Günter A. Böhm

Analysis of seven genes from the eryAI –eryK region of the erythromycin biosynthetic gene cluster in Saccharopolyspora erythraea

Abstract The gene cluster (ery) governing the biosynthesis of the macrolide antibiotic erythromycin A by Saccharopolyspora erythraea contains, in addition to the eryA genes encoding the polyketide synthase, two regions containing genes for later steps in the pathway. The region 5′ of eryA, and lying between eryA and the gene eryK, which is known to encode the C-12 hydroxylase, has been sequenced and shown to contain seven additional open reading frames (ORFs 13–19). On the basis of sequence similarities, roles are proposed for several of these ORFs in the biosynthesis of the deoxysugar mycarose and the deoxyaminosugar desosamine. A chromosomal mutant carrying a deletion in ORF15 has been constructed and shown to accumulate 3-O-mycarosyl-erythronolide B, as expected for an eryC mutant. Similarly, a chromosomal mutant carrying a deletion in ORF16 has been constructed and shown to accumulate erythronolide B, as expected for an eryB mutant.

Analysis of eryBI, eryBIII and eryBVII from the erythromycin biosynthetic gene cluster in Saccharopolyspora erythraea

Abstract The gene cluster (ery) governing the biosynthesis of the macrolide antibiotic erythromycin A by Saccharopolyspora erythraea contains, in addition to the eryA genes encoding the polyketide synthase, two regions containing genes for later steps in the pathway. The region 5′ of eryA that lies between the known genes ermE (encoding the erythromycin resistance methyltransferase) and eryBIII (encoding a putative S-adenosylmethionine-dependent methyltransferase), and that contains the gene eryBI (orf2), has now been sequenced. The inferred product of the eryBI gene shows striking sequence similarity to authentic β-glucosidases. Specific mutants were created in eryBI, and the resulting strains were found to synthesise erythromycin A, showing that this gene, despite its position in the biosynthetic gene cluster, is not essential for erythromycin biosynthesis. A␣mutant in eryBIII and a double mutant in eryBI and eryBIII were obtained and the analysis of novel erythromycins produced by these strains confirmed the proposed function of EryBIII as a C-methyltransferase. Also, a chromosomal mutant was constructed for the previously sequenced ORF19 and shown to accumulate erythronolide B, as expected for an eryB mutant and consistent with its proposed role as an epimerase in dTDP-mycarose biosynthesis.

New rapamycin derivatives by precursor-directed biosynthesis

Rapamycin (1) is a polyketide macrolide produced by Streptomyces hygroscopicus that displays potent immunosuppressant activity. In recent years there has been great interest in the chemistry and biology of rapamycin, and of the structurally similar immunosuppressants FK506 and FK520. We now describe results that promise to broaden the scope for biosynthetic engineering of new rapamycin and FK506/FK520 derivatives. Feeding studies have confirmed the largely polyketide origin of rapamycin, and have shown that the unusual trisubstituted cyclohexane ring arises from shikimate. The entire biosynthetic gene cluster for rapamycin was sequenced by Leadlay, Staunton and co-workers in 1994. The rapamycin polyketide synthase (PKS) is a type I mixed PKS/non-ribosomal peptide synthetase (NRPS) system in which 14 polyketide chain-extension modules are housed in three giant multimodular proteins (RAPS1 ±3). An NRPS-related multienzyme (pipecolate-incorporating enzyme) inserts pipecolate and (probably) ensures closure of the macrocycle. The genes encoding the rapamycin PKS (RAPS) have proved an excellent resource for combinatorial biosynthesis, owing in part to the diverse chemical functionality of the polyketide product. DNA from the rap PKS genes encoding individual and multiple enzyme activities and even whole chain-extension modules, has been spliced into the genes for other PKSs to generate functional, hybrid PKSs. In contrast, despite the potential clinical value of the products, this technology has not yet been extensively applied to engineer the biosynthesis of novel rapamycins. We previously identified 4,5-dihydroxycyclohex-1-enecarboxylic acid (2) as the most likely starter unit for the rapamycin PKS (Scheme 1). We have proposed that this substrate is activated

A Polylinker Approach to Reductive Loop Swaps in Modular Polyketide Synthases

Multiple versions of the DEBS 1‐TE gene, which encodes a truncated bimodular polyketide synthase (PKS) derived from the erythromycin‐producing PKS, were created by replacing the DNA encoding the ketoreductase (KR) domain in the second extension module by either of two synthetic oligonucleotide linkers. This made available a total of nine unique restriction sites for engineering. The DNA for donor “reductive loops,” which are sets of contiguous domains comprising either KR or KR and dehydratase (DH), or KR, DH and enoylreductase (ER) domains, was cloned from selected modules of five natural PKS multienzymes and spliced into module 2 of DEBS 1‐TE using alternative polylinker sites. The resulting hybrid PKSs were tested for triketide production in vivo. Most of the hybrid multienzymes were active, vindicating the treatment of the reductive loop as a single structural unit, but yields were dependent on the restriction sites used. Further, different donor reductive loops worked optimally with different splice sites. For those reductive loops comprising DH, ER and KR domains, premature TE‐catalysed release of partially reduced intermediates was sometimes seen, which provided further insight into the overall stereochemistry of reduction in those modules. Analysis of loops containing KR only, which should generate stereocentres at both C‐2 and C‐3, revealed that the 3‐hydroxy configuration (but not the 2‐methyl configuration) could be altered by appropriate choice of a donor loop. The successful swapping of reductive loops provides an interesting parallel to a recently suggested pathway for the natural evolution of modular PKSs by recombination.