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Ben Shen

Publications and source records attributed to Ben Shen.

At least 19 recordsLinked to original sources
PubMed2026

Discovery of Sphaeriaurantins as Rapid-Acting Antiplasmodials with Dual Activity in Blood and Liver Stages.

The rapid emergence of resistance in the malaria-causing protozoan Plasmodium falciparum has heightened the demand for treatments with novel modes of action. Having evolved to produce a myriad of structurally diverse natural products (NPs) as defenses against soil-dwelling parasites including protozoa, Actinomycetota strains are a promising source for the discovery of NPs as antiplasmodial drug leads. Herein, the selective inhibition of P. falciparum is reported for five distinct NP families from Actinomycetota, including an unprecedented family of glycosylated type II polyketides termed sphaeriaurantins (SPAs). The structures of SPAs were established through the combination of MS and NMR spectroscopic data analysis, derivatization and comparison of the deoxyhexose moieties to authentic standards, and quantum chemical calculations, including 1H and 13C NMR chemical shifts and electronic circular dichroism (ECD) spectra. The three isolated SPA congeners reveal that the characteristic pseudodimeric structure of the SPA family of NPs, likely introduced at a late stage of the SPA biosynthesis, is highly relevant for the observed low nanomolar activity. SPA A exhibits a rapid killing profile, with activities across all intraerythrocytic stages, and potent liver stage efficacy, as well as a low propensity for resistance development. Taken together, these results suggest a mode of action that most likely is distinct from the existing antimalarials, supporting SPA A as a promising antimalarial drug lead for further development.

Plasmodium falciparum↗
PubMed2025

Logical Exploration of Cinnamoyl-Containing Nonribosomal Peptides via Metabologenomic Targeting and Regulator Overexpression.

A targeted method for discovering cinnamoyl-containing nonribosomal peptides (CCNPs), a unique class of bioactive compounds, was devised by using cinnamoyl isomerase, a key enzyme in the biosynthesis of the cinnamoyl moiety, as a genome mining probe. A total of 39 hit strains were obtained, including 35 from polymerase chain reaction-based screening of the in-house bacterial library (2.5% of 1400 strains) targeting the cinnamoyl isomerase-encoding gene and 4 from the genome mining of online databases. Sequence similarity networking and phylogenetic analyses of the isomerase amplicons (∼530 bp) classified the CCNPs into three major substructure-based groups (Z-, E-, and M-type CCNPs) and revealed distinct clade-structure relationships (13 clades). To overcome the challenge of silent biosynthetic gene clusters, we activated these clusters by overexpressing conserved cluster-situated LuxR regulators combined with extensive culture optimization. CCNP production was metabolomically detected in the bacterial extracts by using the characteristic UV absorption and MS/MS fragments of cinnamoyl moieties. CCNP production was observed in 20 of the 39 hit strains, resulting in the isolation of 6 new CCNPs, including oxy-skyllamycin B (2), gwanacinnamycin (3), and luxocinnamycins A-D (4-7), with high structural novelty. Their structures were elucidated using comprehensive spectroscopic analyses and multiple-step chemical derivatizations, and the putative biosynthetic pathways were bioinformatically proposed. Gwanacinnamycin (3) exhibited significant antimycobacterial activity, whereas luxocinnamycin A (4) displayed moderate antiproliferative activity against stomach cancer cells. Our findings highlight a targeted metabologenomic approach combined with transcriptional regulator overexpression as a logical and efficient platform for the discovery of bioactive compounds from nature.

Peptides↗
PubMed2006

Thermolysis of isomigrastatin and its congeners via [3,3]-sigmatropic rearrangement: a new route to the synthesis of migrastatin and its analogues.

Thermolysis of isomigrastatin (1) under neat heating conditions afforded migrastatin (1a). The reaction is proposed to proceed via a concerted [3,3]-sigmatropic rearrangement by which ring expansion is achieved regio- and enantiospecifically. The general applicability of this reaction was demonstrated with six additional isomigrastatin congeners (3-8), providing a new route to the synthesis of migrastatin analogues (3a-8a). [reaction: see text]

Chromatography, High Pressure Liquid↗
PubMed2006

The tallysomycin biosynthetic gene cluster from Streptoalloteichus hindustanus E465-94 ATCC 31158 unveiling new insights into the biosynthesis of the bleomycin family of antitumor antibiotics.

The tallysomycins (TLMs) belong to the bleomycin (BLM) family of antitumor antibiotics. The BLM biosynthetic gene cluster has been cloned and characterized previously from Streptomyces verticillus ATCC 15003, but engineering BLM biosynthesis for novel analogs has been hampered by the lack of a genetic system for S. verticillus. We now report the cloning and sequencing of the TLM biosynthetic gene cluster from Streptoalloteichus hindustanus E465-94 ATCC 31158 and the development of a genetic system for S. hindustanus, demonstrating the feasibility to manipulate TLM biosynthesis in S. hindustanus by gene inactivation and mutant complementation. Sequence analysis of the cloned 80.2 kb region revealed 40 open reading frames (ORFs), 30 of which were assigned to the TLM biosynthetic gene cluster. The TLM gene cluster consists of nonribosomal peptide synthetase (NRPS) genes encoding nine NRPS modules, a polyketide synthase (PKS) gene encoding one PKS module, genes encoding seven enzymes for deoxysugar biosynthesis and attachment, as well as genes encoding other biosynthesis, resistance, and regulatory proteins. The involvement of the cloned gene cluster in TLM biosynthesis was confirmed by inactivating the tlmE glycosyltransferase gene to generate a TLM non-producing mutant and by restoring TLM production to the DeltatlmE::ermE mutant strain upon expressing a functional copy of tlmE. The TLM gene cluster is highly homologous to the BLM cluster, with 25 of the 30 ORFs identified within the two clusters exhibiting striking similarities. The structural similarities and differences between TLM and BLM were reflected remarkably well by the genes and their organization in their respective biosynthetic gene clusters.

Amino Acid Sequence↗
PubMed2006

The bifunctional glyceryl transferase/phosphatase OzmB belonging to the HAD superfamily that diverts 1,3-bisphosphoglycerate into polyketide biosynthesis.

The HAD superfamily protein OzmB from the oxazolomycin biosynthetic pathway is shown to divert the primary metabolite 1,3-diphosphoglycerate into the polyketide biosynthetic pathway as glycerate via loading of a carrier protein. Each of the steps-activation of d-3-phosphoglycerate, dephosphorylation while attached to a cysteine on OzmB, and subsequent transfer of glycerate to the phosphopantetheinyl thiol of an acyl carrier protein-was monitored by nanospray Fourier transform mass spectrometry. This activation of phosphoglycerate represents a general mechanism of diverting glycolytic metabolites into glyceryl-derived polyketides.

Amino Acid Sequence↗
PubMed2006

A dedicated phosphopantetheinyl transferase for the fredericamycin polyketide synthase from Streptomyces griseus.

Polyketide synthases cannot be functional unless their apo-acyl carrier proteins (apo-ACPs) are post-translationally modified by covalent attachment of the 4'-phosphopantetheine group to the highly conserved serine residue, and this reaction is catalyzed by phosphopantetheinyl transferases (PPTases). Cloning and sequence analysis of the 33-kb fredericamycin (FDM) biosynthetic gene cluster from Streptomyces griseus revealed fdmW, whose deduced gene product showed significant sequence homology to known PPTases. Biochemical characterization of FdmW in vitro confirmed that it is a PPTase. Inactivation of fdmW resulted in approximately 93% reduction of FDM production, and complementation of the fdmW::aac (3)IV mutant by expressing fdmW in trans restored FDM production to a level comparable with that of the wild-type strain. Although FdmW can phosphopantetheinylate various ACPs, it prefers its cognate substrate, the FdmH ACP, with a K(m) of 5.8 microM and a k(cat)/K(m) of 8.1 microM(-1) x min(-1), to heterologous ACPs, such as the TcmM ACP with a K(m) of 1.0 x 10(2) microM and a k(cat) /K(m) of 0.6 microM(-1) x min(-1). These findings suggest that FdmW is specific for FDM biosynthesis. FdmW therefore represents the first holo-ACP synthase-type PPTase identified from an aromatic polyketide biosynthetic gene cluster.

Alkenes↗
PubMed2006

Substrate specificity of the adenylation enzyme SgcC1 involved in the biosynthesis of the enediyne antitumor antibiotic C-1027.

C-1027 is an enediyne antitumor antibiotic composed of a chromophore with four distinct chemical moieties, including an (S)-3-chloro-4,5-dihydroxy-beta-phenylalanine moiety that is derived from l-alpha-tyrosine. SgcC4, a novel aminomutase requiring no added co-factor that catalyzes the formation of the first intermediate (S)-beta-tyrosine and subsequently SgcC1 homologous to adenylation domains of nonribosomal peptide synthetases, was identified as specific for the SgcC4 product and did not recognize any alpha-amino acids. To definitively establish the substrate for SgcC1, a full kinetic characterization of the enzyme was performed using amino acid-dependent ATP-[(32)P]PP(i) exchange assay to monitor amino acid activation and electrospray ionization-Fourier transform mass spectroscopy to follow the loading of the activated beta-amino acid substrate to the peptidyl carrier protein SgcC2. The data establish (S)-beta-tyrosine as the preferred substrate, although SgcC1 shows promiscuous activity toward aromatic beta-amino acids such as beta-phenylalanine, 3-chloro-beta-tyrosine, and 3-hydroxy-beta-tyrosine, but all were <50-fold efficient. A putative active site mutant P571A adjacent to the invariant aspartic acid residue of all alpha-amino acid-specific adenylation domains known to date was prepared as a preliminary attempt to probe the substrate specificity of SgcC1; however the mutation resulted in a loss of activity with all substrates except (S)-beta-tyrosine, which was 142-fold less efficient relative to the wild-type enzyme. In total, SgcC1 is now confirmed to catalyze the second step in the biosynthesis of the (S)-3-chloro-4,5-dihydroxy-beta-phenylalanine moiety of C-1027, presenting downstream enzymes with an (S)-beta-tyrosyl-S-SgcC2 thioester substrate, and represents the first beta-amino acid-specific adenylation enzyme characterized biochemically.

Adenine Nucleotides↗
PubMed2006

Microbial genomics for the improvement of natural product discovery.

The quest for the discovery of novel natural products has entered a new chapter with the enormous wealth of genetic data that is now available. This information has been exploited by using whole-genome sequence mining to uncover cryptic pathways, or biosynthetic pathways for previously undetected metabolites. Alternatively, using known paradigms for secondary metabolite biosynthesis, genetic information has been 'fished out' of DNA libraries resulting in the discovery of new natural products and isolation of gene clusters for known metabolites. Novel natural products have been discovered by expressing genetic data from uncultured organisms or difficult-to-manipulate strains in heterologous hosts. Furthermore, improvements in heterologous expression have not only helped to identify gene clusters but have also made it easier to manipulate these genes in order to generate new compounds. Finally, and perhaps the most crucial aspect of the efficient and prosperous use of the abundance of genetic information, novel enzyme chemistry continues to be discovered, which has aided our understanding of how natural products are biosynthesized de novo, and enabled us to rework the current paradigms for natural product biosynthesis.

Bacteria↗
PubMed2006

Polyketide chain skipping mechanism in the biosynthesis of the hybrid nonribosomal peptide-polyketide antitumor antibiotic leinamycin in Streptomyces atroolivaceus S-140.

A fundamental feature of modular polyketide synthases (PKSs) is the highly predictable relationship between the domain order and the chemical functional groups of resultant polyketide products. Sequence analysis and biochemical characterization of the leinamycin (LNM) biosynthetic gene cluster from Streptomyces atroolivaceus S-140 has revealed a gene, lnmJ, that encodes five PKS modules but with six acyl carrier protein (ACP) domains. The LnmJ PKS module 6 contains two ACP domains, ACP(6-1) and ACP(6-2), separated by a C-methyltransferase domain. Site-directed mutagenesis experiments were carried out with each of these ACPs to test alternative mechanisms proposed for their role in polyketide chain elongation. The in vivo results revealed a new type of polyketide chain "skipping" mechanism, in which either ACP is sufficient for LNM biosynthesis. Biochemical characterization in vitro showed that both ACPs can be loaded with a malonate extender unit by the LnmG acyl transferase; however, ACP(6-2) appears to be preferred because the loading efficiency is about 5-fold that of ACP(6-1). The results are consistent with ACP(6-2) being used for the initial chain elongation step wth ACP(6-1) being involved in the ensuing C-methylation process. These findings provide new insights into the polyketide chain skipping mechanism for modular PKSs.

Antibiotics, Antineoplastic↗
PubMed2006

Utilization of the methoxymalonyl-acyl carrier protein biosynthesis locus for cloning of the tautomycin biosynthetic gene cluster from Streptomyces spiroverticillatus.

Tautomycin (TTM), a potent protein phosphatase inhibitor, consists of a polyketide chain containing a spiroketal moiety and an acyl chain bearing a dialkylmaleic anhydride structure. PCR using degenerate primers was used to clone genes from Streptomyces spiroverticillatus for formation of the methoxymalonyl-acyl carrier protein. This locus was found to contain five genes (ttmC, ttmA, ttmD, ttmB, and ttmE), one of which was used as a probe to clone the 110-kb TTM biosynthetic gene cluster. The involvement of the ttmA gene in TTM biosynthesis was confirmed by gene inactivation and mutation complementation experiments.

Acyl Carrier Protein↗
PubMed2006

Utilization of the methoxymalonyl-acyl carrier protein biosynthesis locus for cloning the oxazolomycin biosynthetic gene cluster from Streptomyces albus JA3453.

Oxazolomycin (OZM), a hybrid peptide-polyketide antibiotic, exhibits potent antitumor and antiviral activities. Using degenerate primers to clone genes encoding methoxymalonyl-acyl carrier protein (ACP) biosynthesis as probes, a 135-kb DNA region from Streptomyces albus JA3453 was cloned and found to cover the entire OZM biosynthetic gene cluster. The involvement of the cloned genes in OZM biosynthesis was confirmed by deletion of a 12-kb DNA fragment containing six genes for methoxymalonyl-ACP biosynthesis from the specific region of the chromosome, as well as deletion of the ozmC gene within this region, to generate OZM-nonproducing mutants.

Acyl Carrier Protein↗
PubMed2005

Cloning, sequencing, analysis, and heterologous expression of the fredericamycin biosynthetic gene cluster from Streptomyces griseus.

Fredericamycin (FDM) A, a pentadecaketide featuring two sets of peri-hydroxy tricyclic aromatic moieties connected through a unique chiral spiro carbon center, exhibits potent cytotoxicity and has been studied as a new type of anticancer drug lead because of its novel molecular architecture. The fdm gene cluster was localized to 33-kb DNA segment of Streptomyces griseus ATCC 49344, and its involvement in FDM A biosynthesis was proven by gene inactivation, complementation, and heterologous expression experiments. The fdm cluster consists of 28 open reading frames (ORFs), encoding a type II polyketide synthase (PKS) and tailoring enzymes as well as several regulatory and resistance proteins. The FDM PKS features a KSalpha subunit with heretofore unseen tandem cysteines at its active site, a KSbeta subunit that is distinct phylogenetically from KSbeta of hexa-, octa-, or decaketide PKSs, and a dedicated phosphopantetheinyl transferase. Further study of the FDM PKS could provide new insight into how a type II PKS controls chain length in aromatic polyketide biosynthesis. The availability of the fdm genes, in vivo characterization of the fdm cluster in S. griseus, and heterologous expression of the fdm cluster in Streptomyces albus set the stage to investigate FDM A biosynthesis and engineer the FDM biosynthetic machinery for the production of novel FDM A analogues.

Antibiotics, Antineoplastic↗
PubMed2005

Iso-migrastatin congeners from Streptomyces platensis and generation of a glutarimide polyketide library featuring the dorrigocin, lactimidomycin, migrastatin, and NK30424 scaffolds.

Iso-Migrastatin (10) has been shown to be the main natural product of Streptomyces platensis, which undergoes a facile, H2O-mediated rearrangement into dorrigocin A (2), 13-epi-dorrigocin A (11), dorrigocin B (3), and migrastatin (1). Eight new congeners (12-19) of 10 were characterized. They can undergo the same H2O-mediated rearrangement into the corresponding 1, 2, 3, and 11 analogues (20-43) or 1,4-Michael addition with cysteine to afford the corresponding analogues (44-51) of NK30424 A and B (5, 6). This study generated a 47-member library of glutarimide polyketides, setting the stage to investigate the SAR for this family of natural products. These results also established the absolute stereochemistry of 5 and 6 and shed new light into the post-polyketide synthase steps for 10 biosynthesis.

Antibiotics, Antineoplastic↗
PubMed2005

Biosynthesis of the beta-amino acid moiety of the enediyne antitumor antibiotic C-1027 featuring beta-amino acyl-S-carrier protein intermediates.

The enediyne antitumor antibiotic C-1027 chromoprotein is produced by Streptomyces globisporus. The biosynthesis of the (S)-3-chloro-4,5-dihydroxy-beta-phenylalanine moiety (boxed) of the C-1027 chromophore (1) from l-tyrosine (3) and its incorporation into 1 are catalyzed by six enzymes: SgcC, SgcC1, SgcC2, SgcC3, SgcC4, ShcC5. In vivo and in vitro characterization of these enzymes delineated this pathway, unveiling a novel strategy for beta-amino acid modification featuring beta-amino acyl-S-carrier protein intermediates. These findings shed new light into beta-amino acid biosynthesis and present a new opportunity to engineer the C-1027 biosynthetic machinery for the production of novel analogues as exemplified by 20-deschloro-C-1027 (4), 20-deschro-22-deshydroxy-C-1027 (5), and 22-deshydroxy-C-1027 (6).

Acyl Carrier Protein↗
PubMed2005

Migrastatin and dorrigocins are shunt metabolites of iso-migrastatin.

Fermentation of Streptomyces platensis NRRL18993 typically accumulated migrastation (1), dorrigocin A (2) and B (3), and 13-epi-dorrigocin A (5). Supplement of XAD-16 resin to the fermentation, in contrast, resulted in exclusive production of iso-migrastatin (4). In vitro studies showed that 1, 2, 3, and 5 are stable in aqueous solution but 4 undergoes rapid conversion into 1, 2, 3, and 5 under the same condition. These results revealed that 4 is the only bona fide natural product biosynthesized by S. platensis, and 1, 2, 3, and 5 are shunt metabolites of 4. This study also established the stereochemistry of 2-5 with the exception of C-11 for 3 and 4. A mechanism for H2O-mediated regio- and stereospecific rearrangement of 4 to 1, 2, 3, and 5 is proposed and supported by incorporation of 18O from H218O.

Chromatography, High Pressure Liquid↗
PubMed2005

The neocarzinostatin biosynthetic gene cluster from Streptomyces carzinostaticus ATCC 15944 involving two iterative type I polyketide synthases.

The biosynthetic gene cluster for the enediyne antitumor antibiotic neocarzinostatin (NCS) was localized to 130 kb continuous DNA from Streptomyces carzinostaticus ATCC15944 and confirmed by gene inactivation. DNA sequence analysis of 92 kb of the cloned region revealed 68 open reading frames (ORFs), 47 of which were determined to constitute the NCS cluster. Sequence analysis of the genes within the NCS cluster suggested dNDP-D-mannose as a precursor for the deoxy aminosugar, revealed two distinct type I polyketide synthases (PKSs), and supported a convergent model for NCS chromophore biosynthesis from the deoxy aminosugar, naphthoic acid, and enediyne core building blocks. These findings shed light into deoxysugar biosynthesis, further support the iterative type I PKS paradigm for enediyne core biosynthesis, and unveil a mechanism for microbial polycyclic aromatic polyketide biosynthesis by an iterative type I PKS.

Cloning, Molecular↗