
| PhaB (KR) | Efficient reduction | Efficient reduction of fluorinated product | Downprocessing compatible with fluorine |
Using the acetate kinase (AckA)-phosphotransacetylase (Pta) enzyme pair to activate fluoroacetate to fluoroacetyl-CoA, followed by carboxylation using acetyl-CoA carboxylase (ACCase) to produce fluoromalonyl-CoA.
Employing malonyl-CoA synthetase (MatB) to directly attach CoA to fluoromalonate. Both systems showed promise, with the AckA-Pta route achieving nearly quantitative conversion of fluoroacetate to fluoroacetyl-CoA 1 .
The groundbreaking work to expand fluorine chemistry required a sophisticated set of molecular tools and reagents. The table below highlights key components used in these experiments and their roles in the research 1 4 6 .
| Reagent/Tool | Type | Function in Research |
|---|---|---|
| Fluoroacetate | Chemical Substrate | Starting material for fluorinated building blocks |
| AckA-Pta System | Enzyme Pair | Activates fluoroacetate to fluoroacetyl-CoA |
| ACCase (AccABCD) | Enzyme Complex | Carboxylates fluoroacetyl-CoA to fluoromalonyl-CoA |
| MatB | Enzyme | Directly ligates fluoromalonate to CoA |
| NphT7 | Ketosynthase | Catalyzes decarboxylative Claisen condensation with fluoromalonyl-CoA |
| PhaB | Ketoreductase | Reduces fluorinated β-keto products |
| FlK | Thioesterase | Hydrolyzes fluoroacetyl-CoA as protective mechanism |
| Solubility Biosensor | Reporter System | Identifies stable, functional PKS variants during engineering |
Relative efficiency of enzymes with fluorinated vs natural substrates
The successful incorporation of fluorine into polyketides represents more than just a technical achievement—it opens new avenues for drug discovery and development. By making biofluorination possible, researchers have created a platform for generating molecular diversity that combines the structural complexity of natural products with the optimized properties of fluorinated compounds 1 9 .
This approach could lead to:
As the field progresses, researchers are working to overcome remaining challenges. The relatively low efficiency of fluorinated substrate incorporation presents an opportunity for enzyme engineering through directed evolution or rational design 6 .
New computational tools are emerging to support these efforts. The BioPKS pipeline, for example, combines retrobiosynthesis for both PKSs and monofunctional enzymes, enabling the in silico design of pathways for fluorinated compounds .
Such computational approaches may dramatically accelerate the engineering cycle and bring us closer to programmable cellular factories for fluorinated therapeutics.
"The ongoing integration of fluorine chemistry with synthetic biology represents a powerful convergence of disciplines. This work highlights the prospects for the production of complex fluorinated natural products using synthetic biology" 1 . By learning from nature's molecular blueprints while expanding them with chemical ingenuity, scientists are developing new capabilities to address pressing human health challenges.
References will be listed here in the final version.