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Studies of enzymes catalyzing carbon-fluorine (C-F) bond cleavage have focused largely on a limited number of native microbial hydrolases that are reactive with the natural product fluoroacetate. Driven by widespread interest in biodegrading commercial fluorinated compounds, many of which are known as per- and polyfluorinated alkyl substances (PFAS), it is necessary to identify and engineer new enzymes. For example, some hydrolases react with -CF 2 - moieties, a common functionality in PFAS. Additional enzymatic C-F cleaving mechanisms catalyzed by reductases, lyases, and oxygenases have been identified via screening. Screening and evolving PFAS defluorination in bacteria is inhibited by the obligate release of toxic fluoride from C-F cleavage. Engineering greater fluoride tolerance in bacteria is a problem that must be solved in tandem with enzyme improvement., Competing Interests: Declaration of interests No competing interests are declared., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Per- and polyfluorinated chemicals (PFAS) are of rising concern due to environmental persistence and emerging evidence of health risks to humans. Environmental persistence is largely attributed to a failure of microbes to degrade PFAS. PFAS recalcitrance has been proposed to result from chemistry, specifically C-F bond strength, or biology, largely negative selection from fluoride toxicity. Given natural evolution has many hurdles, this review advocates for a strategy of laboratory engineering and evolution. Enzymes identified to participate in defluorination reactions have been discovered in all Enzyme Commission classes, providing a palette for metabolic engineering. In vivo PFAS biodegradation will require multiple types of reactions and powerful fluoride mitigation mechanisms to act in concert. The necessary steps are to: (1) engineer bacteria that survive very high, unnatural levels of fluoride, (2) design, evolve, and screen for enzymes that cleave C-F bonds in a broader array of substrates, and (3) create overall physiological conditions that make for positive selective pressure with PFAS substrates., (© 2024 The Author(s).)

A major factor limiting the biodegradation of organofluorine compounds has been highlighted as fluoride anion toxicity produced by defluorinating enzymes. Here, two highly active defluorinases with different activities were constitutively expressed in Pseudomonas putida ATCC 12633 to examine adaption to fluoride stress. Each strain was grown on α-fluorophenylacetic acid as the sole carbon source via defluorination to mandelic acid, and each showed immediate fluoride release and delayed growth. Adaptive evolution was performed for each recombinant strain by serial transfer. Both strains adapted to show a much shorter lag and a higher growth yield. The observed adaptation occurred rapidly and reproducibly, within 50 generations each time. After adaption, growth with 50-70 mM α-fluorophenylacetic acid was significantly faster with more fluoride release than a preadapted culture due to larger cell populations. Genomic sequencing of both pre- and postadapted strain pairs revealed decreases in the defluorinase gene content. With both defluorinases, adaption produced a 56%-57% decrease in the plasmid copy number. Additionally, during adaption of the strain expressing the faster defluorinase, two plasmids were present: the original and a derivative in which the defluorinase gene was deleted. An examination of the enzyme rates in the pathway suggested that the defluorinase rate was concurrently optimised for pathway flux and minimising fluoride toxicity. The rapid alteration of plasmid copy number and mutation was consistent with other studies on microbial responses to stresses such as antibiotics. The data presented here support the idea that fluoride stress is significant during the biodegradation of organofluorine compounds and suggest engineered strains will be under strong selective pressure to decrease fluoride stress., (© 2024 The Author(s). Microbial Biotechnology published by John Wiley & Sons Ltd.)

The acetogen Acetobacterium woodii couples caffeate reduction with ferredoxin reduction and NADH oxidation via electron bifurcation, providing additional reduced ferredoxin for energy conservation and cell synthesis. Caffeate is first activated by an acyl-CoA synthetase (CarB), which ligates CoA to caffeate at the expense of ATP. After caffeoyl-CoA is reduced to hydrocaffeoyl-CoA, the CoA moiety in hydrocaffeoyl-CoA could be recycled for caffeoyl-CoA synthesis by an ATP-independent CoA transferase (CarA) to save energy. However, given that CarA and CarB are co-expressed, it was not well understood how ATP could be saved when both two competitive pathways of caffeate activation are present. Here, we reported a dual feedback inhibition of the CarB-mediated caffeate activation by the intermediate hydrocaffeoyl-CoA and the end-product hydrocaffeate. As the product of CarA, hydrocaffeate inhibited CarB-mediated caffeate activation by serving as another substrate of CarB with hydrocaffeoyl-CoA produced. It effectively competed with caffeate even at a concentration much lower than caffeate. Hydrocaffeoyl-CoA formed in this process can also inhibit CarB-mediated caffeate activation. Thus, the dual feedback inhibition of CarB, together with the faster kinetics of CarA, makes the ATP-independent CarA-mediated CoA loop the major route for caffeoyl-CoA synthesis, further saving ATP in the caffeate-dependent electron-bifurcating pathway. A genetic architecture similar to carABC has been found in other anaerobic bacteria, suggesting that the feedback inhibition of acyl-CoA ligases could be a widely employed strategy for ATP conservation in those pathways requiring substrate activation by CoA., Importance: This study reports a dual feedback inhibition of caffeoyl-CoA synthetase by two downstream products, hydrocaffeate and hydrocaffeoyl-CoA. It elucidates how such dual feedback inhibition suppresses ATP-dependent caffeoyl-CoA synthesis, hence making the ATP-independent route the main pathway of caffeate activation. This newly discovered mechanism contributes to our current understanding of ATP conservation during the caffeate-dependent electron-bifurcating pathway in the ecologically important acetogen Acetobacterium woodii . Bioinformatic mining of microbial genomes revealed contiguous genes homologous to carABC within the genomes of other anaerobes from various environments, suggesting this mechanism may be widely used in other CoA-dependent electron-bifurcating pathways., Competing Interests: The authors declare no conflict of interest.

In this study, we propose a self-limiting growth model for Bacillus subtilis spores confined within porous polyacrylamide (PA) hydrogels. We observed that B. subtilis spores germinate into vegetative cells within the hydrogel matrix, forming spherical colonies. These colonies expand until the mechanical stress they exert on their environment surpasses the yield stress of the hydrogel, leading to formation of a nonpermeable layer that halts nutrient diffusion and forces the bacteria to resporulate. These novel observations suggest a model to explain why bacterial growth in confined environments and material interfaces may be limited, providing insight for natural phenomena and biotechnological applications involving bacterial encapsulation.

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., (© 2024. The Author(s).)

The fate of organic matter in the environment, including anthropogenic chemicals, is largely predicated on the enzymatic capabilities of microorganisms. Microbes readily degrade, and thus recycle, most of the ~100,000 commercial chemicals used in modern society. Per- and polyfluorinated compounds (PFAS) are different. Many research papers posit that the general resistance of PFAS to microbial degradation is based in chemistry and that argument relates to the strength of the C-F bond. Here, I advance the opinion that the low biodegradability of PFAS is best formulated as a biological optimization problem, hence evolution. The framing of the problem is important. If it is framed around C-F bond strength, the major effort should focus on finding and engineering new C-F cleaving enzymes. The alternative, and preferred approach suggested here, is to focus on the directed evolution of biological systems containing known C-F cleaving systems. There are now reports of bacteria degrading and/or growing on multiply fluorinated arenes, alkenoic and alkanoic acids. The impediment to more efficient and widespread biodegradation in these systems is biological, not chemical. The rationale for this argument is made in the five sections below that follow the Introduction., (© 2024 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd.)

Metformin is the first-line treatment for type II diabetes patients and a pervasive pollutant with more than 180 million kg ingested globally and entering wastewater. The drug's direct mode of action is currently unknown but is linked to effects on gut microbiomes and may involve specific gut microbial reactions to the drug. In wastewater treatment plants, metformin is known to be transformed by microbes to guanylurea, although genes encoding this metabolism had not been elucidated. In the present study, we revealed the function of two genes responsible for metformin decomposition ( mfmA and mfmB ) found in isolated bacteria from activated sludge. MfmA and MfmB form an active heterocomplex (MfmAB) and are members of the ureohydrolase protein superfamily with binuclear metal-dependent activity. MfmAB is nickel-dependent and catalyzes the hydrolysis of metformin to dimethylamine and guanylurea with a catalytic efficiency (k cat /K M ) of 9.6 × 10 3 M -1 s -1 and K M for metformin of 0.82 mM. MfmAB shows preferential activity for metformin, being able to discriminate other close substrates by several orders of magnitude. Crystal structures of MfmAB show coordination of binuclear nickel bound in the active site of the MfmA subunit but not MfmB subunits, indicating that MfmA is the active site for the MfmAB complex. Mutagenesis of residues conserved in the MfmA active site revealed those critical to metformin hydrolase activity and its small substrate binding pocket allowed for modeling of bound metformin. This study characterizes the products of the mfmAB genes identified in wastewater treatment plants on three continents, suggesting that metformin hydrolase is widespread globally in wastewater., Competing Interests: Competing interests statement:The authors declare no competing interest.

Importance: Society uses thousands of organofluorine compounds, sometimes denoted per- and polyfluoroalkyl substances (PFAS), in hundreds of products, but recent studies have shown some to manifest human and environmental health effects. As a class, they are recalcitrant to biodegradation, partly due to the paucity of fluorinated natural products to which microbes have been exposed. Another limit to PFAS biodegradation is the intracellular toxicity of fluoride anion generated from C-F bond cleavage. The present study identified a broader substrate specificity in an enzyme originally studied for its activity on the natural product fluoroacetate. A recombinant Pseudomonas expressing this enzyme was used here as a model system to better understand the limits and effects of a high level of intracellular fluoride generation. A fluoride stress response has evolved in bacteria and has been described in Pseudomonas spp. The present study is highly relevant to organofluorine compound degradation or engineered biosynthesis in which fluoride anion is a substrate., Competing Interests: The authors declare no conflict of interest.

Metformin is the first-line treatment for type 2 diabetes, yet its mechanism of action is not fully understood. Recent studies suggest metformin's interactions with gut microbiota are responsible for exerting therapeutic effects. In this study, we report that metformin targets the gut microbial enzyme agmatinase, as a competitive inhibitor, which may impair gut agmatine catabolism. The metformin inhibition constant (K i ) of E. coli agmatinase is 1 mM and relevant in the gut where the drug concentration is 1-10 mM. Metformin analogs phenformin, buformin, and galegine are even more potent inhibitors of E. coli agmatinase (K i  = 0.6, 0.1, and 0.007 mM, respectively) suggesting a shared mechanism. Agmatine is a known effector of human host metabolism and has been reported to augment metformin's therapeutic effects for type 2 diabetes. This gut-derived inhibition mechanism gives new insights on metformin's action in the gut and may lead to significant discoveries in improving metformin therapy., Competing Interests: The authors declare no competing interests., (© 2024 The Authors.)

There is intense interest in removing fluorinated compounds from the environment, environments are most efficiently remediated by microbial enzymes, and defluorinating enzymes are readily monitored by fluoride determination. Fluorine is the most electronegative element. Consequently, all mechanisms of enzymatic C-F bond cleavage produce fluoride anion, F - . Therefore, methods for the determination of fluoride are critical for C-F enzymology and apply to any fluorinated organic compounds, including PFAS, or per- and polyfluorinated alkyl substances. The biodegradation of most PFAS chemicals is rare or unknown. Accordingly, identifying new enzymes, or re-engineering the known defluorinases, will require rapid and sensitive methods for measuring fluoride in aqueous media. Most studies currently use ion chromatography or fluoride specific electrodes which are relatively sensitive but low throughput. The methods here describe refashioning a drinking water test to efficiently determine fluoride in enzyme and cell culture reaction mixtures. The method is based on lanthanum alizarin complexone binding of fluoride. Reworking the method to a microtiter well plate format allows detection of as little as 4 nmol of fluoride in 200 μL of assay buffer. The method is amenable to color imaging, spectrophotometric plate reading and automated liquid handling to expedite assays with thousands of enzymes and/or substrates for discovering and improving enzymatic defluorination., (Copyright © 2024. Published by Elsevier Inc.)

With the continued population increase, more sustainable use of water, land, air and chemicals is imperative. Microorganisms will need to be called upon to aid in many sustainability efforts. Prokaryotes are the fastest-evolving cellular life, and most manipulatable via synthetic biology. Moreover, their natural diversity in processing organic and inorganic chemicals, and their survivability in extreme niches, make them prime agents to enlist for solving many of society's pressing problems., (© 2023 The Author. Microbial Biotechnology published by Applied Microbiology International and John Wiley & Sons Ltd.)

Metformin is used globally to treat type II diabetes, has demonstrated anti-ageing and COVID mitigation effects and is a major anthropogenic pollutant to be bioremediated by wastewater treatment plants (WWTPs). Metformin is not adsorbed well by activated carbon and toxic N-chloro derivatives can form in chlorinated water. Most earlier studies on metformin biodegradation have used wastewater consortia and details of the genomes, relevant genes, metabolic products, and potential for horizontal gene transfer are lacking. Here, two metformin-biodegrading bacteria from a WWTP were isolated and their biodegradation characterized. Aminobacter sp. MET metabolized metformin stoichiometrically to guanylurea, an intermediate known to accumulate in some environments including WWTPs. Pseudomonas mendocina MET completely metabolized metformin and utilized all the nitrogen atoms for growth. Pseudomonas mendocina MET also metabolized metformin breakdown products sometimes observed in WWTPs: 1-N-methylbiguanide, biguanide, guanylurea, and guanidine. The genome of each bacterium was obtained. Genes involved in the transport of guanylurea in Aminobacter sp. MET were expressed heterologously and shown to serve as an antiporter to expel the toxic guanidinium compound. A novel guanylurea hydrolase enzyme was identified in Pseudomonas mendocina MET, purified, and characterized. The Aminobacter and Pseudomonas each contained one plasmid of 160 kb and 90 kb, respectively. In total, these studies are significant for the bioremediation of a major pollutant in WWTPs today., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Martinez-Vaz, Dodge, Lucero, Stockbridge, Robinson, Tassoulas and Wackett.)

The present study examined the regulatory and metabolic response of the aromatic degrader Pseudomonas putida F1 and its tod operon, controlling toluene degradation, to fluorinated aromatic and aliphatic compounds. The tod operon is upregulated by inducer binding to the TodS sensing domain of a two-component regulator. The induced enzymes include toluene dioxygenase that initiates catabolic assimilation of benzenoid hydrocarbons. Toluene dioxygenase was shown to oxidize 6-fluoroindole to a meta-stable fluorescent product, 6-fluoroindoxyl. The fluorescent output allowed monitoring relative levels of tod operon induction in whole cells using microtiter well plates. Mono- and polyfluorinated aromatic compounds were shown to induce toluene dioxygenase, in some cases to a greater extent than compounds serving as growth substrates. Compounds that are oxidized by toluene dioxygenase and undergoing defluorination were shown to induce their own metabolism. 1,2,4-Trifluorobenzene caused significant induction and computational modelling indicated productive binding to the TodS sensor domain of the TodST regulator. Toluene dioxygenase also showed preferential binding of 1,2,4-trifluorobenzene such that defluorination was favoured. Fluorinated aliphatic compounds were shown to induce toluene dioxygenase. An aliphatic ether with seven fluorine atoms, 1,1,1,2-tetrafluoro-2-trifluoromethoxy-4-iodobutane (TTIB), was an excellent inducer of toluene dioxygenase activity and shown to undergo transformation in cultures of P. putida F1., (© 2022 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Engineered materials to improve the shelf-life of desiccated microbial strains are needed for cost-effective bioaugmentation strategies. High temperatures and humidity of legume-growing regions challenge long-term cell stabilization at the desiccated state. A thermostable xeroprotectant core and hydrophobic water vapour barrier shell encapsulation technique was developed to protect desiccated cells from the environment. A trehalose core matrix increased the stability of desiccated Bradyrhizobium by three orders of magnitude over 20 days at 32°C and 50% relative humidity (RH) compared to buffer alone; however, the improvement was not deemed sufficient for a shelf-stable bioproduct. We tested common additives (skim milk, albumin, gelatin and dextran) to increase the glass transition temperature of the desiccated product to provide further stabilization. Albumin increased the glass transition temperature of the trehalose-based core by 40°C and stabilized desiccated Bradyrhizobium for 4 months during storage at high temperature (32°C) and moderate humidity (50% RH) with only 1 log loss of viability. Although the albumin-trehalose core provided exceptional protection against high temperature, it was ineffective at higher humidity conditions (75%). We therefore incorporated a paraffin shell, which protected desiccated cells against 75% RH providing proof of concept that core and shell encapsulation is an effective strategy to stabilize desiccated cells., (© 2022 The Authors. Microbial Biotechnology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Many cite the strength of C-F bonds for the poor microbial biodegradability of polyfluorinated organic compounds (PFCs). However, commercial PFCs almost invariably contain more functionality than fluorine. The additional functionality provides a weak entry point for reactions that activate C-F bonds and lead to their eventual cleavage. This metabolic activation strategy is common in microbial biodegradation pathways and is observed with aromatic hydrocarbons, chlorinated compounds, phosphonates and many other compounds. Initial metabolic activation precedes critical bond breakage and assimilation of nutrients. A similar strategy with commercial PFCs proceeds via initial attack at the non-fluorinated functionalities: sulfonates, carboxylates, chlorines, phenyl rings, or phosphonates. Metabolic transformation of these non-fluorinated groups can activate the C-F bonds, allowing more facile cleavage than a direct attack on the C-F bonds. Given that virtually all compounds denoted as "PFAS" are not perfluorinated and are not alkanes, it is posited here that considering their individual chemical classes is more useful for both chemical and microbiological considerations of their fate., Competing Interests: The author declares no conflict of interest.