Gabriele Weitnauer

Biosynthesis of the orthosomycin antibiotic avilamycin A: deductions from the molecular analysis of the avi biosynthetic gene cluster of Streptomyces viridochromogenes Tü57 and production of new antibiotics

BACKGROUND Streptomyces viridochromogenes Tü57 is the producer of avilamycin A. The antibiotic consists of a heptasaccharide side chain and a polyketide-derived dichloroisoeverninic acid as aglycone. Molecular cloning and characterization of the genes governing the avilamycin A biosynthesis is of major interest as this information might set the direction for the development of new antimicrobial agents. RESULTS A 60-kb section of the S. viridochromogenes Tü57 chromosome containing genes involved in avilamycin biosynthesis was sequenced. Analysis of the DNA sequence revealed 54 open reading frames. Based on the putative function of the gene products a model for avilamycin biosynthesis is proposed. Inactivation of aviG4 and aviH, encoding a methyltransferase and a halogenase, respectively, prevented the mutant strains from producing the complete dichloroisoeverninic acid moiety resulting in the accumulation of new antibiotics named gavibamycins. CONCLUSIONS The avilamycin A biosynthetic gene cluster represents an interesting system to study the formation and attachment of unusual deoxysugars. Several enzymes putatively responsible for specific steps of this pathway could be assigned. Two genes encoding enzymes involved in post-PKS tailoring reactions were deleted allowing the production of new analogues of avilamycin A.

The avilamycin resistance determinants AviRa and AviRb methylate 23S rRNA at the guanosine 2535 base and the uridine 2479 ribose

Avilamycin is an orthosomycin antibiotic that has shown considerable potential for clinical use, although it is presently used as a growth promoter in animal feed. Avilamycin inhibits bacterial protein synthesis by binding to the 50S ribosomal subunit. The ribosomes of the producer strain, Streptomyces viridochromogenes Tü57, are protected from the drug by the action of three resistance factors located in the avilamycin biosynthetic gene cluster. Two of the resistance factors, aviRa and aviRb, encode rRNA methyltransferases that specifically target 23S rRNA. Recombinant AviRa and AviRb proteins retain their activity after purification, and both specifically methylate in vitro transcripts of 23S rRNA domain V. Reverse transcriptase primer extension indicated that AviRa is an N‐methyltransferase that targets G2535 within helix 91 of the rRNA, whereas AviRb modified the 2′‐O‐ribose position of nucleotide U2479 within helix 89. MALDI mass spectrometry confirmed the exact positions of each of these modifications, and additionally established that a single methyl group is added at each nucleotide. Neither of these two nucleotides have previously been described as a target for enzymatic methylation. Molecular models of the 50S subunit crystal structure show that the N−1 of the G2535 base and the 2′‐hydroxyl of U2479 are separated by approximately 10 Å, a distance that can be spanned by avilamycin. In addition to defining new resistance mechanisms, these data refine our understanding of the probable ribosome contacts made by orthosomycins and of how these antibiotics inhibit protein synthesis.

An ATP-Binding Cassette Transporter and Two rRNA Methyltransferases Are Involved in Resistance to Avilamycin in the Producer Organism Streptomyces viridochromogenes Tü57

ABSTRACT Three different resistance factors from the avilamycin biosynthetic gene cluster of Streptomyces viridochromogenes Tü57, which confer avilamycin resistance when expressed in Streptomyces lividans TK66, were isolated. Analysis of the deduced amino acid sequences showed that AviABC1 is similar to a large family of ATP-binding transporter proteins and that AviABC2 resembles hydrophobic transmembrane proteins known to act jointly with the ATP-binding proteins. The deduced amino acid sequence of aviRb showed similarity to those of other rRNA methyltransferases, and AviRa did not resemble any protein in the databases. Independent expression inS. lividans TK66 of aviABC1 plus aviABC2, aviRa, or aviRb conferred different levels of resistance to avilamycin: 5, 10, or 250 μg/ml, respectively. When either aviRa plus aviRb or aviRaplus aviRb plus aviABC1 plusaviABC2 was coexpressed in S. lividans TK66, avilamycin resistance levels reached more than 250 μg/ml. Avilamycin A inhibited poly(U)-directed polyphenylalanine synthesis in an in vitro system using ribosomes of S. lividans TK66(pUWL201) (GWO),S. lividans TK66(pUWL201-Ra) (GWRa), or S. lividans TK66(pUWL201-Rb) (GWRb), whereas ribosomes of S. lividans TK66 containing pUWL201-Ra+Rb (GWRaRb) were highly resistant. aviRa and aviRb were expressed inEscherichia coli, and both enzymes were purified as fusion proteins to near homogeneity. Both enzymes showed rRNA methyltransferase activity using a mixture of 16S and 23S rRNAs fromE. coli as the substrate. Coincubation experiments revealed that the enzymes methylate different positions of rRNA.

Genes Involved in Formation and Attachment of a Two-Carbon Chain as a Component of Eurekanate, a Branched-Chain Sugar Moiety of Avilamycin A

ABSTRACT Eurekanate belongs to the important class of branched-chain carbohydrates present in a wide variety of natural sources. It is a component of avilamycin A, a potent inhibitor of bacterial protein synthesis targeting the 50S ribosomal subunit. The present work provides experimental proof for the function of two genes of the avilamycin biosynthetic gene cluster, aviB1 and aviO2, that are both involved in avilamycin structure modification. The functions of both genes were identified by gene inactivation experiments and nuclear magnetic resonance analyses of extracts produced by the mutants. We suggest that both AviO2 and AviB1 are involved in the biosynthesis of eurekanate within avilamycin biosynthesis. Moreover, two other genes (aviO1 and aviO3) have been inactivated, resulting in a breakdown of avilamycin production in the mutants ITO1 and ITO3, which clearly shows the essential role of both enzymes in avilamycin biosynthesis. The exact functions of both aviO1 and aviO3 remained unknown.

Analysis of a C-methyltransferase gene (aviG1) involved in avilamycin biosynthesis in streptomyces viridochromogenes Tü57 and complementation of a Saccharopolyspora erythraea eryBIII mutant by aviG1

Streptomyces viridochromogenes Tü57 is the principal producer of avilamycin A. aviG1, a putative methyltransferase gene, was detected in the avilamycin biosynthetic gene cluster. To determine the function of aviG1, a targeted gene inactivation experiment was performed. The resulting chromosomal mutant, carrying an in-frame deletion in aviG1, was deficient in avilamycin production. aviG1 was used to complement an eryBIII mutant of the erythromycin A producer Saccharopolyspora erythraea [Gaisser, S., Bohm, G. A., Doumith, M., Raynal, M. C., Dhillon, N., Cortes, J. & Leadlay, P. F. (1998). Mol Gen Genet 258, 78-88]. The presence of erythromycin A in the culture supernatant of the complemented mutant indicated that L-mycarose biosynthesis could be restored and that AviG1 could take over the function of the C-methyltransferase EryBIII.

Structure alteration of polyketides by recombinant DNA technology in producer organisms--prospects for the generation of novel pharmaceutical drugs.

Actinomycetes are gram-positive bacteria and commercially important microorganisms. They are producers of approximately two thirds of all bioactive compounds known and they produce a great variety of compounds which have clinical application on the basis of their activity against different kinds of organisms and cells as antibacterial (macrolides, avermectins), antitumor (anthracyclines, angucyclines, aureolic acid group) and also compounds showing immunosuppresant activity (rapamycin, FK506). Most of these clinically useful pharmaceuticals produced by actinomycetes belong to the polyketide family. Polyketides comprise a wide family of chemically diverse compounds, many of which have shown bioactivity. The development of recombinant DNA technology has opened a new and exciting field of research for the generation of new bioactive compounds through genetic manipulation of the biosynthetic pathways. Researchers in this area are trying to take advantage of the enormous capability of actinomycetes to produce pharmaceutically useful compounds in order to manipulate the different biosynthetic pathways and subsequently generate novel drugs. Combinatorial biosynthesis is now emerging as a powerful tool to generate novel families of compounds by interchanging secondary metabolism genes between bioactive producing actinomycetes. Novel compounds will be the consequence of the concerted action of enzymes from different, but related, biosynthetic pathways. Insertional inactivation of selected genes and tailoring modification may also produce novel compounds that can be useful pharmaceuticals or lead compounds for further chemical modification. This minireview will present the state of the art in this field showing the different polyketides biosynthetic pathways so far characterized and how the identified genes are being used to generate structural biodiversity. Emphasis will be made on the polyketide family including type I and type II polyketides.

Glycosyltransferases and other tailoring enzymes as tools for the generation of novel compounds.

Glycosyltransferases are a very important class of enzymes which can be found in biosynthetic gene clusters of a variety of natural compounds. Some of these GTs show a remarkable flexibility towards the donor and the acceptor molecules making them most valuable for combinatorial biosynthesis. Future work is expected to focus on learning more about sugar biosynthesis, sugar modification and sugar attachment to support in vivo engineering of novel natural products.