The nucleotide sequence on both sides of the eryA polyketide synthase genes of the erythromycin-producing bacterium S0cch0rop0lysp0ra erythraea reveals the presence of ten genes that are involved in t-mycarose (eryB) and Ddesosamine ( e m biosynthesis or attachment. Mutant strains carrying targeted lesions in eight of these genes indicate that three (eryB/V, eryBVand eryBV/) act in L-mycarose biosynthesis or attachment, while the other five (eryC//, eryC///, eryC/V, e m and e m / ) are devoted to D-desosamine biosynthesis or attachment. The remaining two genes (eryB// and eryBV//) appear to function in L-mycarose biosynthesis based on computer analysis and earlier genetic data. Three of these genes, eryB//, eryC/// and eryC//, lie between the eryA/// and eryG genes on one side of the polyketide synthase genes, while the remaining seven, eryB/V, eryBV, e m / , eryBVI, erycIV, eryCV and eryBV// lie upstream of the eryAI gene on the other side of the gene cluster. The deduced products of these genes show similarities to: aldohexose 4-ketoreductases (eryB/V), aldoketo reductases (eryB//), aldohexose 5-epimerases (eryBV//), the dnml gene of the daunomycin biosynthetic pathway of Streptomyces peucetius (eryBvl), glycosyltransferases (eryBV and eryC///), the AscC 3,bdehydratase from the ascarylose biosynthetic pathway of Yeminis pseudotuberculosis (eryC/V), and mammalian N-methyltransferases ( e m / ) . The eryC// gene resembles a cytochrome P450, but lacks the conserved cysteine residue responsible for coordination of the haem iron, while the eryCV gene displays no meaningful similarity to other known sequences. From the predicted function of these and other known eryB and eryC genes, pathways for the biosynthesis of L-mycarose and D-desosamine have been deduced.

Streptomyces glaucescens, a Gram-positive soil bacterium, produces the polyketide antibiotic tetracenomycin (Tcm) C. To study possible biochemical connections between the biosynthesis of bacterial fatty acids and polyketides, the abundant acyl carrier protein (ACP) detected throughout the growth of the tetracenomycin (Tcm) C-producing S. glaucescens was purified to homogeneity and found to behave like many other ACPs from bacteria and plants (apparent M(r) of 20,000 on gel filtration chromatography, apparent M(r) of 3400-4800 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, and pI approximately 3.8). By using an oligodeoxynucleotide synthesized in accordance with the sequence of residues 25-36 of the ACP, the fabC gene encoding this protein was cloned, and expression of this gene in Escherichia coli yielded the ACP entirely as the active holoenzyme. Sequence analysis of 4.3 kilobases (kb) of DNA flanking fabC revealed the presence of three other genes oriented in the same transcriptional direction in the order fabD, fabH, fabC, and fabB. Each of the four genes is predicted to encode proteins with high sequence similarity to the following components of the E. coli fatty acid synthase (FAS): the FabD malonyl-coenzyme A:ACP acyltransferase (MAT), FabH 3-oxoacyl:ACP synthase III, AcpP ACP, and FabB 3-oxoacyl:ACP synthase I. Expression of the S. glaucescens fabD gene in E. coli produced active MAT able to catalyze in vitro the transfer of radioactive malonate from malonyl-coenzyme A to the E. coli AcpP and S. glaucescens FabC ACPs, as well as to the TcmM ACP component of the Tcm type II polyketide synthase [Shen, B., et al. (1992) J. Bacteriol 174, 3818-3821]. Expression of fabD also restored the high-temperature growth of the E. coli fabD89 mutant that bears a temperature-sensitive MAT. The latter finding and the close similarity between the organization of the S. glaucescens fabDHCB and E. coli FAS-encoding genes (fabH/fabD/fabG/acpP/fabF) suggest that the S. glaucescens genes encode FAS enzymes. Moreover, on the basis of its in vitro activity, it is possible that the S. glaucescens FabD MAT is responsible for charging the TcmM ACP with malonate in vivo, a key step in the synthesis of the deca(polyketide) precursor of Tcm C. This implies the existence of a functional connection between fatty acid and polyketide metabolism in this bacterium.

The methylmalonyl coenzyme A (methylmalonyl-CoA)-specific acyltransferase (AT) domains of modules 1 and 2 of the 6-deoxyerythronolide B synthase (DEBS1) of Saccharopolyspora erythraea ER720 were replaced with three heterologous AT domains that are believed, based on sequence comparisons, to be specific for malonyl-CoA. The three substituted AT domains were "Hyg" AT2 from module 2 of a type I polyketide synthase (PKS)-like gene cluster isolated from the rapamycin producer Streptomyces hygroscopicus ATCC 29253, "Ven" AT isolated from a PKS-like gene cluster of the pikromycin producer Streptomyces venezuelae ATCC 15439, and RAPS AT14 from module 14 of the rapamycin PKS gene cluster of S. hygroscopicus ATCC 29253. These changes led to the production of novel erythromycin derivatives by the engineered strains of S. erythraea ER720. Specifically, 12-desmethyl-12-deoxyerythromycin A, which lacks the methyl group at C-12 of the macrolactone ring, was produced by the strains in which the resident AT1 domain was replaced, and 10-desmethylerythromycin A and 10-desmethyl-12-deoxyerythromycin A, both of which lack the methyl group at C-10 of the macrolactone ring, were produced by the recombinant strains in which the resident AT2 domain was replaced. All of the novel erythromycin derivatives exhibited antibiotic activity against Staphylococcus aureus. The production of the erythromycin derivatives through AT replacements confirms the computer predicted substrate specificities of "Hyg" AT2 and "Ven" AT and the substrate specificity of RAPS AT14 deduced from the structure of rapamycin. Moreover, these experiments demonstrate that at least some AT domains of the complete 6-deoxyerythronolide B synthase of S. erythraea can be replaced by functionally related domains from different organisms to make novel, bioactive compounds.

Mutations in the tcmll-temlV region of the Streptomyces glaucescens chromosome block the C-3 and C-8 0-methylations of the polyketide antibiotic tetracenomycin C (Tcm C). The nucleotide sequence of this region reveals the presence of two genes, tcmN and temO, whose deduced protein products display similarity to the hydroxyindole 0-methyl transferase of the bovine pineal gland, an enzyme that catalyzes a phenolic 0-methylation analogous to those required for the biosynthesis of Tcm C. The deduced product of the tcmN gene also has an N-terminal domain that shows similarity to the putative ActVH and WhiE ORFVI proteins of Streptomyces coelicolor. The tcmN N-terminal domain can be separated from the remainder of the tcmN gene product, and when coupled on a plasmid with the Tcm C polyketide synthase genes (tcmKLM), this domain enables high-level production of an early, partially cyclized intermediate of Tcm C in a Tcm C-null mutant or in a heterologous host (Streptomyces lividans). By analogy to fatty acid biosynthesis, the tcmKLM polyketide synthase gene products are probably sufficient to produce the linear decaketide precursor of Tcm C; thus, the tcmN N-terminal domain is most likely responsible for one or more of the early cyclizations and, perhaps, the attendant dehydrations that lead to the partially cyclized intermediate. The temN gene therefore appears to encode a multifunctional cyclase-dehydratase-3-0-methyl transferase. The tcmO gene encodes the 8-0-methyl transferase.Polyketide metabolites are a structurally diverse family of compounds that encompasses both aromatic and aliphatic members (Fig. 1A). These molecules are commonly synthesized via secondary metabolic pathways in bacteria, fungi, and plants (10). Importantly, many of these compounds have found clinical utility as antibiotics and chemotherapeutic agents (10).The feature that binds the seemingly disparate polyketide family together is the mechanism of biosynthesis. The carbon backbone of a polyketide is synthesized in a manner that is similar to long-chain fatty acid biosynthesis: small fatty acid units (acetate, propionate, butyrate, etc.) are sequentially condensed to yield extended linear precursors. A notable difference distinguishes polyketide biosynthesis from fatty acid biosynthesis, however. The condensation reactions of polyketide chain growth are not always followed by the cycles of reduction and dehydration that characterize the synthesis of fatty acids. As a result, the linear intermediate of polyketide biosynthesis is peppered with reactive carbonyl groups that form the basis for subsequent chemical elaborations.Tetracenomycin C (Tcm C) is a relatively simple aromatic polyketide antibiotic that is produced by Streptomyces glaucescens (25). The tetracyclic backbone of this molecule is most likely formed by cyclization of a 20-carbon linear decaketide (Fig. 1B). Previously, we cloned the genes required for the biosynthesis of Tcm C from S. (14) and reported the DNA sequence of the polyketide synthase (PKS) genes from the tcmIa region t...

The acyl carrier protein (ACP) of the tetracenomycin C polyketide synthase, encoded by the tcmM gene, has been expressed in both Streptomyces glaucescens and Escherichia coli and purified to homogeneity. Expression of the tcmM gene in E. coli results mainly in the TcmM apo-ACP, whereas expression in S. glaucescens yields solely the holo-ACP. The purified holo-TcmM is active in a malonyl coenzyme A:ACP transacylase assay and is labeled by radioactive beta-alanine, confirming that it carries a 4'-phosphopantetheine prosthetic group.

The secretion of /8-lactamase (EC 3.5.2.6) into the periplasm of Escherichia coli has been followed by pulse-chase labeling at 15'C. Though the periplasmic fraction contains only the mature form of the enzyme, the spheroplast fraction contains the completed precursor and a hitherto undocumented processed form. When whole spheroplasts are treated with trypsin, the processed form in this fraction is completely digested. This is in contrast to the native mature enzyme localized in the periplasm, which is trypsin resistant. The (8-lactamase is evidently processed after translocation to a trypsin-sensitive form that is transiently bound to the periplasmic face of the inner membrane. The release of this processed form into the periplasm occurs concomitantly with a conformational change that results in the soluble, catalytically active, trypsin-resistant structure.One of the clearest differences between the two most favored models for the secretion of proteins across membranes, the signal hypothesis and the membrane-trigger hypothesis, concerns the role of conformational changes that occur during the secretion process. The signal hypothesis was first developed to account for the behavior of secreted proteins in eukaryotic systems (1-5). According to this hypothesis, as soon as the signal peptide emerges from the ribosome, it interacts with a signal recognition particle and further elongation is blocked. Subsequent binding of this complex with a membrane-associated docking protein releases the translation blockade, and the resumed polypeptide elongation allows the cotranslational translocation of the molecule through a protein channel in the membrane. The protein is extruded linearly: folding and processing into its mature form occur either during or after translocation across the membrane. The signal hypothesis emphasizes the function of particular polypeptide sequences [including those required for insertion and stop-transfer (6)] in the translocation pathway, while postulating the process to be essentially independent of tertiary structure or folding patterns.In contrast, the essence of the membrane-trigger hypothesis is that the thermodynamics of protein folding govern the secretory process (7,8). This model was originally formulated to account for the assembly of the M13 phage coat protein into bacterial membranes but has been extended to accommodate the phenomena associated with the transport of secreted proteins. The signal peptide is postulated to allow the nascent polypeptide to fold in a manner compatible with the aqueous cytoplasmic environment and, at the same time, to prevent the chain from adopting the mature, water-soluble tertiary structure that could not be translocated. A conformational change, triggered by interaction with the membrane, allows the protein to become embedded in the apolar membrane. Upon removal of the signal peptide by a peptidase located on the outer face of the membrane, the protein refolds into its mature, water-soluble form and is released into the periplasm. Implicit in th...

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6-Deoxyerythronol ide B synthase (DEBS) is a large multifunctional enzyme that catalyses the biosynthesis of the erythromycin polyketide aglycone. DEBS is organized into six modules, each containing the enzymic domains required for a single condensation of carboxylic acid residues which make up the growing polyketide chain. Module 1 is preceded by loading acyltransferase (AT-L) and acyl carrier protein (ACP-L) domains, hypothesized to initiate polyketide chain growth with a propionate-derived moiety. Using recombinant DNA technology several mutant strains of Saccharopolyspora erythraea were constructed that lack the initial AT-L domain or that lack both the AT4 and ACP-L domains. These strains were still able to produce erythromycin, although a t much lower levels than that produced by the wild-type strain. In addition, the AT-L domain expressed as a monofunctional enzyme was able to complement the deletion of this domain from the PKS, resulting in increased levels of erythromycin production. These findings indicate that neither the initial AT-L nor the ACP-L domains are required to initiate erythromycin biosynthesis; however, without these domains the efficiency of erythromycin biosynthesis is decreased significantly. It is proposed that in these mutants the first step in erythromycin biosynthesis is the charging of KSI with propionate directly from propionyl-CoA.