An asymmetric sp3-sp3 cross-electrophile coupling using 'ene'-reductases.

The catalytic asymmetric construction of Csp3-Csp3 bonds remains one of the foremost challenges in organic synthesis1. Metal-catalysed cross-electrophile couplings (XECs) have emerged as a powerful tool for C-C bond formation2-5. However, coupling two distinct Csp3 electrophiles with high cross-selectivity and stereoselectivity continues as an unmet challenge. Here we report a highly chemoselective and enantioselective Csp3-Csp3 XEC between alkyl halides and nitroalkanes catalysed by flavin-dependent 'ene'-reductases (EREDs). Photoexcitation of the enzyme-templated charge-transfer complex between an alkyl halide and a flavin cofactor enables the chemoselective reduction of alkyl halide over the thermodynamically favoured nitroalkane partner. The key C-C bond-forming step occurs by means of the reaction of an alkyl radical with an in situ-generated nitronate to form a nitro radical anion that collapses to form nitrite and an alkyl radical. An enzyme-controlled hydrogen atom transfer (HAT) affords high levels of enantioselectivity. This reactivity is unknown in small-molecule catalysis and highlights the potential for enzymes to use new mechanisms to address long-standing synthetic challenges.

Non-syntrophic methanogenic hydrocarbon degradation by an archaeal species.

The methanogenic degradation of oil hydrocarbons can proceed through syntrophic partnerships of hydrocarbon-degrading bacteria and methanogenic archaea1-3. However, recent culture-independent studies have suggested that the archaeon 'Candidatus Methanoliparum' alone can combine the degradation of long-chain alkanes with methanogenesis4,5. Here we cultured Ca. Methanoliparum from a subsurface oil reservoir. Molecular analyses revealed that Ca. Methanoliparum contains and overexpresses genes encoding alkyl-coenzyme M reductases and methyl-coenzyme M reductases, the marker genes for archaeal multicarbon alkane and methane metabolism. Incubation experiments with different substrates and mass spectrometric detection of coenzyme-M-bound intermediates confirm that Ca. Methanoliparum thrives not only on a variety of long-chain alkanes, but also on n-alkylcyclohexanes and n-alkylbenzenes with long n-alkyl (C≥13) moieties. By contrast, short-chain alkanes (such as ethane to octane) or aromatics with short alkyl chains (C≤12) were not consumed. The wide distribution of Ca. Methanoliparum4-6 in oil-rich environments indicates that this alkylotrophic methanogen may have a crucial role in the transformation of hydrocarbons into methane.

Anaerobic Degradation of Alkanes by Marine Archaea.

Alkanes are saturated apolar hydrocarbons that range from their simplest form, methane, to high-molecular-weight compounds. Although alkanes were once considered biologically recalcitrant under anaerobic conditions, microbiological investigations have now identified several microbial taxa that can anaerobically degrade alkanes. Here we review recent discoveries in the anaerobic oxidation of alkanes with a specific focus on archaea that use specific methyl coenzyme M reductases to activate their substrates. Our understanding of the diversity of uncultured alkane-oxidizing archaea has expanded through the use of environmental metagenomics and enrichment cultures of syntrophic methane-, ethane-, propane-, and butane-oxidizing marine archaea with sulfate-reducing bacteria. A recently cultured group of archaea directly couples long-chain alkane degradation with methane formation, expanding the range of substrates used for methanogenesis. This article summarizes the rapidly growing knowledge of the diversity, physiology, and habitat distribution of alkane-degrading archaea.

Milk of ruminants in ceramic baby bottles from prehistoric child graves.

The study of childhood diet, including breastfeeding and weaning, has important implications for our understanding of infant mortality and fertility in past societies1. Stable isotope analyses of nitrogen from bone collagen and dentine samples of infants have provided information on the timing of weaning2; however, little is known about which foods were consumed by infants in prehistory. The earliest known clay vessels that were possibly used for feeding infants appear in Neolithic Europe, and become more common throughout the Bronze and Iron Ages. However, these vessels-which include a spout through which liquid could be poured-have also been suggested to be feeding vessels for the sick or infirm3,4. Here we report evidence for the foods that were contained in such vessels, based on analyses of the lipid 'fingerprints' and the compound-specific δ13C and Δ13C values of the major fatty acids of residues from three small, spouted vessels that were found in Bronze and Iron Age graves of infants in Bavaria. The results suggest that the vessels were used to feed infants with milk products derived from ruminants. This evidence of the foodstuffs that were used to either feed or wean prehistoric infants confirms the importance of milk from domesticated animals for these early communities, and provides information on the infant-feeding behaviours that were practised by prehistoric human groups.

On the mechanisms of ion adsorption to aqueous interfaces: air-water vs. oil-water.

The adsorption of ions to water-hydrophobe interfaces influences a wide range of phenomena, including chemical reaction rates, ion transport across biological membranes, and electrochemical and many catalytic processes; hence, developing a detailed understanding of the behavior of ions at water-hydrophobe interfaces is of central interest. Here, we characterize the adsorption of the chaotropic thiocyanate anion (SCN-) to two prototypical liquid hydrophobic surfaces, water-toluene and water-decane, by surface-sensitive nonlinear spectroscopy and compare the results against our previous studies of SCN- adsorption to the air-water interface. For these systems, we observe no spectral shift in the charge transfer to solvent spectrum of SCN-, and the Gibb's free energies of adsorption for these three different interfaces all agree within error. We employed molecular dynamics simulations to develop a molecular-level understanding of the adsorption mechanism and found that the adsorption for SCN- to both water-toluene and water-decane interfaces is driven by an increase in entropy, with very little enthalpic contribution. This is a qualitatively different mechanism than reported for SCN- adsorption to the air-water and graphene-water interfaces, wherein a favorable enthalpy change was the main driving force, against an unfavorable entropy change.

Discovery of a Fungal P450 with an Unusual Two-Step Mechanism for Constructing a Bicyclo[3.2.2]nonane Skeleton.

The successful biomimetic or chemoenzymatic synthesis of target natural products (NPs) and their derivatives relies on enzyme discovery. Herein, we discover a fungal P450 BTG5 that can catalyze the formation of a bicyclo[3.2.2]nonane structure through an unusual two-step mechanism of dimerization and cyclization in the biosynthesis of beticolin 1, whose bicyclo[3.2.2]nonane skeleton connects an anthraquinone moiety and a xanthone moiety. Further investigation reveals that BTG5-T318 not only determines the substrate selectivity but also alters the catalytic reactions, which allows the separation of the reaction to two individual steps, thereby understanding its catalytic mechanism. It reveals that the first heterodimerization undergoes the common oxidation process for P450s, while the second uncommon formal redox-neutral cyclization step is proved as a redox-mediated reaction, which has never been reported. Therefore, this work advances our understanding of P450-catalyzed reactions and paves the way for expansion of the diversity of this class of NPs through synthetic biology.

Broad Chain-Length Specificity of the Alkane-Forming Enzymes NoCER1A and NoCER3A/B in Nymphaea odorata.

Many terrestrial plants produce large quantities of alkanes for use in epicuticular wax and the pollen coat. However, their carbon chains must be long to be useful as fuel or as a petrochemical feedstock. Here, we focus on Nymphaea odorata, which produces relatively short alkanes in its anthers. We identified orthologs of the Arabidopsis alkane biosynthesis genes AtCER1 and AtCER3 in N. odorata and designated them NoCER1A, NoCER3A and NoCER3B. Expression analysis of NoCER1A and NoCER3A/B in Arabidopsis cer mutants revealed that the N. odorata enzymes cooperated with the Arabidopsis enzymes and that the NoCER1A produced shorter alkanes than AtCER1, regardless of which CER3 protein it interacted with. These results indicate that AtCER1 frequently uses a C30 substrate, whereas NoCER1A, NoCER3A/B and AtCER3 react with a broad range of substrate chain lengths. The incorporation of shorter alkanes disturbed the formation of wax crystals required for water-repellent activity in stems, suggesting that chain-length specificity is important for surface cleaning. Moreover, cultured tobacco cells expressing NoCER1A and NoCER3A/B effectively produced C19-C23 alkanes, indicating that the introduction of the two enzymes is sufficient to produce alkanes. Taken together, our findings suggest that these N. odorata enzymes may be useful for the biological production of alkanes of specific lengths. 3D modeling revealed that CER1s and CER3s share a similar structure that consists of N- and C-terminal domains, in which their predicted active sites are respectively located. We predicted the complex structure of both enzymes and found a cavity that connects their active sites.

Sample-to-Answer Detection of SARS-CoV-2 Viremia Using Thermally Responsive Alkane Partitions.

The diagnosis of bloodborne viral infections (viremia) is currently relegated to central laboratories because of the complex procedures required to detect viruses in blood samples. The development of point-of-care diagnostics for viremia would enable patients to receive a diagnosis and begin treatment immediately instead of waiting days for results. Point-of-care systems for viremia have been limited by the challenges of integrating multiple precise steps into a fully automated (i.e., sample-to-answer), compact, low-cost system. We recently reported the development of thermally responsive alkane partitions (TRAPs), which enable the complete automation of diagnostic assays with complex samples. Here we report the use of TRAPs for the sample-to-answer detection of viruses in blood using a low-cost portable device and easily manufacturable cassettes. Specifically, we demonstrate the detection of SARS-CoV-2 in spiked blood samples, and we show that our system detects viremia in COVID-19 patient samples with good agreement to conventional RT-qPCR. We anticipate that our sample-to-answer system can be used to rapidly diagnose SARS-CoV-2 viremia at the point of care, leading to better health outcomes for patients with severe COVID-19 disease, and that our system can be applied to the diagnosis of other life-threatening bloodborne viral diseases, including Hepatitis C and HIV.

Mar1, a high mobility group box protein, regulates n-alkane adsorption and cell morphology of the dimorphic yeast Yarrowia lipolytica.

The dimorphic yeast Yarrowia lipolytica possesses an excellent ability to utilize n-alkane as a sole carbon and energy source. Although there are detailed studies on the enzymes that catalyze the reactions in the metabolic processes of n-alkane in Y. lipolytica, the molecular mechanism underlying the incorporation of n-alkane into the cells remains to be elucidated. Because Y. lipolytica adsorbs n-alkane, we postulated that Y. lipolytica incorporates n-alkane through direct interaction with it. We isolated and characterized mutants defective in adsorption to n-hexadecane. One of the mutants harbored a nonsense mutation in MAR1 (Morphology and n-alkane Adsorption Regulator 1) encoding a protein containing a high mobility group box. The deletion mutant of MAR1 exhibited defects in adsorption to n-hexadecane and filamentous growth on solid media, whereas the strain that overexpressed MAR1 exhibited hyperfilamentous growth. Fluorescence microscopic observations suggested that Mar1 localizes in the nucleus. RNA-sequencing analysis revealed the alteration of the transcript levels of several genes, including those encoding transcription factors and cell surface proteins, by the deletion of MAR1. These findings suggest that MAR1 is involved in the transcriptional regulation of the genes required for n-alkane adsorption and cell morphology transition.IMPORTANCEYarrowia lipolytica, a dimorphic yeast capable of assimilating n-alkane as a carbon and energy source, has been extensively studied as a promising host for bioconversion of n-alkane into useful chemicals and bioremediation of soil and water contaminated by petroleum. While the metabolic pathway of n-alkane in this yeast and the enzymes involved in this pathway have been well characterized, the molecular mechanism to incorporate n-alkane into the cells is yet to be fully understood. Due to the ability of Y. lipolytica to adsorb n-alkane, it has been hypothesized that Y. lipolytica incorporates n-alkane through direct interaction with it. In this study, we identified a gene, MAR1, which plays a crucial role in the transcriptional regulation of the genes necessary for the adsorption to n-alkane and the transition of the cell morphology in Y. lipolytica. Our findings provide valuable insights that could lead to advanced applications of Y. lipolytica in n-alkane bioconversion and bioremediation.

Toward the development of a molecular toolkit for the microbial remediation of per-and polyfluoroalkyl substances.

Per- and polyfluoroalkyl substances (PFAS) are highly fluorinated synthetic organic compounds that have been used extensively in various industries owing to their unique properties. The PFAS family encompasses diverse classes, with only a fraction being commercially relevant. These substances are found in the environment, including in water sources, soil, and wildlife, leading to human exposure and fueling concerns about potential human health impacts. Although PFAS degradation is challenging, biodegradation offers a promising, eco-friendly solution. Biodegradation has been effective for a variety of organic contaminants but is yet to be successful for PFAS due to a paucity of identified microbial species capable of transforming these compounds. Recent studies have investigated PFAS biotransformation and fluoride release; however, the number of specific microorganisms and enzymes with demonstrable activity with PFAS remains limited. This review discusses enzymes that could be used in PFAS metabolism, including haloacid dehalogenases, reductive dehalogenases, cytochromes P450, alkane and butane monooxygenases, peroxidases, laccases, desulfonases, and the mechanisms of microbial resistance to intracellular fluoride. Finally, we emphasize the potential of enzyme and microbial engineering to advance PFAS degradation strategies and provide insights for future research in this field.

AlmA involved in the long-chain n-alkane degradation pathway in Acinetobacter baylyi ADP1 is a Baeyer-Villiger monooxygenase.

Many Acinetobacter species can grow on n-alkanes of varying lengths (≤C40). AlmA, a unique flavoprotein in these Acinetobacter strains, is the only enzyme proven to be required for the degradation of long-chain (LC) n-alkanes, including C32 and C36 alkanes. Although it is commonly presumed to be a terminal hydroxylase, its role in n-alkane degradation remains elusive. In this study, we conducted physiological, biochemical, and bioinformatics analyses of AlmA to determine its role in n-alkane degradation by Acinetobacter baylyi ADP1. Consistent with previous reports, gene deletion analysis showed that almA was vital for the degradation of LC n-alkanes (C26-C36). Additionally, enzymatic analysis revealed that AlmA catalyzed the conversion of aliphatic 2-ketones (C10-C16) to their corresponding esters, but it did not conduct n-alkane hydroxylation under the same conditions, thus suggesting that AlmA in strain ADP1 possesses Baeyer-Villiger monooxygenase (BVMO) activity. These results were further confirmed by bioinformatics analysis, which revealed that AlmA was closer to functionally identified BVMOs than to hydroxylases. Altogether, the results of our study suggest that LC n-alkane degradation by strain ADP1 possibly follows a novel subterminal oxidation pathway that is distinct from the terminal oxidation pathway followed for short-chain n-alkane degradation. Furthermore, our findings suggest that AlmA catalyzes the third reaction in the LC n-alkane degradation pathway.IMPORTANCEMany microbial studies on n-alkane degradation are focused on the genes involved in short-chain n-alkane (≤C16) degradation; however, reports on the genes involved in long-chain (LC) n-alkane (>C20) degradation are limited. Thus far, only AlmA has been reported to be involved in LC n-alkane degradation by Acinetobacter spp.; however, its role in the n-alkane degradation pathway remains elusive. In this study, we conducted a detailed characterization of AlmA in A. baylyi ADP1 and found that AlmA exhibits Baeyer-Villiger monooxygenase activity, thus indicating the presence of a novel LC n-alkane biodegradation mechanism in strain ADP1.

Fatty alcohols, a minor component of the tree tobacco surface wax, are associated with defence against caterpillar herbivory.

Despite decades of research resulting in a comprehensive understanding of epicuticular wax metabolism, the function of these almost ubiquitous metabolites in plant-herbivore interactions remains unresolved. In this study, we examined the effects of CRISPR-induced knockout mutations in four Nicotiana glauca (tree tobacco) wax metabolism genes. These mutations cause a wide range of changes in epicuticular wax composition, leading to altered interactions with insects and snails. Three interaction classes were examined: chewing herbivory by seven caterpillars and one snail species, phloem feeding by Myzus persicae (green peach aphid) and oviposition by Bemisia tabaci (whitefly). Although total wax load and alkane abundance did not affect caterpillar growth, a correlation across species, showed that fatty alcohols, a minor component of N. glauca surface waxes, negatively affected the growth of both a generalist caterpillar (Spodoptera littoralis) and a tobacco-feeding specialist (Manduca sexta). This negative correlation was overshadowed by the stronger effect of anabasine, a nicotine isomer, and was apparent when fatty alcohols were added to an artificial lepidopteran diet. By contrast, snails fed more on waxy leaves. Aphid reproduction and feeding activity were unaffected by wax composition but were potentially affected by altered cutin composition. Wax crystal morphology could explain the preference of B. tabaci to lay eggs on waxy wild-type plants relative to both alkane and fatty alcohol-deficient mutants. Together, our results suggest that the varied responses among herbivore classes and species are likely to be a consequence of the co-evolution that shaped the specific effects of different surface wax components in plant-herbivore interactions.

Structure and Function of Alkane Monooxygenase (AlkB).

ConspectusEvery year, perhaps as much as 800 million tons of hydrocarbons enters the environment; alkanes make up a large percentage of it. Most are transformed by organisms that utilize these molecules as sources of energy and carbon. Both aerobic and anaerobic alkane transformation chemistries exist, capitalizing on the presence of alkanes in both oxic and anoxic environments. Over the past 40 years, tremendous progress has been made in understanding the structure and mechanism of enzymes that catalyze the transformation of methane. By contrast, progress involving enzymes that transform liquid alkanes has been slower with the first structures of AlkB, the predominant aerobic alkane hydroxylase in the environment, appearing in 2023. Because of the fundamental importance of C-H bond activation chemistries, interest in understanding how biology activates and transforms alkanes is high.In this Account, we focus on steps we have taken to understand the mechanism and structure of alkane monooxygenase (AlkB), the metalloenzyme that dominates the transformation of liquid alkanes in the environment (not to be confused with another AlkB that is an α-ketogluturate-dependent enzyme involved in DNA repair). First, we briefly describe what is known about the prevalence of AlkB in the environment and its role in the carbon cycle. Then we review the key findings from our recent high-resolution cryoEM structure of AlkB and highlight important similarities and differences in the structures of members of class III diiron enzymes. Functional studies, which we summarize, from a number of single residue variants enable us to say a great deal about how the structure of AlkB facilitates its function. Next, we overview work from our laboratories using mechanistically diagnostic radical clock substrates to characterize the mechanism of AlkB and contextualize the results we have obtained on AlkB with results we have obtained on other alkane-oxidizing enzymes and explain these results in light of the enzyme's structure. Finally, we integrate recent work in our laboratories with information from prior studies of AlkB, and relevant model systems, to create a holistic picture of the enzyme. We end by pointing to critical questions that still need to be answered, questions about the electronic structure of the active site of the enzyme throughout the reaction cycle and about whether and to what extent the enzyme plays functional roles in biology beyond simply initiating the degradation of alkanes.

Exoproteome analysis of Pseudomonas aeruginosa response to high alkane stress.

Microbial biodegradation serves as an effective approach to treat oil pollution. However, the application of such methods for the degrading long-chain alkanes still encounters significant challenges. Comparative proteomics has extensively studied the intracellular proteins of bacteria that degrade short- and medium-chain alkanes, but the role and mechanism of extracellular proteins in many microorganism remain unclear. To enhance our understanding of the roles of extracellular proteins in the adaptation to long-chain alkanes, a label-free LC-MS/MS strategy was applied for the relative quantification of extracellular proteins of Pseudomonas aeruginosa SJTD-1-M (ProteomeXchange identifier PXD014638). 444 alkane-sentitive proteins were acquired and their cell localization analysis was performed using the Pseudomonas Genome Database. Among them, 111 proteins were found to be located in extracellular or Outer Membrane Vesicles (OMVs). The alkane-induced abundance of 11 extracellular or OMV target proteins was confirmed by parallel reaction monitoring (PRM). Furthermore, we observed that the expression levels of three proteins (Pra, PA2815, and FliC) were associated with the carbon chain length of the added alkane in the culture medium. The roles of these proteins in cell mobility, alkane emulsification, assimilation, and degradation were further discussed. OMVs were found to contain a number of enzymes involved in alkane metabolism, fatty acid beta-oxidation, and the TCA cycle, suggesting their potential as sites for facilitated alkane degradation. In this sense, this exoproteome analysis contributes to a better understanding of the role of extracellular proteins in the hydrocarbon treatment process.

Surfactants Influencing the Biocatalytic Performance of Natural Alkane-Degrading Bacteria via Interfacial Biocatalysis in Pickering Emulsions.

Setting superhydrophobic Mycobacterium sp. as an example, the hydrophobic bacteria acting as demulsifying agents of surfactant-stabilized conventional emulsions, vice versa, the synergistic/antagonistic influence of nonionic surfactants (Tween 80 or Span 80) on the stability of the bacteria-stabilized Pickering emulsions was investigated. At the same time, the activated/suppression effect of nonionic surfactants on microbial degradation of tetradecane, which exhibited a dose-response relationship, was also found. The hydrophobic bacteria acting as demulsifying agents and the suppression influence of nonionic surfactants on the biocatalytic performance (indexing as biomass) of natural alkane-degrading bacteria, believed to be totally separated concepts previously, are for the first time found to be closely related to in situ surface modification of bacteria with nonionic surfactants. During the degradation of tetradecane by Mycobacterium sp. in the presence of nonionic surfactants, demulsification due to the bacteria acting as demulsifying agents and interfacial biocatalysis in the bacteria-stabilized Pickering emulsions are involved, which provides useful information to select optimal dispersants for marine oil spills.

From Antagonism to Enhancement: Triton X-100 Surfactant Affects Phenanthrene Interfacial Biodegradation by Mycobacteria through a Shift in Uptake Mechanisms.

Polycyclic aromatic hydrocarbons (PAHs), as persistent environmental pollutants, often reside in nonaqueous-phase liquids (NAPLs). Mycobacterium sp. WY10, boasting highly hydrophobic surfaces, can adsorb to the oil-water interface, stabilizing the Pickering emulsion and directly accessing PAHs for biodegradation. We investigated the impact of Triton X-100 (TX100) on this interfacial uptake of phenanthrene (PHE) by Mycobacteria, using n-tetradecane (TET) and bis-(2-ethylhexyl) phthalate (DEHP) as NAPLs. Interfacial tension, phase behavior, and emulsion stability studies, alongside confocal laser scanning microscopy and electron microscope observations, unveiled the intricate interplay. In surfactant-free systems, Mycobacteria formed stable W/O Pickering emulsions, directly degrading PHE within the NAPLs because of their intimate contact. Introducing low-dose TX100 disrupted this relationship. Preferentially binding to the cells, the surfactant drastically increased the cell hydrophobicity, triggering desorption from the interface and phase separation. Consequently, PAH degradation plummeted due to hindered NAPL access. Higher TX100 concentrations flipped the script, creating surfactant-stabilized O/W emulsions devoid of interfacial cells. Surprisingly, PAH degradation remained efficient. This paradox can be attributed to NAPL emulsification, driven by the surfactant, which enhanced mass transfer and brought the substrate closer to the cells, despite their absence at the interface. This study sheds light on the complex effect of surfactants on Mycobacteria and PAH uptake, revealing an antagonistic effect at low concentrations that ultimately leads to enhanced degradation through emulsification at higher doses. These findings offer valuable insights into optimizing bioremediation strategies in PAH-contaminated environments.

A novel alkane monooxygenase evolved from a broken piece of ribonucleotide reductase in Geobacillus kaustophilus HTA426 isolated from Mariana Trench.

We have accidentally found that a thermophilic Geobacillus kaustophilus HTA426 is capable of degrading alkanes although it has no alkane oxygenating enzyme genes. Our experimental results revealed that a putative ribonucleotide reductase small subunit GkR2loxI (GK2771) gene encodes a novel heterodinuclear Mn-Fe alkane monooxygenase/hydroxylase. GkR2loxI protein can perform two-electron oxidations similar to homonuclear diiron bacterial multicomponent soluble methane monooxygenases. This finding not only answers a long-standing question about the substrate of the R2lox protein clade, but also expands our understanding of the vast diversity and new evolutionary lineage of the bacterial alkane monooxygenase/hydroxylase family.

The potential of Bacillus species isolated from Cinnamomum camphora for biofuel production.

BACKGROUND: Increasing concerns about climate change and global petroleum supply draw attention to the urgent need for the development of alternative methods to produce fuels. Consequently, the scientific community must devise novel ways to obtain fuels that are both sustainable and eco-friendly. Bacterial alkanes have numerous potential applications in the industry sector. One significant application is biofuel production, where bacterial alkanes can serve as a sustainable eco-friendly alternative to fossil fuels. This study represents the first report on the production of alkanes by endophytic bacteria. RESULTS: In this study, three Bacillus species, namely Bacillus atrophaeus Camph.1 (OR343176.1), Bacillus spizizenii Camph.2 (OR343177.1), and Bacillus aerophilus Camph.3 (OR343178.1), were isolated from the leaves of C. camphora. The isolates were then screened to determine their ability to produce alkanes in different culture media including nutrient broth (NB), Luria-Bertani (LB) broth, and tryptic soy broth (TSB). Depending on the bacterial isolate and the culture media used, different profiles of alkanes ranging from C8 to C31 were detected. CONCLUSIONS: The endophytic B. atrophaeus Camph.1 (OR343176.1), B. spizizenii Camph.2 (OR343177.1), and B. aerophilus Camph.3 (OR343178.1), associated with C. camphora leaves, represent new eco-friendly approaches for biofuel production, aiming towards a sustainable future. Further research is needed to optimize the fermentation process and scale up alkane production by these bacterial isolates.

Divergent Nine-Step Syntheses of Perhydrohistrionicotoxin Analogs and Their Inhibition Activity Toward Chicken α4ß2-Neuronal Nicotinic Acetylcholine Receptors.

Histrionicotoxin (HTX) alkaloids, which are isolated from Colombian poison dart frogs, are analgesic neurotoxins that modulate nicotinic acetylcholine receptors (nAChRs) as antagonists. Perhydrohistrionicotoxin (pHTX) is the potent synthetic analogue of HTX and possesses a 1-azaspiro[5.5]undecane skeleton common to the HTX family. Here, we show for the first time the divergent nine-step synthesis of pHTX and its three stereoisomers from the known aldehyde through a one-step construction of the 1-azaspiro[5.5]undecane framework from a linear amino ynone substrate. Surprisingly, some pHTX diastereomers exhibited antagonistic activities on the chicken α4ß2-neuronal nAChRs that were more potent than pHTX.

Physical impediment to sodium houttuyfonate conversely reinforces ß-glucan exposure stimulated innate immune response to Candida albicans.

Candida albicans is a dimorphic opportunistic pathogen in immunocompromised individuals. We have previously demonstrated that sodium houttuyfonate (SH), a derivative of medicinal herb Houttuynia cordata Thunb, was effective for antifungal purposes. However, the physical impediment of SH by C. albicans ß-glucan may weaken the antifungal activity of SH. In this study, the interactions of SH with cell wall (CW), extracellular matrix (EM), CW ß-glucan, and a commercial ß-glucan zymosan A (ZY) were inspected by XTT assay and total plate count in a standard reference C. albicans SC5314 as well as two clinical fluconazole-resistant strains Z4935 and Z5172. After treatment with SH, the content and exposure of CW ß-glucan, chitin, and mannan were detected, the fungal clearance by phagocytosis of RAW264.7 and THP-1 was examined, and the gene expressions and levels of cytokines TNF-ɑ and IL-10 were also monitored. The results showed that SH could be physically impeded by ß-glucan in CW, EM, and ZY. This impediment subsequently triggered the exposure of CW ß-glucan and chitin with mannan masked in a time-dependent manner. SH-induced ß-glucan exposure could significantly enhance the phagocytosis and inhibit the growth of C. albicans. Meanwhile, the SH-pretreated fungal cells could greatly stimulate the cytokine gene expressions and levels of TNF-ɑ and IL-10 in the macrophages. In sum, the strategy that the instant physical impediment of C. albicans CW to SH, which can induce the exposure of CW ß-glucan may be universal for C. albicans in response to physical deterrent by antifungal drugs.