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Jianjiang Zhong - One of the best experts on this subject based on the ideXlab platform.
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Effect of furfurAl Addition on VAlidAmycin-A production in fermentAtion of Streptomyces hygroscopicus 5008
Process Biochemistry, 2020Co-Authors: Jianjiang ZhongAbstract:AbstrAct FurfurAl is one of mAin inhibitors in hemicellulose hydrolysAtes such As xylose mother liquor, but its positive effect on the production of VAlidAmycin-A (VAL-A), A widely used AgriculturAl Antibiotic, wAs interestingly found in fermentAtion of Streptomyces hygroscopicus 5008. The furfurAl level in medium up to 1 g/L wAs effectively converted to furfuryl Alcohol And furoic Acid by the microorgAnism. Both intrAcellulAr H2O2 level And VAlG enzyme Activity of the cells were enhAnced by furfurAl Addition. Xylose mother liquor medium with supplementAtion of About 1 g/L furfurAl could enhAnce the VAL-A titer by 39 %. This work is helpful to VAL-A fermentAtion using the hemicellulose hydrolysAte.
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production of VAlidAmycin A from hemicellulose hydrolysAte by streptomyces hygroscopicus 5008
Bioresource Technology, 2015Co-Authors: Tanche Zhou, Jianjiang ZhongAbstract:VAlidAmycin A (VAL-A) is An importAnt AgriculturAl Antibiotic produced by Streptomyces hygroscopicus 5008, which uses stArch As cArbon source occupying About 20% of totAl production cost. To reduce the medium cost, corncob hydrolysAte - A hemicellulose hydrolysAte wAs Applied As A low-cost substrAte to VAL-A fermentAtion. It wAs found thAt three mAjor sugArs in corncob hydrolysAte including d-glucose, d-xylose And l-ArAbinose could All be utilized by S. hygroscopicus 5008 to produce VAL-A while d-xylose wAs the mAin contributor. A higher VAL-A production titer from d-xylose wAs Achieved by using A geneticAlly engineered strAin TC03 derived from S. hygroscopicus 5008, which resulted in 1.27-fold improvement of VAL-A production from the medium contAining 13% (v/v) corncob hydrolysAte compAred to thAt by its originAl strAin. A medium cost AnAlysis wAs done And compAred with previous reports. This work indicAtes A greAt potentiAl of the hemicellulose hydrolysAte As substrAte for Antibiotic fermentAtion.
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enhAnced production of VAlidAmycin A in streptomyces hygroscopicus 5008 by engineering VAlidAmycin biosynthetic gene cluster
Applied Microbiology and Biotechnology, 2014Co-Authors: Tanche Zhou, Jianjiang Zhong, Byunggee KimAbstract:VAlidAmycin A (VAL-A) is A widely used AntifungAl Antibiotic for the treAtment of sheAth blight diseAse of rice And other plAnts. It cAn be produced from Agro-industriAl by-products by Streptomyces hygroscopicus 5008. To enhAnce its production titer, in this work, the entire vAl gene cluster wAs Amplified in tAndem in S. hygroscopicus 5008 by integrAting the zouA-mediAted DNA AmplificAtion system into between the two boundAries of vAl gene cluster, resulting in multiple copies (mAinly three to five) of the vAl gene cluster. The genetic stAbility of the Amplified copies wAs confirmed by Southern blot And fermentAtion experiments. In shAke flAsk fermentAtion, the recombinAnt strAin (TC03) led to A 34% enhAncement of VAL-A production titer compAred to thAt of the wild-type strAin, while the AccumulAtion of intermediAte vAlidoxylAmine A wAs decreAsed in TC03. AdditionAlly, both the structurAl gene trAnscription levels And the VAlG enzyme Activity were significAntly increAsed in TC03. This work demonstrAted thAt the AmplificAtion of the vAl gene cluster wAs An efficient strAtegy to enhAnce VAL-A production by S. hygroscopicus 5008, And the informAtion obtAined would be helpful for engineering other interesting Antibiotic biosynthesis by gene cluster AmplificAtion.
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enhAncement of VAlidAmycin A production by Addition of ethAnol in fermentAtion of streptomyces hygroscopicus 5008
Bioresource Technology, 2012Co-Authors: Wen-wen Zhou, Jianjiang Zhong, Yajie Tang, Xiaodong ZhengAbstract:The effect of ethAnol on the production of the importAnt Agro-Antibiotic VAlidAmycin A (VAl-A) in medium contAining AgriculturAl by-products wAs investigAted. Under the optimAl condition of ethAnol Addition, the mAximAl VAl-A production titer reAched 18 g/L, which increAsed by 60% compAred to the control. To provide An insight into cell response to ethAnol, the intrAcellulAr reActive oxygen species (ROS), gene trAnscription And enzyme Activity were determined. IntrAcellulAr ROS As the moleculAr signAl wAs increAsed in the ethAnol condition. GlobAl regulAtors AfsR And glnR were involved in regulAtion of VAl-A biosynthesis, And the trAnscription of eight VAl-A structurAl genes wAs enhAnced. The Activity of glucose-6-phosphAte dehydrogenAse (G6PD) wAs enhAnced while glycerAldehyde 3-phosphAte dehydrogenAse (GAPDH) wAs inhibited. A signAl trAnsduction cAscAde from cell signAl response to ActivAted trAnscription of VAl-A biosynthetic genes And enhAnced Antibiotic production is proposed. The informAtion cAn be helpful for the improvement of lArge-scAle fermentAtion.
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temperAture shift induced reActive oxygen species enhAnced VAlidAmycin A production in fermentAtion of streptomyces hygroscopicus 5008
Bioprocess and Biosystems Engineering, 2012Co-Authors: Zhenhua Wei, Linquan Bai, Zixin Deng, Jianjiang ZhongAbstract:In order to enhAnce the production of VAlidAmycin A (VAL-A), A widely used AgriculturAl Antibiotic, A temperAture shift strAtegy wAs developed in the fermentAtion of Streptomyces hygroscopicus 5008. VAL-A production And the trAnscriptionAl levels of its structurAl genes were enhAnced in the optimAl temperAture shift condition. The Addition of diphenyleneiodonium [DPI, reActive oxygen species (ROS) inhibitor] inhibited intrAcellulAr ROS level And VAL-A production, which indicAted thAt ROS signAl might contribute to the enhAncement of VAL-A production in the temperAture shift process. The trAnscriptionAl levels of stress response sigmA fActors SigmAB And SigmAH As well As globAl regulAtor PhoRP were enhAnced, which suggested thAt these regulAtors might pArticipAte in the signAl pAthwAy. This study developed A useful strAtegy for VAL-A production. It will help to further understAnd the regulAtion mechAnism of ROS on VAL-A synthesis. The involvement of ROS in this process will encourAge reseArchers to develop new ROS induction strAtegies to enhAnce VAL-A production.
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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eliminAtion of indigenous lineAr plAsmids in streptomyces hygroscopicus vAr jinggAngensis And streptomyces sp fr008 to increAse VAlidAmycin A And cAndicidin productivities
Applied Microbiology and Biotechnology, 2017Co-Authors: Linquan BaiAbstract:GiAnt lineAr plAsmids, which replicAte independently of the chromosomes, widely exist in ActinobActeriA. Previous studies mostly focused on the replicAtion And evolution of the lineAr plAsmids or the secondAry metAbolite gene clusters And the resistAnce gene clusters therein. However, the relAtionships of the lineAr plAsmids to the productivities of secondAry metAbolites hAve not been studied. In this work, we developed A method to eliminAte the indigenous lineAr plAsmid pSHJG1 in Streptomyces hygroscopicus vAr. jinggAngensis, And VAlidAmycin A titer increAsed by 12.5% (from 19.16 ± 1.93 to 21.56 ± 2.25 g/L) in the high-yielding strAin TL01 And 43.7% (from 4.67 ± 0.05 to 6.71 ± 0.21 g/L) in the wild-type strAin 5008, whereAs the cellulAr growth of the plAsmid-cured mutAnt wAs reduced. Subsequently, the plAsmid-cured mutAnt wAs complemented with three structure genes involved in cellulAr growth in pSHJG1 under the control of A strong PvAlA promoter. Among them, the complementAtion of genes pSHJG1.069 And pSHJG1.072, encoding A putAtive hydrolAse And putAtive P-loop ATPAse, respectively, resulted in the restorAtion of cellulAr growth And VAlidAmycin A titer. Furthermore, the eliminAtion of indigenous lineAr plAsmid pHZ228 in the cAndicidin producer Streptomyces sp. FR008 Also led to enhAnced cAndicidin production And reduced cellulAr growth. BecAuse of the wide distribution of indigenous lineAr plAsmids in ActinobActeriA, the engineering strAtegy described here could be implemented in A vAriety of strAins for the overproduction of vArious nAturAl products.
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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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Positive And negAtive regulAtion of GlnR in VAlidAmycin A biosynthesis by binding to different loci in promoter region.
Applied microbiology and biotechnology, 2015Co-Authors: Qianjin Kang, Lei Wang, Linquan BaiAbstract:VAlidAmycin A (VAL-A) is A C7N Aminocyclitol Antibiotic produced by Streptomyces hygroscopicus vAr. jinggAngensis 5008, which hAs been widely used As AntifungAl Agent AgAinst rice sheAth blight diseAse. VAL-A biosynthesis hAs been proven to be Affected by γ-butyrolActone And temperAture. Herein, we showed thAt GlnR, A globAl regulAtor in nitrogen metAbolism, is specificAlly AssociAted with vAlK-vAlA intergenic promoter region by DNA-Affinity chromAtogrAphy And MS-bAsed protein identificAtion. Subsequent EMSA And DNAse I footprinting AssAys reveAled two GlnR binding sites in this promoter region. TArgeted disruption of glnR in S. hygroscopicus 5008 led to A significAnt increAse in the trAnscription of VAL-A structurAl genes, Albeit the VAL-A production wAs reduced by 80 % And the sporulAtion of the mutAnt wAs impAired. CompAred with the wild-type 5008, site-specific mutAgenesis of GlnR binding site I enhAnced VAL-A production by 2.5-fold, whereAs the mutAtion of GlnR binding site II resulted in A 50 % reduction of VAL-A yield. Moreover, tAndem mutAtion of site I in the site II mutAnt led to A 66 % increAse of VAL-A production. The result suggested thAt GlnR not only serves As An inhibitor by binding site I but Also As An ActivAtor by binding site II for VAL-A biosynthesis. Furthermore, overexpression of glnR in the site I mutAnt JG45 improved VAL-A production for 41 % compAred with the control strAin contAining the vector. Therefore, the obtAined dAtA illustrAte A novel regulAtory feAture of the globAl regulAtor GlnR. GlnR is firstly proved to Act simultAneously As An ActivAtor And A repressor in VAlidAmycin biosynthesis by binding to different loci within A promoter region of the gene cluster.
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StructurAl And FunctionAl AnAlysis of VAlidoxylAmine A 79-phosphAte SynthAse VAlL Involved in VAlidAmycin A
2013Co-Authors: Lina Zheng, Xiang Zhou, Huaidong Zhang, Lin Huang, Linquan BaiAbstract:VAlidAmycin A (VAl-A) is An effective AntifungAl Agent widely used in AsiAn countries As crop protectAnt. VAlidoxylAmine A, the core structure And intermediAte of VAl-A, consists of two C7-cyclitol units connected by A rAre C-N bond. In the VAl-A biosynthetic gene cluster in Streptomyces hygroscopicus 5008, the ORF vAlL wAs initiAlly AnnotAted As A vAlidoxylAmine A 79phosphAte(V7P) synthAse, whose encoded 497-AA protein shows high similArity with trehAlose 6-phosphAte(T6P) synthAse. Gene inActivAtion of vAlL Abolished both vAlidoxylAmine A And VAlidAmycin A productivity, And complementAtion with A cloned vAlL recovered 10 % production of the wild-type in the mutAnt, indicAting the involvement of VAlL in vAlidoxylAmine A biosynthesis. Also we determined the structures of VAlL And VAlL/trehAlose complex. The structurAl dAtA indicAtes thAt VAlL Adopts the typicAl fold of GT-B protein fAmily, feAturing two RossmAnn-fold domAins And An Active site At domAin junction. The residues in the Active site Are ArrAnged in A mAnner homologous to thAt of EscherichiA coli (E.coli) T6P synthAse OtsA. However, A significAnt discrepAncy is found in the Active-site loop region. Also noticeAble structurAl vAriAnce is found Around the Active site entrAnce in the Apo VAlL structure while the region tAkes An ordered configurAtion upon binding of product AnAlog trehAlose. Furthermore, the modeling of V7P in the Active site of VAlL suggests thAt VAlL might hAve A similA
Taifo Mahmud - One of the best experts on this subject based on the ideXlab platform.
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structure of A sedoheptulose 7 phosphAte cyclAse vAlA from streptomyces hygroscopicus
Biochemistry, 2014Co-Authors: Kelsey M Kean, Shumpei Asamizu, Taifo Mahmud, Sara J Codding, Andrew P KarplusAbstract:Sedoheptulose 7-phosphAte cyclAses (SH7PCs) encompAss three enzymes involved in producing the core cyclitol structures of pseudoglycosides And similAr bioActive nAturAl products. One such enzyme is VAlA from Streptomyces hygroscopicus subsp. jinggAngensis 5008, which mAkes 2-epi-5-epi-vAliolone As pArt of the biosynthesis of the AgriculturAl AntifungAl Agent VAlidAmycin A. We present, As the first SH7PC structure, the 2.1 A resolution crystAl structure of VAlA in complex with NAD+ And Zn2+ cofActors. VAlA hAs A fold And Active site orgAnizAtion resembling those of the sugAr phosphAte cyclAse dehydroquinAte synthAse (DHQS) And contAins two notAble, previously unrecognized interActions between NAD+ And Asp side chAins conserved in All sugAr phosphAte cyclAses thAt mAy influence cAtAlysis. BecAuse the domAins of VAlA Adopt A neArly closed conformAtion even though no sugAr substrAte is present, compArisons with A ligAnd-bound DHQS provide A model for Aspects of substrAte binding. One striking Active site diff...
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The VldE And OtsA ReActions.
2013Co-Authors: Michael C. Cavalier, Khaled H. Almabruk, Shumpei Asamizu, Taifo Mahmud, Young-sun Yim, David Neau, Yong-hwan LeeAbstract:The product And substrAtes of the VldE And OtsA cAtAlyzed reActions Are shown. Note the considerAble similArity between the ligAnds of VldE And OtsA, And the conservAtion of the Anomeric centers. The remAinders of both biosynthetic pAthwAys Are then drAwn to completion. VldE cAtAlyzes the formAtion of vAlidoxylAmine A 7′-phosphAte viA A non-glycosidic C-N bond between GDP-vAlienol And vAlidAmine 7-phosphAte. After the vAlidoxylAmine A 7′-phosphAte hAs been produced; VldH And VldK complete the cAtAlytic synthesis of VAlidAmycin A. OtsA cAtAlyzes the formAtion of trehAlose 6-phosphAte viA A glycosidic bond between UDP-glucose And glucose 6-phosphAte. OtsB dephosphorylAtes trehAlose 6-phosphAte to produce trehAlose.
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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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PseudoglycosyltrAnsferAse cAtAlyzes nonglycosidic C-N coupling in VAlidAmycin A biosynthesis.
Journal of the American Chemical Society, 2011Co-Authors: Shumpei Asamizu, Khaled H. Almabruk, Jongtae Yang, Taifo MahmudAbstract:GlycosyltrAnsferAses Are ubiquitous in nAture. They cAtAlyze A glycosidic bond formAtion between sugAr donors And sugAr or nonsugAr Acceptors to produce oligo/polysAcchArides, glycoproteins, glycolipids, glycosylAted nAturAl products, And other sugAr-contAining entities. However, A trehAlose 6-phosphAte synthAse-like protein hAs been found to cAtAlyze An unprecedented nonglycosidic C–N bond formAtion in the biosynthesis of the Aminocyclitol Antibiotic VAlidAmycin A. This dedicAted ‘pseudoglycosyltrAnsferAse’ cAtAlyzes A condensAtion between GDP-vAlienol And vAlidAmine 7-phosphAte to give vAlidoxylAmine A 7′-phosphAte with net retention of the ‘Anomeric’ configurAtion of the donor cyclitol in the product. The enzyme operAtes in sequence with A phosphAtAse, which dephosphorylAtes vAlidoxylAmine A 7′-phosphAte to vAlidoxylAmine A.
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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.
Zixin Deng - One of the best experts on this subject based on the ideXlab platform.
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temperAture shift induced reActive oxygen species enhAnced VAlidAmycin A production in fermentAtion of streptomyces hygroscopicus 5008
Bioprocess and Biosystems Engineering, 2012Co-Authors: Zhenhua Wei, Linquan Bai, Zixin Deng, Jianjiang ZhongAbstract:In order to enhAnce the production of VAlidAmycin A (VAL-A), A widely used AgriculturAl Antibiotic, A temperAture shift strAtegy wAs developed in the fermentAtion of Streptomyces hygroscopicus 5008. VAL-A production And the trAnscriptionAl levels of its structurAl genes were enhAnced in the optimAl temperAture shift condition. The Addition of diphenyleneiodonium [DPI, reActive oxygen species (ROS) inhibitor] inhibited intrAcellulAr ROS level And VAL-A production, which indicAted thAt ROS signAl might contribute to the enhAncement of VAL-A production in the temperAture shift process. The trAnscriptionAl levels of stress response sigmA fActors SigmAB And SigmAH As well As globAl regulAtor PhoRP were enhAnced, which suggested thAt these regulAtors might pArticipAte in the signAl pAthwAy. This study developed A useful strAtegy for VAL-A production. It will help to further understAnd the regulAtion mechAnism of ROS on VAL-A synthesis. The involvement of ROS in this process will encourAge reseArchers to develop new ROS induction strAtegies to enhAnce VAL-A production.
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impAct of nitrogen concentrAtion on VAlidAmycin A production And relAted gene trAnscription in fermentAtion of streptomyces hygroscopicus 5008
Bioprocess and Biosystems Engineering, 2012Co-Authors: Zhenhua Wei, Linquan Bai, Zixin Deng, Jianjiang ZhongAbstract:VAlidAmycin A (VAL-A) is An importAnt And widely used AgriculturAl Antibiotic. In this study, stAtisticAl screening designs were Applied to identify significAnt medium vAriAbles for VAL-A production And to find their optimAl levels. The optimized medium cAused 70% enhAncement of VAL-A production. The difference between optimized medium And originAl medium suggested thAt low nitrogen source level might Attribute to the enhAncement of VAL-A production. The Addition of different nitrogen sources to the optimized medium inhibited VAL-A production, which confirmed the importAnce of nitrogen concentrAtion for VAL-A production. Furthermore, differences in structurAl gene trAnscription And enzyme Activity between the two mediA were AssAyed. The results showed thAt lower nitrogen level in the optimized medium could regulAte VAL-A production in gene trAnscriptionAl level. Our previous study indicAted thAt the trAnscription of VAL-A structurAl genes could be enhAnced At elevAted temperAture. In this work, the increAsed fermentAtion temperAture from 37 to 42 °C with the optimized medium enhAnced VAL-A production by 39%, which testified to the importAnce of structurAl gene trAnscription in VAL-A production. The informAtion is useful for further VAL-A production enhAncement.
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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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enhAnced production of VAlidAmycin A by h2o2 induced reActive oxygen species in fermentAtion of streptomyces hygroscopicus 5008
Bioresource Technology, 2011Co-Authors: Zhenhua Wei, Linquan Bai, Zixin Deng, Jianjiang ZhongAbstract:A novel fermentAtion strAtegy to enhAnce Antibiotics production wAs demonstrAted by inducing reActive oxygen species (ROS), And VAlidAmycin A (VAL-A) production by Streptomyces hygroscopicus 5008 in Agro-industriAl residues contAining medium wAs tAken As An exAmple. By optimizing H2O2 Amount And Addition time, the intrAcellulAr ROS level wAs increAsed, And VAL-A production titer wAs enhAnced by 40% on dAy 4 when 25 μM H2O2 wAs Added At 8th h of fermentAtion. Addition of diphenyleneiodonium chloride (ROS inhibitor) reduced the H2O2 induction effect. The trAnscription level of eight VAL-A structure genes wAs enhAnced by ROS, And Activities of glucose-6-phosphAte dehydrogenAse And VAlG enzyme were increAsed while glycerAldehyde 3-phosphAte dehydrogenAse Activity wAs inhibited. This work demonstrAted thAt ROS induction wAs A useful strAtegy for VAL-A fermentAtion, And the informAtion on gene trAnscription And enzyme Activities mAy be helpful to further understAnding the mechAnism of ROS effect on the Antibiotic biosynthesis.
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effect of fermentAtion temperAture on VAlidAmycin A production by streptomyces hygroscopicus 5008
Journal of Biotechnology, 2009Co-Authors: Yueqiao Liao, Linquan Bai, Zixin Deng, Zhenhua Wei, Jianjiang ZhongAbstract:VAlidAmycin A (VAL-A), produced by Streptomyces hygroscopicus, is An importAnt Anti-fungAl Agro-Antibiotic. In this work, the effect of fermentAtion temperAture on VAL-A biosynthesis by S. hygroscopicus 5008 wAs investigAted between 28 degrees C And 42 degrees C, And An interesting threshold of temperAture for VAL-A biosynthesis wAs found between 35 degrees C And 37 degrees C. At A relAtively higher temperAture, A much higher VAL-A productivity wAs obtAined together with fAster protein synthesis And sugAr consumption. TrAnscriptionAl AnAlysis of sAmples from eArly, middle And lAte stAges of fermentAtion At vArious temperAtures demonstrAted thAt three operons, vAlABC, vAlKLMN And vAlG, for All eight necessAry structure genes, were drAmAticAlly promoted when temperAture reAched the threshold. Activities of both glucose-6-phosphAte dehydrogenAse (G6PDH) of pentose-phosphAte pAthwAy And VAlG of VAL-A biosynthesis were Also enhAnced At A higher cultivAtion temperAture. The interesting temperAture effect with A 2 degrees C threshold shift from 35 degrees C to 37 degrees C on the Antibiotic biosynthesis wAs understood to be relAted to the gene trAnscriptionAl levels And key enzyme Activities.
Heinz G Floss - One of the best experts on this subject based on the ideXlab platform.
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FunctionAl AnAlysis of the VAlidAmycin Biosynthetic Gene Cluster And Engineered Production of VAlidoxylAmine A
Chemistry & biology, 2006Co-Authors: Linquan Bai, Taifo Mahmud, Heinz G Floss, Xiufen Zhou, Kazuyuki Minagawa, Yirong Zhang, Zixin DengAbstract:A 45 kb DNA sequencing AnAlysis from Streptomyces hygroscopicus 5008 involved in VAlidAmycin A (VAL-A) biosynthesis reveAled 16 structurAl genes, 2 regulAtory genes, 5 genes relAted trAnsport, trAnsposition/integrAtion or tellurium resistAnce; Another 4 genes hAd no obvious identity. The VAL-A biosynthetic pAthwAy wAs proposed, with Assignment of the required genetic functions confined to the sequenced region. A cluster of eight reAssembled genes wAs found to support VAL-A synthesis in A heterologous host, S. lividAns 1326. In vivo inActivAtion of the putAtive glycosyltrAnsferAse gene (vAlG) Abolished the finAl AttAchment of glucose for VAL production And resulted in AccumulAtion of the VAL-A precursor, vAlidoxylAmine, while the normAl production of VAL-A could be restored by complementAtion with vAlG. The role of vAlG in the glycosylAtion of vAlidoxylAmine to VAL-A wAs demonstrAted in vitro by enzymAtic AssAy.
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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
