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Takuya Nihira - One of the best experts on this subject based on the ideXlab platform.
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visg is essential for biosynthesis of Virginiamycin s a streptogramin type b antibiotic as a provider of the nonproteinogenic amino acid phenylglycine
Microbiology, 2011Co-Authors: Fitria Ningsih, Takuya Nihira, Shigeru Kitani, Eriko FukushimaAbstract:A streptogramin type B antibiotic, Virginiamycin S (VS), is produced by Streptomyces virginiae, together with a streptogramin type A antibiotic, Virginiamycin M1 (VM), as its synergistic counterpart. VS is a cyclic hexadepsipeptide containing a nonproteinogenic amino acid, l-phenylglycine (l-pheGly), in its core structure. We have identified, in the left-hand extremity of the Virginiamycin supercluster, two genes that direct VS biosynthesis with l-pheGly incorporation. Transcriptional analysis revealed that visF, encoding a nonribosomal peptide synthetase, and visG, encoding a protein with homology to a hydroxyphenylacetyl-CoA dioxygenase, are under the transcriptional regulation of virginiae butanolide (VB), a small diffusing signalling molecule that governs Virginiamycin production. Gene deletion of visG resulted in complete loss of VS production without any changes in VM production, suggesting that visG is required for VS biosynthesis. The abolished VS production in the visG disruptant was fully recovered either by the external addition of pheGly or by gene complementation, which indicates that VisG is involved in VS biosynthesis as the provider of an l-pheGly molecule. A feeding experiment with l-pheGly analogues suggested that VisF, which is responsible for the last condensation step, has high substrate specificity toward l-pheGly.
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Hierarchical control of Virginiamycin production in Streptomyces virginiae by three pathway-specific regulators: VmsS, VmsT and VmsR.
Microbiology, 2009Co-Authors: Nattika Pulsawat, Shigeru Kitani, Eriko Fukushima, Takuya NihiraAbstract:Two regulatory genes encoding a Streptomyces antibiotic regulatory protein (vmsS) and a response regulator (vmsT) of a bacterial two-component signal transduction system are present in the left-hand region of the biosynthetic gene cluster of the antibiotic Virginiamycin, which is composed of Virginiamycin M (VM) and Virginiamycin S (VS), in Streptomyces virginiae. Disruption of vmsS abolished both VM and VS biosynthesis, with drastic alteration of the transcriptional profile for Virginiamycin biosynthetic genes, whereas disruption of vmsT resulted in only a loss of VM biosynthesis, suggesting that vmsS is a pathway-specific regulator for both VM and VS biosynthesis, and that vmsT is a pathway-specific regulator for VM biosynthesis alone. Gene expression profiles determined by semiquantitative RT-PCR on the Virginiamycin biosynthetic gene cluster demonstrated that vmsS controls the biosynthetic genes for VM and VS, and vmsT controls unidentified gene(s) of VM biosynthesis located outside the biosynthetic gene cluster. In addition, transcriptional analysis of a deletion mutant of vmsR located in the clustered regulatory region in the Virginiamycin cluster (and which also acts as a SARP-family activator for both VM and VS biosynthesis) indicated that the expression of vmsS and vmsT is under the control of vmsR, and vmsR also contributes to the expression of VM and VS biosynthetic genes, independent of vmsS and vmsT. Therefore, coordinated Virginiamycin biosynthesis is controlled by three pathway-specific regulators which hierarchically control the expression of the biosynthetic gene cluster.
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Identification of the bkdAB gene cluster, a plausible source of the starter-unit for Virginiamycin M production in Streptomyces virginiae
Archives of Microbiology, 2007Co-Authors: Nattika Pulsawat, Shigeru Kitani, Hiroshi Kinoshita, Chang Kwon Lee, Takuya NihiraAbstract:The bkdAB gene cluster, which encodes plausible E1 and E2 components of the branched-chain α-keto acid dehydrogenase (BCDH) complex, was isolated from Streptomyces virginiae in the vicinity of a regulatory island for Virginiamycin production. Gene disruption of bkdA completely abolished the production of Virginiamycin M (a polyketide-peptide antibiotic), while the production of Virginiamycin S (a cyclodepsipeptide antibiotic) was unaffected. Complementation of the bkdA disruptant by genome-integration of intact bkdA completely restored the Virginiamycin M production, indicating that the bkdAB cluster is essential for Virginiamycin M biosynthesis, plausibly via the provision of isobutyryl-CoA as a primer unit. In contrast to a feature usually seen in the Streptomyces E1 component, namely, the separate encoding of the α and β subunits, S. virginiae bkdA seemed to encode the fused form of the α and β subunits, which was verified by the actual catalytic activity of the fused protein in vitro using recombinant BkdA overexpressed in Escherichia coli . Supply of an additional bkdA gene under the strong and constitutive promoter ermE * in the wild-type strain of S. virginiae resulted in enhanced production of Virginiamycin M, suggesting that the supply of isobutyryl-CoA is one of the rate-limiting factors in the biosynthesis of Virginiamycin M.
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identification by gene deletion analysis of barb as a negative regulator controlling an early process of Virginiamycin biosynthesis in streptomyces virginiae
Archives of Microbiology, 2004Co-Authors: Chang Kwon Lee, Yasuhiro Yamada, Kiyoshi Matsuno, Takuya NihiraAbstract:The Streptomyces virginiae γ-butyrolactone autoregulator virginiae butanolide is a low-molecular-weight Streptomyces hormone eliciting Virginiamycin biosynthesis through its binding to the specific receptor protein, BarA. Immediately downstream of barA lies barB, the transcription of which is tightly repressed by BarA in the absence of virginiae butanolide and derepressed in its presence. Thus, BarB is next to BarA on the virginiae butanolide-BarA signaling cascade. An in-frame 279-bp deletion was introduced into the barB allele, which rendered it inactive by eliminating the majority of the coding region, including the helix-turn-helix DNA-binding motif. No significant change was observed with the ΔbarB mutant with respect to the timing or amount of virginiae butanolide production, or the morphological differentiation on solid media, indicating that barB neither participates in virginiae butanolide biosynthesis nor in cytodifferentiation. In contrast, analysis of Virginiamycin production in the ΔbarB mutant revealed that production of both Virginiamycin M1 and Virginiamycin S occurred immediately after virginiae butanolide production, 2–3 h earlier than in the wild-type strain, indicating that BarB participates in the temporal retardation of Virginiamycin production after virginiae butanolide inactivates the repressor function of BarA. RT-PCR analysis of the transcription of several genes surrounding barA–barB by the ΔbarB mutant indicated that BarB plays a negative regulatory role, directly or indirectly, in the transcription of barZ, vmsR, and orf5 located upstream of barB.
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Identification and In Vivo Functional Analysis of a Virginiamycin S Resistance Gene (varS) from Streptomyces virginiae
Journal of bacteriology, 1999Co-Authors: Chang Kwon Lee, Takuya Nihira, Yuka Kamitani, Yasuhiro YamadaAbstract:BarA of Streptomyces virginiae is a specific receptor protein for virginiae butanolide (VB), one of the γ-butyrolactone autoregulators of the Streptomyces species, and acts as a transcriptional regulator controlling both Virginiamycin production and VB biosynthesis. The downstream gene barB, the transcription of which is under the tight control of the VB-BarA system, was found to be transcribed as a polycistronic mRNA with its downstream region, and DNA sequencing revealed a 1,554-bp open reading frame (ORF) beginning at 161 bp downstream of the barB termination codon. The ORF product showed high homology (68 to 73%) to drug efflux proteins having 14 transmembrane segments and was named varS (for S. virginiae antibiotic resistance). Heterologous expression of varS with S. lividans as a host resulted in Virginiamycin S-specific resistance, suggesting that varS encoded a Virginiamycin S-specific transport protein. Northern blot analysis indicated that the bicistronic transcript of barB-varS appeared 1 to 2 h before the onset of Virginiamycin M1 and S production, at which time VB was produced, while exogenously added Virginiamycin S apparently induced the monocistronic varS transcript.
Yasuhiro Yamada - One of the best experts on this subject based on the ideXlab platform.
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identification by gene deletion analysis of barb as a negative regulator controlling an early process of Virginiamycin biosynthesis in streptomyces virginiae
Archives of Microbiology, 2004Co-Authors: Chang Kwon Lee, Yasuhiro Yamada, Kiyoshi Matsuno, Takuya NihiraAbstract:The Streptomyces virginiae γ-butyrolactone autoregulator virginiae butanolide is a low-molecular-weight Streptomyces hormone eliciting Virginiamycin biosynthesis through its binding to the specific receptor protein, BarA. Immediately downstream of barA lies barB, the transcription of which is tightly repressed by BarA in the absence of virginiae butanolide and derepressed in its presence. Thus, BarB is next to BarA on the virginiae butanolide-BarA signaling cascade. An in-frame 279-bp deletion was introduced into the barB allele, which rendered it inactive by eliminating the majority of the coding region, including the helix-turn-helix DNA-binding motif. No significant change was observed with the ΔbarB mutant with respect to the timing or amount of virginiae butanolide production, or the morphological differentiation on solid media, indicating that barB neither participates in virginiae butanolide biosynthesis nor in cytodifferentiation. In contrast, analysis of Virginiamycin production in the ΔbarB mutant revealed that production of both Virginiamycin M1 and Virginiamycin S occurred immediately after virginiae butanolide production, 2–3 h earlier than in the wild-type strain, indicating that BarB participates in the temporal retardation of Virginiamycin production after virginiae butanolide inactivates the repressor function of BarA. RT-PCR analysis of the transcription of several genes surrounding barA–barB by the ΔbarB mutant indicated that BarB plays a negative regulatory role, directly or indirectly, in the transcription of barZ, vmsR, and orf5 located upstream of barB.
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Identification and In Vivo Functional Analysis of a Virginiamycin S Resistance Gene (varS) from Streptomyces virginiae
Journal of bacteriology, 1999Co-Authors: Chang Kwon Lee, Takuya Nihira, Yuka Kamitani, Yasuhiro YamadaAbstract:BarA of Streptomyces virginiae is a specific receptor protein for virginiae butanolide (VB), one of the γ-butyrolactone autoregulators of the Streptomyces species, and acts as a transcriptional regulator controlling both Virginiamycin production and VB biosynthesis. The downstream gene barB, the transcription of which is under the tight control of the VB-BarA system, was found to be transcribed as a polycistronic mRNA with its downstream region, and DNA sequencing revealed a 1,554-bp open reading frame (ORF) beginning at 161 bp downstream of the barB termination codon. The ORF product showed high homology (68 to 73%) to drug efflux proteins having 14 transmembrane segments and was named varS (for S. virginiae antibiotic resistance). Heterologous expression of varS with S. lividans as a host resulted in Virginiamycin S-specific resistance, suggesting that varS encoded a Virginiamycin S-specific transport protein. Northern blot analysis indicated that the bicistronic transcript of barB-varS appeared 1 to 2 h before the onset of Virginiamycin M1 and S production, at which time VB was produced, while exogenously added Virginiamycin S apparently induced the monocistronic varS transcript.
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gene replacement analysis of the streptomyces virginiae bara gene encoding the butyrolactone autoregulator receptor reveals that bara acts as a repressor in Virginiamycin biosynthesis
Journal of Bacteriology, 1998Co-Authors: Hiroko Nakano, Takuya Nihira, Emio Takehara, Yasuhiro YamadaAbstract:Virginiae butanolides (VBs), which are among the butyrolactone autoregulators of Streptomyces species, act as a primary signal in Streptomyces virginiae to trigger Virginiamycin biosynthesis and possess a specific binding protein, BarA. To clarify the in vivo function of BarA in the VB-mediated signal pathway that leads to Virginiamycin biosynthesis, two barA mutant strains (strains NH1 and NH2) were created by homologous recombination. In strain NH1, an internal 99-bp EcoT14I fragment of barA was deleted, resulting in an in-frame deletion of 33 amino acid residues, including the second helix of the probable helix-turn-helix DNA-binding motif. With the same growth rate as wild-type S. virginiae on both solid and liquid media, strain NH1 showed no apparent changes in its morphological behavior, indicating that the VB-BarA pathway does not participate in morphological control in S. virginiae. In contrast, Virginiamycin production started 6 h earlier in strain NH1 than in the wild-type strain, demonstrating for the first time that BarA is actively engaged in the control of Virginiamycin production and implying that BarA acts as a repressor in Virginiamycin biosynthesis. In strain NH2, an internal EcoNI-SmaI fragment of barA was replaced with a divergently oriented neomycin resistance gene cassette, resulting in the C-terminally truncated BarA retaining the intact helix-turn-helix motif. In strain NH2 and in a plasmid-integrated strain containing both intact and mutated barA genes, Virginiamycin production was abolished irrespective of the presence of VB, suggesting that the mutated BarA retaining the intact DNA-binding motif was dominant over the wild-type BarA. These results further support the hypothesis that BarA works as a repressor in Virginiamycin production and suggests that the helix-turn-helix motif is essential to its function. In strain NH1, VB production was also abolished, thus indicating that BarA is a pleiotropic regulatory protein controlling not only Virginiamycin production but also autoregulator biosynthesis.
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Optimum autoregulator addition strategy for maximum Virginiamycin production in batch culture of Streptomyces virginiae
Biotechnology and bioengineering, 1995Co-Authors: Young Kook Yang, Takuya Nihira, Hiroshi Shimizu, Suteaki Shioya, Ken-ichi Suga, Yasuhiro YamadaAbstract:Virginiae butanolides (VBs) are autoregulators of Streptomyces virginiae, which induce Virginiamycin biosynthesis. Generally, autoregulators are synthesized by the microorganism itself during culture. Addition of chemically synthesized virginiae butanolide-C (VB-C), which is one of the VBs, can also control the induction time and the amount of Virginiamycin production. The optimum concentration and shot-feeding time of VB-C for the maximum production of Virginiamycins M and S were investigated in flasks and jar-fermentor batch cultures. VB-C addition later than 8 h from the start of culture induced not only Virginiamycin M and S synthesis but also VB synthesis. Virginiamycin M and S production increased with the decrease of total VBs (produced VBs and added VB-C) concentration. That is, although VBs are needed to induce Virginiamycin M and S synthesis, the amount of VB-C added should be such that as small an amount as possible of VBs is synthesized to achieve the maximum production of Virginiamycins M and S. However, the VB-C addition earlier than 8 h from the start of culture showed no clear relationship between the amounts of VBs and Virginiamycins M and S produced. In conclusion, the maximum production of Virginiamycins M and S was attained by the shot addition of 5 mug/L VB-C at 8 h from the start of culture. The maximum value was about twofold that without VB-C addition. The optimum addition strategy of VB-C was confirmed by the jar-fermentor experiments. (c) 1995 John Wiley & Sons, Inc.
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New inducing factors for Virginiamycin production from Streptomyces antibioticus
Journal of Fermentation and Bioengineering, 1992Co-Authors: Takuya Nihira, Shohei Sakuda, Takuo Nishida, Yasuhiro YamadaAbstract:Abstract Four new butyrolactone derivatives, named NFX-1, -2, -3 and -4, were isolated from the culture broth of Streptomyces antibioticus as inducers of Virginiamycin in Streptomyces virginiae . The mode of Virginiamycin induction by NFX was identical with that of virginiae butanolides (VB), although the Virginiamycin-inducing activities of the new factors were hundreds fold lower than those of VB. The structures of NFX-2 and -4 were determined, including their stereochemistry.
Chang Kwon Lee - One of the best experts on this subject based on the ideXlab platform.
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Identification of the bkdAB gene cluster, a plausible source of the starter-unit for Virginiamycin M production in Streptomyces virginiae
Archives of Microbiology, 2007Co-Authors: Nattika Pulsawat, Shigeru Kitani, Hiroshi Kinoshita, Chang Kwon Lee, Takuya NihiraAbstract:The bkdAB gene cluster, which encodes plausible E1 and E2 components of the branched-chain α-keto acid dehydrogenase (BCDH) complex, was isolated from Streptomyces virginiae in the vicinity of a regulatory island for Virginiamycin production. Gene disruption of bkdA completely abolished the production of Virginiamycin M (a polyketide-peptide antibiotic), while the production of Virginiamycin S (a cyclodepsipeptide antibiotic) was unaffected. Complementation of the bkdA disruptant by genome-integration of intact bkdA completely restored the Virginiamycin M production, indicating that the bkdAB cluster is essential for Virginiamycin M biosynthesis, plausibly via the provision of isobutyryl-CoA as a primer unit. In contrast to a feature usually seen in the Streptomyces E1 component, namely, the separate encoding of the α and β subunits, S. virginiae bkdA seemed to encode the fused form of the α and β subunits, which was verified by the actual catalytic activity of the fused protein in vitro using recombinant BkdA overexpressed in Escherichia coli . Supply of an additional bkdA gene under the strong and constitutive promoter ermE * in the wild-type strain of S. virginiae resulted in enhanced production of Virginiamycin M, suggesting that the supply of isobutyryl-CoA is one of the rate-limiting factors in the biosynthesis of Virginiamycin M.
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identification by gene deletion analysis of barb as a negative regulator controlling an early process of Virginiamycin biosynthesis in streptomyces virginiae
Archives of Microbiology, 2004Co-Authors: Chang Kwon Lee, Yasuhiro Yamada, Kiyoshi Matsuno, Takuya NihiraAbstract:The Streptomyces virginiae γ-butyrolactone autoregulator virginiae butanolide is a low-molecular-weight Streptomyces hormone eliciting Virginiamycin biosynthesis through its binding to the specific receptor protein, BarA. Immediately downstream of barA lies barB, the transcription of which is tightly repressed by BarA in the absence of virginiae butanolide and derepressed in its presence. Thus, BarB is next to BarA on the virginiae butanolide-BarA signaling cascade. An in-frame 279-bp deletion was introduced into the barB allele, which rendered it inactive by eliminating the majority of the coding region, including the helix-turn-helix DNA-binding motif. No significant change was observed with the ΔbarB mutant with respect to the timing or amount of virginiae butanolide production, or the morphological differentiation on solid media, indicating that barB neither participates in virginiae butanolide biosynthesis nor in cytodifferentiation. In contrast, analysis of Virginiamycin production in the ΔbarB mutant revealed that production of both Virginiamycin M1 and Virginiamycin S occurred immediately after virginiae butanolide production, 2–3 h earlier than in the wild-type strain, indicating that BarB participates in the temporal retardation of Virginiamycin production after virginiae butanolide inactivates the repressor function of BarA. RT-PCR analysis of the transcription of several genes surrounding barA–barB by the ΔbarB mutant indicated that BarB plays a negative regulatory role, directly or indirectly, in the transcription of barZ, vmsR, and orf5 located upstream of barB.
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Identification and In Vivo Functional Analysis of a Virginiamycin S Resistance Gene (varS) from Streptomyces virginiae
Journal of bacteriology, 1999Co-Authors: Chang Kwon Lee, Takuya Nihira, Yuka Kamitani, Yasuhiro YamadaAbstract:BarA of Streptomyces virginiae is a specific receptor protein for virginiae butanolide (VB), one of the γ-butyrolactone autoregulators of the Streptomyces species, and acts as a transcriptional regulator controlling both Virginiamycin production and VB biosynthesis. The downstream gene barB, the transcription of which is under the tight control of the VB-BarA system, was found to be transcribed as a polycistronic mRNA with its downstream region, and DNA sequencing revealed a 1,554-bp open reading frame (ORF) beginning at 161 bp downstream of the barB termination codon. The ORF product showed high homology (68 to 73%) to drug efflux proteins having 14 transmembrane segments and was named varS (for S. virginiae antibiotic resistance). Heterologous expression of varS with S. lividans as a host resulted in Virginiamycin S-specific resistance, suggesting that varS encoded a Virginiamycin S-specific transport protein. Northern blot analysis indicated that the bicistronic transcript of barB-varS appeared 1 to 2 h before the onset of Virginiamycin M1 and S production, at which time VB was produced, while exogenously added Virginiamycin S apparently induced the monocistronic varS transcript.
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Stereospecific reduction of Virginiamycin M1 as the Virginiamycin resistance pathway in Streptomyces virginiae.
Antimicrobial agents and chemotherapy, 1996Co-Authors: Chang Kwon Lee, Takuya Nihira, Shohei Sakuda, M. Minami, Yusuke YamadaAbstract:In a cell extract of Streptomyces virginiae, Virginiamycin M1 was inactivated in the presence of NADPH, while Virginiamycin S remained intact. The inactivated product of Virginiamycin M1 was isolated, and structure analysis revealed that the inactivation involves reduction of a C-16 carbonyl group leading to the formation of 16-dihydroVirginiamycin M1. Acetonide and benzylidene acetal derivatives were synthesized from the two hydroxyl groups on C-14 and C-16, and the C-16 stereochemistry was determined by 13C nuclear magnetic resonance spectroscopy. Two methyl groups of the acetonide derivative gave 13C signals of 20.1 and 30.1 ppm, indicating that the relative stereochemistry of the C-14 and C-16 hydroxy groups is syn. Furthermore, irradiation of the benzylidene methine proton gave clear nuclear Overhauser effect enhancement of the C-14 or C-16 methine protons, indicating that H-14 and H-16 were in an axial configuration. From the (14S) absolute configuration of natural Virginiamycin M1 and the syn relative configuration for the C-14 and C-16 hydroxyl groups of the inactivated product, the C-16 absolute configuration of the inactivated product was thus identified as R.
Yusuke Yamada - One of the best experts on this subject based on the ideXlab platform.
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Stereospecific reduction of Virginiamycin M1 as the Virginiamycin resistance pathway in Streptomyces virginiae.
Antimicrobial agents and chemotherapy, 1996Co-Authors: Chang Kwon Lee, Takuya Nihira, Shohei Sakuda, M. Minami, Yusuke YamadaAbstract:In a cell extract of Streptomyces virginiae, Virginiamycin M1 was inactivated in the presence of NADPH, while Virginiamycin S remained intact. The inactivated product of Virginiamycin M1 was isolated, and structure analysis revealed that the inactivation involves reduction of a C-16 carbonyl group leading to the formation of 16-dihydroVirginiamycin M1. Acetonide and benzylidene acetal derivatives were synthesized from the two hydroxyl groups on C-14 and C-16, and the C-16 stereochemistry was determined by 13C nuclear magnetic resonance spectroscopy. Two methyl groups of the acetonide derivative gave 13C signals of 20.1 and 30.1 ppm, indicating that the relative stereochemistry of the C-14 and C-16 hydroxy groups is syn. Furthermore, irradiation of the benzylidene methine proton gave clear nuclear Overhauser effect enhancement of the C-14 or C-16 methine protons, indicating that H-14 and H-16 were in an axial configuration. From the (14S) absolute configuration of natural Virginiamycin M1 and the syn relative configuration for the C-14 and C-16 hydroxyl groups of the inactivated product, the C-16 absolute configuration of the inactivated product was thus identified as R.
George A. O'doherty - One of the best experts on this subject based on the ideXlab platform.
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De novo formal synthesis of (-)-Virginiamycin M2 via the asymmetric hydration of dienoates.
Organic letters, 2007Co-Authors: Matthew S. Mortensen, Joshua M. Osbourn, George A. O'dohertyAbstract:A de novo approach to the formal total synthesis of the macrolide natural product (—)-Virginiamycin M2 has been achieved via a convergent approach. The absolute and relative stereochemistry of the non-peptide portion of (—)-Virginiamycin M2 was introduced by two Sharpless asymmetric dihydroxylation reactions.
