b-Ketoacyl Acyl Carrier Protein Synthase III ( FabH ) Is Essential for Fatty Acid Biosynthesis in Streptomyces coelicolor A 3 ( 2 )

Streptomyces spp. synthesize the majority of their fatty acids from branched starters such as isobutyryl, isovaleryl, and anteisovaleryl units to give oddand even-numbered fatty acids with a methyl branch at the v-terminus (80 to 90% of total fatty acid content); the remainder are synthesized from straight starters such as acetyl and butyryl units (11, 21). The fatty acid synthase (FAS) of Streptomyces spp. is, like that found in many other bacteria (including the best-studied example, that of Escherichia coli), a type II or dissociable system (13, 18). The type II FAS consists of several discrete proteins that form loose associations to synthesize the fatty acid. The assembly of fatty acids is initiated by the condensation of an acyl coenzyme A (acyl-CoA) starter unit and a malonyl-acyl carrier protein (malonyl-ACP) extender unit; this condensation is catalyzed by b-ketoacyl ACP synthase III (FabH), the product of the fabH gene. In vitro biochemical studies suggest that FabH determines the choice of starter unit to be used. E. coli FabH is specific for an acetyl-CoA starter unit, whereas Bacillus subtilis and Streptomyces glaucescens FabHs can accept a broader range of substrates, including branchedand straight-chain units (2, 8). In the case of the S. glaucescens FabH, the order of reactivity towards the different starters is isobutyryl-CoA . butyryl-CoA . acetyl-CoA. If FabH were solely responsible for the initiation of fatty acid biosynthesis in Streptomyces species, then one could hypothesize that its biochemical activity, together with the relative pool sizes of the different starter units in vivo, would account for the mix of branched and straight fatty acids (8). But when S. glaucescens was grown in the presence of high concentrations (480 mM) of the FAS inhibitor thiolactomycin, branched-chain fatty acid biosynthesis was inhibited and the proportion of straight-chain fatty acids increased (50% inhibitory concentration [IC50] for purified S. glaucescens FabH 20 mM) (8). This result may be interpreted as evidence for a second; FabH-independent mechanism for fatty acid initiation in Streptomyces spp. Based on this second hypothesis, fabH should be dispensable to Streptomyces spp. In this study we provide further biochemical evidence for the role of a small cluster of presumed fab genes (which includes fabH) in Streptomyces coelicolor and a transcriptional analysis of the fab cluster, and we have attempted to disrupt fabH to determine if it is essential for the viability of the cells. The acpP gene product stimulates long-chain fatty acid biosynthesis in vitro. The S. coelicolor FAS is still relatively poorly understood; a cluster of four fab-like genes has been identified on the S. coelicolor chromosome in the order fabD-fabH-acpPfabF (cosmid SC4A7, S. coelicolor genome project [http://www .sanger.ac.uk/Projects/S_coelicolor/]; nucleotide sequence accession number AL133423). The deduced amino acid sequences of the fab genes are highly similar to components of the E. coli FAS, and at least some of the S. coelicolor genes are essential (13). We used a biochemical assay of fatty acid biosynthesis, dependent on the acpP gene product (ACP), to strengthen the evidence that these genes do encode the FAS of S. coelicolor. Cell extracts were prepared from S. coelicolor M145 grown for 20 h in YEME medium (12) and broken as previously described (3), with an additional clearing step by ultracentrifugation for 1 h at 100,000 3 g. The supernatant was adjusted to 5 mg of protein/ml, a fresh ice-cold 10% (wt/vol) solution of streptomycin sulfate was added slowly while stirring on ice water to a final concentration of 1%; the mixture was then stirred for a further 20 min and centrifuged for 20 min at 14,000 3 g. Endogenous ACP was removed from the cell extract by fractionation with a 60 to 80% ammonium sulfate cut as previously described (4), and this cut was dialyzed overnight against 1 liter of cell disruption buffer containing 2 mM dithiothreitol (DTT). Pure FAS holo-ACP was prepared as previously described (14) and reduced to the active monomeric form just prior to each assay by incubation at 30°C for 30 min in a solution containing 50 mM potassium phosphate, pH 7.2, and 10 mM DTT. Typical assay incubation conditions were as follows: 0.5 mg of S. coelicolor (60 to 80% ammonium sulfate cut) per ml, 100 mM potassium phosphate (pH 7.2), 2 mM DTT, 100 mM NADH, 100 mM NADPH, 20 mM ACP, and 100 mM [2-C]malonyl-CoA (0.02 Bq/pmol) in a total volume of 18 ml were preincubated for 10 min at 30°C; then 2 ml of isobutyryl-CoA was added (20 mM final concentration) to initiate the reaction (alternatively, buffer alone was added as a * Corresponding author. Present address: Kosan Biosciences, 3832 Bay Center Pl., Hayward, CA 94545. Phone: (510) 732-8400. Fax: (510) 732-8401. E-mail: revill@kosan.com. † Present address: C/Garcı́a Morato no. 14, 28805-Alcalá de Henares, Madrid, Spain.