Ascomycin

Engineering of the LysR family transcriptional regulator FkbR1 and its target gene to improve ascomycin production.[Pubmed:28349163]

Appl Microbiol Biotechnol. 2017 Jun;101(11):4581-4592.

Ascomycin (FK520), a macrocyclic polyketide natural antibiotic, displays high anti-fungal and immunosuppressive activity. In this study, the LysR family transcriptional regulator FkbR1 was characterized, and its role in Ascomycin biosynthesis was explored by gene deletion, complementation, and overexpression. Inactivation of fkbR1 led to 67.5% reduction of Ascomycin production, which was restored by complementation of fkbR1. Overexpression of fkbR1 resulted in a 33.5% increase in Ascomycin production compared with the parent strain FS35. These findings indicated that FkbR1 was a positive regulator for Ascomycin production. Quantitative RT-PCR analysis revealed that the expressions of fkbE, fkbF, fkbS, and fkbU were downregulated in the fkbR1 deletion strain and upregulated in the fkbR1 overexpression strain. Electrophoretic mobility shift assays (EMSAs) in vitro and chromatin immunoprecipitation (ChIP)-qPCR assays in vivo indicated that FkbR1 bound to the intergenic region of fkbR1-fkbE. To investigate the roles of the target genes fkbE and fkbF in Ascomycin production, the deletion and overexpressions of fkbE and fkbF were implemented, respectively. Overexpression of fkbE resulted in a 45.6% increase in Ascomycin production, but little change was observed in fkbF overexpression strain. To further enhance Ascomycin production, the fkbR1 and fkbE combinatorial overexpression strain OfkbRE was constructed with the Ascomycin yield increased by 69.9% to 536.7 mg/L compared with that of the parent strain. Our research provided a helpful strategy to increase Ascomycin production via engineering FkbR1 and its target gene.

Application of volumetric absorptive microsampling device for quantification of tacrolimus in human blood as a model drug of high blood cell partition.[Pubmed:28601765]

J Pharm Biomed Anal. 2017 Sep 5;143:168-175.

Volumetric absorptive microsampling device (VAMS) was evaluated for bioanalysis of tacrolimus, which was used as a model drug with high blood cell partition. Aliquots of blood (ca. 10muL) with different hematocrits and fortified with tacrolimus were wicked up by VAMS and tacrolimus was extracted with a methanol-water mixture (1:1, v/v) via sonication. After chromatography on an AQUITY UPLC HSS T3 column (100x2.1 i.d., mm, 1.8mum), tacrolimus and the internal standard Ascomycin, were detected in the positive ion mode with electrospray ionization by monitoring of transitions m/z 826.6-->616.4 and m/z 814.6-->604.0, respectively. An assay method to quantify tacrolimus from 1 to 250ng/mL in whole blood was qualified by ensuring that linearity, selectivity, intra- and inter-batch reproducibility, and stability were within the acceptance criteria. Consistent and high extraction recovery of tacrolimus was ensured from blood with low- (20%), mid- (45%), and high-hematocrit (65%) levels with minimal matrix effects. Apparent instability at ambient temperature or 4 degrees C possibly due to reduced recovery suggests that tacrolimus in VAMS should be stored at -25 degrees C until assay. Potential reduced recovery over time from VAMS should be taken into consideration in method optimization.

FKBP12-Dependent Inhibition of Calcineurin Mediates Immunosuppressive Antifungal Drug Action in Malassezia.[Pubmed:29066552]

MBio. 2017 Oct 24;8(5). pii: mBio.01752-17.

The genus Malassezia includes yeasts that are commonly found on the skin or hair of animals and humans as commensals and are associated with a number of skin disorders. We have previously developed an Agrobacterium tumefaciens transformation system effective for both targeted gene deletion and insertional mutagenesis in Malassezia furfur and M. sympodialis In the present study, these molecular resources were applied to characterize the immunophilin FKBP12 as the target of tacrolimus (FK506), Ascomycin, and pimecrolimus, which are calcineurin inhibitors that are used as alternatives to corticosteroids in the treatment of inflammatory skin disorders such as those associated with Malassezia species. While M. furfur and M. sympodialis showed in vitro sensitivity to these agents, fkb1Delta mutants displayed full resistance to all three of them, confirming that FKBP12 is the target of these calcineurin inhibitors and is essential for their activity. We found that calcineurin inhibitors act additively with fluconazole through an FKBP12-dependent mechanism. Spontaneous M. sympodialis isolates resistant to calcineurin inhibitors had mutations in the gene encoding FKBP12 in regions predicted to affect the interactions between FKBP12 and FK506 based on structural modeling. Due to the presence of homopolymer nucleotide repeats in the gene encoding FKBP12, an msh2Delta hypermutator of M. sympodialis was engineered and exhibited an increase of more than 20-fold in the rate of emergence of resistance to FK506 compared to that of the wild-type strain, with the majority of the mutations found in these repeats.IMPORTANCEMalassezia species are the most abundant fungal components of the mammalian and human skin microbiome. Although they belong to the natural skin commensal flora of humans, they are also associated with a variety of clinical skin disorders. The standard treatment for Malassezia-associated inflammatory skin infections is topical corticosteroids, although their use has adverse side effects and is not recommended for long treatment periods. Calcineurin inhibitors have been proposed as a suitable alternative to treat patients affected by skin lesions caused by Malassezia Although calcineurin inhibitors are well-known as immunosuppressive drugs, they are also characterized by potent antimicrobial activity. In the present study, we investigated the mechanism of action of FK506 (tacrolimus), Ascomycin (FK520), and pimecrolimus in M. furfur and M. sympodialis and found that the conserved immunophilin FKBP12 is the target of these drugs with which it forms a complex that directly binds calcineurin and inhibits its signaling activity. We found that FKBP12 is also required for the additive activity of calcineurin inhibitors with fluconazole. Furthermore, the increasing natural occurrence in fungal pathogen populations of mutator strains poses a high risk for the rapid emergence of drug resistance and adaptation to host defense. This led us to generate an engineered hypermutator msh2Delta mutant strain of M. sympodialis and genetically evaluate mutational events resulting in a substantially increased rate of resistance to FK506 compared to that of the wild type. Our study paves the way for the novel clinical use of calcineurin inhibitors with lower immunosuppressive activity that could be used clinically to treat a broad range of fungal infections, including skin disorders caused by Malassezia.

Metabolic network model guided engineering ethylmalonyl-CoA pathway to improve ascomycin production in Streptomyces hygroscopicus var. ascomyceticus.[Pubmed:28974216]

Microb Cell Fact. 2017 Oct 3;16(1):169.

BACKGROUND: Ascomycin is a 23-membered polyketide macrolide with high immunosuppressant and antifungal activity. As the lower production in bio-fermentation, global metabolic analysis is required to further explore its biosynthetic network and determine the key limiting steps for rationally engineering. To achieve this goal, an engineering approach guided by a metabolic network model was implemented to better understand Ascomycin biosynthesis and improve its production. RESULTS: The metabolic conservation of Streptomyces species was first investigated by comparing the metabolic enzymes of Streptomyces coelicolor A3(2) with those of 31 Streptomyces strains, the results showed that more than 72% of the examined proteins had high sequence similarity with counterparts in every surveyed strain. And it was found that metabolic reactions are more highly conserved than the enzymes themselves because of its lower diversity of metabolic functions than that of genes. The main source of the observed metabolic differences was from the diversity of secondary metabolism. According to the high conservation of primary metabolic reactions in Streptomyces species, the metabolic network model of Streptomyces hygroscopicus var. ascomyceticus was constructed based on the latest reported metabolic model of S. coelicolor A3(2) and validated experimentally. By coupling with flux balance analysis and using minimization of metabolic adjustment algorithm, potential targets for Ascomycin overproduction were predicted. Since several of the preferred targets were highly associated with ethylmalonyl-CoA biosynthesis, two target genes hcd (encoding 3-hydroxybutyryl-CoA dehydrogenase) and ccr (encoding crotonyl-CoA carboxylase/reductase) were selected for overexpression in S. hygroscopicus var. ascomyceticus FS35. Both the mutants HA-Hcd and HA-Ccr showed higher Ascomycin titer, which was consistent with the model predictions. Furthermore, the combined effects of the two genes were evaluated and the strain HA-Hcd-Ccr with hcd and ccr overexpression exhibited the highest Ascomycin production (up to 438.95 mg/L), 1.43-folds improvement than that of the parent strain FS35 (305.56 mg/L). CONCLUSIONS: The successful constructing and experimental validation of the metabolic model of S. hygroscopicus var. ascomyceticus showed that the general metabolic network model of Streptomyces species could be used to analyze the intracellular metabolism and predict the potential key limiting steps for target metabolites overproduction. The corresponding overexpression strains of the two identified genes (hcd and ccr) using the constructed model all displayed higher Ascomycin titer. The strategy for yield improvement developed here could also be extended to the improvement of other secondary metabolites in Streptomyces species.

UPLC-MS/MS assay validation for tacrolimus quantitative determination in peripheral blood T CD4+ and B CD19+ lymphocytes.[Pubmed:29471254]

J Pharm Biomed Anal. 2018 Apr 15;152:306-314.

Monitoring tacrolimus (Tac) exposure in cell matrices enriched with lymphocytes can improve Tac therapeutic drug monitoring (TDM) in solid organ transplant recipients. An UPLC-MS/MS based assay for Tac quantification in peripheral blood T CD4+ and B CD19+ lymphocytes was developed. Peripheral blood mononuclear cells (PBMC) were obtained by density gradient centrifugation and highly purified (purity >90%) T CD4+ and B CD19+ cell suspensions were acquired by magnetic negative selection from whole blood of 6 healthy volunteers. The purity of lymphocyte suspensions was checked by flow cytometry. Tac extraction was performed in a liquid-liquid zinc sulfate, methanol and acetonitrile based medium. Ascomycin was used as internal standard. The equipment used was a Waters((R)) Acquity UPLC system (Waters Corporation, Milford, MA, USA). The chromatographic run was performed on a Waters((R)) MassTrak TDM C18 (2.1x10mm) column (Waters Corporation, Milford, MA, USA). at a flow rate of 0.4mL/min. The instrument was set in electrospray positive ionization mode. The method was validated according to Clinical Laboratory Standard Institute (CLSI) guidelines and showed a high sensitivity and specificity over a range of 0-5.2 ng/mL in PBMC, 0-5.0 ng/mL in T CD4+ Lymphocytes and 0-5.3 ng/mL in B CD19+ lymphocytes. Precision was appropriate with CV of intra-assay quantifications ranging from 4.9 to 7.4%, and of inter-assay quantifications from 7.2 to 13.9%. Limit of detection and quantification were 0.100 and 0.115 ng/mL in PBMC, 0.058 and 0.109 ng/mL in T CD4+ and 0.017 and 0.150 ng/mL in B CD19+ cells. Matrix effect was not significant among all the studied matrices. Samples showed stability for Tac quantification over a period of 90days either at room temperature or at -30 degrees C storage conditions. The method was applied to clinical samples of 20 kidney transplant recipients. Concentrations ranged from 2.200 to 11.900 ng/mL in whole blood, from 0.005 to 0.570 ng/10(6) cells in PBMC, from 0.081 to 1.432 ng/10(6) cells in T CD4+, and from 0.197 to 1.564 ng/10(6) cells in B CD19+ cell matrices. The method has potential applicability for Tac TDM in solid organ transplant recipients.

First UHPLC-MS/MS method coupled with automated online SPE for quantification both of tacrolimus and everolimus in peripheral blood mononuclear cells and its application on samples from co-treated pediatric patients.[Pubmed:28098395]

J Mass Spectrom. 2017 Mar;52(3):187-195.

Tacrolimus (TAC, FK-506) and everolimus (EVE, RAD001) are immunosuppressors used to treat pediatric patients undergoing liver transplantation. Their hematic TDM by liquid chromatography became standard practice. However, it does not always reflect concentrations at their active site. Our aim was to develop and validate a new method for the simultaneous TAC and EVE quantification into target cells: peripheral blood mononuclear cells (PBMCs). Peripheral blood mononuclear cells were collected using cell preparation tubes; cells number and mean cell volume were evaluated by an automatic cell counter. TAC and EVE were quantified using UHPLC-MS/MS coupled with an automated online solid-phase extraction platform. Chromatographic run was performed on an Acquity UPLC(R) BEH C18 1.7 mum (2.1 x 50 mm) column at 45 degrees C, for 6 min at 0.5 ml/min. Mobile phases were water and methanol, both with 2 mm ammonium acetate and 1 ml/l formic acid). XBridge(R) C8 10 mum (1 x 10 mm) SPE cartridges were used, and the internal standard was Ascomycin. Following Food and Drug Administration guidelines, method validation resulted in high sensitivity and specificity. Calibration curves were linear (r(2) = 0.998) and intra-day and inter-day imprecision and inaccuracy were <15%. A reproducible matrix effect was observed, with a good recovery for all compounds. Drug amounts in 15 'real' PBMCs samples from five pediatric patients in co-treatment resulted within the calibration range (0.039-5 ng). Concentrations from each patient were standardized using their evaluated mean cell volume: intra-PBMCs concentration was meanly 19.23 and 218.61 times higher than the hematic one for TAC and EVE, respectively. This method might be useful in clinical routine, giving reliable data on drugs concentration at the active site. Copyright (c) 2017 John Wiley & Sons, Ltd.