Phenalinolactones are terpene glycosides with antibacterial activity. A striking structural feature is a highly oxidized gamma-butyrolactone of elusive biosynthetic origin. To investigate the genetic basis of the phenalinolactones biosynthesis, we cloned and sequenced the corresponding gene cluster from the producer strain Streptomyces sp. Tü6071. Spanning a 42 kbp region, 35 candidate genes could be assigned to putatively encode biosynthetic, regulatory, and resistance-conferring functions. Targeted gene inactivations were carried out to specifically manipulate the phenalinolactones pathway. The inactivation of a sugar methyltransferase gene and a cytochrome P450 monoxygenase gene led to the production of modified phenalinolactone derivatives. The inactivation of a Fe(II)/alpha-ketoglutarate-dependent dioxygenase gene disrupted the biosynthetic pathway within gamma-butyrolactone formation. The structure elucidation of the accumulating intermediate indicated that pyruvate is the biosynthetic precursor of the gamma butyrolactone moiety.
Detailed studies on the biosynthesis of the hexasaccharide side chain of landomycin A, produced by S. cyanogenus S136, revealed the function of each glycosyltransferase gene of the biosynthetic gene cluster. Analyses of generated mutants as well as feeding experiments allowed us to determine that LanGT2 and LanGT3 catalyze the attachment of one sugar, whereas LanGT1 and LanGT4 attach two sugars during landomycin A biosynthesis. The generation of a lanZ2 deletion mutant provided evidence that LanZ2 is controlling the elongation of the saccharide side chain.
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.
Two genes from Streptomyces cyanogenous S136 that encode the reductase LanZ4 and the hydroxylase LanZ5, which are involved in landomycin A biosynthesis, were characterized by targeted gene inactivation. Analyses of the corresponding mutants as well as complementation experiments have allowed us to show that LanZ4 and LanZ5 are responsible for the unique C-11-hydroxylation that occurs during landomycin biosynthesis. Compounds accumulated by the lanZ4/Z5 mutants are the previously described landomycin F and the new landomycins M and O.
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).
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