Construction of a new class of tetracycline lead structures with potent antibacterial activity through biosynthetic engineering.

Antimicrobial resistance and the shortage of novel antibiotics have led to an urgent need for new antibacterial drug leads. Several existing natural product scaffolds (including chelocardins) have not been developed because their suboptimal pharmacological properties could not be addressed at the time. It is demonstrated here that reviving such compounds through the application of biosynthetic engineering can deliver novel drug candidates. Through a rational approach, the carboxamido moiety of tetracyclines (an important structural feature for their bioactivity) was introduced into the chelocardins, which are atypical tetracyclines with an unknown mode of action. A broad-spectrum antibiotic lead was generated with significantly improved activity, including against all Gram-negative pathogens of the ESKAPE panel. Since the lead structure is also amenable to further chemical modification, it is a platform for further development through medicinal chemistry and genetic engineering.

Identification of the chelocardin biosynthetic gene cluster from Amycolatopsis sulphurea: a platform for producing novel tetracycline antibiotics.

Tetracyclines (TCs) are medically important antibiotics from the polyketide family of natural products. Chelocardin (CHD), produced by Amycolatopsis sulphurea, is a broad-spectrum tetracyclic antibiotic with potent bacteriolytic activity against a number of Gram-positive and Gram-negative multi-resistant pathogens. CHD has an unknown mode of action that is different from TCs. It has some structural features that define it as 'atypical' and, notably, is active against tetracycline-resistant pathogens. Identification and characterization of the chelocardin biosynthetic gene cluster from A. sulphurea revealed 18 putative open reading frames including a type II polyketide synthase. Compared to typical TCs, the chd cluster contains a number of features that relate to its classification as 'atypical': an additional gene for a putative two-component cyclase/aromatase that may be responsible for the different aromatization pattern, a gene for a putative aminotransferase for C-4 with the opposite stereochemistry to TCs and a gene for a putative C-9 methylase that is a unique feature of this biosynthetic cluster within the TCs. Collectively, these enzymes deliver a molecule with different aromatization of ring C that results in an unusual planar structure of the TC backbone. This is a likely contributor to its different mode of action. In addition CHD biosynthesis is primed with acetate, unlike the TCs, which are primed with malonamate, and offers a biosynthetic engineering platform that represents a unique opportunity for efficient generation of novel tetracyclic backbones using combinatorial biosynthesis.

Engineering Atypical Tetracycline Formation in Amycolatopsis sulphurea for the Production of Modified Chelocardin Antibiotics.

To combat the increasing spread of antimicrobial resistance and the shortage of novel anti-infectives, one strategy for the development of new antibiotics is to optimize known chemical scaffolds. Here, we focus on the biosynthetic engineering of Amycolatopsis sulphurea for derivatization of the atypical tetracycline chelocardin and its potent broad-spectrum derivative 2-carboxamido-2-deacetyl-chelocardin. Heterologous biosynthetic genes were introduced into this chelocardin producer to modify functional groups and generate new derivatives. We demonstrate cooperation of chelocardin polyketide synthase with tailoring enzymes involved in biosynthesis of oxytetracycline from Streptomyces rimosus. An interesting feature of chelocardin, compared with oxytetracycline, is the opposite stereochemistry of the C4 amino group. Genes involved in C4 transamination and N,N-dimethylation of oxytetracycline were heterologously expressed in an A. sulphurea mutant lacking C4-aminotransferase. Chelocardin derivatives with opposite stereochemistry of the C4 amino group, as N,N-dimethyl- epi-chelocardin and N,N-dimethyl-2-carboxamido-2-deacetyl- epi-chelocardin, were produced only when the aminotransferase from oxytetracycline was coexpressed with the N-methyltransferase OxyT. Surprisingly, OxyT exclusively accepted intermediates carrying an S-configured amino group at C4 in chelocardin. Applying medicinal chemistry approaches, several 2-carboxamido-2-deacetyl- epi-chelocardin derivatives modified at C4 were produced. Analysis of the antimicrobial activities of the modified compounds demonstrated that the primary amine in the R configuration is a crucial structural feature for activity of chelocardin. Unexpectedly, C10 glycosylated chelocardin analogues were identified, thus revealing the glycosylation potential of A. sulphurea. However, efficient glycosylation of the chelocardin backbone occurred only after engineering of a dimethylated amino group at the C4 position in the opposite S configuration, which suggests some evolutionary remains of chelocardin glycosylation.

The Saccharomyces cerevisiae gene ECM11 is a positive effector of meiosis.

Ecm11 is classified as a protein involved in yeast cell wall biogenesis and organization, but in this paper, we provide evidence that it is involved in meiosis as well. Mutants with deleted ECM11 exhibit complex defects in meiosis: replication, recombination and chromosome segregation are affected. The ecm11Delta diploid strains sporulate more slowly and less efficiently than parental strains with wild type copies of ECM11. Fluorescence activated cell sorter scans of DNA content during sporulation showed that meiotic DNA synthesis is initiated at the same time in parental and ecm11Delta strains, but is less efficient in the knockout strain. By recombination tests, we demonstrated that ECM11 is required for crossing-over, but not for gene conversion. In the absence of ECM11 gene product, viability of spores is reduced to 50% and predominantly two viable spores per tetrad are formed. Our results suggest that ECM11 is required in early stages of meiosis where its function is related to DNA replication and crossing-over.

Comparison of Saccharomyces cerevisiae strains of clinical and nonclinical origin by molecular typing and determination of putative virulence traits.

Saccharomyces cerevisiae strains of clinical and nonclinical origin were compared by pulse field gel electrophoresis. Complete separation between strains of clinical origin and food strains by their chromosome length polymorphism was not obtained even though there was a tendency for the clinical and food strains to cluster separately. All the investigated strains, except for one food strain, were able to grow at temperatures > or =37 degrees C but not at 42 degrees C. Great strain variations were observed in pseudohyphal growth and invasiveness, but the characters were not linked to strains of clinical origin. The adhesion capacities of the yeast strains to a human intestinal epithelial cell line (Caco-2) in response to different nutritional availabilities were determined, as were the effects of the strains on the transepithelial electrical resistance (TER) across polarized monolayers of Caco-2 cells. The yeast strains displayed very low adhesion capacities to Caco-2 cells (0.6-6.2%), and no significant difference was observed between the strains of clinical and nonclinical origin. Both S. cerevisiae strains of clinical and non-clinical origin increased the TER of polarized monolayers of Caco-2 cells. Based on the results obtained in this study, no specific virulence factor was found that clearly separated the strains of clinical origin from the strains of nonclinical origin. On the contrary, all investigated strains of S. cerevisiae were found to strengthen the epithelial barrier function.