R. G. Summers

Sequencing and mutagenesis of genes from the erythromycin biosynthetic gene cluster of Saccharopolyspora erythraea that are involved in L-mycarose and D-desosamine production

The nucleotide sequence on both sides of the eryA polyketide synthase genes of the erythromycin-producing bacterium Saccharopolyspora erythraea reveals the presence of ten genes that are involved in L-mycarose (eryB) and D-desosamine (eryC) biosynthesis or attachment. Mutant strains carrying targeted lesions in eight of these genes indicate that three (eryBIV, eryBV and eryBVI) act in L-mycarose biosynthesis or attachment, while the other five (eryCII, eryCIII, eryCIV, eryCV and eryCVI) are devoted to D-desosamine biosynthesis or attachment. The remaining two genes (eryBII and eryBVII) appear to function in L-mycarose biosynthesis based on computer analysis and earlier genetic data. Three of these genes, eryBII, eryCIII and eryCII, lie between the eryAIII and eryG genes on one side of the polyketide synthase genes, while the remaining seven, eryBIV, eryBV, eryCVI, eryBVI, eryCIV, eryCV and eryBVII lie upstream of the eryAI gene on the other side of the gene cluster. The deduced products of these genes show similarities to: aldohexose 4-ketoreductases (eryBIV), aldoketo reductases (eryBII), aldohexose 5-epimerases (eryBVII), the dnmT gene of the daunomycin biosynthetic pathway of Streptomyces peucetius (eryBVI), glycosyltransferases (eryBV and eryCIII), the AscC 3,4-dehydratase from the ascarylose biosynthetic pathway of Yersinia pseudotuberculosis (eryCIV), and mammalian N-methyltransferases (eryCVI). The eryCII gene resembles a cytochrome P450, but lacks the conserved cysteine residue responsible for coordination of the haem iron, while the eryCV gene displays no meaningful similarity to other known sequences. From the predicted function of these and other known eryB and eryC genes, pathways for the biosynthesis of L-mycarose and D-desosamine have been deduced.

Polyketide Synthases: Enzyme Complexes and Multifunctional Proteins Directing the Biosynthesis of Bacterial Metabolites from Fatty Acids

Microorganisms and plants produce from low-molecular weight fatty acids a collection of metabolites called polyketides that represent perhaps the largest group of secondary natural products1. These structurally diverse compounds typically contain oxygen atoms at alternate positions that are derived from the carbonyl groups of the fatty acid precursors by way of poly-β-ketoacylthioester intermediates. In fact, the name “polyketide” was coined about 100 years ago by Collie2,3 as the signature of a concept in which he imagined that poly- β -ketone intermediates could account for the products produced upon treatment of polyacetyl compounds with weak alkali, and for the characteristic hydroxylation pattern of some aromatic metabolites whose structures were known at that time. Biochemical support of his idea was not provided until 1953 by the insightful studies of Birch and co-workers4,5, who deduced from the isotopic labeling pattern of several fungal metabolites that they must have been made from acetic and malonic acids by a process like the biosynthesis of long-chain fatty acids. Polyketide chain growth must differ from fatty acid biosynthesis, however, because it lacks the faithful removal of each β -keto group, introduced by the condensation of acylSR (R = protein) and malonylSR intermediates, by an iterative reduction-dehydration-reduction process as in fatty acid biosynthesis. Further applications of the isotopic labeling method, augmented by the development of sophisticated nuclear magnetic resonance spectroscopic techniques6,7, led by the end of the 1980’s to a probable mechanism for the assembly and processing of poly- β -ketone intermediates in the early steps of polyketide biosynthesis. Synthesis of poly- β -ketones and -esters and studies of their behavior in solution when treated with acid or base, largely carried out by the Harris group8, provided important insights about the chemical reactivity of such compounds in vitro and additionally resulted in the total synthesis of several important natural products8,9.