Biological production of epicoccamide-aglycone and its cytotoxicity.

Epicoccamide (EPC) is an O-d-mannosylated acyltetramic acid of Epicoccum origin and is a bolaamphiphilic fungal polyketide. EPC displays weak toxicity against Staphylococcus aureus and HeLa cell lines. The EPC biosynthetic gene cluster was previously identified in Epicoccum nigrum and knockout of the glycosyltransferase gene (epcB) abolished EPC production. EPC-aglycone was expected in the epcB knockout but was not found. This study demonstrates that extractive culture using the hydrophobic resin Diaion HP-20 resulted in the production of EPC-aglycone, which was isolated using chromatographic separation techniques, and its structural identity was substantiated by chemical analyses. EPC-aglycone displayed strong antibacterial activity against Staphylococcus aureus, with the minimal inhibitory concentration of 1 µg/mL (64 µg/mL for EPC). EPC-aglycone displayed higher levels of growth inhibition against HeLa cell line (the half inhibitory concentration, 19 µM) and WI-38 (15 µM) cell line than EPC (76 µM and 38 µM vs. HeLa and WI-38, respectively). The dose-response curve fit of growth inhibition indicated that EPC-aglycone adopted a shallow curve (low slope factor), which was different from that of EPC, suggesting that their cellular targets are distinct from each other. This study substantiates that the d-mannose attachment is the final step in EPC biosynthesis, showcasing a glycosylation-mediated modulation of the biological activity of simple acyltetramic acid. This study also highlights the usefulness of extractive cultures in mining cryptic microbial natural products.

Genetic localization of the orevactaene/epipyrone biosynthetic gene cluster in Epicoccum nigrum.

Epipyrone (EPN)-A (syn. orevactaene) is a polyketide compound of 3-d-galactosyl-4-hydroxy-2-pyrone with a modified heptaene acyl moiety, produced from Epicoccum nigrum and was reported to have various biological activities. Genome analysis identified a hypothetical EPN biosynthetic gene cluster (BGC) composed of the four genes epnABCD, which encode a highly-reducing fungal polyketide synthase, a glycosyltransferase, a cytochrome P450, and a transporter. The individual gene inactivation of epnABC resulted in the total loss of EPN production, while the inactivation of a nearby transcription factor-encoding gene had no effect on the production of EPN, substantiating that epnABCD is the EPN BGC. mRNA expression indicated no epnA transcription in the epnB knockout mutant and the occurrence of the bicistronic transcription of epnAB. This study defined an EPN BGC, which is the first blueprint reported for glycosylated 2-pyrone polyketide biosynthesis.

A reductase gene mppE controls yellow component production in azaphilone polyketide pathway of Monascus.

OBJECTIVES: To characterize a biosynthetic gene that is selectively involved in the biosynthesis of yellow or orange components in the azaphilone polyketide pathway of Monascus. RESULTS: A reductive modification is predicted to control the relative levels of reduced (yellow) and oxidized (orange and red) components in the pathway of azaphilone pigment biosynthesis in Monascus. Targeted inactivation of a reductase gene mppE enhanced orange and red pigment production whereas overexpression of the gene promoted yellow pigment production. The effect of mppE overexpression was dependent on culture methods, and augmented yellow pigmentation was evident in a submerged culture employing a chemically defined medium. CONCLUSIONS: MppE controls the biosynthesis of the yellow pigments, ankaflavin and monascin, as a reductive enzyme in the azaphilone polyketide pathway.

Delineating citrinin biosynthesis: Ctn-ORF3 dioxygenase-mediated multi-step methyl oxidation precedes a reduction-mediated pyran ring cyclization.

Citrinin (3) is a polyketide-derived mycotoxin, that is, produced by Monascus, Penicillium, and Aspergillus spp. and is a common contaminant in a number of agricultural products. ctPKS, a non-reducing type iterative polyketide synthase with a C-terminal reductive domain, is proposed to generate the polyketide backbone of 3. The targeted gene inactivation of ctn-orf1 or ctn-orf3 gene resulted in the accumulation of a benzaldehyde derivative 6, and the ectopic expression of ctPKS/ctnB in yeast produced 6, demonstrating that ctPKS generates 6 with the support of CtnB and suggesting that Ctn-ORF1/Ctn-ORF3 converts 6 into 3. The Δctn-orf1 mutant also produced a novel benzdialdehyde derivative 10. When either 6 or 10 was fed into a ΔctPKS mutant, 3 was readily detected, which confirms that both 6 and 10 are involved in the biosynthesis of 3. A bioconversion experiment of 6 in the ectopic expression system demonstrated that ctn-orf3 expression, but not ctn-orf1 expression, efficiently consumed 6. The resulting metabolite(s) of 6 could not be identified, however. A recombinant Ctn-ORF3 enzyme was demonstrated to convert 6 into 10 and a hypothetical carboxylic derivative 8, which substantiates that Ctn-ORF3 oxidizes the exocyclic methyl moiety of 6. Ctn-ORF1 is thus proposed to reduce 8 and the subsequent non-enzymatic reactions to complete the biosynthesis of 3. The present study delineates the biosynthetic route of 3, proposing the biochemical mechanism, that is, involved in producing the natural dihydropyranoquinone structure.

A single module type I polyketide synthase directs de novo macrolactone biogenesis during galbonolide biosynthesis in Streptomyces galbus.

Galbonolide (GAL) A and B are antifungal macrolactone polyketides produced by Streptomyces galbus. During their polyketide chain assembly, GAL-A and -B incorporate methoxymalonate and methylmalonate, respectively, in the fourth chain extension step. The methoxymalonyl-acyl carrier protein biosynthesis locus (galG to K) is specifically involved in GAL-A biosynthesis, and this locus is neighbored by a gene cluster composed of galA-E. GalA-C constitute a single module, highly reducing type I polyketide synthase (PKS). GalD and GalE are cytochrome P450 and Rieske domain protein, respectively. Gene knock-out experiments verified that galB, -C, and -D are essential for GAL biosynthesis. A galD mutant accumulated a GAL-C that lacked two hydroxyl groups and a double bond when compared with GAL-B. A [U-(13)C]propionate feeding experiment indicated that no rare precursor other than methoxymalonate was incorporated during GAL biogenesis. A search of the S. galbus genome for a modular type I PKS system, the type that was expected to direct GAL biosynthesis, resulted in the identification of only one modular type I PKS gene cluster. Homology analysis indicated that this PKS gene cluster is the locus for vicenistatin biosynthesis. This cluster was previously reported in Streptomyces halstedii. A gene deletion of the vinP2 ortholog clearly demonstrated that this modular type I PKS system is not involved in GAL biosynthesis. Therefore, we propose that GalA-C direct macrolactone polyketide formation for GAL. Our studies provide a glimpse into a novel biochemical strategy used for polyketide synthesis; that is, the iterative assembly of propionates with highly programmed ß-keto group modifications.

Genetic localization and in vivo characterization of a Monascus azaphilone pigment biosynthetic gene cluster.

Monascus spp. produce several well-known polyketides such as monacolin K, citrinin, and azaphilone pigments. In this study, the azaphilone pigment biosynthetic gene cluster was identified through T-DNA random mutagenesis in Monascus purpureus. The albino mutant W13 bears a T-DNA insertion upstream of a transcriptional regulator gene (mppR1). The transcription of mppR1 and the nearby polyketide synthase gene (MpPKS5) was significantly repressed in the W13 mutant. Targeted inactivation of MpPKS5 also gave rise to an albino mutant, confirming that mppR1 and MpPKS5 belong to an azaphilone pigment biosynthetic gene cluster. This M. purpureus sequence was used to identify the whole biosynthetic gene cluster in the Monascus pilosus genome. MpPKS5 contains SAT/KS/AT/PT/ACP/MT/R domains, and this domain organization is preserved in other azaphilone polyketide synthases. This biosynthetic gene cluster also encodes fatty acid synthase (FAS), which is predicted to assist the synthesis of 3-oxooactanoyl-CoA and 3-oxodecanoyl-CoA. These 3-oxoacyl compounds are proposed to be incorporated into the azaphilone backbone to complete the pigment biosynthesis. A monooxygenase gene (an azaH and tropB homolog) that is located far downstream of the FAS gene is proposed to be involved in pyrone ring formation. A homology search on other fungal genome sequences suggests that this azaphilone pigment gene cluster also exists in the Penicillium marneffei and Talaromyces stipitatus genomes.

A new route to dTDP-6-deoxy-l-talose and dTDP-L-rhamnose: dTDP-L-rhamnose 4-epimerase in Burkholderia thailandensis.

dTDP-L-rhamnose (dTDP-Rha)-synthesizing dTDP-6-deoxy-L-lyxo-4-hexulose reductase (4-KR) and dTDP-Rha 4-epimerase were characterized from Burkholderia thailandensis E264 by utilizing rmlD(Bth) (BTH_I1472) and wbiB(Bth) (BTH_I1476), respectively. Incubation of the recombinant WbiB(Bth) with RmlA/RmlB/RmlC/Tal, which has previously been shown to generate dTDP-6-deoxy-L-talose (dTDP-6dTal) from α-D-glucose-1-phosphate, dTTP, and NADPH, produced dTDP-Rha. (1)H NMR measurements confirmed that both RmlA/RmlB/RmlC/Tal/WbiB(Bth) and RmlA/RmlB/RmlC/RmlD produced dTDP-Rha. WbiB(Bth) alone produced dTDP-Rha when incubated with dTDP-6dTal. This is the first report to demonstrate epimerase activity interconverting between dTDP-Rha and dTDP-6dTal.

Characterization of 2-octenoyl-CoA carboxylase/reductase utilizing pteB from Streptomyce avermitilis.

The filipin biosynthetic gene cluster of Streptomyces avermitilis contains pteB, a homolog of crotonyl-CoA carboxylase/reductase. PteB was predicted to be 2-octenoyl-CoA carboxylase/reductase, supplying hexylmalonyl-CoA to filipin biosynthesis. Recombinant PteB displayed selective reductase activity toward 2-octenoyl-CoA while generating a broad range of alkylmalonyl-CoAs in the presence of bicarbonate.

A DNA-binding factor, ArfA, interacts with the bldH promoter and affects undecylprodigiosin production in Streptomyces lividans.

The fact that adpA promoter activity is enhanced by S-adenosylmethionine without the involvement of the A-factor/ArpA regulatory cascade suggests the existence of additional transcriptional regulators for adpA expression in Streptomyces griseus. In this study, an additional adpA promoter regulatory protein, named ArfA, that is conserved among many bacteria was identified using DNA affinity purification from the cell extracts of Streptomyces lividans. The interactions of ArfA with the adpA promoter from S. griseus and with the bldH promoter from S. lividans were specific and both adpA and bldH promoters required ArfA for the wild-type level of their expressions in S. lividans. bldH of S. lividans is a homolog of adpA of S. lividans. ArfA-deletion mutant had only 70% of the normal undecylprodigiosin production. This result was confirmed by reduced redD promoter activity in the ArfA-deletion mutant. These results suggest that ArfA is a new type of DNA-binding regulator.

Polyaromatic Resin HP-20 Induced Accumulation of Intermediate Azaphilones in Monascus purpureus ∆mppC and ∆mpp7 Strains.

Monascus purpureus recombinant mppC and mpp7 knockout strains were subjected to extractive fermentation in the context of azaphilone pigment production. Inclusion of Diaion HP-20 resin resulted in the selective production of unreduced azaphilone congeners, in addition to the early intermediate FK17-P2a, from ∆mppC and ∆mpp7 strains that would otherwise mainly produce reduced congeners. Structural determination of two novel unreduced azaphilones from the ∆mpp7 strain was accomplished. The unreduced azaphilone compound was converted into the cognate reduced congener in recombinant M. purpureus strains, demonstrating its intermediate role in azaphilone biosynthesis. This study demonstrates the possibility that extractive fermentation with Diaion HP-20 resin can be used to obtain cryptic azaphilone metabolites.

Gene inactivation study of gntE reveals its role in the first step of pseudotrisaccharide modifications in gentamicin biosynthesis.

A gene inactivation study was performed on gntE, a member of the gentamicin biosynthetic gene cluster in Micromonospora echinospora. Computer-aided homology analysis predicts a methyltransferase-related cobalamin-binding domain and a radical S-adenosylmethionine domain in GntE. It is also found that there is no gntE homolog within other aminoglycoside biosynthetic gene clusters. Inactivation of gntE was achieved in both M. echinospora ATCC 15835 and a gentamicin high-producer GMC106. High-performance liquid chromatographic analysis, coupled with mass spectrometry, revealed that gntE mutants accumulated gentamicin A2 and its derivative with a methyl group installed on the glucoamine moiety. This result substantiated that GntE participates in the first step of pseudotrisaccharide modifications in gentamicin biosynthesis, though the catalytic nature of this unusual oxidoreductase/methyltransferase candidate is not resolved. The present gene inactivation study also demonstrates that targeted genetic engineering can be applied to produce specific gentamicin structures and potentially new gentamicin derivatives in M. echinospora.

Effects of extracellular ATP on the physiology of Streptomyces coelicolor A3(2).

Because ATP is an extracellular effector in animal and plant systems and derivatives of ATP, such as S-adenosylmethionine and cAMP, can control antibiotic production and morphological differentiation in Streptomyces, we hypothesized that extracellular ATP (exATP) can also affect physiologies of Streptomyces. We found that the addition of 10 microM exATP to Streptomyces coelicolor A3(2) cultures resulted in enhanced actinorhodin and undecylprodigiosin production and morphological differentiation on solid medium. However, these phenotypes were reduced by the addition of a 10-fold higher concentration of exATP (100 microM). Intracellular ATP concentrations were also modulated in response to changes in exATP. ATP analogs, added at a 100-fold lower concentration, affected Streptomyces similarly to that seen for 10 microM exATP. The enhanced promoter activity of actII-orf4 indicated that 10 microM exATP affect the transcriptional level for actinorhodin production. Results from this study suggest that exATP is an effector for the physiology of S. coelicolor and careful manipulation of exATP may significantly enhance the high-yield production of antibiotics by S. coelicolor.

Elucidation of the kijanimicin gene cluster: insights into the biosynthesis of spirotetronate antibiotics and nitrosugars.

The antibiotic kijanimicin produced by the actinomycete Actinomadura kijaniata has a broad spectrum of bioactivities as well as a number of interesting biosynthetic features. To understand the molecular basis for its formation and to develop a combinatorial biosynthetic system for this class of compounds, a 107.6 kb segment of the A. kijaniata chromosome containing the kijanimicin biosynthetic locus was identified, cloned, and sequenced. The complete pathway for the formation of TDP-l-digitoxose, one of the two sugar donors used in construction of kijanimicin, was elucidated through biochemical analysis of four enzymes encoded in the gene cluster. Sequence analysis indicates that the aglycone kijanolide is formed by the combined action of a modular Type-I polyketide synthase, a conserved set of enzymes involved in formation, attachment, and intramolecular cyclization of a glycerate-derived three-carbon unit, which forms the core of the spirotetronate moiety. The genes involved in the biosynthesis of the unusual deoxysugar d-kijanose [2,3,4,6-tetradeoxy-4-(methylcarbamyl)-3-C-methyl-3-nitro-d-xylo-hexopyranose], including one encoding a flavoenzyme predicted to catalyze the formation of the nitro group, have also been identified. This work has implications for the biosynthesis of other spirotetronate antibiotics and nitrosugar-bearing natural products, as well as for future mechanistic and biosynthetic engineering efforts.

Glucoamylase gene, vldI, is linked to validamycin biosynthesis in Streptomyces hygroscopicus var. limoneus, and vldADEFG confers validamycin production in Streptomyces lividans, revealing the role of VldE in glucose attachment.

The validamycin biosynthetic gene cluster in Streptomyces hygroscopicus var. limoneus contains vldI, the gene encoding a glucoamylase (1,4-alpha-D-glucan glucohydrolase). The knock-out of vldI (vldI::neo) reduced the yield of validamycin-A, thus indicating that VldI contributes to validamycin-A productivity by supplying glucose with the hydrolysis of 1,4-alpha-D-glucan(s). Promoter-probe assays employing xylE fusions indicated that the transcription of vldI correlates to those of other biosynthetic genes, which are organized with two divergently arranged vldABC and vldDEFGH sets. These results reveal that the contiguous region covering nine genes of vldABCDEFGHI represents the core of the validamycin biosynthetic cluster. After confirming that genes vldABCDEFGH confer validamycin production ability to Streptomyces lividans, genes vldBCHI were eliminated from the expression construct and the remaining genes, vldADEFG, were tested for the ability to confer validamycin-A production to S. lividans. Ion-exchange chromatographic purification and a subsequent HPLC analysis confirmed that S. lividans/vldADEFG yielded a 75 microg/l of validamycin-A, showing that the validamycin pathway involves a single NDP-sugar glycosyltransferase reaction. It was also demonstrated that VldE is capable of coupling validoxylamine-A and UDP-glucose to generate validamycin-A. The proposal that VldADEFG catalyze the biosynthesis of validamycin-A from sedo-heptulose 7-phosphate and UDP-glucose and include a N-bridge-forming catalyst will serve as a guideline for future biochemical studies and a platform to explore this m-C7N cyclitol pathway for biotechnological applications.

Two threonine residues required for role of AfsKav in controlling morphogenesis and avermectin production in Streptomyces avermitilis.

AfsKav is a eukaryotic-type serine/threonine protein kinase, required for sporulation and avermectin production in Streptomyces avermitilis. In terms of their ability to complement SJW4001 (DeltaafsK-av), afsK-av mutants T165A and T168A were not functional, whereas mutants T165D and T168D retained their ability, indicating that Thr-165 and Thr-168 are the phosphorylation sites required for the role of AfsKav. Expression of the S-adenosylmethione synthetase gene promoted avermectin production in the wild-type S. avermitilis, yet not in the mutant harboring T168D or T165D, demonstrating that tandem phosphorylation on Thr-165 and Thr-168 in AfsKav is the mechanism modulating avermectin production in response to S-adenosylmethione accumulation in S. avermitilis.

Genetic characterization of two S-adenosylmethionine-induced ABC transporters reveals their roles in modulations of secondary metabolism and sporulation in Streptomyces coelicolor M145.

S-Adenosylmethionine (SAM) was previously documented to activate secondary metabolism in a variety of Streptomyces spp. and to promote actinorhodin (ACT) and undecylprodigiosin (RED) in Streptomyces coelicolor. The SAM-induced proteins in S. coelicolor include several ABC transporter components (SCO5260 and SCO5477) including BldKB, the component of a well-known regulatory factor for differentiations. In order to assess the role of these ABC transporter complexes in differentiation of Streptomyces, SCO5260 and SCO5476, the first genes from the cognate complex clusters, were individually inactivated by gene replacement. Inactivation of either SCO5260 or SCO5476 led to impaired sporulation on agar medium, with the more drastic defect in the SCO5260 null mutant (ASCO5260). ASCO5260 displayed growth retardation and reduced yields of ACT and RED in liquid cultures. In addition, SAM supplementation failed in promoting the production of ACT and RED in ASCO5260. Inactivation of SCO5476 gave no significant change in growth and production of ACT and RED, but impaired the promoting effect of SAM on ACT production without interfering with the effect on RED production. The present study suggests that SAM induces several ABC transporters to modulate secondary metabolism and morphological development in S. coelicolor.

Genetic localization and heterologous expression of validamycin biosynthetic gene cluster isolated from Streptomyces hygroscopicus var. limoneus KCCM 11405 (IFO 12704).

The validamycin biosynthetic gene cluster was isolated from Streptomyces hygroscopicus var. limoneus KTCC 1715 (IFO 12704) using a pair of degenerated PCR primers designed from the sequence of AcbC, 2-epi-5-epi-valiolone synthase in the acarbose biosynthesis. The nucleotide sequence analysis of the 37-kb DNA region revealed 22 complete ORFs including vldA, the acbC ortholog. Located around vldA, vldB to K were predicted to encode adenyltransferase, kinase, ketoreductase (or epimerase/dehydratase), glycosyltransferase, aminotransferase, dehydrogenase, phosphatase/phosphomutase, glycosyl hydrolase, transport protein, and glycosyltransferase, respectively. Apparently absent were any regulatory components within the sequenced region. The disruption of vldA abolished the validamycin biosynthesis and the plasmid-based complementation with vldABC restored production to the vldA-mutant; this substantiated that vldABC are essential to validamycin biosynthesis. This finding enabled us to discover the complete validamycin biosynthetic cluster. The cosmid clone of pJWS3001 harboring the 37-kb DNA region conferred validamycin-accumulation to Streptomyces lividans, indicating that the entire gene cluster of validamycin biosynthesis had been isolated. Additionally, Streptomyces albus, transformed with pJWS3001, produced a high level of alpha-glucosidase inhibitory activity in a R2YE liquid culture, which highlights the portability of the cluster within Streptomyces. The product of vldI was characterized as a glucoamylase (kcat, 32 s(-1); K(m), 5 mg/ml of starch) that does not play any apparent role in the validamycin biosynthesis. In order to characterize the upstream region, a vldW knockout was achieved via gene-replacement. A phenotypic study of the resulting mutant revealed that vldW is not essential for the host's ability to control Pellicularia filamentosa growth. The current information suggests that vldA to vldH is the genetic region essential to validamycin biosynthesis. This promises excellent opportunities to elucidate biosynthetic route(s) to the validamycin complex and to engineer the pathway for industrial application.

S-adenosylmethionine activates adpA transcription and promotes streptomycin biosynthesis in Streptomyces griseus.

S-adenosylmethionine (SAM), the major methyl donor in diverse biological processes, was previously documented as a regulator for secondary metabolism in Streptomyces. The present study demonstrates that exogenous SAM, in a quantity as low as 10 muM, enhanced streptomycin production and adpA transcription in both Streptomyces griseus wild-type strain and mutant HO1, which displays no ArpA repression on the adpA promoter. SAM also enhanced xylE expression driven by the promoter of adpA or strR in a heterologous host, S. lividans. This report substantiates that exogenous SAM promotes adpA transcription in S. griseus, which is involved in the SAM-mediated promotion of streptomycin, and that the mechanism underlying this event is shared in S. lividans.

Cloning and functional characterization of 2-C-methyl-D-erythritol 4-phosphate cytidyltransferase (GbMECT) gene from Ginkgo biloba.

2-C-methyl-D-erythritol 4-phosphate cytidyltransferase (MECT), the third enzyme of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway, catalyzes formation of 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol from MEP. GbMECT, presumably involved in ginkgolide biosynthesis, was cloned and characterized from Ginkgo biloba embryonic roots. The protein containing the N-terminal chloroplast transit peptide consisted of 327 amino acid residues. Complementation of GbMECT with Escherichia coli NMW33, ygbP (EcMECT) knock-out mutant, rescued the mutant, confirming the function of the protein. Transcription levels of GbMECT remained generally constant in embryonic roots and leaves for 1 month. Full 88 N-terminal residues were necessary to deliver the protein into the chloroplast as shown by protein-targeting analysis with GFP as a reporter protein in Arabidopsis thaliana protoplasts.

Talosins A and B: new isoflavonol glycosides with potent antifungal activity from Kitasatospora kifunensis MJM341. II. Physicochemical properties and structure determination.

In our screening program for new antifungal agents from microbial secondary metabolites, two new isoflavonol glycosides, with potent antifungal activity, talosins A and B, were isolated from the culture broth of Kitasatospora kifunensis MJM341. Talosins A and B were determined to be genistein 7-alpha-L-6-deoxy-talopyranoside and genistein 4',7-di-alpha-L-6-deoxy-talopyranoside, respectively, by spectroscopic studies. They are the first flavonoid glycosides incorporating 6-deoxy-talose as a sugar component.