Generation of New Landomycins by Combinatorial Biosynthetic Manipulation of the LndGT4 Gene of the Landomycin E Cluster in S. globisporus

Abstract

A 3 kb DNA fragment from the Streptomyces globisporus 1912 landomycin E (LaE) biosynthetic gene cluster (lnd) was completely sequenced. Three open reading frames were identified, lndGT4, lndZ4, and lndZ5, whose probable translation products resemble a glycosyltransferase, a reductase, and a hydroxylase, respectively. Studies of generated mutants from disruption and complementation experiments involving the lndGT4 gene allowed us to determine that LndGT4 controls the terminal L-rhodinose sugar attachment during LaE biosynthesis and that LndZ4/LndZ5 are responsible for the unique C11-hydroxylation of the landomycins. Generation of the novel landomycins F, G, and H in the course of these studies provided evidence for the flexibility of lnd glycosyltransferases toward their acceptor substrates and a basis for initial structure-activity relationships within the landomycin family of antibiotics.

Identification of the Function of Gene lndM2 Encoding a Bifunctional Oxygenase-Reductase Involved in the Biosynthesis of the Antitumor Antibiotic Landomycin E by Streptomyces globisporus 1912 Supports the Originally Assigned Structure for Landomycinone

“…The disappearance of the fed metabolites 5 and 9 as well as the appearance of landomycin E (1) was monitored by HPLC-MS; a peak not observable in a control experiment (in which no compound was added) after feeding with tetrangomycin or rabelomycin was identified to be landomycin E according to its HPLC relative retention time and its UV and MS spectra. 12 No such transformation was detected in the corresponding cultures after feeding with the same amount of the other three compounds. Accordingly, it was concluded that tetrangomycin (5) and rabelomycin (9) are intermediates in the biosynthesis of landomycin E, while the other three metabolites (compounds 6-8) yielded by S. globisporus M12 strain are shunt products.…”

“…Whereas the formation of tetrangulol (8) is most likely due to a spontaneous dehydration and aromatization of ring A of 5 22,23 and the formation of shunt product 6 can be explained by a certain flexibility of the enzymes LndZ4/Z5, which are responsible for the 11-hydroxylation, 12 the formation of angucyclinone 7 cannot readily be explained, because a 4-hydroxylation has never been observed before in any of the landomycins, neither from S. globisporus nor from S. cyanogenus. Nevertheless, this shunt pathway toward 7 might be caused by one of the two remaining oxygenases of the lnd gene cluster, e.g., LndE, or more likely by an oxygenase of S. globisporus, which is not part of the lnd pathway.…”

“…The fact that S. globisporus M12 does not produce any glycosylated metabolites suggests that the 6-oxygenation and de-aromatization of ring B are crucial early biosynthetic steps, shaping the landomycin aglycon for its recognition by the first glycosyltransferase, Lnd/LanGT2. 12 However, at this stage it could not be distinguished whether the monooxygenation of the 6-position is preceded or followed by a reduction step in order to yield the partly saturated ring B. This reduction step is most likely catalyzed by the reductase domain of LndM2, which therefore acts either as a ketoreductase (Scheme 4, path A via rabelomycin) or as saturase (or enereductase, path B, Scheme 4), the former being more likely than the latter as a result of the sequential arrangement in the gene lndM2.…”

“…2,11 Only recently we could identify the reductase/oxygenase pair LndZ4/Z5 as being responsible for the introduction of the 11-hydroxy function of the landomycin aglycon. 12 The incorporation experiments left some ambiguity particularly with respect to the oxygen in 6-position, because the 6-oxygen, although in a suitable position for being acetate-derived, clearly was not 18 O-labeled by [1-13 C, 18 O 2 ] acetate, while the fermentation in an 18 O 2 -enriched atmosphere yielded anhydrolandomycin A (4) lacking the 6-oxygen (Scheme 2). 2 In this context it is interesting that Roush et al recently suggested a revised structure of landomycin A with the hydroxy group in the 5-position instead of in the 6-position.…”

Inactivation of gilGT, Encoding a C‐Glycosyltransferase, and gilOIII, Encoding a P450 Enzyme, Allows the Details of the Late Biosynthetic Pathway to Gilvocarcin V to be Delineated

“…The highest homologies were found with LanGT2 (38% amino-acid identity), a GT presumed to be involved in attaching the first D-olivose to landomycin aglycon, [13][14][15] and UrdGT2 (31% amino-acid identity), which is the C-GT of the urdamycin biosynthetic pathway. [16][17][18] The previously reported putative GilGT protein had 495 amino acids, which is approximately 120 amino acids longer than any other polyketide GT found so far.…”