Anil Shrestha

Overexpression of a pathway specific negative regulator enhances production of daunorubicin in bldA deficient Streptomyces peucetius ATCC 27952.

The dnrO gene is the first regulator to be activated in the daunorubicin (DNR) biosynthesis pathway of Streptomyces peucetius ATCC 27952. DnrO is known for its self-repression capability while it activates rest of the DNR biosynthesis pathway through cascades of regulatory events. S. peucetius was found to contain no functional copy of bldA-tRNA while a detailed examination of dnrO codons reveals the presence of TTA codon, which is rarely encoded by bldA-tRNA. Therefore, for evaluating the role of dnrO in DNR production, multiple engineered strains of S. peucetius were generated by heterologously expressing bldA, dnrO and combination of bldA and dnrO. Using these strains, the effects of heterologously expressed bldA and overexpressed dnrO were evaluated on pathway specific regulators, mycelial densities and production of DNR. The results showed that the transcription level of dnrO and master regulator dnrI, was found to be elevated in bldA containing strain in comparison to dnrO overexpressed strain. The bldA containing strain produces 45.7% higher DNR than bldA deficient wild type strain from culture broth with OD600 of 1.45 at 72h. Heterologous expression of bldA-tRNA is accounted for increased transcription levels of the DNR pathway specific regulators and enhanced DNR production.

Genetic Manipulation of Nocardia Species.

Nocardia spp. are aerobic, Gram‐positive, catalase positive, and non‐motile actinomycetes. They are associated with human infections. However, some species produce important natural products, degrade toxic chemicals, and are involved in biotransformation of valuable products. The lack of robust genetic tools has hindered detailed studies and advanced research. This unit describes the major genetic engineering approaches using Nocardia sp. CS682 as a prototype. These methods will certainly help in understanding the basis of their pathogenicity as well as biosynthetic and biotransforming abilities. It can be expected that knowledge of the biochemistry behind their pathogenicity will be crucial in developing effective treatment strategies. These genetic tools can be utilized to develop rational metabolic engineering approaches for crafting host strains with higher production or biotransformation ability.

Structural characterization of cyclosporin A, C and microbial bio-transformed cyclosporin A analog AM6 using HPLC-ESI-ion trap-mass spectrometry.

Cyclosporin A (CyA), a cyclic undecapeptide produced by a number of fungi, contains 11 unusual amino acids, and has been one of the most commonly prescribed immunosuppressive drugs. To date, there are over sixty different analogs reported as congeners and analogs resulting from precursor-directed biosynthesis, human CYP-mediated metabolites, or microbial bio-transformed analogs. However, there is still a need for more structurally diverse CyA analogs in order to discover new biological potentials and/or improve the physicochemical properties of the existing cyclosporins. As a result of the complexity of the resulting mass spectrometric (MS) data caused by its unusual amino acid composition and its cyclic nature, structural characterization of these cyclic peptides based on fragmentation patterns using multiple tandem MS analyses is challenging task. Here, we describe, an efficient HPLC-ESI-ion trap MS(n) (up to MS(8)) was developed for the identification of CyA and CyC, a (Thr(2))CyA congener in which L-aminobutyric acid (Abu) is replaced by L-threonine (Thr). In addition, we examined the fragmentation patterns of a CyA analog obtained from the cultivation of a recombinant Streptomyces venezuelae strain fed with CyA, assigning this analog as (γ-hydroxy-MeLeu(6))CyA (otherwise, known as an human CYP metabolite AM6). This is the first report on both the MS(n)-aided identification of CyC and the structural characterization of a CyA analog by employing HPLC-ESI-ion trap MS(n) analysis.

Ultra-High Performance Liquid Chromatography with Electrospray Ionization Tandem Mass Spectrometry for the Determination of Caffeine in Energy Drinks

A simple and highly sensitive method for the determination of caffeine in energy beverages was developed and validated. Sample preparation utilizing solid-phase extraction (SPE) was simple and reliable. Separation by isocratic ultra-high performance liquid chromatography (UPLC) with a reversed-phase C18 column was performed within 6 min. The use of SPE with UPLC coupled with electrospray ionization-multiple tandem mass spectrometry detection (ESI-MSn) was accurate, reproducible, and validated for the determination of caffeine in energy drink matrices. The limit of quantification for caffeine was approximately 2.1 ng mL−1. The intra-day and inter-day precisions were less than 4%, and the accuracy of the measurements was between 85.1% and 93.2%. Results for caffeine concentrations in eighteen beverages were compared to the values on the labels. This paper describes the first use of the UPLC–ESI-ion trap MSn technique for quality-control purposes of caffeine present in energy drinks.

Ultra-Performance Liquid Chromatography with Electrospray Ionization Mass Spectrometry for the Determination of Coenzyme Q10 as an Anti-Aging Ingredient in Edible Cosmetics

A simple and rapid method for the determination of coenzyme Q10 in edible cosmetics was developed and validated. Cleanup utilizing a solid-phase extraction technique provided a simple and reliable method for extracting both coenzyme Q10 and its internal standard coenzyme Q9 from the lecithin-rich samples with efficiencies of 92 ± 4% and 89 ± 3%, respectively. The separation of analytes through a reversed-phase C18 column was carried out within 10 min. By employing authentic coenzyme Q10 and the internal standard spiked into blank matrices, the combined use of solid phase extraction cleanup and chromatographic separation coupled with electrospray ionization-tandem mass spectrometry detection was shown to be sufficiently accurate to detect coenzyme Q10 in edible cosmetic matrices. The limit of quantification of coenzyme Q10 determined by the validated method was 54 ng/g in the matrices. In addition, the intra- and inter-day precision was <4% and the accuracy ranged from 87.1 to 92.3%. Quantitative results for coenzyme Q10 levels in two different edible cosmetic samples obtained by the established analytical protocol were reproducible. This is the first report of the determination of coenzyme Q10 in lipophilic edible cosmetic matrices by liquid chromatography–electrospray ionization tandem mass spectrometry.

Regio-selectively reduced streptogramin A analogue, 5,6-dihydrovirginiamycin M1 exhibits improved potency against MRSA

A newly reduced macrocyclic lactone antibiotic streptogramin A, 5,6‐dihydrovirginiamycin M1 was created by feeding virginiamycin M1 into a culture of recombinant Streptomyces venezuelae. Its chemical structure was spectroscopically elucidated, and this streptogramin A analogue showed twofold higher antibacterial activities against methicillin‐resistant Staphylococcus aureus (MRSA) compared with its parent molecule virginiamycin M1. Docking studies using the model of streptogramin A acetyltransferase (VatA) suggested that the newly generated analogue binds tighter with overall lower free energy compared with the parent molecule virginiamycin M1. This hypothesis was validated experimentally through the improvement of efficacy of the new analogue against MRSA strains. The biotransformation approach presented herein could have a broad application in the production of reduced macrocyclic molecules.

Ultra-Performance Liquid Chromatography with Electrospray Ionization Tandem Mass Spectrometry for the Determination of Ketoconazole in Anti-Dandruff Shampoo

A fast and highly sensitive method for the quantification of ketoconazole in antidandruff emulsion formulas was developed and validated. Sample preparation utilizing solid-phase extraction was a simple and reliable method for extracting both ketoconazole and its internal standard miconazole from the samples: 97 ± 3% for ketoconazole and 93 ± 4% for the internal standard. The separation by isocratic ultra-high performance liquid chromatography and multiple-stage mass spectrometry with a reversed-phase C18 column was performed within 10 min. The method, which operated in a selective reaction monitoring mode specific to ketoconazole, was validated for quantitative use. The intra- and inter-day precision values were <5% and their accuracies ranged from 89.6 to 96.2%. The ketoconazole concentrations in two samples obtained by the established protocol were comparable to the concentrations indicated on the labels of the formulations. Thus, this paper describes the first use of ultra-high performance liquid chromatography and multiple-stage mass spectrometry for the determination of ketoconazole.