Microbial transformation of aklanonic acid, a potential early intermediate in the biosynthesis of anthracyclines.
Sir: Recently, we isolated a new pigment from fermentations of Streptomyces sp. ZIMET 43717". The close structural relationship of this compound (aklanonic acid, Ia, Fig. 1) to anthracyclinones induced us to attempt its biotransformation to anthracyclines by daunorubicin-negative mutants derived from different daunorubicin producing organisms. The results of our experiments provided evidence that aklanonic acid very probably is an early intermediate in the biosynthetic pathway of anthracyclines. Thus, aklanonic acid, its methyl ester, and aklanone, a chemical conversion product of aklanonic acid (Fig. 1)", were fed to fermentations of the mutant strains O3P; and IP,. Both mutant strains were isolated after N-methyl-N'nitro-N'-nitrosoguanidine treatment of daunorubicin-producing Streptomyces griseaus strains. Strain O1P; was derived from IMET JA 5142. Strain IP, is a negative mutant of strain 1P which produces a mixture of new red and blue pigments"'. The chemical structures of the two main red components designated 1P/I and IP/II3) are shown in Fig. 1. Fermentations of the strains O1P; and 1P, were carried out as described2) in 500-m1 flasks each containing 80 ml of medium for 24 hours. Then 3 ml of a solution of substrate in methanol (1 mg/ml) were added to each flask and cultivation continued for 24 hours. Extracts of mycelium with acetone and chloroform (1: 1) were purified by chromatography on KH2PO4 buffered silica gel (chloroform acetone, 10: 2). Identification of the biotransformation products was performed as indicated in Table 1. Glycosides extracted from the culture filtrate were hydrolyzed with 0.1 N HCI. Compound IP/II was purified by column chromatography on silica gel with benzene. The results are listed in Table 1. Aklanonic acid as well as its methyl ester were transformed by mutant O,P, to give e-rhodomycinone, 7deoxy-e-rhodomycinone and glycosides of dauno-
Nucleotide sequence analysis of five putative Streptomyces griseus genes, one of which complements an early function in daunorubicin biosynthesis that is linked to a putative gene cluster involved in TDP-daunosamine formation
Sequence analysis of the lkmB region of the daunorubicin biosynthetic gene cluster of Streptomyces griseus JA3933 revealed two contiguous open reading frames (ORF) in the same orientation, and three ORFs in the opposite orientation together extending over a 4.6 kb region adjacent to a homologue of the S. peucetius dnrJ gene. ORF1 complemented in trans the lkmB mutation, which seems to affect an early step in daunorubicin biosynthesis. Its deduced product showed no similarity to any known enzyme in the databases. The mutation in ORF1 was localised to a C-T transition at position 1172, leading to the change from a glycine to aspartic acid in the deduced protein. The lack of any homology to known polyketide synthesis enzymes indicates a regulatory role for the product of ORF1, despite the ability of lkmB mutants to further metabolise aklanonic acid. The genes of the oppositely oriented cluster seem to be involved in sugar metabolism. The putative ORF3 protein revealed strong homology to eukaryotic acyl CoA dehydrogenases and might encode an enzyme for the oxidoreduction preceding the introduction of the amino group into daunosamine, and the ORF4 protein is homologous to several epimerases, central enzymes in the formation of the l,-2,3,6-trideoxy-3-aminohexoses from TDP-d-glucose. ORF5 seems also to be related to enzymes metabolising nucleotide-activated hexoses.
A study of the local tissue reactions in man to cortisone and compound F. VI. Histopathological studies of the local effect of compound F in normal and pathologic skin of man.
After our previous negative studies (1) of the local effect of cortisone acetate in the skin of man, a year ago, a parallel study of 17-hydroxycorticosterone-21-acetate, Compound F, was outlined. Theoretically, Compound F should be the active steroid. It was so suggested by Rothman (2), that in all probability Compound F and not cortisone would be active locally in the skin of man.