Optimization, characterization and biosafety of carotenoids produced from whey using Micrococcus luteus.

This study aimed to optimize the production of carotenoid pigments from Micrococcus luteus (ATCC 9341) through the statistical screening of media components and the characterization of antimicrobial, antioxidant, cytogenetic and cytotoxic activities. A BOX-Behnken design was used to assess the effects of whey concentration, inoculum size, pH, temperature, and agitation speed on carotenoid yield. The optimum combination increased production to 2.19 g/L, with a productivity of 0.045 g L-1 h-1 and a productivity yield of 0.644 g/g, as confirmed by an observed carotene production of 2.19 g/L. The final response surface model fitting the data had an R2 of 0.9461. High-performance liquid chromatography (HPLC) analysis identified 12 carotenoid pigment compounds produced by M. luteus. The extracts displayed moderate antimicrobial efficacy against Gram-positive bacteria such as Bacillus cereus (ATCC 11778), Staphylococcus aureus (ATCC 6538), and E. faecalis (ATCC 19433), with inhibition zone diameters (IZD) of 29.0, 14.0, and 37.0 mm, respectively, at 1000 µg/mL. However, its effectiveness against Gram-negative bacteria is limited. In comparison, tetracycline exhibited greater antimicrobial potency. The IC50 value of carotenoids was used to indicate the antioxidant activity. IC50 value from the DPPH assay was 152.80 mg/100mL. An IC50 cytotoxicity value greater than 300 µg/mL was found against normal mouse liver cells, with over 68% cell viability even at 300 µg/mL, indicating low toxicity. Histological structure studies revealed normal myocardial muscle tissue, lung tissue, and kidney tissue sections, whereas liver tissue sections revealed ballooning degeneration of hepatocytes and disorganization of hepatic cords. Cytogenetic parameters revealed that the carotene treatment group had a mitotic index (70%) lower than that of the control but higher than that of the positive control, mitomycin, and did not substantially increase numerical (1.2%) or structural aberrations compared with those of the control, suggesting a lack of genotoxic effects under the experimental conditions. In conclusion, optimized culture conditions enhanced carotenoid yields from M. luteus, and the extracts displayed promising bioactivity as moderate antibiotics against certain gram-positive bacteria and as antioxidants. The high IC50 values demonstrate biosafety. Overall, this bioprocess for enhanced carotenoid production coupled with bioactivity profiling and low cytotoxicity support the application of M. luteus carotenoids.

Phage-resistant Streptomyces abietis and its telomycin bioactive metabolite as a possible alternative to antibiotics.

Multidrug-resistant pathogens are now thought to be the primary global causes of disease and death. Therefore, it is imperative to develop new effective bioactive compounds from microbial sources, such as Streptomyces species. Nevertheless, the pharmaceutical industry suffered financial losses and low-quality end products as a result of Streptomyces bacteriophage contamination. To reduce the likelihood of phage-induced issues in the medical industry, it is crucial to develop a method for finding phage-resistant strains. Hence, we aimed to isolate and characterize Streptomyces spp. and Streptomyces phages from various rhizospheric soil samples in Egypt and to investigate their antibacterial activities. Moreover, we targeted development of a Streptomyces phage-resistant strain to extract its active metabolites and further testing its antibacterial activity. Herein, the antibacterial activities of the isolated 58 Streptomyces isolates showed that 10 (17.2 %) Streptomyces isolates had antibacterial activities against the tested bacteria including Listeria monocytogenes, E. coli O157, Acinetobacter baumannii, methicillin resistant-vancomycin-intermediate Staphylococcus aureus (MRSA-VISA) and Micrococcus luteus. Three lytic bacteriophages (ϕPRSC1, ϕPRSC2, and ϕPRSC4) belonging to the families Siphoviridae and Podoviridae were obtained from the rhizospheric soil samples using the most potent S. abietis isolate as the host strain. The three isolated Streptomyces phages were thermostable, ultraviolet stable, infectious, and had a wide range of hosts against the 10 tested Streptomyces isolates with antibacterial activities. The DNA of the ϕPRSC1 and ϕPRSC4 phages were resistant to digestion by EcoRI and HindIII, but the DNA of ϕPRSC2 was resistant to digestion by EcoRI and sensitive to digestion by HindIII. Of note, we developed a S. abietis strain resistant to the three isolated phages and its antibacterial activities were twice that of the wild strain. Finally, telomycin was recognized as an antibacterial metabolite extracted from phage-resistant S. abietis strain, which was potent against the tested Gram-positive bacteria including L. monocytogenes, MRSA-VISA, and M. luteus. Thus, our findings open new horizons for researching substitute antimicrobial medications for both existing and reemerging illnesses.

Utilisation of acid-tolerant bacteria for base metal recovery under strongly acidic conditions.

Hydrometallurgical bioprocesses for base metal recovery in environmentally friendly electronic device waste (e-waste) recycling are typically studied under neutral pH conditions to avoid competition between metals and hydrogen ions. However, metal leachate is generally strongly acidic, thus necessitating a neutralisation process in the application of these bioprocesses to e-waste recycling. To solve this pH disparity, we focused on acid-tolerant bacteria for metal recovery under strongly acidic conditions. Four acid-tolerant bacterial strains were isolated from neutral pH environments to recover base metals from simulated waste metal leachate (pH 1.5, containing 100 or 1000 mg L-1 of Co, Cu, Li, Mn, and Ni) without neutralisation. The laboratory setting for sequential metal recovery was established using these strains and a reported metal-adsorbing bacterium, Micrococcus luteus JCM1464. The metal species were successfully recovered from 100 mg L-1 metal mixtures at the following rates: Co (8.95%), Cu (21.23%), Li (5.49%), Mn (13.18%), and Ni (9.91%). From 1000 mg L-1 metal mixtures, Co (7.23%), Cu (6.82%), Li (5.85%), Mn (7.64%), and Ni (7.52%) were recovered. These results indicated the amenability of acid-tolerant bacteria to environmentally friendly base metal recycling, contributing to the development of novel industrial application of the beneficial but unutilised bioresource comprising acid-tolerant bacteria.

Maduraflavacins A-E, Unusual Phenyl Polyene Metabolites Produced by a Rare Marine-Derived Actinomadura sp.

Phenyl polyenes comprise a small family of bacterial natural products with broad and potent bioactivities, primarily found in actinobacteria. Here we report the discovery of five new phenyl polyene metabolites, maduraflavacins A-E (1-5), from a rare, marine-derived actinobacteria strain Actinomadura glauciflava NA03286. The structures of these natural products were determined by NMR spectroscopy, HRESIMS, LC-MS/MS, and chemical derivatization. All of these new maduraflavacins feature methyl substitutions at the polyene side chain, and maduraflavacins A-C (1-3) possessed a 1-N-ß-d-glucosamine-(3 → 1)-O-ß-d-glucopyranosyl-(3 → 1)-O-ß-d-glucopyranosyl-(6 → 1)-O-ß-d-glucopyranoside tetrasaccharide moiety via an amido linkage with a phenyl polyene skeleton. Compounds 1 and 2 showed weak antibacterial activities against the Gram-positive bacteria Staphylococcus aureus Sau 16339 and Micrococcus luteus, respectively.

Secondary metabolite profile of Streptomyces spp. changes when grown with the sub-lethal concentration of silver nanoparticles: possible implication in novel compound discovery.

The decline of new antibiotics and the emergence of multidrug resistance in pathogens necessitates a revisit of strategies used for lead compound discovery. This study proposes to induce the production of bioactive compounds with sub-lethal concentrations of silver nanoparticles (Ag-NPs). A total of Forty-two Actinobacteria isolates from four Saudi soil samples were grown with and without sub-lethal concentration of Ag-NPs (50 µg ml-1). The spent broth grown with Ag-NPs, or without Ag-NPs were screened for antimicrobial activity against four bacteria. Interestingly, out of 42 strains, broths of three strains grown with sub-lethal concentration of Ag-NPs exhibit antimicrobial activity against Staphylococcus aureus and Micrococcus luteus. Among these, two strains S4-4 and S4-21 identified as Streptomyces labedae and Streptomyces tirandamycinicus based on 16S rRNA gene sequence were selected for detailed study. The change in the secondary metabolites profile in the presence of Ag-NPs was evaluated using GC-MS and LC-MS analyses. Butanol extracts of spent broth grown with Ag-NPs exhibit strong antimicrobial activity against M. luteus and S. aureus. While the extracts of the controls with the same concentration of Ag-NPs do not show any activity. GC-analysis revealed a clear change in the secondary metabolite profile when grown with Ag-NPs. Similarly, the LC-MS patterns also differ significantly. Results of this study, strongly suggest that sub-lethal concentrations of Ag-NPs influence the production of secondary metabolites by Streptomyces. Besides, LC-MS results identified possible secondary metabolites, associated with oxidative stress and antimicrobial activities. This strategy can be used to possibly induce cryptic biosynthetic gene clusters for the discovery of new lead compounds.

Secretion of the arthrodial membrane gland of a harvester (Arachnida: Opiliones): Antimicrobial activity.

Because of the exoskeleton, arthropods must have flexible areas to be able to move. Such regions are called arthrodial membranes and are particularly vulnerable to bacteria and fungi. Here, we analyzed the secretion in the glands underneath it in a Neotropical harvester Mischonyx squalidus (Arachnida: Opiliones) and tested whether it has antiseptical properties. Wepuncturedthemembrane,collectedand quantified ina spectrophotometer. We also fractionated and analyzed the samples in reversed-phase high-performance liquid chromatography (RP-HPLC) and then incubated the treated fractions and determined growth inhibition by measuring absorbance. The secretions resulted in 100 fractions, among which two had activity against the Gram-positive bacteria Micrococcus luteus and against the yeast Candida albicans. The low concentrations at which the secretions were active are relevant from a biotechnological point of view. For the organism, the secretions possibly prevent infections, including when they are attacked in these regions by predators that pick that spot to bite.

Phytochemical Profiling and Biological Activity of the Methanolic Extracts of Cirsium Monspessulanum (L.) Hill.

The study of new plant species and the identification of their chemical composition may contribute to the discovery of a new breakthrough substances for pharmacotherapeutical applications. For the first time, we examined antioxidant and antimicrobial activity of 70 % v/v methanolic extracts from inflorescences and roots of Cirsium monspessulanum (L.) Hill. obtained by the ASE method. In the (2,2-diphenyl-1-picrylhydrazyl) DPPH analysis, tested extract of inflorescences showed antioxidant activity with an EC50=0.223±0.0479 mg/mL, and (Cupric Ion Reducting Antioxidant Capacity) CUPRAC test assessed the antiradical activity on 14.95±0.13 mgTE/g and for roots the values were EC50=0.307±0.0554 mg/mL and 11.18±0.49 mgTE/g, respectively. Furthermore, extract from the inflorescences possessed the highest antimicrobial activity against Staphylococcus aureus, Staphylococcus epidermidis and Micrococcus luteus with MIC=1.25 mg/mL for each. HPLC/ESI-QTOF-MS/MS method identified 7 phenolic acids and 14 flavonoids in inflorescences extract and only 7 phenolic acids in roots extract. To the best of our knowledge, this is the first qualitative analysis of Cirsium monspessulanum (L.) Hill. and all substances were described for the first time.

Synthesis of Alkyl/Aryloxymethyl Derivatives of 1,2,4-Triazole-3-Carboxamides and Their Biological Activities.

Ribavirin and its analogues exhibit an in vitro antiproliferative effect in cancer cells. In this work, we studied the biological activities of a number of alkyl/aryloxymethyl derivatives of ribavirin's aglycon-1,2,4-triazole-3-carboxamide. Alkyl/arylxymethyl derivatives of 1,2,4-triazole-3-carboxamide with substitutions at the fifth or first position of the triazole ring, were synthesized and their antiproliferative and antimicrobial effects were assessed. For both series, the presence of an antiproliferative effect was investigated, and 1-alkyl/aryloxymethyl derivatives were shown an antimicrobial potential against a Gram-positive bacteria Micrococcus luteus and Gram-negative bacterium Pseudomonas aeruginosa. The obtained results showed that the n-decyloxymethyl derivatives induced leukemia cell death at low micromolar concentrations. We confirmed that n-decyloxymethyl derivatives of ribavirin inhibited the cell cycle progression and induced an accumulation of leukemia cells in the subG1-phase. The molecular docking results suggest that alkyl/aryloxymethyl derivatives may act by inhibiting translation initiation, due to interference with eIF4E assembly. The outcome results revealed that active derivatives (1- or 5-n-decyloxymethyl-1,2,4-triazole-3-carboxamides) can be considered as a lead compound for anticancer treatments.

Multistep optimization of a cell-penetrating peptide towards its antimicrobial activity.

Multidrug resistant (MDR) bacteria have adapted to most clinical antibiotics and are a growing threat to human health. One promising type of candidates for the everlasting demand of new antibiotic compounds constitute antimicrobial peptides (AMPs). These peptides act against different types of microbes by permeabilizing pathogen cell membranes, whereas being harmless to mammalian cells. Contrarily, another class of membrane-active peptides, namely cell-penetrating peptides (CPPs), is known to translocate in eukaryotic cells without substantially affecting the cell membrane. Since CPPs and AMPs share several physicochemical characteristics, we hypothesized if we can rationally direct the activity of a CPP towards antimicrobial activity. Herein, we describe the screening of a synthetic library, based on the CPP sC18, including structure-based design to identify the active residues within a CPP sequence and to discover novel AMPs with high activity. Peptides with increased hydrophobicity were tested against various bacterial strains, and hits were further optimized leading to four generations of peptides, with the last also comprising fluorinated amino acid building blocks. Interestingly, beside strong antibacterial activities, we also detected activity in cancer cells, while non-cancerous cells remained unharmed. The results highlight our new candidates, particularly those from generation 4, as a valuable and promising source for the development of future therapeutics with antibacterial activity and beyond.

Expression analysis of tissue factor pathway inhibitors TFPI-1 and TFPI-2 in Paralichthys olivaceus and antibacterial and anticancer activity of derived peptides.

Tissue factor pathway inhibitors (TFPI), including TFPI-1 and TFPI-2, are Kunitz-type serine protease inhibitors that mainly inhibit the blood coagulation induced by tissue factors. Previous reports on teleost proved TFPI play important roles in innate immunity. In this study, two TFPI (PoTFPI-1 and PoTFPI-2) molecules from Japanese flounder (Paralichthys olivaceus) were analyzed and characterized for their expression patterns, antibacterial and anticancer activities of the C-terminal derived peptides. Quantitative real time RT-PCR analysis shows that constitutive PoTFPI-1 expression occurred, in increasing order, in the brain, muscle, spleen, gills, head kidney, blood, intestine, heart, and liver; PoTFPI-2 was expressed, in increasing order, in the brain, gills, head kidney, muscle, intestine, spleen, liver, heart, and blood. Under the stimulation of fish pathogens, both PoTFPI-1 and PoTFPI-2 expressions increased significantly in a manner that depended on the pathogens, tissue type, and infection stage. Furthermore, C-terminal peptides TP25 and TP26, derived from PoTFPI-1 and PoTFPI-2, respectively, were synthesized and proved to be active against Micrococcus luteus (for TP25 and TP26) and Staphylococcus aureus (for TP25) via retardation effects on bacterial nucleic acids. In addition, TP25 and TP26 also displayed significant inhibitory effects on human colon cancer cell line HT-29. These results reveal that both PoTFPI-1 and PoTFPI-2 play important roles in host innate immunity. The antibacterial activity and anticancer cells function of TP25 and TP26 will add new insights into the roles of teleost TFPI.

Title: insoluble proteins catch heterologous soluble proteins into inclusion bodies by intermolecular interaction of aggregating peptides.

BACKGROUND: Protein aggregation is a biological event observed in expression systems in which the recombinant protein is produced under stressful conditions surpassing the homeostasis of the protein quality control system. In addition, protein aggregation is also related to conformational diseases in animals as transmissible prion diseases or non-transmissible neurodegenerative diseases including Alzheimer, Parkinson's disease, amyloidosis and multiple system atrophy among others. At the molecular level, the presence of aggregation-prone domains in protein molecules act as seeding igniters to induce the accumulation of protein molecules in protease-resistant clusters by intermolecular interactions. RESULTS: In this work we have studied the aggregating-prone performance of a small peptide (L6K2) with additional antimicrobial activity and we have elucidated the relevance of the accompanying scaffold protein to enhance the aggregating profile of the fusion protein. Furthermore, we demonstrated that the fusion of L6K2 to highly soluble recombinant proteins directs the protein to inclusion bodies (IBs) in E. coli through stereospecific interactions in the presence of an insoluble protein displaying the same aggregating-prone peptide (APP). CONCLUSIONS: These data suggest that the molecular bases of protein aggregation are related to the net balance of protein aggregation potential and not only to the presence of APPs. This is then presented as a generic platform to generate hybrid protein aggregates in microbial cell factories for biopharmaceutical and biotechnological applications.

One-pot sequential coupling reactions as a new practical protocol for the synthesis of unsymmetrical 2,3-diethynyl quinoxalines and 4-ethynyl-substituted pyrrolo[1,2-a]quinoxalines.

One palladium-catalyzed sequential coupling reactions were successfully used as a new protocol for the synthesis of unsymmetrical 2,3-diethynyl quinoxalines and 4-ethynyl-substituted pyrrolo[1,2-a]quinoxalines. The one-pot two coupling reactions of 2,3-dichloroquinoxaline, with two different terminal alkynes, under controlled conditions produced selectively unsymmetrical 2,3-diethynyl quinoxalines with high yields. When one of the two terminal alkynes was 3-propyne-1-ol, in the presence of secondary amines, cyclization occurred and 4-ethynyl-substituted pyrrolo[1,2-a]quinoxalines were successfully formed. All synthesized compounds were tested against the two bacterial strains including Micrococcus luteus and Pseudomonas aeruginosa.

Antibacterial, antifungal and enzymatic activities of azithromycin-heavy metal complexes: Newly synthesized and characterized.

As part of our continuous research to understand the interaction mechanism of drug and metallo-elements, heavy metal complexes of azithromycin (AZI) were synthesized with arsenic oxide, lead carbonate and silver chloride salts in molar ratio of 2: 1 (L: M). Synthesized heavy metal complexes have shown good percent yield and characterized through spectroscopic parameters including UV-Visible, TLC, FT-IR, NMR and elemental analysis (CHN). Spectroscopic characterization reveals the binding of ligand AZI with heavy metals in bi-dentate manner involving the hydroxide and 9a-NCH3 group of the aglycone ring of AZI. These newly synthesized heavy metal complexes were evaluated for their antimicrobial response against selected gram positive and gram negative organisms and antifungal species. It was noted that all newly synthesized complexes exhibits increased activity against B.subtilus whereas, AZI itself didn't show any activity, while synthesized complexes have low to moderate response against all the studied organisms. Complex A-M12 possess greater enzymatic response against both urease and alpha chymotrypsin among all the studied complexes. Results obtained were then statistically analyzed through one way ANOVA and Dunnett's test by using SPSS version 20.0 suggesting the significant response of complexes against selected organisms.

Antimicrobial activity and secondary structure of a novel peptide derived from ovalbumin.

A novel antimicrobial peptide derived from ovalbumin has been discovered. First, the peptide fragment RKIKVYLPRMK (TK9.1) was identified based on computerized predictions of the secondary structure of peptides in a protein data bank. Using HeliQuest, the sequence was developed into RKIKRYLRRMI (TK9.1.3), which was synthesized using Fmoc-solid phase peptide synthesis, and found to have strongly antimicrobial activity against Gram-positive and Gram-negative bacteria, and fungi but not cytotoxic to HeLa cells and hemolysis in mouse red blood cells. Although ovalbumin itself does not have an antibacterial activity, our results suggest that it may supply the organisms that consume it with antimicrobial peptides, in support of their immunodefence.

The rational design, synthesis, and antimicrobial investigation of 2-Amino-4-Methylthiazole analogues inhibitors of GlcN-6-P synthase.

A series of novel 2-Amino-4-Methylthiazole analogs were developed via three-step reaction encompassing hydrazine-1-carboximidamide motif to combat Gram-positive and Gram-negative bacterial and fungal infections. Noticeably, the thiazole-carboximidamide derivatives 4a-d displayed excellent antimicrobial activity and the most efficacious analogue 4d with MIC/MBC values of 0.5 and 4 µg/mL, compared to reference drugs with very low toxicity to mammalian cells, resulting in a prominent selectivity more than 100 folds. Microscopic investigation of 4d biphenyl analogue showed cell wall lysis and promote rapid bactericidal activity though disrupting the bacterial membrane. In addition, an interesting in vitro investigation against GlcN-6-P Synthase Inhibition was done which showed potency in the nanomolar range. Meanwhile, this is the first study deploying a biomimicking strategy to design potent thiazole-carboximidamides that targeting GlcN-6-P Synthase as antimicrobial agents. Importantly, Molecular modeling simulation was done for the most active 4d analogue to study the interaction of this analogue which showed good binding propensity to glucosamine binding site which support the in vitro data.

New phenylspirodrimane metabolites MBJ-0030, MBJ-0031, and MBJ-0032 isolated from the soil fungal strain Stachybotrys sp. f23793.

Chemical screening of culture medium from the soil fungus Stachybotrys sp. resulted in the isolation of the three new phenylspirodrimanes MBJ-0030 (1), MBJ-0031 (2) and MBJ-0032 (3). Their structures were determined by detailed analysis of spectroscopic data. The absolute configurations of 1-3 were determined by modified Mosher's and Marfey's methods. In addition, cytotoxic and antimicrobial evaluations of the compounds were conducted.

Julichrome Monomers from Marine Gastropod Mollusk-Associated Streptomyces and Stereochemical Revision of Julichromes Q3 ⋠5 and Q3 ⋠3.

Two julichrome monomers, julichromes Q11 (1) and Q12 (2), along with five known julichromes (Q10 , Q3 ⋅ 5 , Q3 ⋅ 3 , Q6 ⋅ 6 , Q6 , 3-7) and four known anthraquinones (chrysophanol, 4-acetylchrysophanol, islandicin, huanglongmycin A, 8-11), were isolated from the marine gastropod mollusk Batillaria zonalis-associated Streptomyces sampsonii SCSIO 054. This is the first report of julichromes isolated from a marine source. Extensive dissection of 1D and 2D NMR datasets combined with X-ray crystallography enabled rigorous elucidation of the previously reported configurations of julichrome Q3 ⋅ 5 (4) and related julichrome Q3 ⋅ 3 (5); both of the configuration at C(9) needs to be revised. In addition, julichrome Q12 (2) was found to display antibacterial activity against Micrococcus luteus and Bacillus subtilis with MICs of 2.0 and 8.0 µg mL-1 ; four compounds (1, 3, 6, 7) also showed inhibitory activities against an array of methicillin-resistant Staphylococcus aureus, S. aureus and S. simulans AKA1 with MIC values ranging from 8 to 64 µg mL-1 .

Polyketides and Terpenoids with Potent Antibacterial Activities from the Artemisia argyi-Derived Fungus Trichoderma koningiopsis QA-3.

The AcOEt extract of Artemisia argyi-derived fungus Trichoderma koningiopsis QA-3 showed potent inhibitory activity against pathogenic bacteria. Fractionation of the extract resulted in the isolation of three new polyketides (1-3) and two new terpenoids (4 and 5), together with three known metabolites (6-8). Their chemical structures were analyzed by NMR spectra, ECD, HR-ESI-MS or HR-EI-MS, optical rotation, and X-ray crystallographic data, as well as by comparison with literature reports. In the antibacterial assays, 3-hydroxyharziandione (4) showed potent activity against human pathogen Escherichia coli with an MIC value of 0.5 µg/mL, while 6-(3-hydroxypent-1-en-1-yl)-2H-pyran-2-one exhibited strong activity against marine-derived aquatic pathogen Micrococcus luteus with an MIC value of 1.0 µg/mL.

Bacterial Biotransformation of Oleic Acid: New Findings on the Formation of γ-Dodecalactone and 10-Ketostearic Acid in the Culture of Micrococcus luteus.

Microbial conversion of oleic acid (1) to form value-added industrial products has gained increasing scientific and economic interest. So far, the production of natural lactones with flavor and fragrance properties from fatty acids by non-genetically modified organisms (non-GMO) involves whole cells of bacteria catalyzing the hydration of unsaturated fatty acids as well as yeast strains responsible for further ß-oxidation processes. Development of a non-GMO process, involving a sole strain possessing both enzymatic activities, significantly lowers the costs of the process and constitutes a better method from the customers' point of view regarding biosafety issues. Twenty bacteria from the genus of Bacillus, Comamonas, Dietzia, Gordonia, Micrococcus, Pseudomonas, Rhodococcus and Streptomyces were screened for oxidative functionalization of oleic acid (1). Micrococcus luteus PCM525 was selected as the sole strain catalyzing the one-pot transformation of oleic acid (1) into natural valuable peach and strawberry-flavored γ-dodecalactone (6) used in the food, beverage, cosmetics and pharmaceutical industries. Based on the identified products formed during the process of biotransformation, we clearly established a pathway showing that oleic acid (1) is hydrated to 10-hydroxystearic acid (2), then oxidized to 10-ketostearic acid (3), giving 4-ketolauric acid (4) after three cycles of ß-oxidation, which is subsequently reduced and cyclized to γ-dodecalactone (6) (Scheme 1). Moreover, three other strains (Rhodococcus erythropolis DSM44534, Rhodococcus ruber PCM2166, Dietzia sp. DSM44016), with high concomitant activities of oleate hydratase and alcohol dehydrogenase, were identified as efficient producers of 10-ketostearic acid (3), which can be used in lubricant and detergent formulations. Considering the prevalence of γ-dodecalactone (6) and 10-ketostearic acid (3) applications and the economic benefits of sustainable management, microbial bioconversion of oleic acid (1) is an undeniably attractive approach.

A Universal Stress Protein That Controls Bacterial Stress Survival in Micrococcus luteus.

Bacteria have remarkable mechanisms to survive severe external stresses, and one of the most enigmatic is the nonreplicative persistent (NRP) state. Practically, NRP bacteria are difficult to treat, and so inhibiting the proteins underlying this survival state may render such bacteria more susceptible to external stresses, including antibiotics. Unfortunately, we know little about the proteins and mechanisms conferring survival through the NRP state. Here, we report that a universal stress protein (Usp) is a primary regulator of bacterial survival through the NRP state in Micrococcus luteus NCTC 2665, a biosafety level 1 (BSL1) mycobacterial relative. Usps are widely conserved, and bacteria, including Mycobacterium tuberculosis, Mycobacterium smegmatis, and Escherichia coli, have multiple paralogs with overlapping functions that have obscured their functional roles. A kanamycin resistance cassette inserted into the M. luteus universal stress protein A 616 gene (ΔuspA616::kanM. luteus) ablates the UspA616 protein and drastically impairs M. luteus survival under even short-term starvation (survival, 83% wild type versus 32% ΔuspA616::kanM. luteus) and hypoxia (survival, 96% wild type versus 48% ΔuspA616::kanM. luteus). We observed no detrimental UspA616 knockout phenotype in logarithmic growth. Proteomics demonstrated statistically significant log-phase upregulation of glyoxylate pathway enzymes isocitrate lyase and malate synthase in ΔuspA616::kanM. luteus We note that these enzymes and the M. tuberculosis UspA616 homolog (Rv2623) are important in M. tuberculosis virulence and chronic infection, suggesting that Usps are important stress proteins across diverse bacterial species. We propose that UspA616 is a metabolic switch that controls survival by regulating the glyoxylate shunt.IMPORTANCE Bacteria tolerate severe external stresses, including antibiotics, through a nonreplicative persistent (NRP) survival state, yet the proteins regulating this survival state are largely unknown. We show a specific universal stress protein (UspA616) controls the NRP state in Micrococcus luteus Usps are widely conserved across bacteria, but their biological function(s) has remained elusive. UspA616 inactivation renders M. luteus susceptible to stress: bacteria die instead of adapting through the NRP state. UspA616 regulates malate synthase and isocitrate lyase, glyoxylate pathway enzymes important for chronic Mycobacterium tuberculosis infection. These data show that UspA616 regulates NRP stress survival in M. luteus and suggest a function for homologous proteins in other bacteria. Importantly, inhibitors of UspA616 and homologs may render NRP bacteria more susceptible to stresses, including current antibiotics.