Deciphering the late steps in the biosynthesis of the anti-tumour indolocarbazole staurosporine: sugar donor substrate flexibility of the StaG glycosyltransferase

Abstract

The indolocarbazole staurosporine is a potent inhibitor of a variety of protein kinases. It contains a sugar moiety attached through C-N linkages to both indole nitrogen atoms of the indolocarbazole core. Staurosporine biosynthesis was reconstituted in vivo in a heterologous host Streptomyces albus by using two different plasmids: the 'aglycone vector' expressing a set of genes involved in indolocarbazole biosynthesis together with staG (encoding a glycosyltransferase) and/or staN (coding for a P450 oxygenase), and the 'sugar vector' expressing a set of genes responsible for the biosynthesis of the sugar moiety. Attachment of the sugar to the two indole nitrogens of the indolocarbazole core was dependent on the combined action of StaG and StaN. When StaN was absent, the sugar was attached only to one of the nitrogen atoms, through an N-glycosidic linkage, as in the indolocarbazole rebeccamycin. The StaG glycosyltransferase showed flexibility with respect to the sugar donor. When the 'sugar vector' was substituted by constructs directing the biosynthesis of l-rhamnose, L-digitoxose, L-olivose and D-olivose, respectively, StaG and StaN were able to transfer and attach all of these sugars to the indolocarbazole aglycone.

Fig. 1

Chemical structures of indolocarbazoles.

Fig. 2

HPLC analysis of cultures of S. albus cos32D (A), and of S. albus 39GT(pETAS) (B) and chemical structure of holyrine A (only found with sugar in ⁴C₁-conformation) (C).

Fig. 3

Plasmid constructs used in this work. Constructs pRHAM (Rodriguez et al., 2000), pLN2 and pLNR (Rodriguez et al., 2002) and pLNBIV (Fischer et al., 2002) have been already described. They contain different sets of genes involved in the biosynthesis of L-olivose in Streptomyces antibioticus (ole genes), L-mycarose in Saccha-ropolyspora erythraea (eryB gene) or L-rhodinose in Streptomyces fradiae (urdR gene). The black triangle indicate the position of the erythromycin resistance promoter ermE*p.

Fig. 4

HPLC analysis of cultures of recombinant strains expressing different sugar plasmids. A. S. albus 39GNT(pRHAM). B. S. albus 39GNT(pLN2). C. S. albus 39GNT(pLNR). D. S. albus 39GNT(pLNBIV). 1: L-rhamnosyl-2N-K252c; 2: L-rhamnosyl-1N-K252c; 3: L-olivosyl-2N-K252c; 4: L-olivosyl-1N-K252c; 5: D-olivosyl-1N-K252c; 6: L-digitoxosyl-1N-K252c; 7: L-digitoxosyl-2N-K252c.

Fig. 5

Chemical structures of the novel glycosylated staurosporine derivatives. The sugar residues were found exclusively in ⁴C₁-conformation, when singly attached (1 N compounds), but were found in ¹C₄-conformation, when doubly attached (2 N compounds)

Fig. 6

Proposed pathway for the late staurosporine biosynthesis.