Heterologous production of (-)-geosmin in Saccharomyces cerevisiae.

(-)-Geosmin has high demand in perfumes and cosmetic products for its earthy congenial aroma. The current production of (-)-geosmin is either by distillation of sun-baked soil or by inefficient chemical synthesis because of the presence of multiple chiral centers. Fermentation processes are not viable as the titers of the Streptomyces sp. based processes are low. This work presents an alternative route by the heterologous synthesis of (-)-geosmin in Saccharomyces cerevisiae. The enzyme involved is the bifunctional geosmin synthase that catalyzes the conversion of farnesyl diphosphate to germacradienol and germacradienol to geosmin. This study evaluated the activity of many orthologs of geosmin synthase when expressed heterologously in S. cerevisiae. When the well-characterized CAB41566 from Streptomyces coelicolor origin was tested, germacradienol and germacrene D were detected but no geosmin. Bioinformatic analysis based on high/low identities to N-terminal and C-terminal domains of CAB41566 was carried out to identify different orthologs of geosmin synthase proteins from different bacterial and fungal origins. ADO68918 of Stigmatella aurantiaca origin showed the best activity among the tested orthologs, not only in terms of geosmin production but also an order of magnitude higher total abundance of the products of geosmin synthase as compared to CAB41566. This study successfully demonstrated the production of (-)-geosmin in S. cerevisiae and offers an alternative, sustainable and environment-friendly approach to producing (-)-geosmin.

Identification of 1,3,6,8-Tetrahydroxynaphthalene Synthase (ThnA) from Nocardia sp. CS682.

Type III polyketide synthase (PKS) found in bacteria is known as 1,3,6,8-tetrahydroxynaphthalene synthase (THNS). Microbial type III PKSs synthesize various compounds that possess crucial biological functions and significant pharmaceutical activities. Based on our sequence analysis, we have identified a putative type III polyketide synthase from Nocardia sp. CS682 was named as ThnA. The role of ThnA, in Nocardia sp. CS682 during the biosynthesis of 1,3,6,8 tetrahydroxynaphthalene (THN), which is the key intermediate of 1-(α-L-(2-O-methyl)-6-deoxymannopyranosyloxy)-3,6,8-trimethoxynaphthalene (IBR-3) was characterized. ThnA utilized five molecules of malonyl-CoA as a starter substrate to generate the polyketide 1,3,6,8-tetrahydroxynaphthalene, which could spontaneously be oxidized to the red flaviolin compound 2,5,7-trihydroxy-1,4-naphthoquinone. The amino acid sequence alignment of ThnA revealed similarities with a previously identified type III PKS and identified Cys138, Phe188, His270, and Asn303 as four highly conserved active site amino acid residues, as found in other known polyketide synthases. In this study, we report the heterologous expression of the type III polyketide synthase thnA in S. lividans TK24 and the identification of THN production in a mutant strain. We also compared the transcription level of thnA in S. lividans TK24 and S. lividans pIBR25-thnA and found that thnA was only transcribed in the mutant.

Volatile sensation: The chemical ecology of the earthy odorant geosmin.

Geosmin may be the most familiar volatile compound, as it lends the earthy smell to soil. The compound is a member of the largest family of natural products, the terpenoids. The broad distribution of geosmin among bacteria in both terrestrial and aquatic environments suggests that this compound has an important ecological function, for example, as a signal (attractant or repellent) or as a protective specialized metabolite against biotic and abiotic stresses. While geosmin is part of our everyday life, scientists still do not understand the exact biological function of this omnipresent natural product. This minireview summarizes the current general observations regarding geosmin in prokaryotes and introduces new insights into its biosynthesis and regulation, as well as its biological roles in terrestrial and aquatic environments.

SAR study of piperidine derivatives as inhibitors of 1,4-dihydroxy-2-naphthoate isoprenyltransferase (MenA) from Mycobacterium tuberculosis.

The electron transport chain (ETC) in the cell membrane consists of a series of redox complexes that transfer electrons from electron donors to acceptors and couples this electron transfer with the transfer of protons (H+) across a membrane. This process generates proton motive force which is used to produce ATP and a myriad of other functions and is essential for the long-term survival of Mycobacterium tuberculosis (Mtb), the causative organism of tuberculosis (TB), under the hypoxic conditions present within infected granulomas. Menaquinone (MK), an important carrier molecule within the mycobacterial ETC, is synthesized de novo by a cluster of enzymes known as the classic/canonical MK biosynthetic pathway. MenA (1,4-dihydroxy-2-naphthoate prenyltransferase), the antepenultimate enzyme in this pathway, is a verified target for TB therapy. In this study, we explored structure-activity relationships of a previously discovered MenA inhibitor scaffold, seeking to improve potency and drug disposition properties. Focusing our campaign upon three molecular regions, we identified two novel inhibitors with potent activity against MenA and Mtb (IC50 = 13-22 µM, GIC50 = 8-10 µM). These analogs also displayed substantially improved pharmacokinetic parameters and potent synergy with other ETC-targeting agents, achieving nearly complete sterilization of Mtb in combination therapy within two weeks in vivo. These new inhibitors of MK biosynthesis present a promising new strategy to curb the continued spread of TB.

Synthesis and Evaluation of Anti-HIV Activity of Mono- and Di-Substituted Phosphonamidate Conjugates of Tenofovir.

The activity of nucleoside and nucleotide analogs as antiviral agents requires phosphorylation by endogenous enzymes. Phosphate-substituted analogs have low bioavailability due to the presence of ionizable negatively-charged groups. To circumvent these limitations, several prodrug approaches have been proposed. Herein, we hypothesized that the conjugation or combination of the lipophilic amide bond with nucleotide-based tenofovir (TFV) (1) could improve the anti-HIV activity. During the current study, the hydroxyl group of phosphonates in TFV was conjugated with the amino group of L-alanine, L-leucine, L-valine, and glycine amino acids and other long fatty ester hydrocarbon chains to synthesize 43 derivatives. Several classes of derivatives were synthesized. The synthesized compounds were characterized by 1H NMR, IR, UV, and mass spectrometry. In addition, several of the synthesized compounds were evaluated as racemic mixtures for anti-HIV activity in vitro in a single round infection assay using TZM-bl cells at 100 ng/mL. TFV (1) was used as a positive control and inhibited HIV infection by 35%. Among all the evaluated compounds, the disubstituted heptanolyl ester alanine phosphonamidate with naphthol oleate (69), pentanolyl ester alanine phosphonamidate with phenol oleate (62), and butanolyl ester alanine phosphonamidate with naphthol oleate (87) ester conjugates of TFV were more potent than parent drug TFV with 79.0%, 76.5%, 71.5% inhibition, respectively, at 100 ng/mL. Furthermore, two fatty acyl amide conjugates of tenofovir alafenamide (TAF) were synthesized and evaluated for comparative studies with TAF and TFV conjugates. Tetradecanoyl TAF conjugate 95 inhibited HIV infection by 99.6% at 100 ng/mL and showed comparable activity to TAF (97-99% inhibition) at 10-100 ng/mL but was more potent than TAF when compared at molar concentration.

The Ubiquitous Soil Terpene Geosmin Acts as a Warning Chemical.

Known as the smell of earth after rain, geosmin is an odorous terpene detectable by humans at picomolar concentrations. Geosmin production is heavily conserved in actinobacteria, myxobacteria, cyanobacteria, and some fungi, but its biological activity is poorly understood. We theorized that geosmin was an aposematic signal used to indicate the unpalatability of toxin-producing microbes, discouraging predation by eukaryotes. Consistent with this hypothesis, we found that geosmin altered the behavior of the bacteriophagous nematode Caenorhabditis elegans on agar plates in the absence of bacteria. Normal movement was restored in mutant worms lacking differentiated ASE (amphid neurons, single ciliated endings) neurons, suggesting that geosmin is a taste detected by the nematodal gustatory system. In a predation assay, geosmin and the related terpene 2-methylisoborneol reduced grazing on the bacterium Streptomyces coelicolor. Predation was restored by the removal of both terpene biosynthetic pathways or the introduction of C. elegans that lacked differentiated ASE taste neurons, leading to the apparent death of both bacteria and worms. While geosmin and 2-methylisoborneol appeared to be nontoxic, grazing triggered bacterial sporulation and the production of actinorhodin, a pigment coproduced with a number of toxic metabolites. In this system, geosmin thus appears to act as a warning signal indicating the unpalatability of its producers and reducing predation in a manner that benefits predator and prey. This suggests that molecular signaling may affect microbial predator-prey interactions in a manner similar to that of the well-studied visual markers of poisonous animal prey. IMPORTANCE One of the key chemicals that give soil its earthy aroma, geosmin is a frequent water contaminant produced by a range of unrelated microbes. Many animals, including humans, are able to detect geosmin at minute concentrations, but the benefit that this compound provides to its producing organisms is poorly understood. We found that geosmin repelled the bacterial predator Caenorhabditis elegans in the absence of bacteria and reduced contact between the worms and the geosmin-producing bacterium Streptomyces coelicolor in a predation assay. While geosmin itself appears to be nontoxic to C. elegans, these bacteria make a wide range of toxic metabolites, and grazing on them harmed the worms. In this system, geosmin thus appears to indicate unpalatable bacteria, reducing predation and benefiting both predator and prey. Aposematic signals are well known in animals, and this work suggests that metabolites may play a similar role in the microbial world.

Discovery of novel rigid analogs of 2-naphthol with potent anticancer activity through multi-target topoisomerase I & II and tyrosine kinase receptor EGFR & VEGFR-2 inhibition mechanism.

A novel oxygen-containing heterocyclic linked 1H-benzo[f]chromene moieties (4a-g) and (6a-g) with anti-proliferative activity against cancer cell lines was designed, synthesized, and established on the basis of spectral data. All the prepared compounds were evaluated in vitro for their anti-proliferative activity against MCF-7, HCT-116, HepG-2 cell lines and normal cell lines HFL-1, WI-38. Compounds 4a, 4b, and 6e exhibited good activity against MCF-7, HCT-116, and HepG-2 cell lines, comparable to that of Vinblastine and Doxorubicin, and weak active against normal cell lines. Moreover, the potential mechanisms of the cytotoxic activity of the promising compounds 4a, 4b, and 6e on the more sensitive cell line MCF-7 were studied. We found that compounds 4a, 4b, and 6e induce cell cycle arrest at G2/M phases for MCF-7 treated cells compared to untreated cells, which causes apoptosis and inhibits both the topoisomerase I and II enzymes. In addition, compounds 4a and 4b exhibited comparable inhibitory activity on tyrosine kinase receptors EGFR and VEGFR-2 kinases to that of the reference protein kinases inhibitor Sorafenib. The in silico molecular docking of the most active compounds into the active sites of EGFR kinase and Topo I & II enzymes provides us with a reasonable clarification of the interpreted biological data.

The Role of Melanin in the Biology and Ecology of Nematophagous Fungi.

Melanin is a heteropolymer formed by the polymerization of phenolic and indolic compounds. It occurs in organisms across all biological kingdoms and has a range different of functions, thus indicating its important evolutionary role. The presence of melanin offers several protective advantages, including against ultraviolet radiation, traumatic damage, oxidative stress, extreme temperatures, and pressure. For many species of fungi, melanin also participates directly in the process of virulence and pathogenicity. These organisms can synthesize melanin in two main ways: using a substrate of endogenous origin, involving 1,8-dihydroxynaphthalene (DHN); alternatively, in an exogenous manner with the addition of L-3, 4-dihydroxyphenylalanine (L-DOPA or levodopa). As melanin is an amorphous and complex substance, its study requires expensive and inaccessible technologies and analyses are often difficult to perform with conventional biochemical techniques. As such, details about its chemical structure are not yet fully understood, particularly for nematophagous fungi that remain poorly studied. Thus, this review presents an overview of the different types of melanin, with an emphasis on fungi, and discusses the role of melanin in the biology and ecology of nematophagous fungi.

Granulocyte-Macrophage Colony-Stimulating Factor-Activated Neutrophils Express B7-H4 That Correlates with Gastric Cancer Progression and Poor Patient Survival.

Neutrophils are prominent components of gastric cancer (GC) tumors and exhibit distinct phenotypes in GC environment. However, the phenotype, regulation, and clinical relevance of neutrophils in human GC are presently unknown. Here, immunohistochemistry, real-time PCR, and flow cytometry analyses were performed to examine levels and phenotype of neutrophils in samples from 41 patients with GC, and also isolated, stimulated, and/or cultured neutrophils for in vitro regulation assays. Finally, we performed Kaplan-Meier plots for overall survival by using the log-rank test to evaluate the clinical relevance of neutrophils and their subsets. In our study, neutrophils in tumor tissues were significantly higher than those in nontumor tissues and were positively associated with tumor progression but negatively correlated with GC patient survival. Most intratumoral neutrophils showed an activated CD54+ phenotype and expressed high-level immunosuppressive molecule B7-H4. Tumor tissue culture supernatants from GC patients induced neutrophils to express CD54 and B7-H4 in both time-dependent and dose-dependent manners. Locally enriched CD54+ neutrophils and B7-H4+ neutrophils positively correlated with increased granulocyte-macrophage colony-stimulating factor (GM-CSF) detection ex vivo, and in vitro GM-CSF induced the expression of CD54 and B7-H4 on neutrophils in a time-dependent and dose-dependent manner. Moreover, GC tumor-derived GM-CSF activated neutrophils and induced neutrophil B7-H4 expression via Janus kinase (JAK)-signal transducer and activator of transcription 3 (STAT3) signaling pathway activation. Furthermore, higher intratumoral B7-H4+ neutrophil percentage/number was found in GC patients with advanced tumor node metastasis stage and reduced overall survival following surgery. Our results illuminate a novel regulating mechanism of B7-H4 expression on tumor-activated neutrophils in GC, suggesting that functional inhibition of these novel GM-CSF-B7-H4 pathways may be a suitable therapeutic strategy to treat the immune tolerance feature of GC.

Compartmentalization of Melanin Biosynthetic Enzymes Contributes to Self-Defense against Intermediate Compound Scytalone in Botrytis cinerea.

In filamentous fungi, 1,8-dihydroxynaphthalene (DHN) melanin is a major component of the extracellular matrix, endowing fungi with environmental tolerance and some pathogenic species with pathogenicity. However, the subcellular location of the melanin biosynthesis pathway components remains obscure. Using the gray mold pathogen Botrytis cinerea, the DHN melanin intermediate scytalone was characterized via phenotypic and chemical analysis of mutants, and the key enzymes participating in melanin synthesis were fused with fluorescent proteins to observe their subcellular localizations. The Δbcscd1 mutant accumulated scytalone in the culture filtrate rather than in mycelium. Excessive scytalone appears to be self-inhibitory to the fungus, leading to repressed sclerotial germination and sporulation in the Δbcscd1 mutant. The BcBRN1/2 enzymes responsible for synthesizing scytalone were localized in endosomes and found to be trafficked to the cell surface, accompanied by the accumulation of BcSCD1 proteins in the cell wall. In contrast, the early-stage melanin synthesis enzymes BcPKS12/13 and BcYGH1 were localized in peroxisomes. Taken together, the results of this study revealed the subcellular distribution of melanin biosynthetic enzymes in B. cinerea, indicating that the encapsulation and externalization of the melanin synthetic enzymes need to be delicately orchestrated to ensure enzymatic efficiency and protect itself from the adverse effect of the toxic intermediate metabolite.IMPORTANCE The devastating gray mold pathogen Botrytis cinerea propagates via melanized conidia and sclerotia. This study reveals that the sclerotial germination of B. cinerea is differentially affected by different enzymes in the melanin synthesis pathway. Using gene knockout mutants and chemical analysis, we found that excessive accumulation of the melanin intermediate scytalone is inhibitory to B. cinerea. Subcellular localization analysis of the melanin synthesis enzymes of B. cinerea suggested two-stage partitioning of the melanogenesis pathway: the intracellular stage involves the steps until the intermediate scytalone was translocated to the cell surface, whereas the extracellular stage comprises all the steps occurring in the wall from scytalone to final melanin formation. These strategies make the fungus avert self-poisoning during melanin production. This study opens avenues for better understanding the mechanisms of secondary metabolite production in filamentous fungi.

Highly Efficient Production of Menaquinone-7 from Glucose by Metabolically Engineered Escherichia coli.

Menaquinone-7 (MK-7) possesses wide health and medical value, and the market demand for MK-7 has increased. Metabolic engineering for MK-7 production in Escherichia coli still remains challenging due to the characteristics of the competing quinone synthesis, and cells mainly synthesized menaquinones under anaerobic conditions. To increase the production of MK-7 in engineered E. coli strains under aerobic conditions, we divided the whole MK-7 biosynthetic pathway into three modules (MVA pathway, DHNA pathway, and MK-7 pathway) and systematically optimized each of them. First, by screening and enhancing Idi expression, the amounts of MK-7/DMK-7 increased significantly. Then, in the MK-7 pathway, by combinatorial overexpression of endogenous MenA and exogenous UbiE, and fine-tuning the expression of HepPPS, MenA, and UbiE, 70 µM MK-7 was achieved. Third, the DHNA synthetic pathway was enhanced, and 157 µM MK-7 was achieved. By the combinational metabolic engineering strategies and membrane engineering, an efficient metabolic engineered E. coli strain for MK-7 synthesis was developed, and 200 µM (129 mg/L) MK-7 was obtained in shake flask experiment, representing a 306-fold increase compared to the starting strain. In the scale-up fermentation, 2074 µM (1350 mg/L) MK-7 was achieved after 52 h fermentation with a productivity of 26 mg/L/h. This is the highest titer of MK-7 ever reported. This study offers an alternative method for MK-7 production from biorenewable feedstock (glucose) by engineered E. coli. The high titer of our process should make it a promising cost-effective resource for MK-7.

Allomelanin: A Biopolymer of Intrinsic Microporosity.

Melanin is a ubiquitous natural pigment found in a diverse array of organisms. Allomelanin is a class of nitrogen-free melanin often found in fungi. Herein, we find artificial allomelanin analogues exhibit high intrinsic microporosity and describe an approach for further increasing and tuning that porosity. Notably, the synthetic method involves an oxidative polymerization of 1,8-DHN in water, negating the need for multiple complex templating steps and avoiding expensive or complex chemical precursors. The well-defined morphologies of these nanomaterials were elucidated by a combination of electron microscopy and scattering methods, yielding to high-resolution 3D reconstruction based on small-angle X-ray scattering (SAXS) results. Synthetic allomelanin nanoparticles exhibit high BET areas, up to 860 m2/g, and are capable of ammonia capture up to 17.0 mmol/g at 1 bar. In addition, these nanomaterials can adsorb nerve agent simulants in solution and as a coating on fabrics with high breathability where they prevent breakthrough. We also confirmed that naturally derived fungal melanin can adsorb nerve gas simulants in solution efficiently despite lower porosity than synthetic analogues. Our approach inspires further analysis of yet to be discovered biological materials of this class where melanins with intrinsic microporosity may be linked to evolutionary advantages in relevant organisms and may in turn inspire the design of new high surface area materials.

Coinducible Catabolism of 1-Naphthol via Synergistic Regulation of the Initial Hydroxylase Genes in Sphingobium sp. Strain B2.

1-Naphthol, a widely used raw material for organic synthesis, is also a well-known organic pollutant. Due to its high toxicity, 1-naphthol is rarely used by microorganisms as the sole carbon source for growth. In this study, catabolism of 1-naphthol by Sphingobium sp. strain B2 was found to be greatly enhanced by additional supplementation with primary carbon sources (e.g., glucose, maltose, and sucrose), and 1-naphthol was even used as the carbon source for growth when strain B2 cells had been preinduced by both 1-naphthol and glucose. A distinct two-component flavin-dependent monooxygenase, NdcA1A2, was found to be responsible for the initial hydroxylation of 1-naphthol to 1,2-dihydroxynaphthalene, a more toxic compound. Transcriptional levels of ndcA1A2 genes were significantly upregulated when strain B2 cells were cultured with both 1-naphthol and glucose compared to cells cultured with only 1-naphthol or glucose. Two transcriptional regulators, the activator NdcS and the inhibitor NdcR, were found to play key roles in the synergistic regulation of the transcription of the 1-naphthol initial catabolism genes ndcA1A2IMPORTANCE Cometabolism is a widely observed phenomenon, especially in the field of microbial catabolism of highly toxic xenobiotics. However, the mechanisms of cometabolism are ambiguous, and the roles of the obligately coexisting growth substrates remain largely unknown. In this study, we revealed that the roles of the coexisting primary carbon sources (e.g., glucose) in the enhanced catabolism of the toxic compound 1-naphthol in Sphingobium sp. strain B2 were not solely because they were used as growth substrates to support cell growth but, more importantly, because they acted as coinducers to interact with two transcriptional regulators, the activator NdcS and the inhibitor NdcR, to synergistically regulate the transcription of the 1-naphthol initial catabolism genes ndcA1A2 Our findings provide new insights into the cometabolic mechanism of highly toxic compounds in microorganisms.

Limitation of nutrients stimulates musty odor production by Streptomyces sp. isolated from a tropical environment.

Musty odor production by actinomycetes is usually related to the presence of geosmin and 2-methylisoborneol (2-MIB), which are synthesized by enzymes encoded by the geoA and tpc genes, respectively. Streptomyces spp. strain S10, which was isolated from a water reservoir in Malaysia, has the ability to produce geosmin when cultivated in a basal salt (BS) solid medium, but no 2-MIB production occurred during growth in BS medium. Strain S10 could produce higher levels of geosmin when the phosphate concentration was limited to 0.05 mg/L, with a yield of 17.53 ± 3.12 ✕ 105 ng/L, compared with growth in BS medium. Interestingly, 2-MIB production was suddenly detected when the nitrate concentration was limited to 1.0 mg/L, with a yield of 1.4 ± 0.11 ✕ 105 ng/L. Therefore, it was concluded that phosphate- and nitrate-limiting conditions could induce the initial production of geosmin and 2-MIB by strain S10. Furthermore, a positive amplicon of geoA was detected in strain S10, but no tpc amplicon was detected by PCR analysis. Draft genome sequence analysis showed that one open reading frame (ORF) contained a conserved motif of geosmin synthase with 95% identity with geoA in Streptomyces coelicolor A3 (2). In the case of the tpc genes, it was found that one ORF showed 23% identity to the known tpc gene in S. coelicolor A3(2), but strain S10 lacked one motif in the N-terminus.

Expression and kinetic analysis of carboxylesterase LmCesA1 from Locusta migratoria.

OBJECTIVE: To investigate the biochemical characterization of the carboxylesterase LmCesA1 from Locusta migratoria. RESULTS: We expressed recombinant LmCesA1 in Sf9 cells by using the Bac-to-bac baculovirus expression system. Enzyme kinetic assays showed that the Km values of LmCesA1 for α-naphthyl acetate (α-NA) and ß-naphthyl acetate (ß-NA) were 0.08 ± 0.01 mM and 0.22 ± 0.03 mM, respectively, suggesting that LmCesA1 has a higher affinity for α-NA. LmCesA1 retained its enzymatic activity during incubations at pH 7-10 and at 10-30 °C. In an inhibition experiment, two organophosphate pesticides (malaoxon and malathion) and one pyrethroid pesticide (deltamethrin) showed different inhibition profiles against purified LmCesA1. Recombinant LmCesA1 activity was significantly inhibited by malaoxon in vitro. UPLC analysis showed that no metabolites were detected. CONCLUSIONS: These results suggest that overexpression of LmCesA1 enhances malathion sequestration to confer malathion tolerance in L. migratoria.

Targeting the menaquinol binding loop of mycobacterial cytochrome bd oxidase.

Mycobacteria have shown enormous resilience to survive and persist by remodeling and altering metabolic requirements. Under stringent conditions or exposure to drugs, mycobacteria have adapted to rescue themselves by shutting down their major metabolic activity and elevate certain survival factor levels and efflux pathways to survive and evade the effects of drug treatments. A fundamental feature in this adaptation is the ability of mycobacteria to vary the enzyme composition of the electron transport chain (ETC), which generates the proton motive force for the synthesis of adenosine triphosphate via oxidative phosphorylation. Mycobacteria harbor dehydrogenases to fuel the ETC, and two terminal respiratory oxidases, an aa3-type cytochrome c oxidase (cyt-bcc-aa3) and a bacterial specific cytochrome bd-type menaquinol oxidase (cyt-bd). In this study, we employed homology modeling and structure-based virtual screening studies to target mycobacteria-specific residues anchoring the b558 menaquinol binding region of Mycobacterium tuberculosis cyt-bd oxidase to obtain a focused library. Furthermore, ATP synthesis inhibition assays were carried out. One of the ligands MQL-H2 inhibited both NADH2- and succinate-driven ATP synthesis inhibition of Mycobacterium smegmatis inside-out vesicles in micromolar potency. Similarly, MQL-H2 also inhibited NADH2-driven ATP synthesis in inside-out vesicles of the cytochrome-bcc oxidase deficient M. smegmatis strain. Since neither varying the electron donor substrates nor deletion of the cyt-bcc oxidase, a major source of protons, hindered the inhibitory effects of the MQL-H2, reflecting that MQL-H2 targets the terminal oxidase cytochrome bd oxidase, which was consistent with molecular docking studies. Characterization of novel cytochrome bd oxidase Menaquinol binding domain inhibitor (MQL-H2) using virtual screening and ATP synthesis inhibition assays.

Biosynthesis of ambrein in ambergris: evidence from isotopic data and identification of possible intermediates.

Ambrein is found in ambergris, a coprolith occurring in the rectum of the sperm whale. In vitro, ambrein is produced by enzymatic cyclisation of squalene, via a monocyclic intermediate. However, little is known of the in vivo process. In order to find evidence for the reaction in vivo, a comparison was made of the δ13C relative isotopic ratios of ambrein in ambergris with those of co-occurring sterols. A statistically significant difference was noted. This suggests that ambrein originates via a different biosynthetic mechanism from that of the sterols. Examination of the minor constituents of a hydrogenolysed extract of ambergris revealed compounds with a bicyclic polypodane nucleus, rather than those with monocyclic structures. It is hypothesised that in vivo biosynthesis of ambrein proceeds, at least in some cases, via bacterial production of bicyclic polypodenols. The latter are known products of non-concerted squalene (or squalene oxide) cyclisations in other organisms.

Hydrolase CehA and a Novel Two-Component 1-Naphthol Hydroxylase CehC1C2 are Responsible for the Two Initial Steps of Carbaryl Degradation in Rhizobium sp. X9.

Carbaryl is a widely used carbamate pesticide in agriculture. The strain Rhizobium sp. X9 possesses the typical carbaryl degradation pathway in which carbaryl is mineralized via 1-naphthol, salicylate, and gentisate. In this study, we cloned a carbaryl hydrolase gene cehA and a novel two-component 1-naphthol hydroxylase gene cehC1C2. CehA mediates carbaryl hydrolysis to 1-naphthol and CehC1, an FMNH2 or FADH2-dependent monooxygenase belonging to the HpaB superfamily, and hydroxylates 1-naphthol in the presence of reduced nicotinamide-adenine dinucleotide (FMN)/flavin adenine dinucleotide (FAD), and the reductase CehC2. CehC1 has the highest amino acid similarity (58%) with the oxygenase component of a two-component 4-nitrophenol 2-monooxygenase, while CehC2 has the highest amino acid similarity (46%) with its reductase component. CehC1C2 could utilize both FAD and FMN as the cofactor during the hydroxylation, although higher catalytic activity was observed with FAD as the cofactor. The optimal molar ratio of CehC1 to CehC2 was 2:1. The Km and Kcat/Km values of CehC1 for 1-naphthol were 74.71 ± 16.07 µM and (8.29 ± 2.44) × 10-4 s-1·µM-1, respectively. Moreover, the enzyme activities and substrate spectrum between CehC1C2 and previously reported 1-naphthol hydroxylase McbC were compared. The results suggested that McbC had a higher 1-naphthol hydroxylation activity, while CehC1C2 had a broader substrate spectrum.

Atropselective Hydrolysis of Chiral Binol-Phosphate Esters Catalyzed by the Phosphotriesterase from Sphingobium sp. TCM1.

The phosphotriesterase from Sphingobium sp. TCM1 (Sb-PTE) is notable for its ability to hydrolyze a broad spectrum of organophosphate triesters, including organophosphorus flame retardants and plasticizers such as triphenyl phosphate and tris(2-chloroethyl) phosphate that are not substrates for other enzymes. This enzyme is also capable of hydrolyzing any one of the three ester groups attached to the central phosphorus core. The enantiomeric isomers of 1,1'-bi-2-naphthol (BINOL) have become among the most widely used chiral auxiliaries for the chemical synthesis of chiral carbon centers. PTE was tested for its ability to hydrolyze a series of biaryl phosphate esters, including mono- and bis-phosphorylated BINOL derivatives and cyclic phosphate triesters. Sb-PTE was shown to be able to catalyze the hydrolysis of the chiral phosphate triesters with significant stereoselectivity. The catalytic efficiency, kcat/Km, of Sb-PTE toward the test phosphate triesters ranged from ∼10 to 105 M-1 s-1. The product ratios and stereoselectivities were determined for four pairs of phosphorylated BINOL derivatives.

Improved cis-Abienol production through increasing precursor supply in Escherichia coli.

cis-Abienol, a natural diterpene-diol isolated from balsam fir (Abies balsamea), can be employed as precursors for the semi-synthesis of amber compounds, which are sustainable replacement for ambergris and widely used in the fragmented industry. This study combinatorially co-expressed geranyl diphosphate synthase, geranylgeranyl diphosphate synthase, Labda-13-en-8-ol diphosphate synthase and diterpene synthase, with the best combination achieving ~ 0.3 mg/L of cis-abienol. An additional enhancement of cis-abienol production (up to 8.6 mg/L) was achieved by introducing an exogenous mevalonate pathway which was divided into the upper pathway containing acetyl-CoA acetyltransferase/HMG-CoA reductase and HMG-CoA synthase and the lower pathway containing mevalonate kinase, phosphomevalonate kinase, pyrophosphate mevalonate decarboxylase and isopentenyl pyrophosphate isomerase. The genetically modified strain carrying chromosomal copy of low genes of the mevalonate with the trc promoter accumulated cis-abienol up to 9.2 mg/L in shake flask. Finally, cis-abienol titers of ~ 220 mg/L could be achieved directly from glucose using this de novo cis-abienol-producing E. coli in high-cell-density fermentation. This study demonstrates a microbial process to apply the E. coli cell factory in the biosynthesis of cis-abienol.