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Yin-chu Shen - One of the best experts on this subject based on the ideXlab platform.
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Bioactivities of Validamycins and Related Natural Compounds
Validamycin and its Derivatives, 2017Co-Authors: Xiaolong Chen, Yongxian Fan, Yin-chu ShenAbstract:Abstract Validamycins and related natural compounds have good bioactivities, such as antifungal activities, enzymatic inhibitory activities, and insecticidal activities. Validamycins has good antifungal activity against Rhizoctonia Solani. The antifungal mechanism was complex. Validamycin A and validoxylamine A could also be used to control Fusarium wilt of tomato. As to other fungi, all the Basidiomycotina were sensitive to validamycin A. With the exception of Fusarium culmorum, Chaetomium globosum, and Chaetomium bostrychoides, all Ascomycotina, Oocyectes, and Mucorales tested were insensitive to validamycin A. Since the validoxylamines and Validamycins are structurally similar to trehalose, they have good inhibitory activity for trehalase. Valienamine and related natural compounds has strong inhibitory activity for glucoside hydrolases. Most important of all, validoxylamine A has good insecticidal activity in vitro.
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An Introduction to Validamycins and Their Derivatives
Validamycin and its Derivatives, 2017Co-Authors: Xiaolong Chen, Yongxian Fan, Yin-chu ShenAbstract:Abstract Validamycin is a magic agricultural antibiotic due to its outstanding merits such as excellent control effect, low price, low drug-resistance and low toxicity. Thus it has widely been used in Asia as the rice and wheat protectant against Rhizoctonia solani for decades. In this chapter, the simple introduction of antibiotics, agricultural antibiotics, and Validamycins was described. Most of all, the aim of the book was provided in the context.
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Production of Validamycins
Validamycin and its Derivatives, 2017Co-Authors: Xiaolong Chen, Yongxian Fan, Yin-chu ShenAbstract:Validamycins have been used to efficiently prevent and treat sheath blight disease of corps including rice, wheat, potatoes, vegetables, strawberries, tobacco, ginger, and other crops. Thus it has large markets in China and eastern Asia with annual revenue of billions of Chinese dollars. In this chapter, the discovery of Validamycins was first described. Then the microbes for producing Validamycins were compared, including general characteristics and complete genomes. Later, production, isolation, structures, characterization and properties, biosynthesis, analysis, and microbial degradation of Validamycins were presented. And gene engineering, including cloning, expression, and deficiency of genes in the biosynthesis, was reviewed, too. At last, fermentation process for production of Validamycins in large scale was focused.
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Chemical Synthesis of Validamycin and Related Natural Compounds
Validamycin and its Derivatives, 2017Co-Authors: Xiaolong Chen, Yongxian Fan, Yin-chu ShenAbstract:Because of their good biological activities, Validamycins and their related natural compounds have attracted more and more researchers to synthesize them. Most of the research has focused on chemical synthesis of valienamine and valiolamine, important intermediates for voglibose, N -octyl-β-valienamine, and N -octyl-4- epi -β-valienamine. In addition, the total synthesis of Validamycins and validoxylamines has been carried out by many research groups. Different kinds of chemicals, including glucose, L -quebranchitol, 2,3,4,6- tetra - O -benzyl- d -glucose, d -xylose, etc., have been employed as the starting materials.
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cloning expression and medium optimization of validamycin glycosyltransferase from streptomyces hygroscopicus var jinggangensis for the biotransformation of validoxylamine a to produce validamycin a using free resting cells
Bioresource Technology, 2013Co-Authors: Yongxian Fan, Xiaolong Chen, Xiaoqin Jia, Yin-chu ShenAbstract:Validamycin A is widely used to control Basidiomycetes, which causes sheath blight disease in rice, potatoes, vegetables, and other crops as well as dumping-off disease in vegetable seedlings, cotton, sugar beets, and other plants. In order to improve the content of validamycin A in the commercial products, valG from Streptomyces hygroscopicus was successfully cloned into Escherichia coli BL21(DE3) and was directly employed as the biocatalyst in the biotransformation from validoxylamine A to validamycin A with the existence of d-cellobiose using the free resting cells in the present study. The fermentation medium was optimized through single factor experiment and response surface method. With the optimized medium, which contained lactose 4.7g/L, yeast extract 49.5g/L, ammonium chloride 2.7g/L, potassium phosphate buffer solution 110mL/L, Ca(2+) 0.0352g/L, the biomass yield and enzyme activity reached 5.5g/L and 1.49U/mL, respectively, which were nearly twice higher than those with initial medium.
Taifo Mahmud - One of the best experts on this subject based on the ideXlab platform.
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The α‐Ketoglutarate/FeII‐Dependent Dioxygenase VldW Is Responsible for the Formation of Validamycin B
Chembiochem : a European journal of chemical biology, 2012Co-Authors: Khaled H. Almabruk, Shumpei Asamizu, Ada Chang, Sheril G. Varghese, Taifo MahmudAbstract:Validamycin A (1), an antifungal agent used widely as a crop protectant, is the main component of the validamycin complex produced by Streptomyces hygroscopicus subsp. limoneus.[1, 2] The antifungal activity of 1 has been attributed to its core structure, validoxylamine A (4), which consists of two pseudosugar units, valienamine (7) and validamine (8) (Scheme 1). Commercially available validamycin usually contains ~60% validamycin A (1), ~15% validamycin B (2), and other minor analogues. In contrast to 1, the hydroxylated analogue 2 is significantly less active against fungal pathogens. Therefore, it is desirable to abolish the production of 2 while increasing the yield of 1. On the other hand, validamycin G (3), another hydroxylated analogue of 1, has great potential to be used as a direct source of valiolamine (10), the precursor of the antidiabetic drug voglibose (11). However, the production yield of 3 by S. hygroscopicus subsp. limoneus is extremely low (0.008% of the crude Validamycins),[3] making it impractical to produce voglibose from this natural product. Efforts to control or improve the production of these hydroxylated Validamycins have been hampered by the lack of knowledge of their biosynthesis. Scheme 1 Chemical structures of the Validamycins and related compounds. While the biosynthesis of 1 has been studied extensively, the modes of formation of the hydroxylated Validamycins were not clearly understood. Early speculations suggested that the formation of 2 and 3 may involve hydroxylation of early cyclitol intermediates in the pathway.[4] However, no experimental data were available to support that notion. The identification of the biosynthetic gene clusters of validamycin in several strains of S. hygroscopicus, e.g., S. hygroscopicus subsp. jinggangensis 5008 and S. hygroscopicus subsp. limoneus KCCM 11405 (IFO 12704),[5, 6] however, provides new opportunities to investigate the modes of formation of these compounds. Direct comparison of the former (the val cluster) and the latter (the vld cluster) has shown that both clusters share similar sets of genes necessary for the biosynthesis of validamycin A (Figure S1).[7] However, no candidate genes for the formation of 2 and 3 were identified. To this end, we first investigated two genes within the val cluster (valE and valJ) from S. hygroscopicus subsp. jinggangensis 5008 that may be involved in the formation of hydroxylated Validamycins. ValE and ValJ are homologous enzymes (67% identity) that show high identity to α-ketoglutarate/Fe(II)-dependent dioxygenases, non-heme enzymes that catalyze a variety of oxidative transformations. This family of enzymes catalyze a diverse array of biotransformations in primary and secondary metabolism, including many bioactive natural products such as penicillin,[8] clavulanic acid,[9, 10] viomycin,[11, 12] morphine,[13] and flavonoids.[14] Both ValE and ValJ contain a highly conserved Fe(II) binding HXD/E…H triad motif (Figure S2). However, attempts to express valJ in Escherichia coli did not yield soluble protein, whereas overexpression of valE gave a moderately soluble recombinant protein. However, no catalytic activity of ValE was observed when 1 and 4 were used as substrate (data not shown). Interestingly, while there are two α-ketoglutarate/Fe(II)-dependent dioxygenase genes in the val cluster, only one homologous gene, vldW, is present in the vld cluster. Multiple amino acid sequence alignment of VldW, ValE, and ValJ revealed that the N-terminal sequence of VldW is highly similar to that of ValJ and the C-terminal sequence is more similar to that of ValE (Figure S2), suggesting that VldW may be a hybrid protein originated from ValE and ValJ. Inactivation of vldW in S. hygroscopicus subsp. limoneus has been reported to have no effects in the production of 1.[6] However, the study did not show whether this inactivation had any effects to the production of hydroxylated Validamycins. To examine if VldW is involved in the formation of hydroxylated Validamycins, we cloned the gene from the chromosome of S. hygroscopicus subsp. limoneus and the product was inserted into an expression vector pRSET B to give pTMS005. The plasmid was then used to transform E. coli BL21(DE3)pLysS. The expression of the gene was induced by isopropyl-β-D-thiogalactopyranoside (IPTG) to give a 43.4 kDa soluble His6-tagged protein (Figure S3A). Incubation of the enzyme with validamycin A in the presence of α-ketoglutarate and Fe(NH4)2(SO4)2 showed that the enzyme can catalyze the conversion of 1 (m/z 498 [M+H]+) to its hydroxylated product (m/z 514 [M+H]+) (Figure S3C–S3E). TLC analysis showed that the product has an Rf value comparable to that of 2 (Figure S3B). However, the results cannot rule out 3 as a possible product, as, due to the lack of an authentic sample, no direct comparison could be made with the latter compound. To determine the chemical structure of the VldW product, we scaled up the enzymatic reaction and purified the product using ion-exchange resin [DOWEX 1 (OH− form)] and gel filtration (Sephadex LH-20) column chromatography. 1H and 13C NMR spectra of the pure compound showed signals identical to those of the authentic 2, suggesting that VldW is indeed a validamycin B synthase (Figures S4 and S5). In addition, the C-6′ methylene protons of the substrate 1 [δH 1.39 ppm (brt) and 2.01 ppm (brd)] (Figure S4C) are missing in the VldW product (Figure S4B), indicating that the hydroxylation occurs at the C-6′ position. Moreover, DEPT-135 experiments with the product showed that it has only three methylene carbons at around 60 ppm (Figure S5B), which are attributed to the primary alcohol carbons C-7, C-7′, and C-6″. All together the data provided convincing evidence that the product of VldW is 2 and there is no indication that 3 is coproduced during the biotransformation. Because there is only one α-ketoglutarate/Fe(II)-dependent dioxygenase gene present in the vld cluster in S. hygroscopicus subsp. limoneus, we speculate that 3 is a shunt product of an unspecific cellular dioxygenase or cytochrome P450 monooxygenase enzyme. In addition, its low yield production may be due to an unfavorable hydroxylation of the less reactive C-5′ position. Previously, it was proposed that the formation of 2 and 3 might involve earlier hydroxylated cyclitol intermediates, e.g., hydroxyvalidamine (9) or valiolamine (10). However, no experimental evidence was available to suggest that the hydroxylation occurs early in the pathway. To determine the timing of the hydroxylation reaction and the substrate specificity of VldW, we tested validamine (8), validamine 7-phosphate (12), validoxylamine A 7′-phosphate (13), 4, and 1 as substrates (Scheme 1). Compounds 8, 12, 13, and 4 were prepared by chemical transformations as reported previously.[15] Whereas the involvement of 8 in validamycin biosynthesis is still obscure, 12, 13, and 4 have recently been biochemically demonstrated to be involved in 1 biosynthesis.[15] As shown in Figure 1, among the compounds tested using cell-free extracts of E. coli harboring vldW, 1 appears to be the true substrate for VldW. Parallel experiments using cell-free extract of E. coli harboring empty vector (pRSET B) did not give any product (Figure S6). VldW is also able to convert 4 (m/z 336 [M+H]+) to its hydroxylated derivative (m/z 352 [M+H]+) (Figure 1D), albeit much less efficiently than the conversion of 1 to 2 (Figure 1E). It is most likely that 4 is not the natural substrate for VldW. No products were observed when 8, 12, or 13 were used as substrate (Figures 1A, 1B, 1C). The results confirm that 2 is derived from 1 and the hydroxylation reaction occurs late in the pathway. Figure 1 Mass spectral analyses of VldW reactions with various substrates. (A) with validamine, (B) with validamine 7-phosphate, (C) with validoxylamine A 7′-phosphate, (D) with validoxylamine A, and (E) with validamycin A. Whereas VldW is a relatively stable enzyme, its catalytic activity is affected by Ni2+ ions. A significant reduction of activity was observed when the protein was purified using Ni-NTA column. However, the activity can be restored by dialysis of the protein in a buffer solution containing 0.1 mM EDTA, followed by the addition of 0.2 mM of Fe2+ to the protein solution. To determine the optimal conditions for enzyme catalysis, four different buffers (HEPES buffer pH 7.5, MOPS buffer pH 7.5, Tris-HCl buffer pH 7.5, potassium phosphate buffer pH 7.5) were used to incubate VldW and 1. The results showed that VldW is most active in potassium phosphate buffer (Figure S7A). The hydroxylation of 1 by VldW was found to have a pH optimum at approximately 7.2 (Figure S7B). The kinetics values were determined by using a succinyl-CoA synthetase (SCS), pyruvate kinase (PK) and lactate dehydrogenase (LDH) coupled enzyme assay (Scheme 2).[16] Oxidation of NADH to NAD+ was monitored at 340 nm in 96-well plates using a spectrophotometric microplate reader. The apparent kinetic parameters, obtained from Hanes-Woolf plots, were Km of 303 ± 36 μM and 19 ± 3.5 μM for 1 and α-ketoglutarate, respectively, and a Kcat of 0.97 ± 0.14 min−1 (Figure 2). Figure 2 Kinetic parameters of VldW. (A) Michaelis-Menten curve for validamycin A; (B) Michaelis-Menten curve for α-ketoglutarate. Scheme 2 Biochemical characterization of VldW. PEP, phosphoenolpyruvate. The present study demonstrated that validamycin B (2) is derived from validamycin A (1) by the action of VldW, an α-ketoglutarate/Fe(II)-dependent dioxygenase that regioselectively hydroxylates the C-6′ position of 1. The result suggests that inactivation of the vldW gene in the producing strains may abolish the production of 2 and 5, which in turn may lead to an increased overall production of the important crop protectant validamycin A.
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Nucleotidylation of unsaturated carbasugar in validamycin biosynthesis
Organic & biomolecular chemistry, 2010Co-Authors: Jongtae Yang, Zixin Deng, Linquan Bai, Yirong Zhang, Taifo MahmudAbstract:Validamycin A is a member of microbial-derived C7N-aminocyclitol family of natural products that is widely used as crop protectant and the precursor of the antidiabetic drug voglibose. Its biosynthetic gene clusters have been identified in several Streptomyces hygroscopicus strains, and a number of genes within the clusters have been functionally analyzed. Of these genes, valB, which encodes a sugar nucleotidyltransferase, was found through inactivation study to be essential for validamycin biosynthesis, but its role was unclear. To characterize the role of ValB in validamycin biosynthesis, four carbasugar phosphate analogues were synthesized and tested as substrate for ValB. The results showed that ValB efficiently catalyzes the conversion of valienol 1-phosphate to its nucleotidyl diphosphate derivatives, whereas other unsaturated carbasugar phosphates were found to be not the preferred substrate. ValB requires Mg2+, Mn2+, or Co2+ for its optimal activity and uses the purine-based nucleotidyltriphosphates (ATP and GTP) more efficiently than the pyrimidine-based NTPs (CTP, dTTP, and UTP) as nucleotidyl donor. ValB represents the first member of unsaturated carbasugar nucleotidyltransferases involved in natural products biosynthesis. Its characterization not only expands our understanding of aminocyclitol-derived natural products biosynthesis, but may also facilitate the development of new tools for chemoenzymatic synthesis of carbohydrate mimetics.
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genetically engineered production of 1 1 bis valienamine and validienamycin in streptomyces hygroscopicus and their conversion to valienamine
Applied Microbiology and Biotechnology, 2009Co-Authors: Jongtae Yang, Zixin Deng, Linquan Bai, Taifo MahmudAbstract:The antifungal agent validamycin A is an important crop protectant and the source of valienamine, the precursor of the antidiabetic drug voglibose. Inactivation of the valN gene in the validamycin A producer, Streptomyces hygroscopicus subsp. jinggangensis 5008, resulted in a mutant strain that produces new secondary metabolites 1,1′-bis-valienamine and validienamycin. The chemical structures of 1,1′-bis-valienamine and validienamycin were elucidated by 1D and 2D nuclear magnetic resonance (NMR) spectroscopy in conjunction with mass spectrometry and bioconversion employing a glycosyltransferase enzyme, ValG. 1,1′-Bis-valienamine and validienamycin exhibit a moderate antifungal activity against Pellicularia sasakii. Chemical degradation of 1,1′-bis-valienamine using N-bromosuccinimide followed by purification of the products with ion-exchange column chromatography only resulted in valienamine, whereas parallel treatments of validoxylamine A, the aglycon of validamycin A, resulted in an approximately 1:1 mixture of valienamine and validamine, underscoring the advantage of 1,1′-bis-valienamine over validoxylamine A as a commercial source of valienamine.
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Catalytic analysis of the validamycin glycosyltransferase (ValG) and enzymatic production of 4''-epi-validamycin A.
Journal of natural products, 2008Co-Authors: Kazuyuki Minagawa, Zixin Deng, Linquan Bai, Taifo MahmudAbstract:Combinatorial biocatalysis is an emerging technology in the field of drug discovery and development. Some of the approaches include the use of enzymatic, chemoenzymatic, and microbial transformations to generate libraries of new chemical entities from lead compounds.1-3 Since many bioactive natural products contain sugar moieties, which play a critical role in their pharmacological activities,4, 5 glycosylation processes of natural products have become a prime subject of investigation.6 Glycosyltransferases (GTs) catalyze the transfer of a sugar moiety from a nucleotidyldiphosphate (NDP)-sugar to an acceptor, which could be a growing oligosaccharide, a lipid, a protein, or a small molecule. Based on tertiary structure analysis, GTs have been divided into two superfamilies, known as GT-A and GT-B.7 Members of the GT-A superfamily contain two dissimilar domains, one involved in the recognition of the NDP-sugar and the other in the recognition of the acceptor molecule. Most of the GTs in the Leloir pathway that reside in the Golgi apparatus and the endoplasmic reticulum belong to this family. The GT-B superfamily is remarkably diverse and contains members that are extremely promiscuous to their NDP-sugar donors.8, 9 The GT-B family consists of most of prokaryotic enzymes that glycosylate secondary metabolites to produce active natural products, as well as some glycosyltranferases from the primary pathways. Validamycin A (2), a commercially used agricultural antifungal antibiotic, is a microbial-derived pseudotrisaccharide, which contains a core aminocyclitol moiety, validoxylamine A (1), and glucose. The core aminocyclitol moiety is derived from two units of 2-epi-5-epi-valiolone, each of which is a cyclization product of the C7-sugar phosphate, sedoheptulose 7-phosphate. The glucose attachment is critical for the intake of the drug by the fungal cells through the common oligosaccharide transport system. Inside the cells, the compound is hydrolyzed to 1, and serves as a competitive inhibitor of trehalase.10 In fungi, trehalose is commonly used as a storage carbohydrate, which can be hydrolyzed by trehalase to two molecules of glucose for energy supply and other physiological purposes. Therefore, inhibition of the trehalase activity is detrimental to fungal growth. As part of our ongoing study on the biosynthesis of validamycin, we have identified the complete biosynthetic gene cluster in Streptomyces hygroscopicus subsp. jinggangensis 5008.11 Among the proteins believed to be directly involved in the biosynthesis, ValG has been characterized both in vivo and in vitro as a glycosyltransferase that catalyzes the conversion of 1 to 2 using UDP-glucose as the sugar donor (Fig. 1).11 Interestingly, ValG belongs to the GT-A family of glycosyltransferases, even though it functions in secondary metabolism. ValG is also unique in that it has an unusual DTG motif instead of the DXD motif common in closely related proteins. While generally there is no significant identity between different GT families, the acidic DXD motif is highly conserved in almost all GTs,12 and is predicted to participate in the coordination of a divalent metal ion (most commonly Mn2+) and/or in the binding of the NDP-sugar. Mutagenesis of a mannosyltransferase has showed that altering either of these aspartates completely eliminates the enzymatic activity without causing the protein to misfold or denature.13 In this study, we investigated the utilization of ValG as a tool for generating analogs of validamycin by testing a number of commercially available NDP-sugars as substrates. The involvement of the DTG sequence of ValG in its catalytic activity or substrate specificity was explored by replacing the DTG sequence with a DCD motif. Figure 1 Biosynthesis of validamycin A.
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valc a new type of c7 cyclitol kinase involved in the biosynthesis of the antifungal agent validamycin a
ChemBioChem, 2007Co-Authors: Kazuyuki Minagawa, Linquan Bai, Zixin Deng, Yirong Zhang, Takuya Ito, Taifo MahmudAbstract:The gene valC encoding an enzyme homologous to the 2-epi-5-epi-valiolone kinase (AcbM) of the acarbose biosynthetic pathway was identified in the validamycin A biosynthetic gene cluster. Inactivation of valC resulted in mutants that lack the ability to produce validamycin A. Complementation experiments with a replicating plasmid harboring full-length valC restored the production of validamycin A, suggesting a critical function of valC in validamycin biosynthesis. In vitro characterization of ValC revealed a new type of C7-cyclitol kinase, which phosphorylates valienone and validone, but not 2-epi-5-epi-valiolone, 5-epi-valiolone, and glucose, to their 7-phosphate derivatives. The results provide new insights about this enzyme activity and also confirm the existence of two different pathways leading to the same end-product, the valienamine moiety of acarbose and validamycin A.
Linquan Bai - One of the best experts on this subject based on the ideXlab platform.
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a validamycin shunt pathway for valienamine synthesis in engineered streptomyces hygroscopicus 5008
ACS Synthetic Biology, 2020Co-Authors: Li Cui, Linquan Bai, Xiaodong Wei, Xinran Wang, Shuangjun Lin, Yan FengAbstract:Valienamine is the key functional component of many natural glycosidase inhibitors, including the crop protectant validamycin A and the clinical antidiabetic agent acarbose. Due to its important biomedical activity, it is also the prominent lead compound for the exploration of therapeutic agents, such as the stronger α-glucosidase inhibitor voglibose. Currently, the main route for obtaining valienamine is a multistep biosynthetic process involving the synthesis and degradation of validamycin A. Here, we established an alternative, vastly simplified shunt pathway for the direct synthesis of valienamine based on an envisioned non-natural transamination in the validamycin A producer Streptomyces hygroscopicus 5008. We first identified candidate aminotransferases for the non-natural ketone substrate valienone and conducted molecular evolution in vitro. The WecE enzyme from Escherichia coli was verified to complete the envisioned step with >99.9% enantiomeric excess and was further engineered to produce a 32.6-fold more active mutant, VarB, through protein evolution. Subsequently, two copies of VarB were introduced into the host, and the new shunt pathway produced 0.52 mg/L valienamine after a 96-h fermentation. Our study thus illustrates a dramatically simplified alternative shunt pathway for valienamine production and introduces a promising foundational platform for increasing the production of valienamine and its valuable N-modified derivatives for use in pharmaceutical applications.
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Effects of pigment gene deletions on validamycin A production in Streptomyces hygroscopicus var. jinggangensis
Wei sheng wu xue bao = Acta microbiologica Sinica, 2016Co-Authors: Yao Peng, Linquan BaiAbstract:Objective We studied the contributions of four pigment biosynthetic genes to validamycin A yield, biomass accumulation, and the color of fermentation broth via individual gene deletions. Methods The deletion mutants were obtained via homologous recombination. The titer of validamycin A was detected by HPLC. The transcription of validamycin biosynthetic genes was quantified by qRT-PCR, and the growth was measured with dry cell weight. Results Compared with the parent strain, the deletion of DOPA melanin genes increased the validamycin A titer from 20.6 to 23.1 g/L (by 12%), whereas the deletion of type Ⅲ polyketide synthase melanin genes showed no effect. The inactivation of type Ⅱ polyketide synthase spore pigment genes and ochronotic pigment genes decreased validamycin A production by 11.7% and 17.2%, respectively. All these mutant strains had no significant change in transcriptional level and the color of supernatant. Conclusion Pigment biosynthetic gene deletions showed different effects on validamycin yield and biomass accumulation, and the deletion of DOPA melanin biosynthetic genes redirected the precursor flux and successfully increased the yield of validamycin A.
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engineering validamycin production by tandem deletion of γ butyrolactone receptor genes in streptomyces hygroscopicus 5008
Metabolic Engineering, 2015Co-Authors: Gaoyi Tan, Yao Peng, Linquan Bai, Jianjiang ZhongAbstract:Paired homologs of γ-butyrolactone (GBL) biosynthesis gene afsA and GBL receptor gene arpA are located at different positions in genome of Streptomyces hygroscopicus 5008. Inactivation of afsA homologs dramatically decreased biosynthesis of validamycin, an important anti-fungal antibiotic and a critical substrate for antidiabetic drug synthesis, and the deletion of arpA homologs increased validamycin production by 26% (ΔshbR1) and 20% (ΔshbR3). By double deletion, the ΔshbR1/R3 mutant showed higher transcriptional levels of adpA-H (the S. hygroscopicus ortholog of the global regulatory gene adpA) and validamycin biosynthetic genes, and validamycin production increased by 55%. Furthermore, by engineering a high-producing industrial strain via tandem deletion of GBL receptor genes, validamycin production and productivity were enhanced from 19 to 24 g/L (by 26%) and from 6.7 to 9.7 g/L(-1) d(-1) (by 45%), respectively, which was the highest ever reported. The strategy demonstrated here may be useful to engineering other Streptomyces spp. with multiple pairs of afsA-arpA homologs.
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genomic and transcriptomic insights into the thermo regulated biosynthesis of validamycin in streptomyces hygroscopicus 5008
BMC Genomics, 2012Co-Authors: Huajun Zheng, Linquan Bai, Xiufen Zhou, Zixin DengAbstract:Background: Streptomyces hygroscopicus 5008 has been used for the production of the antifungal validamycin/ jinggangmycin for more than 40 years. A high yield of validamycin is achieved by culturing the strain at 37°C, rather than at 30°C for normal growth and sporulation. The mechanism(s) of its thermo-regulated biosynthesis was largely unknown. Results: The 10,383,684-bp genome of strain 5008 was completely sequenced and composed of a linear chromosome, a 164.57-kb linear plasmid, and a 73.28-kb circular plasmid. Compared with other Streptomyces genomes, the chromosome of strain 5008 has a smaller core region and shorter terminal inverted repeats, encodes more α/β hydrolases, major facilitator superfamily transporters, and Mg 2+ /Mn 2+ -dependent regulatory phosphatases. Transcriptomic analysis revealed that the expression of 7.5% of coding sequences was increased at 37°C, including biosynthetic genes for validamycin and other three secondary metabolites. At 37°C, a glutamate dehydrogenase was transcriptionally up-regulated, and further proved its involvement in validamycin production by gene replacement. Moreover, efficient synthesis and utilization of intracellular glutamate were noticed in strain 5008 at 37°C, revealing glutamate as the nitrogen source for validamycin biosynthesis. Furthermore, a SARP-family regulatory gene with enhanced transcription at 37°C was identified and confirmed to be positively involved in the thermo-regulation of validamycin production by gene inactivation and transcriptional analysis. Conclusions: Strain 5008 seemed to have evolved with specific genomic components to facilitate the thermoregulated validamycin biosynthesis. The data obtained here will facilitate future studies for validamycin yield improvement and industrial bioprocess optimization.
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over expression of udp glucose pyrophosphorylase increases validamycin a but decreases validoxylamine a production in streptomyces hygroscopicus var jinggangensis 5008
Metabolic Engineering, 2011Co-Authors: Xiang Zhou, Linquan Bai, Xiufen Zhou, Zixin DengAbstract:During the fermentation of Streptomyces hygroscopicus TL01 to produce validamycin A (18 g/L), a considerable amount of an intermediate validoxylamine A (4.0 g/L) is accumulated. Chemical or enzymatic hydrolysis of validamycin A was not observed during the fermentation process. Over-expression of glucosyltransferase ValG in TL01 did not increase the efficiency of glycosylation. However, increased validamycin A and decreased validoxylamine A production were observed in both the cell-free extract and fermentation broth of TL01 supplemented with a high concentration of UDP-glucose. The enzymatic activity of UDP-glucose pyrophosphorylase (Ugp) in TL01, which catalyzes UDP-glucose formation, was found to be much lower than the activities of other enzymes involved in the biosynthesis of UDP-glucose and the glucosyltransferase ValG. An ugp gene was cloned from S. hygroscopicus 5008 and verified to code for Ugp. In TL01 with an extra copy of ugp, the transcription of ugp was increased for 1.5 times, and Ugp activity was increased by 100%. Moreover, 22 g/L validamycin A and 2.5 g/L validoxylamine A were produced, and the validamycin A/validoxylamine A ratio was increased from 3.15 in TL01 to 5.75. These data prove that validamycin A biosynthesis is limited by the supply of UDP-glucose, which can be relieved by Ugp over-expression.
Zixin Deng - One of the best experts on this subject based on the ideXlab platform.
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genomic and transcriptomic insights into the thermo regulated biosynthesis of validamycin in streptomyces hygroscopicus 5008
BMC Genomics, 2012Co-Authors: Huajun Zheng, Linquan Bai, Xiufen Zhou, Zixin DengAbstract:Background: Streptomyces hygroscopicus 5008 has been used for the production of the antifungal validamycin/ jinggangmycin for more than 40 years. A high yield of validamycin is achieved by culturing the strain at 37°C, rather than at 30°C for normal growth and sporulation. The mechanism(s) of its thermo-regulated biosynthesis was largely unknown. Results: The 10,383,684-bp genome of strain 5008 was completely sequenced and composed of a linear chromosome, a 164.57-kb linear plasmid, and a 73.28-kb circular plasmid. Compared with other Streptomyces genomes, the chromosome of strain 5008 has a smaller core region and shorter terminal inverted repeats, encodes more α/β hydrolases, major facilitator superfamily transporters, and Mg 2+ /Mn 2+ -dependent regulatory phosphatases. Transcriptomic analysis revealed that the expression of 7.5% of coding sequences was increased at 37°C, including biosynthetic genes for validamycin and other three secondary metabolites. At 37°C, a glutamate dehydrogenase was transcriptionally up-regulated, and further proved its involvement in validamycin production by gene replacement. Moreover, efficient synthesis and utilization of intracellular glutamate were noticed in strain 5008 at 37°C, revealing glutamate as the nitrogen source for validamycin biosynthesis. Furthermore, a SARP-family regulatory gene with enhanced transcription at 37°C was identified and confirmed to be positively involved in the thermo-regulation of validamycin production by gene inactivation and transcriptional analysis. Conclusions: Strain 5008 seemed to have evolved with specific genomic components to facilitate the thermoregulated validamycin biosynthesis. The data obtained here will facilitate future studies for validamycin yield improvement and industrial bioprocess optimization.
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over expression of udp glucose pyrophosphorylase increases validamycin a but decreases validoxylamine a production in streptomyces hygroscopicus var jinggangensis 5008
Metabolic Engineering, 2011Co-Authors: Xiang Zhou, Linquan Bai, Xiufen Zhou, Zixin DengAbstract:During the fermentation of Streptomyces hygroscopicus TL01 to produce validamycin A (18 g/L), a considerable amount of an intermediate validoxylamine A (4.0 g/L) is accumulated. Chemical or enzymatic hydrolysis of validamycin A was not observed during the fermentation process. Over-expression of glucosyltransferase ValG in TL01 did not increase the efficiency of glycosylation. However, increased validamycin A and decreased validoxylamine A production were observed in both the cell-free extract and fermentation broth of TL01 supplemented with a high concentration of UDP-glucose. The enzymatic activity of UDP-glucose pyrophosphorylase (Ugp) in TL01, which catalyzes UDP-glucose formation, was found to be much lower than the activities of other enzymes involved in the biosynthesis of UDP-glucose and the glucosyltransferase ValG. An ugp gene was cloned from S. hygroscopicus 5008 and verified to code for Ugp. In TL01 with an extra copy of ugp, the transcription of ugp was increased for 1.5 times, and Ugp activity was increased by 100%. Moreover, 22 g/L validamycin A and 2.5 g/L validoxylamine A were produced, and the validamycin A/validoxylamine A ratio was increased from 3.15 in TL01 to 5.75. These data prove that validamycin A biosynthesis is limited by the supply of UDP-glucose, which can be relieved by Ugp over-expression.
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Nucleotidylation of unsaturated carbasugar in validamycin biosynthesis
Organic & biomolecular chemistry, 2010Co-Authors: Jongtae Yang, Zixin Deng, Linquan Bai, Yirong Zhang, Taifo MahmudAbstract:Validamycin A is a member of microbial-derived C7N-aminocyclitol family of natural products that is widely used as crop protectant and the precursor of the antidiabetic drug voglibose. Its biosynthetic gene clusters have been identified in several Streptomyces hygroscopicus strains, and a number of genes within the clusters have been functionally analyzed. Of these genes, valB, which encodes a sugar nucleotidyltransferase, was found through inactivation study to be essential for validamycin biosynthesis, but its role was unclear. To characterize the role of ValB in validamycin biosynthesis, four carbasugar phosphate analogues were synthesized and tested as substrate for ValB. The results showed that ValB efficiently catalyzes the conversion of valienol 1-phosphate to its nucleotidyl diphosphate derivatives, whereas other unsaturated carbasugar phosphates were found to be not the preferred substrate. ValB requires Mg2+, Mn2+, or Co2+ for its optimal activity and uses the purine-based nucleotidyltriphosphates (ATP and GTP) more efficiently than the pyrimidine-based NTPs (CTP, dTTP, and UTP) as nucleotidyl donor. ValB represents the first member of unsaturated carbasugar nucleotidyltransferases involved in natural products biosynthesis. Its characterization not only expands our understanding of aminocyclitol-derived natural products biosynthesis, but may also facilitate the development of new tools for chemoenzymatic synthesis of carbohydrate mimetics.
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Enhanced validamycin production and gene expression at elevated temperature in Streptomyces hygroscopicus subsp. jingangensis 5008
Science Bulletin, 2009Co-Authors: Linquan Bai, Xiufen Zhou, Zixin DengAbstract:Cultivation shift from 30° to 37° significantly enhanced validamycin (VAL) production. Analyzed by reverse-transcription PCR, the transcription of three val genes, valA, valK and valG, representing the three operons of the cluster was simultaneously increased at elevated temperature. Furthermore, the transcription of valP and valQ, a pair of two-component regulators in validamycin biosynthetic gene cluster, was also increased at 37°. Inactivation of valP and valQ reduced validamycin production at 37° to the yield level of wild type strain at 30°, and the val genes showed reduced expression in the mutant LL-8 at 37°. These results revealed that the two-component regulator valP and valQ contribute to the elevated validamycin production.
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genetically engineered production of 1 1 bis valienamine and validienamycin in streptomyces hygroscopicus and their conversion to valienamine
Applied Microbiology and Biotechnology, 2009Co-Authors: Jongtae Yang, Zixin Deng, Linquan Bai, Taifo MahmudAbstract:The antifungal agent validamycin A is an important crop protectant and the source of valienamine, the precursor of the antidiabetic drug voglibose. Inactivation of the valN gene in the validamycin A producer, Streptomyces hygroscopicus subsp. jinggangensis 5008, resulted in a mutant strain that produces new secondary metabolites 1,1′-bis-valienamine and validienamycin. The chemical structures of 1,1′-bis-valienamine and validienamycin were elucidated by 1D and 2D nuclear magnetic resonance (NMR) spectroscopy in conjunction with mass spectrometry and bioconversion employing a glycosyltransferase enzyme, ValG. 1,1′-Bis-valienamine and validienamycin exhibit a moderate antifungal activity against Pellicularia sasakii. Chemical degradation of 1,1′-bis-valienamine using N-bromosuccinimide followed by purification of the products with ion-exchange column chromatography only resulted in valienamine, whereas parallel treatments of validoxylamine A, the aglycon of validamycin A, resulted in an approximately 1:1 mixture of valienamine and validamine, underscoring the advantage of 1,1′-bis-valienamine over validoxylamine A as a commercial source of valienamine.
Heinz G Floss - One of the best experts on this subject based on the ideXlab platform.
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gene cluster responsible for validamycin biosynthesis in streptomyces hygroscopicus subsp jinggangensis 5008
Applied and Environmental Microbiology, 2005Co-Authors: Linquan Bai, Taifo Mahmud, Heinz G Floss, Xiufen Zhou, Kazuyuki Minagawa, Xiaohong Jian, Shuangya Chen, Erhu Cao, Zixin DengAbstract:A gene cluster responsible for the biosynthesis of validamycin, an aminocyclitol antibiotic widely used as a control agent for sheath blight disease of rice plants, was identified from Streptomyces hygroscopicus subsp. jinggangensis 5008 using heterologous probe acbC, a gene involved in the cyclization of D-sedoheptulose 7-phosphate to 2-epi-5-epi-valiolone of the acarbose biosynthetic gene cluster originated from Actinoplanes sp. strain SE50/110. Deletion of a 30-kb DNA fragment from this cluster in the chromosome resulted in loss of validamycin production, confirming a direct involvement of the gene cluster in the biosynthesis of this important plant protectant. A sequenced 6-kb fragment contained valA (an acbC homologue encoding a putative cyclase) as well as two additional complete open reading frames (valB and valC, encoding a putative adenyltransferase and a kinase, respectively), which are organized as an operon. The function of ValA was genetically demonstrated to be essential for validamycin production and biochemically shown to be responsible specifically for the cyclization of D-sedoheptulose 7-phosphate to 2-epi-5-epi-valiolone in vitro using the ValA protein heterologously overexpressed in E. coli. The information obtained should pave the way for further detailed analysis of the complete biosynthetic pathway, which would lead to a complete understanding of validamycin biosynthesis.
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Biosynthesis of the Validamycins: Identification of Intermediates in the Biosynthesis of Validamycin A by Streptomyces hygroscopicus var. limoneus
Journal of the American Chemical Society, 2001Co-Authors: Haijun Dong, Taifo Mahmud, Ingo Tornus, Sungsook Lee, Heinz G FlossAbstract:To study the biosynthesis of the pseudotrisaccharide antibiotic, validamycin A (1), a number of potential precursors of the antibiotic were synthesized in (2)H-, (3)H-, or (13)C-labeled form and fed to cultures of Streptomyces hygroscopicus var. limoneus. The resulting validamycin A from each of these feeding experiments was isolated, purified and analyzed by liquid scintillation counting, (2)H- or (13)C NMR or selective ion monitoring mass spectrometry (SIM-MS) techniques. The results demonstrate that 2-epi-5-epi-valiolone (9) is specifically incorporated into 1 and labels both cyclitol moieties. This suggests that 9 is the initial cyclization product generated from an open-chain C(7) precursor, D-sedoheptulose 7-phosphate (5), by a DHQ synthase-like cyclization mechanism. A more proximate precursor of 1 is valienone (11), which is also incorporated into both cyclitol moieties. The conversion of 9 into 11 involves first epimerization to 5-epi-valiolone (10), which is efficiently incorporated into 1, followed by dehydration, although a low level of incorporation of 2-epi-valienone (15) is also observed. Reduction of 11 affords validone (12), which is also incorporated specifically into 1, but labels only the reduced cyclitol moiety. The mode of introduction of the nitrogen atom linking the two pseudosaccharide moieties is not clear yet. 7-Tritiated valiolamine (8), valienamine (2), and validamine (3) were all not incorporated into 1, although each of these amines has been isolated from the fermentation, with 3 being most prevalent. Demonstration of in vivo formation of [7-(3)H]validamine ([7-(3)H]-3) from [7-(3)H]-12 suggests that 3 may be a pathway intermediate and that the nonincorporation of [7-(3)H]-3 into 1 is due to a lack of cellular uptake. We thus propose that 3, formed by amination of 12, and 11 condense to form a Schiff base, which is reduced to the pseudodisaccharide unit, validoxylamine A (13). Transfer of a D-glucose unit to the 4'-position of 13 then completes the biosynthesis of 1. Other possibilities for the mechanism of formation of the nitrogen bridge between the two pseudosaccharide units are also discussed.
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biosynthesis of the Validamycins identification of intermediates in the biosynthesis of validamycin a by streptomyces hygroscopicus var limoneus
Journal of the American Chemical Society, 2001Co-Authors: Haijun Dong, Taifo Mahmud, Ingo Tornus, Sungsook Lee, Heinz G FlossAbstract:To study the biosynthesis of the pseudotrisaccharide antibiotic, validamycin A (1), a number of potential precursors of the antibiotic were synthesized in 2H-, 3H-, or 13C-labeled form and fed to c...
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The biosynthesis of acarbose and validamycin.
Chemical record (New York N.Y.), 2001Co-Authors: Taifo Mahmud, Sungsook Lee, Heinz G FlossAbstract:The studies reported here have established the biosynthetic origin of the mC7N units of acarbose and validamycin from sedo-heptulose 7-phosphate, and have identified 2-epi-5-epi-valiolone as the initial cyclization product. The deoxyhexose moiety of acarbose arises from glucose with deoxythymidyl-diphospho-4-keto-6-deoxy-D-glucose (dTDP-4-keto-6-deoxy-D-glucose) as a proximate intermediate. However, despite the identical origin of the aminocyclitol moieties in acarbose and validamycin A, the pathways of their formation seem to be substantially different. Validamycin A formation involves a number of discrete ketocyclitol intermediates, 5-epi-valiolone, valienone, and validone, whereas no free intermediates have been identified on the pathway from 2-epi-5-epi-valiolone to the pseudodisaccharide moiety of acarbose. The stage is now set for unraveling the mechanism or mechanisms by which the two components of the pseudodisaccharide moieties of acarbose and validamycin are uniquely coupled to each other via a nitrogen bridge.
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The biosynthesis of acarbose and validamycin.
Chemical record (New York N.Y.), 2001Co-Authors: Taifo Mahmud, Sungsook Lee, Heinz G FlossAbstract:The studies reported here have established the biosynthetic origin of the mC7N units of acarbose and validamycin from sedo-heptulose 7-phosphate, and have identified 2-epi-5-epi-valiolone as the initial cyclization product. The deoxyhexose moiety of acarbose arises from glucose with deoxythymidyl-diphospho-4-keto-6-deoxy-D-glucose (dTDP-4-keto-6-deoxy-D-glucose) as a proximate intermediate. However, despite the identical origin of the aminocyclitol moieties in acarbose and validamycin A, the pathways of their formation seem to be substantially different. Validamycin A formation involves a number of discrete ketocyclitol intermediates, 5-epi-valiolone, valienone, and validone, whereas no free intermediates have been identified on the pathway from 2-epi-5-epi-valiolone to the pseudodisaccharide moiety of acarbose. The stage is now set for unraveling the mechanism or mechanisms by which the two components of the pseudodisaccharide moieties of acarbose and validamycin are uniquely coupled to each other via a nitrogen bridge. © 2001 John Wiley & Sons, Inc. and The Japan Chemical Journal Forum Chem Rec 1: 300–310, 2001
