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The mannopeptimycins are a novel class of lipoglycopeptide antibiotics active against multidrug-resistant pathogens with potential as clinically useful antibacterials. This report is the first to describe the biosynthesis of this novel class of mannosylated lipoglycopeptides. Included here are the cloning, sequencing, annotation, and manipulation of the mannopeptimycin biosynthetic gene cluster from Streptomyces hygroscopicus NRRL 30439. Encoded by genes within the mannopeptimycin biosynthetic gene cluster are enzymes responsible for the generation of the hexapeptide core (nonribosomal peptide synthetases [NRPS]) and tailoring reactions (mannosylation, isovalerylation, hydroxylation, and methylation). The NRPS system is noncanonical in that it has six modules utilizing only five amino acid-specific adenylation domains and it lacks a prototypical NRPS macrocyclizing thioesterase domain. Analysis of the mannopeptimycin gene cluster and its engineering has elucidated the mannopeptimycin biosynthetic pathway and provides the framework to make new and improved mannopeptimycins biosynthetically.Multidrug-resistant gram-positive bacterial pathogens now exist, and their rising numbers are a major concern. The prevalence of methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) exemplifies the seriousness of this concern (22, 29). Finding novel antibiotics active against such drug-resistant gram-positive pathogens, however, is problematic (32, 42). The mannopeptimycins ( Fig. 1), which are produced by Streptomyces hygroscopicus NRRL 30439, represent a new class of lipoglycopeptide antibiotics with exceptional in vitro and in vivo antibacterial activities against MRSA, VRE, and penicillin-resistant Streptococcus pneumoniae (17,36). Studies with mannopeptimycin-␦ demonstrated that this antibiotic acts by blocking the transglycosylation reaction in cell wall biosynthesis mediated by lipid II binding. Mannopeptimycin-␦ inhibits gram-positive cell wall biosynthesis through a mechanism which does not compete or render it ineffective due to crossresistance with the vancomycin-type antibiotics (33, 36).The unique structure, mode of action, and bioactivity of the mannopeptimycins have generated much interest in identifying a therapeutic candidate from this new class of molecules (17,33,36). One semisynthetic analog, AC98-6446, of the natural mannopeptimycins is significantly more potent and effective than the natural mannopeptimycins, with MICs in the 15-to 60-ng/ml range against MRSA, VRE, penicillin-resistant Streptococcus pneumoniae, and glycopeptide-intermediate Staphylococcus aureus (10, 31). The success in improving on the natural mannopeptimycins argues strongly for broadening the effort of generating additional new mannopeptimycins through promising strategies such as combinatorial biosynthesis, mutasynthesis, and chemoenzymatic synthesis (4, 28). The establishment and success of such approaches hinge on the cloning of the mannopeptimycin biosynthetic gene cluster and elucidation of ...

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Mannopeptimycins alpha, beta, gamma, delta, and epsilon are new cyclic glycopeptide antibiotics produced by Streptomyces hygroscopicus LL-AC98. Mannopeptimycins gamma, delta, and epsilon, which have an isovaleryl substitution at various positions on the terminal mannose of the disaccharide moiety, demonstrated moderate to good antibacterial activities. Mannopeptimycin epsilon was the most active component against methicillin-resistant staphylococci and vancomycin-resistant enterococci (MICs, 2 to 4 micro g/ml for staphylococci and streptococci and 4 to 32 micro g/ml for enterococci), while mannopeptimycins gamma and delta were two- to fourfold less active. Mannopeptimycins alpha and beta, which lack the isovaleryl substitution and the disaccharide moiety, respectively, had poor antibacterial activities. The in vivo efficacies of the mannopeptimycins in Staphylococcus aureus mouse protection studies paralleled their in vitro activities. The median effective doses of mannopeptimycins gamma, delta, and epsilon were 3.8, 2.6, and 0.59 mg/kg of body weight, respectively. The mannopeptimycins were inactive against cell wall-deficient S. aureus and caused spheroplasting of Escherichia coli imp similar to that observed with penicillin G in an osmotically protective medium. Mannopeptimycin delta rapidly inhibited [(3)H]N-acetylglucosamine incorporation into peptidoglycan in Bacillus subtilis and had no effect on DNA, RNA, or protein biosynthesis. On the basis of the observations presented above, an effect on cell wall biosynthesis was suggested as the primary mode of action for mannopeptimycin delta. The mannopeptimycins were inactive against Candida albicans, did not initiate hemolysis of human erythrocytes, and did not promote potassium ion leakage from E. coli imp, suggesting a lack of membrane damage to prokaryotic or eukaryotic cells.

Fermentation of a marine fungal species obtained from a tissue sample of a marine sponge collected in Indonesia in October 1996, yielded the novel hexaketide compounds iso-cladospolide B (1); seco-patulolide C (2); the 12-membered macrolides, pandangolide 1 (3) and pandangolide 2 (4); and the known terrestrial fungal metabolite, cladospolide B (5).

Two new isomalabaricane triterpenes, stellettin H (1) and stellettin I (2), have been isolated from the marine sponge Rhabdastrella globostellata, collected from the Philippines. Stellettins A-D (3-6), (-)-stellettin E (7), and rhabdastrellic acid-A (8) were also isolated and characterized. Stellettin B (4) and (-)-stellettin E (7) showed selective cytotoxicity toward p21(WAF1/Cip1)-deficient human colon tumor (HCT-116) cells with IC(50) values of 0.043 and 0.039 microM, respectively.

Fermentation extracts of culture CR11 5, an unknown plant endophyte originally isolated from Costa Rica, were found to be active against antibiotic-resistant bacteria. The metabolite responsible for activity was identified as a novel diterpenoid antibiotic guanacastepene (mol. wt. 374.47 and mol. formula C22H30O5). Mechanistic studies done in an E. coli imp strain suggested membranedamage as the primary modeof bactericidal action. This compoundalso lysed human RBCsand caused leakage of intracellular potassium from E. coli imp. Screening of unusual fungi from various ecological niches around the world has been productive in yielding new bioactive metabolitesl~7\ During our screening of fungal fermentation extracts, an unidentified terrestrial fungus CR1 15 was found to exhibit antibacterial activity against methicillin-resistant Staphylococcus aureus and