EMILIO AGUINALDO COLLEGE

Gov. D. Mangubat Ave., Brgy. Burol Main, City of Dasmariñas, Cavite 4114, Philippines
Tel. Nos. (046) 416-4339/41 www.eac.edu.ph

SCHOOL OF MEDICAL TECHNOLOGY

JOURNAL ARTICLE CRITIQUE

Calero, P., Volke, D. C., Lowe, P. T., Gotfredsen, C. H., O’Hagan, D., & Nikel, P. I. (2020). A
fluoride-responsive genetic circuit enables in vivo biofluorination in engineered pseudomonas
putida. Nature Communications. https://doi.org/10.1038/s41467-020-18813-x

I. TITLE

A Fluoride-Responsive Genetic Circuit Enables In Vivo Biofluorination In Engineered

Pseudomonas Putida

II. RELEVANCE OF THE OBJECTIVE TO THE STUDY

The objective of this research is to develop a genetic circuit that enables the
biofluorination of organic compounds in living cells. The article aims to address the
challenge of synthesizing fluorinated organic compounds, which are important in
several industries such as pharmaceuticals, agrochemicals, and materials science.
Fluorine is a unique element that imparts desirable properties to organic molecules
such as increased stability and lipophilicity. However, the synthesis of fluorinated
compounds is often difficult and expensive. The researchers developed a genetic
circuit that enables the expression of a fluoride-responsive enzyme in Pseudomonas
putida, a bacterium commonly used in biotechnology. The enzyme can convert a non-
fluorinated precursor molecule into a fluorinated product when exposed to fluoride
ions. The significance of this research lies in its potential to provide a more sustainable
and cost-effective method for synthesizing fluorinated compounds. By using living
cells as biocatalysts, the need for harsh chemical reagents and high temperatures can
be reduced or eliminated. This approach could also enable the production of novel
fluorinated molecules that are currently difficult or impossible to synthesize using
traditional methods.

III. SUMMARY OF THE METHODOLOGY

The researchers embarked on a multi-step process to create an effective

genetic circuit for in vivo biofluorination using engineered Pseudomonas putida cells.
Initially, they designed the circuit, which incorporated a promoter responsive to
fluoride ions, a gene encoding the fluorinase enzyme, and a reporter gene for enzyme
activity detection. Employing standard molecular biology techniques like PCR, cloning,
and transformation, they successfully constructed the circuit. Subsequently, the
researchers focused on optimizing its performance. They experimented with varying
the strength of the promoter and the gene's copy number to enhance the expression

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of the fluorinase enzyme. Additionally, they evaluated different strains of
Pseudomonas putida to determine the most suitable host organism. To assess the
activity of the fluorinase enzyme, the researchers conducted biofluorination assays in
vivo. They exposed the engineered cells to fluoride ions and monitored the production
of fluorinated compounds using analytical methods like HPLC and NMR spectroscopy.
The researchers also investigated the substrate specificity of the enzyme by testing its
ability to fluorinate various non-fluorinated precursor molecules. To demonstrate
scalability, they performed scale-up experiments utilizing a bioreactor system to grow
the cells and produce larger quantities of fluorinated compounds. In conclusion, these
comprehensive methodologies enabled the development and optimization of a
genetic circuit that facilitates in vivo biofluorination in engineered Pseudomonas
putida cells. Implementing this circuit could offer a more sustainable and cost-
effective approach for synthesizing fluorinated compounds.

IV. SUMMARY OF THE RESULTS, DISCUSSIONS, AND CONCLUSIONS

The researchers achieved successful development of a genetic circuit facilitating

in vivo biofluorination in engineered Pseudomonas putida cells. The circuit comprised
a fluoride-responsive promoter, a gene encoding a fluorinase enzyme, and a reporter
gene for enzyme activity detection. Optimization efforts involved adjusting the
promoter strength and gene copy number, while different Pseudomonas putida
strains were evaluated to determine the optimal host. Results demonstrated that the
engineered cells could produce fluorinated compounds upon exposure to fluoride
ions. Furthermore, the researchers established that the fluorinase enzyme exhibited
broad substrate specificity, enabling the fluorination of various non-fluorinated
precursor molecules. Discussions revolved around the potential advantages of using
living cells as biocatalysts for biofluorination, including enhanced sustainability and
cost-effectiveness compared to traditional synthesis methods. The developed genetic
circuit was seen as a valuable tool for producing novel fluorinated molecules that were
previously challenging or impossible to synthesize. The use of a fluoride-responsive
promoter offered precise control over fluorinase enzyme expression, minimizing the
risk of toxicity or undesirable side reactions. Additionally, the enzyme's broad
substrate specificity opened the door to synthesizing diverse fluorinated compounds
with varied properties and applications.

The researchers concluded that the developed genetic circuit represented a

significant advancement in biocatalysis, holding promise for various industries.
Employing engineered Pseudomonas putida cells for biofluorination could offer a
more sustainable and cost-effective approach to synthesizing fluorinated compounds.
Leveraging the fluorinase enzyme's broad substrate specificity and the fluoride-
responsive promoter allowed for precise control and expanded possibilities in
fluorination processes.

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V. LIMITATIONS AND SUGGESTIONS

While the research study has several strengths and potential applications, there are
also some limitations that should be considered. These include:

1. Limited substrate scope: While the fluorinase enzyme has broad substrate
specificity, it may not be able to fluorinate all non-fluorinated precursor
molecules. The researchers tested a limited number of substrates in this study and
it is possible that other substrates may not be compatible with the enzyme.
2. Limited scale-up experiments: While the researchers demonstrated the feasibility
of using the engineered cells for large-scale biofluorination, they did not perform
extensive scale-up experiments or evaluate the economic feasibility of this
approach.
3. Limited in vivo testing: The researchers tested the activity of the fluorinase
enzyme in vivo using Pseudomonas putida cells, but they did not test the toxicity
or immunogenicity of the fluorinated compounds produced by these cells.
4. Limited comparison to traditional methods: While the use of living cells as
biocatalysts for biofluorination could provide a more sustainable and cost-
effective method for synthesizing fluorinated compounds, the researchers did not
compare this approach to traditional chemical methods in terms of cost,
efficiency, or environmental impact.

To address these limitations, future studies could:

1. Expand the substrate scope of the fluorinase enzyme by testing its activity on a
wider range of non-fluorinated precursor molecules.
2. Perform more extensive scale-up experiments and evaluate the economic
feasibility of using engineered cells for large-scale biofluorination.
3. Test the toxicity and immunogenicity of the fluorinated compounds produced by
engineered cells and compare them to traditional chemical methods.
4. Conduct a comprehensive cost-benefit analysis of using living cells as biocatalysts
for biofluorination compared to traditional chemical methods.

Even though the research study has several limitations, it represents an important
step towards developing more sustainable and cost-effective methods for
synthesizing fluorinated compounds

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Tel. Nos. (046) 416-4339/41 www.eac.edu.ph

SCHOOL OF MEDICAL TECHNOLOGY

VI. SOCIETAL IMPACT

The article has several potential societal impacts. These include Advancing sustainable
chemistry by the use of living cells as biocatalysts for biofluorination could provide a more
sustainable and environmentally friendly approach for synthesizing fluorinated
compounds. This could reduce the reliance on traditional chemical methods that often
require harsh reagents and generate toxic waste. Second, by enabling the production of
novel fluorinated compounds; the developed genetic circuit could enable the production
of novel fluorinated molecules that are currently difficult or impossible to synthesize
using traditional methods. This could have significant implications for several industries
such as pharmaceuticals, agrochemicals, and materials science. Third, Contributing to the
development of precision biocatalysis, where the use of a fluoride-responsive promoter
allows for precise control over the expression of the fluorinase enzyme and reduces the
risk of toxicity or unwanted side reactions. This could contribute to the development of
precision biocatalysis, which has applications in several fields such as medicine and
biotechnology. Lastly, by promoting interdisciplinary research. The research study
involves expertise from several fields such as molecular biology, microbiology, and
chemistry. This interdisciplinary approach could promote collaboration between different
scientific disciplines and lead to discoveries and innovations.

In conclusion, the societal impact of this research study lies in its potential to provide
a more sustainable and cost-effective method for synthesizing fluorinated compounds,
enable the production of novel molecules with unique properties, contribute to the
development of precision biocatalysis, and promote interdisciplinary research.

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