Gene clusters actinorhodin

A 26-kb gene cluster encoding enzymes for synthesis of the blue antibiotic actinorhodin by Streptomyces coelicolor has been cloned and sequenced.332337 The three large 10-kb genes required for formation of the broad-spectrum antibiotic erythromycin by Saccharopolyspora erythraea have also been cloned and sequenced.337 339 In both cases, the genes... 

Figure 3 Aromatic PKS gene clusters and actinorhodin biosynthesis. Partial gene clusters for the polyketides actinorhodin (act), tetracenomycin (tcm), and doxorubicin (dps) are shown. Reconstitution of combinations of act genes in a PKS clean host have led to a proposed pathway by which the early stages of actinorhodin biosynthesis occur.

Figure 10 Examples of unnatural aromatic polyketides. Libraries of aromatic poly-ketides have been generated using combinations from the actinorhodin, tetracenomycin, frenolicin, griseusin, and whiE spore pigment gene clusters.

Scheme 3 The actinorhodin PKS gene cluster and proposed "processive assembly and cyclisation pathway catalysed by "minimal PKS and further components...

Figure 4. Organization of representative type 1, II, and III polyketide synthases. Upper modular arrangement of DEBS 1,2,3 subunits Center orientation and arrangement of open reading frames in actinorhodin gene cluster Lower chalcone synthase subunit. AT, acyltransferase ACP, acyl carrier protein KS, ketosynthase KR, ketoreductase DH, dehydratase ER, enoyl reductase TE, thioesterase TA, tailoring enzyme R/T, regulatory/transport related AR aromatase CY, cyclase.

As mentioned above, iterative PKSs have been studied in detail, focusing especially on the biosynthesis of actinorhodin and tetracenomycin. Later, further genes and gene clusters have become available. In this section we describe how knowledge of the function of these enzymatic systems can be used to generate new molecules. The most important results are summarized in Figure 12.1 la and b. 

Gaballero, J.L., Martinez, E., Malpartida, F. Hopwood, D.A. Organisation and functions of the actVA region of the actinorhodin biosynthetic gene cluster of Streptomyces coelicolor. Mol. Gen. Genet. 230, 401-412 (1991). 

Since the PKS (polyketide synthase) gene cluster for actinorhodin (act), an antibiotic produced by Streptomyces coelicolor[ 109], was cloned, more than 20 different gene clusters encoding polyketide biosynthetic enzymes have been isolated from various organisms, mostly actinomycetes, and characterized [98, 100]. Bacterial PKSs are classified into two broad types based on gene organization and biosynthetic mechanisms [98, 100, 102]. In modular PKSs (or type I), discrete multifunctional enzymes control the sequential addition of thioester units and their subsequent modification to produce macrocyclic compounds (or complex polyketides). Type I PKSs are exemplified by 6-deoxyerythronolide B synthase (DEBS), which catalyzes the formation of the macrolactone portion of erythromycin A, an antibiotic produced by Saccharopolyspora erythraea. There are 7 different active-site domains in DEBS, but a given module contains only 3 to 6 active sites. Three domains, acyl carrier protein (ACP), acyltransferase (AT), and P-ketoacyl-ACP synthase (KS), constitute a minimum module. Some modules contain additional domains for reduction of p-carbons, e.g., P-ketoacyl-ACP reductase (KR), dehydratase (DH), and enoyl reductase (ER). The thioesterase-cyclase (TE) protein is present only at the end of module 6. 

In order to efficiently and systematically exchange and recombine biosynthetic genes, the biosynthetic genes responsible for various polyketide natural products must be sequenced, and a host-vector system must be available. Several complete biosynthetic gene clusters have now succumbed to cloning and sequencing (7 -84), and a host-vector system has been developed in Strepto-myces (8S). In initial recombination and truncation efforts, compounds exhibiting dramatically different carbon skeletons have been produced. A sample of some of the hybrid structures produced with enzymes taken from the biosynthetic pathways that normally lead to actinorhodin and tetracenomycin (Fig. 15) are shown in Fig. 16. 

The most well-established system for heterologous expression involves the hosts S. coelicolor or its close relative S. lividans, and a bifimctional actino-myces- . coli vector with control elements for PKS gene expression that have been derived from the actinorhodin gene cluster [59]. This host-vector system has successfuUy been used to reconstitute functionally the polyketide pathways associated with biosynthesis of frenolicin [60], tetracenomycin [59], oxytetra-cycline [61], erythromycin [62], picromycin/methymycin [63], oleandomycin... 

FemSndez Moieno MA, Martinez E, Caballero JL, Ichinose K, Hopwood DA, Malpartida F. DNA sequence and functions of the octVI region of the actinorhodin biosynthetic gene cluster of Streptomyces coelicolor A3(Z). J Biol Chem 1994 269 24854-24863. 

S. Okamoto, T. Taguchi, K. Ochi, K. Ichinose, Biosynthesis of actinorhodin and related antibiotics discovery of alternative routes for quinone formation encoded in the act gene cluster. Chem. Biol. 16, 226-236 (2009)... 

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