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The link between ancient microbial fluoride resistance mechanisms and bioengineering organofluorine degradation or synthesis.
| Nature Communications | Randy B Stockbridge and Lawrence P Wackett. | 15(1):4593.
F-toxicity FEX Fluc PFAS Other
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Abstract

Original full-text study online at
https://www.nature.com/articles/s41467-024-49018-1

Fluorinated organic chemicals, such as per- and polyfluorinated alkyl substances (PFAS) and fluorinated pesticides, are both broadly useful and unusually long-lived. To combat problems related to the accumulation of these compounds, microbial PFAS and organofluorine degradation and biosynthesis of less-fluorinated replacement chemicals are under intense study. Both efforts are undermined by the substantial toxicity of fluoride, an anion that powerfully inhibits metabolism. Microorganisms have contended with environmental mineral fluoride over evolutionary time, evolving a suite of detoxification mechanisms. In this perspective, we synthesize emerging ideas on microbial defluorination/fluorination and fluoride resistance mechanisms and identify best approaches for bioengineering new approaches for degrading and making organofluorine compounds.

Excerpts:

Engineering fluoride resistance

By engineering host bacteria, fluoride resistance could be further improved. Most straightforwardly, genes that are critical to the fluoride stress response, such as fluoride exporters, could be constitutively expressed. During PFAS biodegradation, the release of fluoride anion intracellularly can cause cessation of energy metabolism before a fluoride stress response is mounted63. Constitutive expression of fluoride exporters is one naturally evolved response that improves bacterial fluoride tolerance91. It is also possible that the expression of multiple fluoride exporters could help improve fluoride resistance. Each of the two mechanistically distinct fluoride export proteins has its own advantages, at least in principle. The F–/H+ antiport mechanism couples fluoride export to the proton gradient, sustaining a lower intracellular fluoride concentration at equilibrium, whereas fluoride channels have the advantage of more rapid fluoride removal that does not depend on a proton gradient. However, it is at least theoretically possible that at high external fluoride, if a cell is unable to maintain a membrane potential, a fluoride channel could permit fluoride influx. Protein-level regulation could prevent this outcome. Although synthetic proteins that inhibit fluoride channels have been developed123,124,125, they have not been tested in biological systems, and no such natural regulatory mechanism has been identified. Fluc channels are more widely distributed among diverse bacteria than CLCF transporters (Fig. 3), and Flucs are found more commonly in strains that resist high fluoride or that use fluoride for synthesis. These observations perhaps imply that channels are biology’s favored solution to fluoride export.

We can also follow the example of nature and simply overexpress enzymes like enolase that represent key roadblocks in metabolism, those that respond to oxidative stress, or that, like pyrophosphatase, have homologs that are less sensitive to fluoride inhibition126. For bioremediation, the introduction of genes that contribute to external sequestration may be protective in static natural or engineered bioremediation systems in which exported fluoride might accumulate and reenter cells. One recent study showed that calcium carbonate precipitate generated by Pseudomonas sp. HXF1 could sequester fluoride in the form of CaF2 and Ca5(PO4)3F and diminish fluoride in groundwater127.

Summary and outlook

PFAS accumulation in the environment is an expanding societal problem, and microbial bioengineering shows promise for PFAS remediation or synthesis of less-fluorinated and more biodegradable chemicals to replace undesirable PFAS. Much attention within this field has been directed towards the discovery or engineering of enzymes that can break the famously strong C–F bond. Progress along this front is promising: although such enzymes are rare, recent studies show that they are more diverse than previously thought51, and advancements in metagenomic sequencing and protein engineering will support future discovery and optimization of organisms, genes, and pathways that support organofluorine synthesis and degradation. For example, homologs to a newly discovered reductive defluorinating enzyme system were recently identified in metagenomes found on six continents, greatly expanding the range of enzymes of this type to be studied51. Furthermore, we argue here that defluorination chemistry is only a part of the challenge in this field. By acquiring a deep understanding of the fundamental microbial physiologies — in particular the fluoride stress responses — that support biodegradation or biosynthesis of organofluorine molecules, we can better harness ancient fluoride resistance mechanisms to address this very contemporary biochemical problem. Box 1 describes targeted areas of research that will further advance these fields.

Authors and Affiliations

  1. Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI, 48109, USA

    Randy B. Stockbridge

  2. Department of Biochemistry, Biophysics & Molecular Biology and Biotechnology Institute, University of Minnesota, Minneapolis, MN, 55455, USA

    Lawrence P. Wackett

Contributions

R.B.S. and L.P.W. contributed equally to writing and editing the manuscript and preparing figures.

Corresponding authors

Correspondence to Randy B. Stockbridge or Lawrence P. Wackett.

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Acknowledgements

The authors thank Carla Peralta for assistance in analyzing the distribution of fluoride exporters in GEBA genomes. R.B.S. is supported by the National Institutes of Health grant R35 GM128768. L.P.W. is supported by a grant from MnDRIVE Industry and the Environment Program.