Genetic construction and functional analysis of hybrid polyketide synthases containing heterologous acyl carrier proteins

Abstract

The gene that encodes the acyl carrier protein (ACP) of the actinorhodin polyketide synthase (PKS) of Streptomyces coelicolor A3(2) was replaced with homologs from the granaticin, oxytetracycline, tetracenomycin, and putative frenolicin polyketide synthase gene clusters. All of the replacements led to expression of functional synthases, and the recombinants synthesized aromatic polyketides similar in chromatographic properties to actinorhodin or to shunt products produced by mutants defective in the actinorhodin pathway. Some regions within the ACP were also shown to be interchangeable and allow production of a functional hybrid ACP. Structural analysis of the most abundant polyketide product of one of the recombinants by electrospray mass spectrometry suggested that it is identical to mutactin, a previously characterized shunt product of an actVII mutant (deficient in cyclase and dehydrase activities). Quantitative differences in the product profiles of strains that express the various hybrid synthases were observed. These can be explained, at least in part, by differences in ribosome-binding sites upstream of each ACP gene, implying either that the ACP concentration in some strains is rate limiting to overall PKS activity or that the level of ACP expression also influences the expression of another enzyme(s) encoded by a downstream gene(s) in the same operon as the actinorhodin ACP gene. These results reaffirm the idea that construction of hybrid polyketide synthases will be a useful approach for dissecting the molecular basis of the specificity of PKS-catalyzed reactions. However, they also point to the need for reducing the chemical complexity of the approach by minimizing the diversity of polyketide products synthesized in strains that produce recombinant polyketide synthases.Polyketides include antibiotics such as oxytetracycline and erythromycin made by actinomycetes, fungal toxins such as aflatoxin, and plant anthocyanins. Chemical and biochemical evidence (21, 30) has long pointed to a similarity between (i) the mechanism of fatty acid synthesis, in which the fatty acid synthase (FAS) assembles a long carbon chain by successive decarboxylative condensation of simple carboxylic acids, with a round of P-ketoreduction, dehydration, and enoylreduction after most or all of the condensation steps, and (ii) the mechanism of polyketide synthesis by polyketide synthases (PKS). The chief difference is in the omission of many of the reductive steps in polyketide assembly. The recent sequencing of many polyketide-biosynthetic genes has shown that this similarity actually reflects a clear evolutionary relationship between PKS and FAS genes (see references 13, 14, and 22 for reviews). In particular, there are striking end-to-end similarities in the amino acid sequences of P-ketoacyl-acyl carrier protein (ACP) synthase (condensing enzyme) domains or subunits between and within groups of PKSs and FASs. One also observes a high degree of sequence similarity around the 4

Kinetic Analysis of the Actinorhodin Aromatic Polyketide Synthase

“…The observation that ACP is needed in substantially more than stoichiometric amounts compared with the KS/CLF for maximal activity is consistent with earlier genetic studies where alterations in expression levels of the ACP gene resulted in significant differences in polyketide production in vivo (28). It suggests that the ACP interacts only loosely with the PKS core comprised of the KS and CLF.…”

Tandemly Duplicated Acyl Carrier Proteins, Which Increase Polyketide Antibiotic Production, Can Apparently Function Either in Parallel or in Series

“…Quantitative differences in hybrid PKS products were attributed to the differences in the upstream ribosomal binding sites, which probably affect the level of expression or efficiency of translation. The strong quantitative variation in total aromatic polyketide production was found to be either because of limiting ACP concentration or some level of translational coupling (41). Many in vitro and in vivo studies have confirmed that by increasing ACP concentration the level of antibiotic production can be increased.…”

Isolation and sequence analysis of polyketide synthase genes from the daunomycin-producing Streptomyces sp. strain C5

“…Only in the cases of the tetracenomycin (40) and actinorhodin (5,19,34,55) PKSs, however, have biochemical characterizations also been carried out. Thus, most of the information on type II PKSs is derived from the strong conservation of gene structure among the PKS genes (23,25) and cross-functionality of the components (5,26,34,41). We describe here the structure of a gene region from Streptomyces sp.…”