Zhiguo Yu

Dedicated ent-kaurene and ent-atiserene synthases for platensimycin and platencin biosynthesis

Platensimycin (PTM) and platencin (PTN) are potent and selective inhibitors of bacterial and mammalian fatty acid synthases and have emerged as promising drug leads for both antibacterial and antidiabetic therapies. Comparative analysis of the PTM and PTN biosynthetic machineries in Streptomyces platensis MA7327 and MA7339 revealed that the divergence of PTM and PTN biosynthesis is controlled by dedicated ent-kaurene and ent-atiserene synthases, the latter of which represents a new pathway for diterpenoid biosynthesis. The PTM and PTN biosynthetic machineries provide a rare glimpse at how secondary metabolic pathway evolution increases natural product structural diversity and support the wisdom of applying combinatorial biosynthesis methods for the generation of novel PTM and/or PTN analogues, thereby facilitating drug development efforts based on these privileged natural product scaffolds.

Tirandamycins from Streptomyces sp. 17944 Inhibiting the Parasite Brugia malayi Asparagine tRNA Synthetase

Lymphatic filariasis is caused by the parasitic nematodes Brugia malayi and Wuchereria bancrofti, and asparaginyl-tRNA synthetase (AsnRS) is considered an excellent antifilarial target. The discovery of three new tirandamycins (TAMs), TAM E (1), F (2), and G (3), along with TAM A (4) and B (5), from Streptomyces sp. 17944 was reported. Remarkably, 5 selectively inhibits the B. malayi AsnRS and efficiently kills the adult B. malayi parasite, representing a new lead scaffold to discover and develop antifilarial drugs.

Engineering of Streptomyces platensis MA7339 for overproduction of platencin and congeners.

Platensimycin (1) and platencin (2) are novel antibiotic leads against multidrug resistant pathogens. The production of 2 in Streptomyces platensis MA7339 is under the control of ptnR1, a GntR-like transcriptional regulator. Inactivating ptnR1 afforded S. platensis MA7339 mutant strain SB12600 that overproduces 2 at a titer approximately 100-fold greater than that from the wild-type strain and accumulates platencin A(1) (3) and eight new congeners, platencins A(2)-A(9) (4-11). The isolation, structural elucidation, and antibacterial activity of 4-11, in comparison to 1-3, are described.

Biosynthetic potential-based strain prioritization for natural product discovery: a showcase for diterpenoid-producing actinomycetes.

Natural products remain the best sources of drugs and drug leads and serve as outstanding small-molecule probes to dissect fundamental biological processes. A great challenge for the natural product community is to discover novel natural products efficiently and cost effectively. Here we report the development of a practical method to survey biosynthetic potential in microorganisms, thereby identifying the most promising strains and prioritizing them for natural product discovery. Central to our approach is the innovative preparation, by a two-tiered PCR method, of a pool of pathway-specific probes, thereby allowing the survey of all variants of the biosynthetic machineries for the targeted class of natural products. The utility of the method was demonstrated by surveying 100 strains, randomly selected from our actinomycete collection, for their biosynthetic potential of four classes of natural products, aromatic polyketides, reduced polyketides, nonribosomal peptides, and diterpenoids, identifying 16 talented strains. One of the talented strains, Streptomyces griseus CB00830, was finally chosen to showcase the discovery of the targeted classes of natural products, resulting in the isolation of three diterpenoids, six nonribosomal peptides and related metabolites, and three polyketides. Variations of this method should be applicable to the discovery of other classes of natural products.

Expression of the Platencin Biosynthetic Gene Cluster in Heterologous Hosts Yielding New Platencin Congeners

Platensimycin (PTM) and platencin (PTN) are potent and selective inhibitors of bacterial and mammalian fatty acid synthases and have emerged as promising drug leads for both antibacterial and antidiabetic therapies. We have previously cloned and sequenced the PTM-PTN dual biosynthetic gene cluster from Streptomyces platensis MA7327 and the PTN biosynthetic gene cluster from S. platensis MA7339, the latter of which is composed of 31 genes encoding PTN biosynthesis, regulation, and resistance. We have also demonstrated that PTM or PTN production can be significantly improved upon inactivation of the pathway-specific regulator ptmR1 or ptnR1 in S. platensis MA7327 or MA7339, respectively. We now report engineered production of PTN and congeners in a heterologous Streptomyces host. Expression constructs containing the ptn biosynthetic gene cluster were engineered from SuperCos 1 library clones and introduced into five model Streptomyces hosts, and PTN production was achieved in Streptomyces lividans K4-114. Inactivation of ptnR1 was crucial for expression of the ptn biosynthetic gene cluster, thereby PTN production, in S. lividans K4-114. Six PTN congeners, five of which were new, were also isolated from the recombinant strain S. lividans SB12606, revealing new insights into PTN biosynthesis. Production of PTN in a model Streptomyces host provides new opportunities to apply combinatorial biosynthetic strategies to the PTN biosynthetic machinery for structural diversity.

Bafilomycins produced by an endophytic actinomycete Streptomyces sp. YIM56209

Division of Pharmaceutical Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53705, USA; Hunan Engineering Research Center of Combinatorial Biosynthesis and Natural Product Drug Discovery, Changsha, Hunan 410329, China; Yunnan Institute of Microbiology, Yunnan University, Kunming, Yunnan 650091, China; Department of Comparative Biosciences, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA; University of Wisconsin National Cooperative Drug Discovery Group, University of Wisconsin-Madison, Madison, Wisconsin 53705, USA; and Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53705, USA

Medium optimization of Streptomyces sp. 17944 for tirandamycin B production and isolation and structural elucidation of tirandamycins H, I and J.

We have recently isolated tirandamycin (TAM) B from Streptomyces sp. 17944 as a Brugia malayi AsnRS (BmAsnRS) inhibitor that efficiently kills the adult B. malayi parasites and does not exhibit general cytotoxicity to human hepatic cells. We now report (i) the comparison of metabolite profiles of S. sp. 17944 in six different media, (ii) identification of a medium enabling the production of TAM B as essentially the sole metabolite, and with improved titer, and (iii) isolation and structural elucidation of three new TAM congeners. These findings shed new insights into the structure–activity relationship of TAM B as a BmAsnRS inhibitor, highlighting the δ-hydroxymethyl-α,β-epoxyketone moiety as the critical pharmacophore, and should greatly facilitate the production and isolation of sufficient quantities of TAM B for further mechanistic and preclinical studies to advance the candidacy of TAM B as an antifilarial drug lead. The current study also serves as an excellent reminder that traditional medium and fermentation optimization should continue to be very effective in improving metabolite flux and titer.

Cycloheximide and congeners as inhibitors of eukaryotic protein synthesis from endophytic actinomycetes Streptomyces sps. YIM56132 and YIM56141

Cycloheximide and congeners as inhibitors of eukaryotic protein synthesis from endophytic actinomycetes Streptomyces sps. YIM56132 and YIM56141

Isolation and structural elucidation of glucoside congeners of platencin from Streptomyces platensis SB12600.

Isolation and structural elucidation of glucoside congeners of platencin from Streptomyces platensis SB12600

Identification of antifungal natural products via Saccharomyces cerevisiae bioassay: insights into macrotetrolide drug spectrum, potency and mode of action

Since current antifungal drugs have not kept pace with the escalating medical demands of fungal infections, new, effective medications are required. However, antifungal drug discovery is hindered by the evolutionary similarity of mammalian and fungal cells, which results in fungal drug targets having human homologs and drug non-selectivity. The group III hybrid histidine kinases (HHKs) are an attractive drug target since they are conserved in fungi and absent in mammals. We used a Saccharomyces cerevisiae reporter strain that conditionally expresses HHK to establish a high-throughput bioassay to screen microbial extracts natural products for antifungals. We identified macrotetrolides, a group of related ionophores thought to exhibit restricted antifungal activity. In addition to confirming the use of this bioassay for the discovery of antifungal natural products, we demonstrated broader, more potent fungistatic activity of the macrotetrolides against multiple Candida spp., Cryptococcus spp., and Candida albicans in biofilms. Macrotetrolides were also active in an animal model of C. albicans biofilm, but were found to have inconsistent activity against fluconazole-resistant C. albicans, with most isolates resistant to this natural product. The macrotetrolides do not directly target HHKs, but their selective activity against S. cerevisiae grown in galactose (regardless of Drk1 expression) revealed potential new insight into the role of ion transport in the mode of action of these promising antifungal compounds. Thus, this simple, high-throughput bioassay permitted us to screen microbial extracts, identify natural products as antifungal drugs, and expand our understanding of the activity of macrotetrolides.