The FK520 gene cluster of Streptomyces hygroscopicus var. ascomyceticus (ATCC 14891) contains genes for biosynthesis of unusual polyketide extender units

FK520 (ascomycin) is a macrolide produced by Streptomyces hygroscopicus var. ascomyceticus (ATCC 14891) that has immunosuppressive, neurotrophic and antifungal activities. To further elucidate the biosynthesis of this and related macrolides, we cloned and sequenced an 80kb region encompassing the FK520 gene cluster. Genes encoding the three polyketide synthase (PKS) subunits (fkbB, fkbC and fkbA), the peptide synthetase (fkbP), the 31-O-methyltransferase (fkbM), the C-9 hydroxylase (fkbD) and the 9-hydroxyl oxidase (fkbO) had the same organization as the genes reported in the FK506 gene cluster of Streptomyces sp. MA6548 (Motamedi, H., Shafiee, A., 1998. The biosynthetic gene cluster for the macrolactone ring of the immunosuppressant FK506. Eur. J. Biochem. 256, 528-534). Disruption of a PKS gene in the cluster using the φC31 phage vector, KC515, led to antibiotic non-producing strains, proving the identity of the cluster. Previous labeling data have indicated that FK520 biosynthesis uses novel polyketide extender units (Byrne, K.M., Shafiee, A., Nielson, J., Arison, B., Monaghan, R.L., Kaplan, L., 1993. The biosynthesis and enzymology of an immunosuppressant, immunomycin, produced by Streptomyces hygroscopicus var, ascomyceticus. Dev. Ind. Microbiol. 32, 29-45). Genes in the flanking regions of the FK520 cluster were identified that appear to be involved in synthesis of these extender units. All but two of these genes were homologous to genes with known function. In addition to a crotonyl-CoA reductase gene (fkbS), at least two other genes are proposed to be involved in biosynthesis of the atypical PKS extender unit ethylmalonyl-CoA, which accounts for the ethyl side chain on C-21 of FK520. A set of five contiguous genes (fkbGHIJK) is proposed to be involved in biosynthesis of an unusual PKS extender unit bearing an oxygen on the alpha-carbon, and leading to the 13- and 15-methoxy side chains. These putative precursor synthesis genes in the flanking regions of the FK520 cluster are not found in the flanking regions of the rapamycin cluster (Molnár, I., Aparicio, J.F., Haydock, S.F., Khaw, L.E., Schwecke, T., K?nig, A., Staunton, J., Leadlay, P.F., 1996. Organisation of the biosynthetic gene cluster for rapamycin in Streptomyces hygroscopicus: analysis of genes flanking the polyketide synthase. Gene 169, 1-7), consistent with labeling data showing that rapamycin biosynthesis uses only malonyl and methylmalonyl extender units.     

Draft genome of Streptomyces tsukubaensis NRRL 18488, the producer of the clinically important immunosuppressant tacrolimus (FK506)

The macrocyclic polyketide tacrolimus (FK506) is a potent immunosuppressant that prevents T-cell proliferation produced solely by Streptomyces species. We report here the first draft genome sequence of a true FK506 producer, Streptomyces tsukubaensis NRRL 18488, the first tacrolimus-producing strain that was isolated and that contains the full tacrolimus biosynthesis gene cluster.     

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.     

FK506, a secondary metabolite produced by Streptomyces, presents a novel antiviral activity against Orthopoxvirus infection in cell culture

AIMS: To investigate the antiviral potential of the macrolide FK506, produced by Streptomyces tsukubaensis, against Orthopoxvirus infection in cell culture, and determine the replicative stage of viral cycle affected by the treatment. METHODS AND RESULTS: Cell lines were infected with different Orthopoxviruses and treated with FK506. The macrolide inhibited the replication of the prototypic Orthopoxvirus, vaccinia virus strain WR, with an IC50 of 12.05 micromol l(-1). Progeny production of other Orthopoxviruses was also inhibited by FK506 at noncytotoxic concentrations, as evaluated by the neutral-red uptake assay and metabolic labelling of cellular proteins. By Western blot assay, we detected a severe inhibition (approximately 87.6% +/- 2.78%) of VV strain WR post-replicative protein synthesis. A similar reduction of virus DNA accumulation, as observed by slot-blot assay, probably accounts for the subsequent inhibition of virus late proteins. CONCLUSIONS: The macrolide FK506, isolated from S. tsukubaensis, presents a novel anti-poxvirus activity, probably targeting the stage of DNA replication during Orthopoxvirus infection. SIGNIFICANCE AND IMPACT OF THE STUDY: The secondary metabolite FK506, isolated from the culture filtrate of S. tsukubaensis, shows a pleiotropic range of activities, and might be a valuable tool as a lead structure in the generation of non-immunosuppressant analogues with strong anti-poxvirus activity.     

Enhancement of FK506 production by engineering secondary pathways of Streptomyces tsukubaensis and exogenous feeding strategies

FK506 is a clinically important macrocyclic polyketide with immunosuppressive activity produced by Streptomyces tsukubaensis. However, the low titer at which it is produced is a bottleneck to its application and use in industrial processes. We have overexpressed five potential targets associated with FK506 production (fkbO, fkbL, fkbP, fkbM, fkbD) which were identified in our previous study, with the aim to improve FK506 production. The results of the analysis showed that the constructed strains with an additional copy of each gene increased FK506 production by approximately 10–40 % compared with the wild-type strain D852. The results of the gene expression analysis indicated that each gene was upregulated. Combinatorial overexpression of the five genes resulted in a 146 % increase in the FK506 titer to 353.2 mg/L, in comparison with the titer produced by D852. To further improve the production of FK506 by the engineered strain HT-FKBOPLMD, we supplemented the medium with various nutrients, including soybean oil, lactate, succinate, shikimate, chorismate, lysine, pipecolate, isoleucine and valine. Optimization of feeding concentrations and times resulted in HT-FKBOPLMD being able to produce approximately 70 % more FK506, thereby reaching the maximal titer of 457.5 mg/L, with lower amounts of by-products (FK520 and 37,38-dihydro-FK506). These results demonstrate that the combination of the metabolically engineered secondary pathways and the exogenous feeding strategies developed here was able to be successfully applied to improve the production of industrially and clinically important compounds.     

Enhanced FK506 production in Streptomyces clavuligerus CKD1119 by engineering the supply of methylmalonyl-CoA precursor

FK506 is a 23-membered polyketide macrolide with immunosuppressant activity produced by Streptomyces species. The production of FK506 in S. clavuligerus CKD1119 (KCTC 10561BP) was improved by enhancing the supply of biosynthetic precursors. This improvement was approximately 2.5-fold (15 mg/l) with the supplementation of 10 mM methyl oleate, which is the probable source of acyl-CoAs, to R2YE medium. When the level of FK506 production reached its maximum, the intracellular concentration of methylmalonyl-CoA in S. clavuligerus CKD1119 supplemented with methyl oleate was 12.5-fold higher than that of the unsupplemented strain, suggesting that an increased methylmalonyl-CoA level caused the high-level production of FK506. The following three pathways for the production of (2S)-methylmalonyl-CoA were evaluated to identify the effective precursor supply pathway that can support the high production of FK506 in S. clavuligerus CKD1119: propionyl-CoA carboxylase, methylmalonyl-CoA mutase (MCM), and malonyl/methylmalonyl-CoA ligase. Of the three pathways examined, the MCM pathway supported the highest levels of FK506 production. The expression of MCM in S. clavuligerus CKD1119 led to a threefold and 1.5-fold increase in the methylmalonyl-CoA pool and FK506 production, respectively. Supplementing the culture broth of S. clavuligerus CKD1119 expressing MCM with methyl oleate resulted in an additional twofold increase in the FK506 titer (17.8 mg/l). Overall, these results show that the methylmalonyl-CoA supply is a limiting factor for FK506 biosynthesis and that among the three pathways analyzed, the MCM pathway is the most effective precursor supply pathway supporting the highest titer of FK506 in S. clavuligerus CKD1119.     

Catalysis,specificity, and ACP docking site of Streptomyces coelicolor malonyl-CoA:ACP transacylase

Malonyl-CoA:ACP transacylase (MAT), the fabD gene product of Streptomyces coelicolor A3(2), participates in both fatty acid and polyketide synthesis pathways, transferring malonyl groups that are used as extender units in chain growth from malonyl-CoA to pathway-specific acyl carrier proteins (ACPs). Here, the 2.0 A structure reveals an invariant arginine bound to an acetate that mimics the malonyl carboxylate and helps define the extender unit binding site. Catalysis may only occur when the oxyanion hole is formed through substrate binding, preventing hydrolysis of the acyl-enzyme intermediate. Macromolecular docking simulations with actinorhodin ACP suggest that the majority of the ACP docking surface is formed by a helical flap. These results should help to engineer polyketide synthases (PKSs) that produce novel polyketides.     

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.     

Crystal structure of an Acyl-ACP dehydrogenase from the FK520 polyketide biosynthetic pathway: insights into extender unit biosynthesis

Polyketide synthases (PKSs) synthesize the polyketide cores of pharmacologically important natural products such as the immunosuppressants FK520 and FK506. Understanding polyketide biosynthesis at atomic resolution could present new opportunities for chemo-enzymatic synthesis of complex molecules. The crystal structure of FkbI, an enzyme involved in the biosynthesis of the methoxymalonyl extender unit of FK520, was solved to 2.1A with an R(crys) of 24.4%. FkbI has a similar fold to acyl-CoA dehydrogenases. Notwithstanding this similarity, the surface and substrate-binding site of FkbI reveal key differences from other acyl-CoA dehydrogenases, suggesting that FkbI may recognize an acyl-ACP substrate rather than an acyl-CoA substrate. This structural observation coincided the genetic experiment done by Carroll et al. J. Am. Chem. Soc., 124 (2002) 4176. Although an in vitro assay for FkbI remains elusive, the structural basis for the substrate specificity of FkbI is analyzed by a combination of sequence comparison, docking simulations and structural analysis. A biochemical mechanism for the role of FkbI in the biosynthesis of methoxymalonyl-ACP is proposed.     

Application of a combined approach involving classical random mutagenesis and metabolic engineering to enhance FK506 production in Streptomyces sp. RM7011

FK506 production by a mutant strain (Streptomyces sp. RM7011) induced by N-methyl-N′-nitro-N-nitrosoguanidine and ultraviolet mutagenesis was improved by 11.63-fold (94.24 mg/l) compared to that of the wild-type strain. Among three different metabolic pathways involved in the biosynthesis of methylmalonyl-CoA, only expression of propionyl-CoA carboxylase (PCC) pathway led to a 1.75-fold and 2.5-fold increase in FK506 production and the methylmalonyl-CoA pool, respectively, compared to those of the RM7011 strain. Lipase activity of the high FK506 producer mutant increased in direct proportion to the increase in FK506 yield, from low detection level up to 43.1 U/ml (12.6-fold). The level of specific FK506 production and lipase activity was improved by enhancing the supply of lipase inducers. This improvement was approximately 1.88-fold (71.5 mg/g) with the supplementation of 5 mM Tween 80, which is the probable effective stimulator in lipase production, to the R2YE medium. When 5 mM vinyl propionate was added as a precursor for PCC pathway to R2YE medium, the specific production of FK506 increased approximately 1.9-fold (71.61 mg/g) compared to that under the non-supplemented condition. Moreover, in the presence of 5 mM Tween 80, the specific FK506 production was approximately 2.2-fold (157.44 mg/g) higher than that when only vinyl propionate was added to the R2YE medium. In particular, PCC expression in Streptomyces sp. RM7011 (RM7011/pSJ1003) together with vinyl propionate feeding resulted in an increase in the FK506 titer to as much as 1.6-fold (251.9 mg/g) compared with that in RM7011/pSE34 in R2YE medium with 5 mM Tween 80 supplementation, indicating that the vinyl propionate is more catabolized to propionate by stimulated lipase activity on Tween 80, that propionyl-CoA yielded from propionate generates methylmalonyl-CoA, and that the PCC pathway plays a key role in increasing the methylmalonyl-CoA pool for FK506 biosynthesis in RM7011 strain. Overall, these results show that a combined approach involving classical random mutation and metabolic engineering can be applied to supply the limiting factor for FK506 biosynthesis, and vinyl propionate could be successfully used as a precursor of important methylmalonyl-CoA building blocks.     

Characterization of type II thioesterases involved in natamycin biosynthesis in Streptomyces chattanoogensis L10

The known functions of type II thioesterases (TEIIs) in type I polyketide synthases (PKSs) include selecting of starter acyl units, removal of aberrant extender acyl units, releasing of final products, and dehydration of polyketide intermediates. In this study, we characterized two TEIIs (ScnI and PKSIaTEII) from Streptomyces chattanoogensis L10. Deletion of scnI in S. chattanoogensis L10 decreased the natamycin production by about 43%. Both ScnI and PKSIaTEII could remove acyl units from the acyl carrier proteins (ACPs) involved in the natamycin biosynthesis. Our results show that the TEII could play important roles in both the initiation step and the elongation steps of a polyketide biosynthesis; the intracellular TEIIs involved in different biosynthetic pathways could complement each other.     

Minimal polyketide pathway expression in an actinorhodin cluster-deleted and regulation-stimulated <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis>

Along with traditional random mutagenesis-driven strain improvement, cloning and heterologous expression of Streptomyces secondary metabolite gene clusters have become an attractive complementary approach to increase its production titer, of which regulation is typically under tight control via complex multiple regulatory networks present in a metabolite low-producing wild-type strain. In this study, we generated a polyketide non-producing strain by deleting the entire actinorhodin cluster from the chromosome of a previously generated S. coelicolor mutant strain, which was shown to stimulate actinorhodin biosynthesis through deletion of two antibiotic downregulators as well as a polyketide precursor flux downregulator (Kim et al. in Appl Environ Microbiol 77:1872–1877, 2011). Using this engineered S. coelicolor mutant strain as a surrogate host, a model minimal polyketide pathway for aloesaponarin II, an actinorhodin shunt product, was cloned in a high-copy conjugative plasmid, followed by functional pathway expression and quantitative metabolite analysis. Aloesaponarin II production was detected only in the presence of a pathway-specific regulatory gene, actII-ORF4, and its production level was the highest in the actinorhodin cluster-deleted and downregulator-deleted mutant strain, implying that this engineered polyketide pathway-free and regulation-optimized S. coelicolor mutant strain could be used as a general surrogate host for efficient expression of indigenous or foreign polyketide pathways derived from diverse actinomycetes in nature.     

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