The rapid development of antifungal resistance poses a threat to the health and agricultural sectors. Iturin A, a cyclic lipopeptide with pronounced antifungal properties produced by Bacillus sp., holds promise against several pathogens. Here, a novel synthesis of iturin A is presented, which enables access to different analogs for the study of its mode of action. The route includes the enantioselective synthesis of the β-amino fatty acid present in the lipopeptide structure, followed by solid-phase peptide synthesis and on-resin cyclization. This robust synthesis was used to obtain natural iturin A. Furthermore, the synthesis of two analogs is described: an epimer with an inverted stereochemistry of the β-amino fatty acid, which was designed to shed light on the role of this stereocenter on iturin A's bioactivity, and a monofluorinated analog to assess fluorination's impact on bioactivity and as a fluorine NMR probe for mechanistic studies. Antifungal assays against Candida albicans and Fusarium graminearum showed that the epimer of iturin A lost all bioactivity, while the monofluorinated analog retained the bioactivity of the natural compound, thus confirming its potential as an NMR probe.

A route is developed to (g,g',g''''-trifluoro)neopentyl (TFNP) aryl ethers to extend the methods for the introduction of the tert-butyl group, carrying a fluorine on each of the methyl substituents. The route combines neopentyltosylate 3 with phenols and thiophenols to give efficient substitution reactions to the corresponding TFNP aryl ethers. The three C-F bonds adopt a helical propeller conformation as revealed by computation and single crystal X-ray structure analysis. The LogPs of TFNP ethers are lower (more hydrophilic) than their tert-butyl analogues. The metabolism of selected TFNP ethers was explored in the fungus Cunninghamella elegans.

The pursuit of novel antifungal agents is imperative to tackle the threat of antifungal resistance, which poses major risks to both human health and to food security. Iturin A is a cyclic lipopeptide, produced by Bacillus sp., with pronounced antifungal properties against several pathogens. Its challenging synthesis, mainly due to the laborious synthesis of the β‐amino fatty acid present in its structure, has hindered the study of its mode of action and the development of more potent analogues. In this work, a facile synthesis of bioactive iturin A analogues containing an alkylated cysteine residue is presented. Two analogues with opposite configurations of the alkylated cysteine residue were synthesized, to evaluate the role of the stereochemistry of the newly introduced amino acid on the bioactivity. Antifungal assays, conducted against F. graminearum, showed that the novel analogues are bioactive and can be used as a synthetic model for the design of new analogues and in structure–activity relationship studies. The assays also highlight the importance of the β‐amino acid in the natural structure and the role of the stereochemistry of the amino fatty acid, as the analogue with the D configuration showed stronger antifungal properties than the one with the L configuration.

Some species of the genus Cunninghamella (C. elegans, C. echinulata and C. blaskesleeana) produce the same phase I and phase II metabolites when incubated with xenobiotics as mammals, and thus are considered microbial models of mammalian metabolism. This had made these fungi attractive for metabolism studies with drugs, pesticides and environmental pollutants. As a substantial proportion of pharmaceuticals and agrochemicals are fluorinated, their biotransformation has been studied in Cunninghamella fungi and C. elegans in particular. This article details the methods employed for cultivating the fungi in planktonic and biofilm cultures, and extraction and analysis of fluorinated metabolites. Furthermore, protocols for the heterologous expression of Cunninghamella cytochromes P450 (CYPs), which are the enzymes associated with phase I metabolism, are described.

A route is developed to (γ,γ',γ'''-trifluoro)neopentyl (TFNP) aryl ethers to extend the methods for the introduction of the tert-butyl group, carrying a fluorine on each of the methyl substituents. The route combines neopentyltosylate 3 with phenols and thiophenols to give efficient substitution reactions to the corresponding TFNP aryl ethers. The three C-F bonds adopt a helical propeller conformation as revealed by computation and single crystal X-ray structure analysis. The LogPs of TFNP ethers are lower (more hydrophilic) than their neopentyl analogues. The metabolism of selected TFNP ethers was explored in the fungus Cunninghamella elegans.

Within the circular bioeconomy the production of optically pure LA from 2nd generation feedstocks would be ideal but it is very challenging. In this paper genetically engineered Escherichia coli strains were created to resolve racemic LA solutions synthesised and produced from the fermentation of organic waste or ensiled grass. Refining LA racemic mixtures into either a D- or L-LA was achieved by cells being able to consume one LA isomer as a sole carbon and energy source while not being able to consume the other. A D-LA refining strain JSP0005 was grown on fermented source-sorted organic household waste and different grass silage leachates, which are 2nd generation feedstocks containing up to 33 g/L lactic acid racemate. In all growth experiments, L-LA was completely removed leaving D-LA as the only LA stereoisomer, i.e. resulting in optically pure D-LA, which also increased by as much as 248.6 % from its starting concentration, corresponding to 38 g/L. The strains resulting from this study are a promising first step towards a microbial based LA biorefining process.

EDITORIAL article Front. Microbiol., 17 May 2023Sec. Terrestrial Microbiology Volume 14 - 2023 | https://doi.org/10.3389/fmicb.2023.1206179

In this paper, we report the degradation of perfluorooctanoic acid (PFOA), which is a persistent contaminant in the environment that can severely impact human health, by exposing it to a photocatalyst, bismuth oxyiodide (BiOI), containing both Bi4O5I2 and Bi5O7I phases and a fungal biocatalyst (Cunninghamella elegans). Individually, the photocatalyst (after 3 h) and biocatalyst (after 48 h) degraded 35-40% of 100 ppm PFOA with 20-30% defluorination. There was a marked improvement in the degree of degradation (90%) and defluorination (60%) when PFOA was first photocatalytically treated, then exposed to the fungus. GC- and LC-MS analysis identified the products formed by the different treatments. Photocatalytic degradation of PFOA yielded short-chain perfluorocarboxylic acids, whereas fungal degradation yielded mainly 5:3 fluorotelomer carboxylic acid, which is a known inhibitor of cytochrome P450-catalysed degradation of PFAS in C. elegans. The combined treatment likely resulted in greater degradation because photocatalysis reduced the PFOA concentration without generating the inhibitory 5:3 fluorotelomer carboxylic acid, enabling the fungus to remove most of the remaining substrate. In addition, new fluorometabolites were identified that shed light on the initial catabolic steps involved in PFOA biodegradation.

Cunninghamella elegans is a well-studied fungus that biotransforms a range of xenobiotics owing to impressive cytochrome P450 (CYP) activity. In this paper, we report the biotransformation of 6:2 fluorotelomer alcohol (6:2 FTOH) by the fungus, yielding a range of fluorinated products that were detectable by fluorine-19 nuclear magnetic resonance spectroscopy (19F NMR), gas chromatography-mass spectrometry (GC–MS) and liquid chromatography-mass spectrometry (LC–MS). Upon incubation with the pre-grown cultures, the substrate (100 mg/L) was completely consumed within 48 h, which is faster biotransformation than other fungi that have hitherto been studied. The main metabolite formed was the 5:3 fluorotelomer carboxylic acid (5:3 FTCA), which accumulated in the culture supernatant. When the cytochrome P450 inhibitor 1-aminobenzotriazole was included in the culture flasks, there was no biotransformation of 6:2 FTOH, indicating that these enzymes are key to the catalysis. Furthermore, when exogenous 5:3 FTCA was added to the fungus, the standard biotransformation of the drug flurbiprofen was inhibited, strongly suggesting that the main fluorotelomer alcohol biotransformation product inhibits CYP activity and accounts for its accumulation.

Three peptides comprising mono-, di-, and tri-fluoroethylglycine (MfeGly, DfeGly, and TfeGly) residues alternating with lysine were digested by readily available proteases (elastase, bromelain, trypsin, and proteinase K). The degree of degradation depended on the enzyme employed and the extent of fluorination. Incubation of the peptides with a microbial consortium from garden soil resulted in degradation, yielding fluoride ions. Further biodegradation studies conducted with the individual fluorinated amino acids demonstrated that the degree of defluorination followed the sequence MfeGly > DfeGly > TfeGly. Enrichment of the soil bacteria employing MfeGly as a sole carbon and energy source resulted in the isolation of a bacterium, which was identified as Serratia liquefaciens. Cell-free extracts of this bacterium enzymatically defluorinated MfeGly, yielding fluoride ion and homoserine. In silico analysis of the genome revealed the presence of a gene that putatively codes for a dehalogenase. However, the low overall homology to known enzymes suggests a potentially new hydrolase that can degrade monofluorinated compounds. 19F NMR analysis of aqueous soil extracts revealed the unexpected presence of trifluoroacetate, fluoride ion, and fluoroacetate. Growth of the soil consortium in tryptone soya broth supplemented with fluoride ions resulted in fluoroacetate production; thus, bacteria in the soil produce and degrade organofluorine compounds.

Fungi have been extensively studied for their capacity to biotransform a wide range of natural and xenobiotic compounds. This versatility is a reflection of the broad substrate specificity of fungal enzymes such as laccases, peroxidases and cytochromes P450, which are involved in these reactions. This review gives an account of recent advances in the understanding of fungal metabolism of drugs and pollutants such as dyes, agrochemicals and per- and poly-fluorinated alkyl substances (PFAS), and describes the key enzymes involved in xenobiotic biotransformation. The potential of fungi and their enzymes in the bioremediation of polluted environments and in the biocatalytic production of important compounds is also discussed.

Fungi have long been investigated for their abilities to degrade organic and inorganic pollutants, owing mostly to the diversity in oxidative enzymes that they produce. However, as the chemical complexity of pollutants has increased, the application of fungi in bioremediation has become more challenging. This chapter summarizes the mechanisms by which fungi can degrade a number of pollutants and examines the role of these microorganisms in the green synthesis of nanomaterials, which have a range of potential uses. Significantly, nanoparticles may be used in the remediation of a variety of recalcitrant compounds, and the possibility of using a combination of this technology with the more established bioremediation features of fungi is explored.

The filamentous fungus Cunninghamella echinulata is a model of mammalian xenobiotic metabolism. Under certain conditions it grows as a biofilm, which is a natural form of immobilisation and enables the fungus to catalyse repeated biotransformations. Putative signalling molecules produced by other Cunninghamella spp., such as 3-hydroxytyrosol and tyrosol, do not affect the biofilm growth of C. echinulata, suggesting that it employs a different molecule to regulate biofilm growth. In this paper we report that 2-phenylethanol is produced in higher concentrations in planktonic cultures of C. echinulata than when the fungus is grown as a biofilm. We demonstrate that exogenously added 2-phenylethanol inhibits biofilm growth of C. echinulata but has no effect on planktonic growth. Furthermore, we show that addition of 2-phenylethanol to established C. echinulata biofilm causes detachment. Therefore, we conclude that this molecule is produced by the fungus to regulate biofilm growth.

The (β,β′,β″-trifluoro)-tert-butyl (TFTB) group has received very little attention in the literature. This work presents a direct synthesis of this group and explores its properties. The TFTB group arises when the methyl groups of a tert-butyl moiety are exchanged for fluoromethyl groups. Sequential fluoromethylations result in a decrease of Log P (increasing hydrophilicity), ultimately by 1.7 Log P units in the TFTB group relative to that of tert-butyl benzene itself. A focus is placed on synthetic transformations, conformational analysis, and metabolism of the TFTB group in the context of presenting a favorable profile as a motif for the discovery of bioactives.

The microbial model of mammalian drug metabolism, Cunninghamella elegans, has three cytochrome P450 reductase genes in its genome: g1631 (CPR_A), g4301 (CPR_B), and g7609 (CPR_C). The nitroreductase activity of the encoded enzymes was investigated via expression of the genes in the yeast Pichia pastoris X33. Whole cell assays with the recombinant yeast demonstrated that the reductases converted the anticancer drug flutamide to the nitroreduced metabolite that was also produced from the same substrate when incubated with human NADPH: cytochrome P450 reductase. The nitroreductase activity extended to other substrates such as the related drug nilutamide and the environmental contaminants 1-nitronaphthalene and 1,3-dinitronaphthalene. Comparative experiments with cell lysates of recombinant yeast were conducted under aerobic and reduced oxygen conditions and demonstrated that the reductases are oxygen sensitive.

Biofilms are colonies of microorganisms that adhere to a surface, often in a flowing system. They convey advantages to the community of cells within, such as access to nutrients, protection from physicochemical stress, intercellular communication and horizontal gene transfer. In many instances, biofilms are viewed as problematic, for example, causing difficult-to-treat infections or corroding metal surfaces. However, the properties that make biofilms a challenge in some circumstances are beneficial in others. As biofilms have mechanical stability, are resistant to the toxic effects of xenobiotics and are stable for extended time periods, they have potential applications in the continuous production of chemicals and the biodegradation of pollutants. In this chapter, we will present examples of the research that has been conducted on examining single-species biofilms of bacteria and fungi in the biosynthesis of commodity and fine chemicals and the biodegradation of xenobiotics. The merits and disadvantages of biofilms in different applications are discussed.

Cunninghamella elegans is a long-established microbial model of mammalian drug and xenobiotic metabolism enabled by the actions of cytochrome P450 enzymes that are poorly characterised. In this paper we describe the identification of a new cytochrome P450 (CYP) monooxygenase in the fungus that catalyses the biotransformation of a range of structurally distinct xenobiotic substrates. The fungal enzyme was heterologously expressed in the yeast Pichia pastoris X-33 alone and in combination with previously identified C. elegans CYP reductases (CPRs A, B and C). Enzyme activity was assessed against a panel of drugs (flurbiprofen, diclofenac and ibuprofen), pesticides (transfluthrin, β-cyfluthrin and λ-cyhalothrin) and a perfluoroalkyl substance (6:2 fluorotelomer alcohol) that were incubated with whole yeast cells expressing CYP5208A3. The biotransformation products were determined by gas chromatography-mass spectrometry (GC-MS) revealing the same metabolites that had been previously observed in the fungus. Co-expression of the CPRs improved metabolite production and the degree of improvement depended on the substrate and the CYP/CPR combination. Optimal pyrethroid biotransformation was achieved with CYP/CPR_C, whereas the best combination for non-steroidal anti-inflammatory drug hydroxylation was CYP/CPR_A; fluorotelomer alcohol oxidation was only observed with CYP/CPR_B. The change in substrate specificity observed with CYP5208A3 in combination with the different CPRs might help explain how C. elegans can biotransform such a broad spectrum of xenobiotics.

Wastewater surveillance is a cost-effective tool for monitoring SARS-CoV-2 transmission in a community. However, challenges remain with regard to interpretating such studies, not least in how to compare SARS-CoV-2 levels between different-sized wastewater treatment plants. Viral faecal indicators, including crAssphage and pepper mild mottle virus, have been proposed as population biomarkers to normalise SARS-CoV-2 levels in wastewater. However, as these indicators exhibit variability between individuals and may not be excreted by everyone, their utility as population biomarkers may be limited. Coprostanol, meanwhile, is a bacterial metabolite of cholesterol which is excreted by all individuals. In this study, composite influent samples were collected from a large- and medium-sized wastewater treatment plant in Dublin, Ireland and SARS-CoV-2 N1, crAssphage, pepper mild mottle virus, HF183 and coprostanol levels were determined. SARS-CoV-2 N1 RNA was detected and quantified in all samples from both treatment plants. Regardless of treatment plant size, coprostanol levels exhibited the lowest variation in composite influent samples, while crAssphage exhibited the greatest variation. Moreover, the strongest correlations were observed between SARS-CoV-2 levels and national and Dublin COVID-19 cases when levels were normalised to coprostanol. This work demonstrates the usefulness of coprostanol as a population biomarker for wastewater surveillance studies.

Real-time monitoring of key performance indicator analytes such as acetate and propionate within anaerobic digestors (AD) is required for optimum biogas production. In this paper the further refinement of acetate and propionate whole cell (E. coli) exclusion biosensors is reported following an iterative process in which key metabolites that might interfere with O-2-uptake measurements are identified and genes required for their catabolism are knocked out (exclusion). Analysis of biological leachate from an AD reactor treating lignocellulosic material revealed the presence of formate, which was subsequently shown to elicit a response in previously developed E. coli biosensor strains. P1 phage transduction was employed to delete two genes encoding formate dehydrogenase, fdoH and fdnH, to eliminate formate catabolism. Deletion of these genes from the propionate biosensor strain W:ldgyepak abolished interference from formate and enabled accurate determination of propionate concentrations in biological leachate. However, the acetate biosensing strain E1/pGDR11-acs, despite not having any response to formate, responded to propionate. It was likely that this was a result of the promiscuity of the wild type acetyl CoA synthetase, which was replaced with Acs2 from Saccharomyces cerevisiae, resolving the problem and enabling acetate determination with the biosensor. Acetate and propionate concentrations in authentic leachate influent were estimated to be 26.5 mM and 65.5 mM, respectively, using the biosensor, and 26.6 and 70 mM, respectively, by HPLC, demonstrating the accuracy and specificity of the refined biosensor.