A reinvestigation of the structure of mithramycin, the principal product of Streptomyces argillaceus ATCC 12956, is reported. The structure elucidation was carried out with mithramycin decaacetate (4) using 2D NMR methods, including TOCSY, HMBC, and HSQC experiments. The work resulted in structure 3being confirmed for mithramycin.

The potential of actinomycetes to produce natural products has been exploited for decades. Recent genomic sequence analyses have revealed a previously unrecognized biosynthetic potential and diversity. In order to rationally exploit this potential, we have developed a sequence-guided genetic screening strategy. In this "genome mining" approach, genes that encode tailoring enzymes from natural product biosyntheses pathways serve as indicator genes for the identification of strains that have the genetic potential to produce natural products of interest. We chose halogenases, which are known to be involved in the synthesis of halometabolites as representative examples. From PCR screening of 550 randomly selected actinomycetes strains, we identified 103 novel putative halogenase genes. A phylogenetic analysis of the corresponding putative halogenases, and the determination of their sequential context with mass spectrometric analysis of cultures filtrates revealed a distinct correlation between the sequence and secondary metabolite class of the halometabolite. The described screening strategy allows rapid access to novel natural products with predetermined structural properties.

Sweet success: A natural product glycosyltransferase, UrdGT2 from Streptomyces fradiae Tü2717, is the first glycosyltransferase able to catalyze both C‐glycosidic and O‐glycosidic sugar transfers. These results shed new light on the mechanisms of C‐glycosylation of aromatic compounds, which is significant for chemoenzymatic drug lead derivatization as well as for enzymology.

The glycosyltransferase LanGT2 is involved in the biosynthesis of the hexasaccharide side chain of the angucyclic antibiotic landomycin A. Its function was elucidated by targeted gene inactivation of lanGT2. The main metabolite of the obtained mutant was identified as tetrangulol (4), the progenitor of the landomycin aglycon (7). The lack of the sugar side chain indicates that LanGT2 catalyzes the priming glycosyl transfer in the hexasaccharide biosynthesis: the attachment of a D-olivose to O-8 of the polyketide backbone. Heterologous expression of urdGT2 from S. fradiae Tü2717 in this mutant resulted in the production of a novel C-glycosylated angucycline (6).

The plasmid-based reconstruction of the avr deoxysugar genes for expression in a heterologous system combined with biotransformation has led to new information about the mechanism of 2,6-deoxysugar biosynthesis. The structures of the di-demethyldeoxysugar avermectins accumulated indicate that in the oleandrose pathway the stereochemistry at C-3 is ultimately determined by the 3-O-methyltransferase and not by the 3-ketoreductase or a possible 3,5-epimerase. The AvrF protein is therefore a 5-epimerase and not a 3,5-epimerase. The ability of the AvrB (mono-)glycosyltransferase to accommodate different deoxysugar intermediates is evident from the structures of the novel avermectins produced.

Cosmid 16F4 contains 25 kb of the elloramycin biosynthetic pathway of Streptomyces olivaceus Tü2353. Transformation of this cosmid into a polyketide synthase (PKS)-deleted mutant of the urdamycin producer, Streptomyces fradiae Tü2717/ΔPKS and into the mithramycin producer Streptomyces argillaceus ATCC 12956 resulted in the production of several novel glycosylated tetracenomycins. Four of the structures of these elloramycin analogues (3, 5−7) were elucidated. They carry various deoxysugar moieties (d-olivose, l-rhodinose, d-mycarose, and a disaccharide consisting of two 1,3-linked d-olivoses) attached at C-8-O of the same aglycon, 8-demethyltetracenomycin C (4). The transfer of the sugars is not catalyzed by glycosyltransferases of the S. fradiae or S. argillaceus strains since the novel hybrid tetracenomycins are also produced by a S. argillaceus mutant carrying cosmid 16F4 but lacking all the known mithramycin glycosyltransferases. Furthermore, a Streptomyces lividans strain containing cosmid 16F4 produced the novel tetracenomycins only when a second plasmid containing the cloned mithramycin sugar biosynthetic genes but lacking glycosyltransferase genes was also present. The glycosyl transfer therefore must be catalyzed by an elloramycin glycosyltransferase encoded by cosmid 16F4. Apparently, this glycosyltransferase is able to catalyze the glycosylation of 8-demethyltetracenomycin C (4, = 12a-demethylelloramycinone) using various d- and l-sugars including a disaccharide. Its future use for combinatorial biosynthetic approaches is discussed.

Add sugar…︁ Cloning and heterologous expression of the aranciamycin biosynthetic gene cluster revealed a new flexible glycosyltransferase, AraGT, which accepts different nucleotide‐activated sugars (D‐amicetose, L‐rhodinose, L‐rhamnose and L‐axenose) The newly generated aranciamycin derivatives displayed antiproliferative activity against MaTu and MCF7 cells; this shows that the deoxysugar is important for anticancer activity.

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