The actVl genetic region ofStreptomyces coelicolor A3(2) is part of the biosynthetic gene cluster of actinorhodin (ACT), the act cluster, consisting of six ORFs: ORFB, ORFA, ORF1, ORF2, ORF3, ORF4. A newly devised method of ACTdetection with a combination of HPLC and LC/MSwas applied to the analysis of the disruptants of each OREACTwas produced by those of ORFB, ORFA, ORF3, and ORF4. Instead of ACT, the ORF1disruptant produced 3,8-dihydroxy-l -methylanthraquinone-2-carboxylic acid (DMAC)and aloesaponarin II as shunt products. The ORF2 disruptant gave 4-dihydro-9-hydroxy-l -methyl-1 0-oxo-3-//-naphtho-[2,3- pp.144 -152

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Potential "reagents" for the enantioselective reduction, and other biotransformations, of beta-keto-esters result from the genetic engineering of Streptomyces coelicolor A3(2). For example, incubation of the N-acetylcysteamine thioester 1 with the recombinant strain CH999/pIJ5675 followed by treatment with MeOH/HCl gave the lactone 2 as essentially a single enantiomer.

The use of whole cells for the biotransformation of organic substrates is a well-documented technique [1] which has a number of benefits over conventional, reagent-based methods. For example, bakers yeast is routinely used for the reduction of b-keto-esters, and the resulting b-hydroxy-esters are obtained with predictably high chemo-and enantioselectivities, under mild and neutral conditions. [2] With the huge advances made in molecular biology over the last 20 years it has become possible to manipulate microbial biosynthetic pathways at the genetic level.For a number of years Hopwood et al. [3] and others [4] have been studying the genetics of Streptomyces coelicolor A3(2), a soil bacterium that produces the dimeric benzoisochromanequinone antibiotic actinorhodin (1). The biosynthesis of 1 proceeds by the polyketide pathway outlined in Scheme 1, [5] where key features involve a type II minimal act polyketide synthase (PKS)Ðconsisting of b-ketoacyl synthase (KS), chain length factor (CLF), and acyl carrier protein (ACP)Ð and associated aromatase and cyclase enzymes to assemble the naphthol 2, which undergoes selective reduction to 3 and cyclization to the yellow pigment (S)-DNPA (4). Recently we have provided definitive proof that the actVI genetic region of S. coelicolor A3(2) is involved in the stereospecific construction of the pyran ring of 1. [6] More specifically it was found that the engineered strain CH999/pIJ5660 (a pRM5-based plasmid [4b] containing the act minimal PKS, aromatase (ARO), and cyclase (CYC) genes as well as the actVI-evaporated in vacuo from the filtrate to afford the air-sensitive, analytically pure 4 (4.48 g, 66 %). Colorless, single crystals suitable for X-ray structure analysis were obtained from hexane. Elemental analysis C 72 H 168 N 32 Si 16 Tl 8 4, 1 calcd 1.07 g cm À3 , l(Mo Ka ) 0.71073 , m(Mo Ka ) 2.2 cm À1 . Data for a specimen of dimensions 0.4 Â 0.4 Â 0.4 mm 3 were collected at 173(2) K on an Enraf-Nonius CAD-4 diffractometer, 14 448 unique reflections for 28`q`238, R1 0.078 (for 9400 reflections with I b 2s(I)), wR2 0.235 (all data). In 3 there are two independent molecules of the trimer and one poorly defined cyclohexane solvent molecule. For 4: C 72 H 168 N 32 Si 16 Tl 8 , M r 3566.8, monoclinic, space group C2/c, a 15.6556(4), b 30.3470(9), c 30.2663(3) , b 96.195(1)8, V 14295.6(6) 3 , Z 4, 1 calcd 1.657 g cm À3 , l(Mo Ka ) 0.71073 , m(Mo Ka ) 0.9159 cm À1 . Data for a specimen of dimensions 0.33 Â 0.21 Â 0.12 mm 3 were collected at 193 K in the hemisphere mode on a Siemens P4 instrument fitted with a CCD area detector, 8557 unique reflections for 4.68`q`548, R1 0.0565 (for 6019 reflections with I b 2s(I)), wR2 0.1443 (all data). Each of the structures 3 and 4 was solved by using the heavy atom method and refined with fullmatrix least-squares on all F 2 (SHELXL-93 and 97, respectively) with nonhydrogen atoms anisotropic.Crystallographic data (excluding structure factors) for the structures reported in this paper have been deposited with the Cambridge Crystallographic ...

Potential "reagents" for the enantioselective reduction, and other biotransformations, of beta-keto-esters result from the genetic engineering of Streptomyces coelicolor A3(2). For example, incubation of the N-acetylcysteamine thioester 1 with the recombinant strain CH999/pIJ5675 followed by treatment with MeOH/HCl gave the lactone 2 as essentially a single enantiomer.