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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. 

ABSTRACT 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 inPseudomonas putidaATCC 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. 

Metformin is the first-line treatment for type 2 diabetes, yet its mechanism of action is not fully under- stood. 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 (Ki) of E. coli agmatinase is 1 mM and relevant in the gut where the drug concentration is 1–10 mM. Met- formin analogs phenformin, buformin, and galegine are even more potent inhibitors of E. coli agmatinase (Ki = 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. 

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 (mfmAandmfmB) 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 × 10³M⁻¹s⁻¹and K_Mfor 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 themfmABgenes identified in wastewater treatment plants on three continents, suggesting that metformin hydrolase is widespread globally in wastewater. 

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.