Alteration of the fatty acid profile of Streptomyces coelicolor by replacement of the initiation enzyme 3-ketoacyl acyl carrier protein synthase III (FabH)

Abstract

The first elongation step of fatty acid biosynthesis by a type II dissociated fatty acid synthases is catalyzed by 3-ketoacyl-acyl carrier protein (ACP) synthase III (KASIII, FabH). This enzyme, encoded by the fabH gene, catalyzes a decarboxylative condensation between an acyl coenzyme A (CoA) primer and malonyl-ACP. In organisms such as Escherichia coli, which generate only straight-chain fatty acids (SCFAs), FabH has a substrate preference for acetyl-CoA. In streptomycetes and other organisms which produce a mixture of both SCFAs and branched-chain fatty acids (BCFAs), FabH has been shown to utilize straight- and branched-chain acyl-CoA substrates. We report herein the generation of a Streptomyces coelicolor mutant (YL/ecFabH) in which the chromosomal copy of the fabH gene has been replaced and the essential process of fatty acid biosynthesis is initiated by plasmid-based expression of the E. coli FabH (bearing only 35% amino acid identity to the Streptomyces enzyme). The YL/ecFabH mutant produces predominantly SCFAs (86%). In contrast, BCFAs predominate (approximately 70%) in both the S. coelicolor parental strain and S. coelicolor YL/sgFabH (a deltafabH mutant carrying a plasmid expressing the Streptomyces glaucescens FabH). These results provide the first unequivocal evidence that the substrate specificity of FabH observed in vitro is a determinant of the fatty acid made in an organism. The YL/ecFabH strain grows significantly slower on both solid and liquid media. The levels of FabH activity in cell extracts of YL/ecFabH were also significantly lower than those in cell extracts of YL/sgFabH, suggesting that a decreased rate of fatty acid synthesis may account for the observed decreased growth rate. The production of low levels of BCFAs in YL/ecFabH suggests either that the E. coli FabH is more tolerant of different acyl-CoAs substrates than previously thought or that there is an additional pathway for initiation of BCFA biosynthesis in Streptomyces coelicolor.

FIG. 1.

Organization of four fatty acid biosynthetic genes in S. coelicolor M511 (A) and YL/sgFabH and YL/ecFabH(B).

FIG. 2.

Growth of S. coelicolor M511/pSW7 (▪), M511/pSE4 (♦), YL/sgFabH (○), and YL/ecFabH (▴) in liquid YEME medium.

FIG. 3.

Fatty acid profiles of YL/sgFabH (A) and YL/ecFabH (B). Fatty acid abbreviations: iC14, isomyristate; C14, myristate; iC15, isopentadecanoate; aiC15, anteisopentadecanoate; iC16, isopalmitate; C16:1, palmitoleate; C16:0, palmitate; iC17 isopentadecanoate; aiC17, anteisopentadecanoate; C17, pentadecanoate.