Characterization of mutations in aclacinomycin A-non-producing Streptomyces galilaeus strains with altered glycosylation patterns

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Abstract
In this study a set of Streptomyces galilaeus ATCC 31615 mutants was characterized, which are incapable of synthesizing some or all of the deoxyhexose sugars of aclacinomycin A. Complementation experiments with the the mutant strains H026, H038, H039, H054, H063, H065 and H075 were carried out with glycosylation genes previously derived from the wild-type S. galilaeus. Mutations in strains H038, H063 and H075 were complemented with single PCR-amplified genes. Furthermore, amplification and sequencing of the corresponding genes from the mutant strains revealed single point mutations in the sequences. First, in H038 a transition mutation in aknQ, encoding a putative dTDP-hexose 3-ketoreductase, causes an amino acid substitution from glycine to aspartate, suppressing the biosynthesis of both 2-deoxyfucose and rhodinose and thus leading to the accumulation of aclacinomycin T with rhodosamine as its only sugar. Second, in H063, which accumulates aklavinone without a sugar moiety, amino acid substitution occurs, with threonine being substituted by isoleucine in dTDP-glucose synthase, the first enzyme participating in deoxyhexose biosynthesis, encoded by aknY. Third, a nonsense mutation in aknP leads to truncated dTDP-hexose 3-dehydratase in H075, which is incapable of synthesizing rhodinose. In addition, mutants H054 and H065, which accumulate aclacinomycins without aminosugars, were complemented by a gene for an aminotransferase, aknZ. Characterization of the nature of the mutations adds to the usefulness and value of the mutants in the analysis of gene function and in the creation of novel compounds by combinatorial biosynthesis. Furthermore, these results strengthen the assignments of akn gene products and enlighten the biosynthetic pathway for deoxyhexoses.

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