Cloning, characterization, and high-level expression in Escherichia coli of the Saccharopolyspora erythraea gene encoding an acyl carrier protein potentially involved in fatty acid biosynthesis

Abstract

The erythromycin A-producing polyketide synthase from the gram-positive bacterium Saccharopolyspora erythraea (formerly Streptomyces erythraeus) has evident structural similarity to fatty acid synthases, particularly to the multifunctional fatty acid synthases found in eukaryotic cells. Fatty acid synthesis in S. erythraea has previously been proposed to involve a discrete acyl carrier protein (ACP), as in most prokaryotic fatty acid synthases. We have cloned and sequenced the structural gene for this ACP and find that it does encode a discrete small protein. The gene lies immediately adjacent to an open reading frame whose gene product shows sequence homology to known ,I-ketoacyl-ACP synthases. A convenient expression system for the S. erythraea ACP was obtained by placing the gene in the expression vector pT7-7 in Escherichia coli. In this system the ACP was efficiently expressed at levels 10 to 20% of total cell protein. The recombinant ACP was active in promoting the synthesis of branched-chain acyl-ACP species by extracts of S. erythraea. Electrospray mass spectrometry is shown to be an excellent method for monitoring the efficiency of in vivo posttranslational modification of ACPs.The macrolide antibiotic erythromycin A is produced by the gram-positive filamentous bacterium Saccharopolyspora erythraea (formerly Streptomyces erythraeus [32]), starting from simple fatty acyl-coenzyme A (CoA) precursors. The biosynthesis of erythromycin and other polyketides shows important similarities (25) to the more familiar process of fatty acid biosynthesis, and this has led to renewed interest in the structure, organization, and mechanism of action of fatty acid synthase in S. erythraea and in Streptomyces spp. Initial reports indicated that S. erythraea (39) and Streptomyces coelicolor (17) might contain a type I fatty acid synthase complex like that of Mycobacterium smegmatis (9), Brevibacterium ammoniagenes (30), or Saccharomyces cerevisiae (43, 48), in which multifun.ctional polypeptides are tightly associated in a complex of high molecular weight. More recently, a small discrete acyl carrier protein (ACP) has been identified and purified from S. erythraea (21) on the basis of its ability to stimulate the incorporation of malonylCoA into acyl-ACP in a cell-free system. This implies that the fatty acid synthase of S. erythraea may be patterned on the type II system found generally in prokaryotes, consisting of freely dissociable, monofunctional enzyme components. The best-studied system of this kind is that of Escherichia coli (see reference 52 for a recent review), but a discrete ACP is also apparently involved in fatty acid synthase from gram-positive bacteria more closely related to S. erythraea (1, 27) and which, like S. erythraea, characteristically produce iso-and anteiso-terminally branched fatty acids. Alternatively, there may exist more than one type of fatty acid synthase activity, as in Euglena gracilis (13). The recent (11) finding that the erythromycin-producing polyketide synthase of S. erythraea is a ...

Post‐translational modification of heterologously expressed Streptomyces type II polyketide synthase acyl carrier proteins

“…Our results, and those of Hutchinson [10], show that the type II PKS ACPs are clearly poor in vivo substrates for the endogenous E. coli ACPS. This contrasts with the S. erythrae FAS ACP which appears to be a relatively good in vivo substrate of E. coli ACPS [16,17]. In addition otc ACP could also cause substrate inhibition.…”

“…The results of Leadlay et al would appear to support this hypothesis. They showed that the heterologous expression of a presumed FAS ACP from the Actinomycete Saccharopolyspora erythrae in E. coli yielded high levels (20 mg 1 _1 ) of predominantly (65%) holo-ACP [16,17]. Clearly in this case CoA and Mg 2+ cannot be limiting.…”

In vivo and in vitro effects of thiolactomycin on fatty acid biosynthesis in Streptomyces collinus

“…Such synthases are large, nondissociable, multifunctional complexes which are generally found in yeast and mammalian systems. More recent work has suggested that streptomycetes have a type II FAS; such synthases consist of a series of dissociable enzymes (24,32). The type II FAS is more commonly found in bacterial systems and has been extensively characterized in Escherichia coli (17).…”

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

“…Modular systems like erythromycin have a single ACP/module, one for each condensation cycle the growing polyketide chain undergoes before the completed chain is released, so that each ACP domain is used only once in the course of each biosynthetic cycle (11). Most of the Type II systems in bacteria contain a single ACP that works iteratively with other PKS enzymes performing the dual function of receiving the chain extender unit before condensation and of holding the growing polyketide chain after each condensation to produce the polyketide (12,13).…”