Two acyl-CoA carboxylases from Streptomyces coelicolor have been successfully reconstituted from their purified components. Both complexes shared the same biotinylated ␣ subunit, AccA2. The ␤ and the ⑀ subunits were specific from each of the complexes; thus, for the propionyl-CoA carboxylase complex the ␤ and ⑀ components are PccB and PccE, whereas for the acetyl-CoA carboxylase complex the components are AccB and AccE. The two complexes showed very low activity in the absence of the corresponding ⑀ subunits; addition of PccE or AccE dramatically increased the specific activity of the enzymes. The kinetic properties of the two acyl-CoA carboxylases showed a clear difference in their substrate specificity. The acetyl-CoA carboxylase was able to carboxylate acetyl-, propionyl-, or butyryl-CoA with approximately the same specificity. The propionylCoA carboxylase could not recognize acetyl-CoA as a substrate, whereas the specificity constant for propionyl-CoA was 2-fold higher than for butyryl-CoA. For both enzymes the ⑀ subunits were found to specifically interact with their carboxyltransferase component forming a ␤-⑀ subcomplex; this appears to facilitate the further interaction of these subunits with the ␣ component. The ⑀ subunit has been found genetically linked to several carboxyltransferases of different Streptomyces species; we propose that this subunit reflects a distinctive characteristic of a new group of acyl-CoA carboxylases.The first committed step in the biosynthesis of long-chain fatty acids in all animals, plants, and bacteria is catalyzed by acetyl-CoA carboxylase (ACC) 1 (EC 6.4.1.2) (1). The reaction catalyzed by ACC involves two separate reactions.

An Escherichia coli strain capable of producing the potent antibiotic erythromycin C (Ery C) was developed by expressing 17 new heterologous genes in a 6-deoxyerythronolide B (6dEB) producer strain. The megalomicin gene cluster was used as the source for the construction of two artificial operons that contained the genes encoding the deoxysugar biosynthetic and tailoring enzymes necessary to convert 6dEB to Ery C. The reconstructed mycarose operon contained the seven genes coding for the enzymes that convert glucose-1-phosphate (G-1-P) to TDP-L-mycarose, a 6dEB mycarosyl transferase, and a 6dEB 6-hydroxylase. The activity of the pathway was confirmed by demonstrating conversion of exogenous 6dEB to 3-O-␣-mycarosylerythronolide B (MEB). The reconstructed desosamine operon contained the six genes necessary to convert TDP-4-keto-6-deoxyglucose, an intermediate formed in the mycarose pathway, to TDP-D-desosamine, a desosamine transferase, a 6dEB 12-hydroxylase, and the rRNA methyltransferase ErmE; the last was required to confer resistance to the host cell upon production of mature macrolide antibiotics. The activity of this pathway was demonstrated by conversion of MEB to Ery C. When the mycarose and desosamine operons were expressed in an E. coli strain engineered to synthesize 6dEB, Ery C and Ery D were produced. The successful production of Ery C in E. coli shows the potentiality of this model microorganism to synthesize novel 6-deoxysugars and to produce bioactive glycosylated compounds and also establishes the basis for the future use of E. coli both in the production of new glycosylated polyketides and for the generation of novel bioactive compounds through combinatorial biosynthesis.

Although Lactococcus is one of the most extensively studied lactic acid bacteria and is the paradigm for biochemical studies of citrate metabolism, little information is available on the regulation of the citrate lyase complex. In order to fill this gap, we characterized the genes encoding the subunits of the citrate lyase of Lactococcus lactis CRL264, which are located on an 11.4-kb chromosomal DNA region. Nucleotide sequence analysis revealed a cluster of eight genes in a new type of genetic organization. The citM-citCDEFXG operon (cit operon) is transcribed as a single polycistronic mRNA of 8.6 kb. This operon carries a gene encoding a malic enzyme (CitM, a putative oxaloacetate decarboxylase), the structural genes coding for the citrate lyase subunits (citD, citE, and citF), and the accessory genes required for the synthesis of an active citrate lyase complex (citC, citX, and citG). We have found that the cit operon is induced by natural acidification of the medium during cell growth or by a shift to media buffered at acidic pHs. Between the citM and citC genes is a divergent open reading frame whose expression was also increased at acidic pH, which was designated citI. This inducible response to acid stress takes place at the transcriptional level and correlates with increased activity of citrate lyase. It is suggested that coordinated induction of the citrate transporter, CitP, and citrate lyase by acid stress provides a mechanism to make the cells (more) resistant to the inhibitory effects of the fermentation product (lactate) that accumulates under these conditions. Many bacteria can utilize citrate under fermentative conditions. The citrate pathway has been extensively studied in enterobacteria (4, 26). In all known citrate fermentation pathways, after its uptake into the cell, citrate is split into acetate and oxaloacetate by the enzyme citrate lyase. In Klebsiella pneumoniae the citrate-specific fermentation genes form a cluster of two divergent operons (4, 26). This cluster includes the genes citDEF, encoding the citrate lyase subunits ␥, ␤, and ␣, and the citS gene, encoding a citrate H 2Ϫ /Na 1ϩ proton motive force-dissipating transporter. Associated with this cluster, K. pneumoniae contains the oadGAB genes, encoding the biotin oxaloacetate decarboxylase, which allows growth with citrate as the sole carbon and energy source (Fig. 1). In Escherichia coli, the cit cluster includes the genes encoding citrate lyase and the CitT citrate/succinate antiporter (22) (Fig.