Phage P1-Derived Artificial Chromosomes Facilitate Heterologous Expression of the FK506 Gene Cluster

We describe a procedure for the conjugative transfer of phage P1-derived Artificial Chromosome (PAC) library clones containing large natural product gene clusters (≥70 kilobases) to Streptomyces coelicolor strains that have been engineered for improved heterologous production of natural products. This approach is demonstrated using the gene cluster for FK506 (tacrolimus), a clinically important immunosuppressant of high commercial value. The entire 83.5 kb FK506 gene cluster from Streptomyces tsukubaensis NRRL 18488 present in one 130 kb PAC clone was introduced into four different S. coelicolor derivatives and all produced FK506 and smaller amounts of the related compound FK520. FK506 yields were increased by approximately five-fold (from 1.2 mg L^-1 to 5.5 mg L^-1) in S. coelicolor M1146 containing the FK506 PAC upon over-expression of the FK506 LuxR regulatory gene fkbN. The PAC-based gene cluster conjugation methodology described here provides a tractable means to evaluate and manipulate FK506 biosynthesis and is readily applicable to other large gene clusters encoding natural products of interest to medicine, agriculture and biotechnology.     

Characterization of methyltransferase and hydroxylase genes involved in the biosynthesis of the immunosuppressants FK506 and FK520.

FK506 and FK520 are 23-membered macrocyclic polyketides with potent immunosuppressive and antifungal activities. The gene encoding 31-O-demethyl-FK506 methyltransferase, fkbM, was isolated from Streptomyces sp. strains MA6858 and MA6548, two FK506 producers, and Streptomyces hygroscopicus subsp. ascomyceticus, an FK520 producer. The nucleotide sequence of the fkbM gene revealed an open reading frame encoding a polypeptide of 260 amino acids. Disruption of fkbM in Streptomyces sp. strain MA6548 yielded a mutant that produced 31-O-demethyl-FK506, confirming the involvement of the isolated genes in the biosynthesis of FK506 and FK520. Heterologous expression of fkbM in Streptomyces lividans established that fkbM encodes an O-methyltransferase catalyzing the methylation of the C-31 hydroxyl group of 31-O-demethyl-FK506 and FK520. A second open reading frame, fkbD, was found upstream of fkbM in all three aforementioned species and was predicted to encode a protein of 388 residues that showed a strong resemblance to cytochrome P-450 hydroxylases. Disruption of fkbD had a polar effect on the synthesis of the downstream fkbM gene product and resulted in the formation of 9-deoxo-31-O-demethyl-FK506. This established the product of fkbD as the cytochrome P-450 9-deoxo-FK506 hydroxylase, which is responsible for hydroxylation at position C-9 of the FK506 and FK520 macrolactone ring.     

Trends in the biosynthesis and production of the immunosuppressant tacrolimus (FK506)

The current off-patent state of tacrolimus (FK506) has opened the hunting season for new generic pharmaceutical formulations of this immunosuppressant. This fact has boosted the scientific and industrial research on tacrolimus for the last 5 years in order to improve its production. The fast discovery of tacrolimus producer strains has generated a huge number of producers, which presents the biosynthetic cluster of FK506 as a high promiscuous genetic region. For the first time, the current state-of-the-art on the tacrolimus biosynthesis, production improvements and drug purification is reviewed. On one hand, all the genes involved in the tacrolimus biosynthesis, in addition to the traditional PKS/NRPS, as well as their regulation are analysed. On the other hand, tacrolimus direct and indirect precursors are reviewed as a straight manner to improve the final yield, which is a current trend in the field. Twenty years of industrial and scientific improvements on tacrolimus production are summarised, whereas future trends are also drafted.     

Novel chemobiosynthetic approach for exclusive production of FK506

FK506, a widely used immunosuppressant, is produced by industrial fermentation processes using various Streptomyces species. Independently of the strain, structurally related compound FK520 is co-produced, resulting in complex and costly isolation procedures. In this paper, we report a chemobiosynthetic approach for exclusive biosynthesis of FK506. This approach is based on the Streptomyces tsukubaensis strain with inactivated allR gene, a homologue of crotonyl-CoA carboxylase/reductase, encoded in the FK506 biosynthetic cluster. This strain produces neither FK506 nor FK520; however, if allylmalonyl-S-N-acetylcysteamine precursor is added to cultivation broth, the production of FK506 is reestablished without FK506-related by-products. Using a combination of metabolic engineering and chemobiosynthetic approach, we achieved exclusive production of FK506, representing a significant step towards development of an advanced industrial bioprocess.     

Intravenously injected FK506 failed to inhibit hippocampal calcineurin

FK506 (tacrolimus) is known as an inhibitor for calcineurin and is used in numerous research fields. It is not clear whether intravenously injected FK506 inhibits neuronal calcineurin. We measured the calcineurin activities of normal and postischemic rat hippocampi after intravenous injection of FK506 (3 mg/kg). Intravenously injected FK506 had no inhibitory effect on calcineurin activity in the hippocampi of normal and postischemic rats, whereas FK506 inhibited the calcineurin in vitro (purified enzyme, hippocampal homogenate, and hippocampal slice culture homogenate). Thus, it is considered that intravenously injected FK506 does not act on intraneuronal calcineurin and that several effects of FK506 are not due to the inhibition of neuronal calcineurin.     

FK506 positively regulates the migratory potential of melanocyte‐derived cells by enhancing syndecan‐2 expression

Although topical tacrolimus (FK506) is known to promote repigmentation by increasing the pigmentation and migration of melanocytes, the mechanism through which FK506 regulates cell migration remains unclear. Here, we report that FK506 treatment enhanced cell spreading on laminin‐332 and increased migration in both melanocytes and melanoma cells. Interestingly, FK506 also increased the expression of syndecan‐2, a transmembrane heparan sulfate proteoglycan through c‐jun terminal kinase activation. Moreover, siRNA‐mediated reduction of syndecan‐2 expression decreased FK506‐mediated cell spreading and migration in melanoma cells and decreased focal adhesion kinase phosphorylation in both melanocytes and melanoma cells. Consistent with these effects on syndecan‐2 expression, FK506 enhanced the membrane and melanosome localizations of PKCβII, a regulator of tyrosinase activity. This suggests that FK506 may play a dual regulatory role by affecting both melanogenesis and migration in melanocyte‐derived cells. Interestingly, however, FK506 failed to show any synergistic effect on the migration of UVB‐treated melanocyte‐derived cells. Taken together, these data indicate that FK506 regulates cell migration by enhancing syndecan‐2 expression, further suggesting that syndecan‐2 could be a potential target for the treatment of patients with vitiligo.     

Liquid chromatography-mass spectrometry characterization of FK506 biosynthetic intermediates in Streptomyces clavuligerus KCTC 10561BP

The development of an efficient analytical method for the reliable detection and identification of the biosynthetic intermediates found in microbial cultures, which usually produce complex intermediates of the metabolites of interest, is essential for further biosynthetic investigations. This study developed a simple and highly selective method for detecting the biosynthetic intermediates involved in the FK506 pathway of Streptomyces clavuligerus KCTC 10561BP involving a cleanup procedure using a solid-phase extraction technique to provide reliable extraction of FK506-related compounds from a cell culture broth and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) to separate and detect the FK506-related intermediates at concentrations as low as 0.2 μg/L in the broth. This method enabled the analytical profiling of the intermediates formed during the biosynthesis of FK506 in this S. clavuligerus strain, which produced FK506 as a main product. Eight FK506 intermediates—FK520, 37,38-dihydroFK506, prolylFK506, 9-decarbonyl-9-hydroxylFK506, 9-deoxoFK506, desmethylFK520, prolylFK520, and 9-deoxoFK520—were identified. This is the first report of the LC-ESI-MS/MS characterization of a wide range of FK506 analogs from a bacterial fermentation broth. The protocol employed in this study may be useful for estimating the structure of the metabolites without the need for a time-consuming isolation process and nuclear magnetic resonance (NMR) spectroscopy.     

Characterization of liposomal tacrolimus in lung surfactant-like phospholipids and evaluation of its immunosuppressive activity

Tacrolimus (FK506) is a hydrophobic immunosuppressive agent that rapidly penetrates the plasmatic membrane and inhibits the signal transduction cascade of T lymphocytes. The objective of this study was the characterization of liposomal FK506 with surfactant-like phospholipids to be administered intratracheally after lung transplantation or in inflammatory lung diseases. We evaluated the optimal incorporation of FK506 in dipalmitoylphosphatidylcholine (DPPC) and DPPC/1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) monolayers and bilayers and the effects of FK506 on the physical properties of DPPC and DPPC/POPG (8:2 w/w) vesicles. In addition, we assessed the immunosuppressive effects of surfactant-like phospholipid vesicles containing different amounts of FK506 on T-cell proliferation and interleukin 2 production. From surface pressure measurements of FK506/DPPC and FK506/DPPC/POPG mixed monolayers, we determined that FK506 was embedded into these monolayers up to an FK506 concentration of about 0.4 mol %. Beyond this concentration, FK506 was not quantitatively incorporated into the monolayer, suggesting possible concentration-dependent aggregation of tacrolimus. The incorporation of FK506 into DPPC monolayers, at concentrations 相似文献   

Sustained QT prolongation induced by tacrolimus in guinea pigs.

Recently, clinical cases have been reported of QT prolongation and torsades de pointes associated with the use of tacrolimus (FK506). We examined the relationship between QTc prolongation and the pharmacokinetics of FK506 in guinea pigs in order to evaluate the arrhythmogenicity of FK506 in comparison with quinidine (QND). FK506 (0.1 or 0.01 mg/hr/kg) or QND (30 mg/hr/kg) was intravenously infused to guinea pigs and time profiles of drug concentration in blood and QTc interval were examined during and after infusion. Both FK506 and QND evoked a significant QTc prolongation, and the dose-response relationship showed an anti-clockwise hysteresis, FK506-induced QTc prolongation persisted throughout the duration of the experiment despite a decline in the plasma FK506 concentration, whilst QND-induced QTc prolongation disappeared as plasma concentrations decreased. FK506 induced a sustained QTc prolongation in guinea pigs at drug concentrations in blood that correspond to its therapeutic range in human, suggesting that it might be of clinical significance to monitor the electrocardiogram, especially when patients have congenital or acquired QT-prolonging risk factors.     

FK506 reduces abnormal prion protein through the activation of autolysosomal degradation and prolongs survival in prion-infected mice

Prion diseases are fatal neurodegenerative disorders and no effective treatment has been established to date. In this study, we evaluated the effect of FK506 (tacrolimus), a macrolide that is known to be a mild immunosuppressant, on prion infection, using cell culture and animal models. We found that FK506 markedly reduced the abnormal form of prion protein (PRNP^Sc) in the cell cultures (N2a58 and MG20) infected with Fukuoka-1 prion. The levels of autophagy-related molecules such as LC3-II, ATG12–ATG5 and ATG7 were significantly increased in the FK506-treated cells, and resulted in the increased formation of autolysosomes. Upregulation of the autophagy-related molecules was also seen in the brains of FK506-treated mice and the accumulation of PRNP^Sc was delayed. The survival periods in mice inoculated with Fukuoka-1 were significantly increased when FK506 was administered from day 20 post-inoculation. These findings provide evidence that FK506 could constitute a novel antiprion drug, capable of enhancing the degradation of PRNP^Sc in addition to attenuation of microgliosis and neuroprotection.     

FK506 requires stimulation of the extracellular signal-regulated kinase 1/2 and the steroid receptor chaperone protein p23 for neurite elongation

The immunosuppressant drug FK506 (tacrolimus) accelerates nerve regeneration in vivo and increases neurite elongation in vitro. We have proposed that the mechanism involves binding to the FK506-binding protein 52, a chaperone component of mature steroid receptor complexes, and a subsequent 'gain-of-function' involving p23 dissociation from Hsp-90 in the complex and extracellular signal-regulated kinase (ERK) activation. Here, we tested the involvement of the ERK and p23 in neurite elongation by FK506 in human SH-SY5Y cells. FK506 (10 nM) increased ERK1/2 phosphorylation at 12 and 24 h, eliciting a 3.5-fold increase at 24 h, which was inhibited in a concentration-dependent manner by an antibody (JJ3) to recombinant human p23. Neurite elongation by FK506 (10 nM), determined by measuring neurite lengths at 96 and 168 h, was completely blocked by the mitogen-activated protein kinase inhibitor PD 098059 (10 microM) and prevented, in a concentration-dependent fashion, by the p23 antibody. Taken together, the results demonstrate the functional role for ERK and p23 in the neurite elongation activity of FK506 and reveal a novel signal transduction pathway involving p23 activation of ERK. We suggest that compounds that stimulate or mimic p23 may be useful for accelerating nerve regeneration.     

Equilibrium studies of a fluorescent tacrolimus binding to surfactant protein A

Tacrolimus (FK506) is a hydrophobic immunosuppressive agent used in kidney, liver, and lung transplantation. The objective of this study was to characterize the binding of FK506 to surfactant protein A (SP-A), an abundant lipoprotein found in the alveolar fluid that functions as part of the innate immune system in the lung. We have synthesized a novel derivative of FK506 in which a dansyl moiety was covalently bound via cadaverine to the C22 position of the FK506 molecule (DNS-FK). Using the fluorescence and anisotropy properties of DNS-FK, we demonstrated that tacrolimus avidly binds to SP-A with an apparent equilibrium association constant (K(app)) of 10(7)M(-1) and a Gibbs binding free energy of -40 kJ mol(-1)K(-1). Derivatization of FK506 at the C22 position did not block FK506 binding to the cytosolic immunophilin FK506-binding protein (FK-BP) or human serum albumin (HSA), both used as controls of tacrolimus-binding proteins. K(app) for FK-BP/DNS-FK and HSA/DNS-FK complexes were 1.5 x 10(7) and 10(7)M(-1), respectively. The high sensitivity of this analytical technique makes it suitable for binding analysis of FK506 to proteins.     

Regulation of the Ca2+-activated chloride channel Anoctamin-1 (TMEM16A) by Ca2+-induced interaction with FKBP12 and calcineurin

Chloride fluxes through the calcium-gated chloride channel Anoctamin-1 (TMEM16A) control blood pressure, secretion of saliva, mucin, insulin, and melatonin, gastrointestinal motility, sperm capacitation and motility, and pain sensation. Calcium activates a myriad of regulatory proteins but how these proteins affect TMEM16A activity is unresolved. Here we show by co-immunoprecipitation that increasing intracellular calcium with ionomycin or by activating sphingosine-1-phosphate receptors, induces coupling of calcium/calmodulin-dependent phosphatase calcineurin and prolyl isomerase FK506-binding protein 12 (FKBP12) to TMEM16A in HEK-293 cells. Application of drugs that target either calcineurin (cyclosporine A) or FKBP12 (tacrolimus known as FK506 and sirolimus known as rapamycin) caused a decrease in TMEM16A activity. In addition, FK506 and BAPTA-AM prevented co-immunoprecipitation between FKBP12 and TMEM16A. FK506 rendered the channel insensitive to cyclosporine A without altering its apparent calcium sensitivity whereas zero intracellular calcium blocked the effect of FK506. Rapamycin decreased TMEM16A activity in cells pre-treated with cyclosporine A or FK506. These results suggest the formation of a TMEM16A-FKBP12-calcineurin complex that regulates channel function. We conclude that upon a cytosolic calcium increase the TMEM16A-FKPB12-calcineurin trimers are assembled. Such hetero-oligomerization enhances TMEM16A channel activity but is not mandatory for activation by calcium.     

免疫抑制剂他克莫司生物合成的研究进展

他克莫司(FK506)是一种具有免疫抑制活性的23元大环内酯类化合物,临床上广泛用于防止器官移植术后的免疫排斥反应。生物合成法是他克莫司制备方法的研究热点,但他克莫司生物合成的研究还存在一定生产技术上的瓶颈。基于此,本文主要从他克莫司代谢途径改造和发酵过程控制等方面对他克莫司生物合成进展进行综述,以期为今后突破他克莫司生物合成的技术瓶颈提供参考,进而利用代谢工程、发酵工程等技术提升他克莫司的生物合成水平。     

Prevention of antigen-induced bronchial hyperreactivity and airway inflammation in sensitized guinea-pigs by tacrolimus

We examined the effect of the immunosuppressive agent, tacrolimus (FK506), on antigen-induced bronchial hyperreactivity to acetylcholine and leukocyte infiltration into the airways of ovalbumin-challenged guinea-pigs. Subcutaneous injection of 0.5 mg/kg of FK506, 1 h before and 5 h after intra-nasal antigen challenge prevented bronchial hyperreactivity to aerosolized acetylcholine, eosinophilia in bronchoalveolar lavage (BAL) fluid and bronchial tissue and the invasion of the bronchial wall by CD4+ T-lymphocytes. FK506 also suppressed ovalbumin-induced increase in the number of leukocytes adhering to the pulmonary vascular endothelium and expressing alpha4-integrins. Inhibition by FK506 of antigen-induced bronchial hyperreactivity in sensitized guinea-pigs may thus relate to its ability to prevent the emergence of important inflammatory components of airway inflammation, such as eosinophil accumulation, as well as CD4+ T-lymphocyte infiltration into the bronchial tissue.