Diane L. Stassi

A second type-I PKS gene cluster isolated from Streptomyces hygroscopicus ATCC 29253, a rapamycin-producing strain

Analysis of a 32.8-kb segment of DNA from the rapamycin (Rp) producer, Streptomyces hygroscopicus ATCC 29253, revealed a new type-I polyketide synthase (PKS) cluster consisting of four open reading frames (ORF 1-4), each encoding a single PKS module. The four ORFs are transcribed in the same direction and are flanked by several smaller ORFs (ORF 5-9), which may be related to the PKS cluster. The first PKS-containing ORF has a ligase domain at the N-terminus of the polypeptide. This domain has 55% aa identity to the CoA ligase domain of the Rp PKS (Schwecke et al., 1995. Proc. Natl. Acad. Sci. 92, 7839-7843) which is also encoded in this strain (Lowden et al., 1996. Angew. Chem. Int. Ed. Engl. 35, 2249-2251). ORF5 (340 aa) and ORF6 (924 aa) were found to be homologous to RapK (41% aa identity) and RapH (35% aa identity), which are hypothesized to be a pteridine-dependent dioxygenase and a regulatory protein, respectively (Molnar et al., 1996. Gene 169, 1-7). In addition, ORF7 (391 aa) was found to have up to 42% aa identity to a number of plant 3-deoxy-D-arabino-heptulosonate-7-phosphate synthases (DAHPS) and 47% aa identity to PhzF, a bacterial DAHPS involved in phenazine antibiotic synthesis. The proximity of the DAHPS-encoding gene to the PKS cluster containing a Rp-like ligase domain suggests that a derivative of shikimate may be used as the PKS starter. ORF8 (283 aa) was found to have homology (32% aa identity) to a Synechocystis sp. gene of unknown function. The N-terminal portion of ORF9 was found to be similar to a tetracycline 6-hydroxylase (34% aa identity) from Streptomyces aureofaciens.

Effect of strong promoters on the cloning in Escherichia coli of DNA fragments from Streptococcus pneumoniae

Attempts to clone wild-type DNA containing the malM gene of Streptococcus pneumoniae in plasmid pBR322 of Escherichia coli were unsuccessful. However, it was possible to clone a PstI fragment of DNA containing this gene in a plasmid of S. pneumoniae. Cells carrying the recombinant plasmid produced large amounts of the malM product, amylomaltase, and a fragment of the protein coded by the adjacent malX gene, apparently as a result of transcription in opposite directions from strong promoters located between the two genes in the plasmid insert. Under derepressed conditions these products represented 10% of the total protein. No transcription terminators appeared to be included within the cloned segment. The effect of various mutations in the segment on its ability to be cloned in pBR322 was examined. Of those tested, only a down promoter mutation that affected production of both the amylomaltase and the X-protein rendered the segment clonable in E. coli. Fragments of the S. pneumoniae vector, pMV158, which appear to lack strong promoters, were readily cloned in the pBR322-E. coli system. Although it is possible that large amounts of the X-fragment are toxic for E. coli, a more general explanation would be that excessive transcription of the pBR322 vector portion interferes with maintenance of the recombinant plasmid.

The loading domain of the erythromycin polyketide synthase is not essential for erythromycin biosynthesis in Saccharopolyspora erythraea

6-Deoxyerythronolide 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 AT-L and ACP-L domains. These strains were still able to produce erythromycin, although at 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 KS1 with propionate directly from propionyl-CoA.

Nucleotide sequence of DNA controlling expression of genes for maltosaccharide utilization in Streptococcus pneumoniae.

An analysis of previous data indicated that four structural genes concerned with maltosaccharide utilization in Streptococcus pneumoniae are organized in two operons that are transcribed in opposite directions from a central control region. This region contains two strong promoters subject to repression by a regulatory gene product in the absence of maltose. The nucleotide sequence of the 554-bp control region DNA and adjacent portions of the malX and malM structural genes was determined. Unique reading frames and initiation codons allowed identification of the oppositely oriented structural genes. Putative ribosome binding sites and -10 and -35 RNA-polymerase-binding sites, as well as AT-rich regions farther upstream, were observed proximal to both the X and M genes. The similarity of these sequences to sites found in Escherichia coli and Bacillus subtilis indicated the conservation of control signals in bacteria, both Gram-negative and Gram-positive. A pair of 17-bp hyphenated repeat sequences in the control region may represent repressor binding sites. Two down promoter mutations, VII and 69, were shown to be deletions in the control region. The VII mutation, which affected only the MP operon, deleted the promoter adjacent to the M gene. Mutation 69, which reduced both X and M gene functions, deleted the entire segment between the promoters so that they now overlap at their -35 binding sites. As a consequence of this deletion, the AT-rich regions proximal to the promoters were lost. This suggests that the AT-rich regions are important for promoter strength.