Ethyl-substituted erythromycin derivatives produced by directed metabolic engineering.

A previously unknown chemical structure, 6-desmethyl-6-ethylerythromycin A (6-ethylErA), was produced through directed genetic manipulation of the erythromycin (Er)-producing organism Saccharopolyspora erythraea. In an attempt to replace the methyl side chain at the C-6 position of the Er polyketide backbone with an ethyl moiety, the methylmalonate-specific acyltransferase (AT) domain of the Er polyketide synthase was replaced with an ethylmalonate-specific AT domain from the polyketide synthase involved in the synthesis of the 16-member macrolide niddamycin. The genetically altered strain was found to produce ErA, however, and not the ethyl-substituted derivative. When the strain was provided with precursors of ethylmalonate, a small quantity of a macrolide with the mass of 6-ethylErA was produced in addition to ErA. Because substrate for the heterologous AT seemed to be limiting, crotonyl-CoA reductase, a primary metabolic enzyme involved in butyryl-CoA production in streptomycetes, was expressed in the strain. The primary macrolide produced by the reengineered strain was 6-ethylErA.

Acyltransferase domain substitutions in erythromycin polyketide synthase yield novel erythromycin derivatives.

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.

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.

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.

Facilitation of plasmid transfer in Streptococcus pneumoniae by chromosomal homology.

The frequency of plasmid establishment in the transformation of Streptococcus pneumoniae by plasmid DNA was increased more than 10-fold when the plasmid carried DNA homologous to the host chromosome. Perfect homology was not necessary for such facilitation; small additions or deletions were tolerated, but extensive deletions in the homologous segment of either plasmid or chromosome reduced or eliminated facilitation. The facilitated plasmid transfer showed a linear dependence on monomeric plasmid concentration rather than the quadratic dependence found in the absence of homology, which indicated that entering plasmid fragments interacted with the chromosome rather than with each other to establish a plasmid replicon. Restriction enzyme cleavage of the plasmid in the nonhomologous segment destroyed its activity, but cleavage in the homologous segment or even enzymatic removal of part of that segment did not prevent plasmid transfer, and plasmids of the original size were established. In facilitated transfer, chromosomal markers (additions and deletions as well as single-site mutations) entered the plasmid with a frequency ranging from 10 to 90% depending on the marker location. Several possible mechanisms for the establishment of plasmids in the presence of chromosomal homology and for the transfer of chromosomal information are considered. They depend on synapsis of the newly entered single-strand plasmid fragment with the host chromosome and subsequent copying of, donation from, or integration into the homologous chromosomal segment. After plasmid establishment, equilibration of donor and chromosomal markers between the chromosome and the plasmid pool, presumably by homologous recombination events, was observed.

Cloning of chromosomal genes in Streptococcus pneumoniae.

A system for molecular cloning in Streptococcus pneumoniae was developed. The multicopy plasmids pMV158 (5.4 kilobases) and pLS1 (4.3 kilobases), which confer tetracycline resistance, were used as vectors to clone chromosomal genes of S. pneumoniae in host cells of this species. A 3.3-kilobase restriction fragment containing the malM gene, which codes for amylomaltase, was cloned in a deletion mutant lacking chromosomal homology with the fragment. The recombinant plasmid pLS70, could transform over 50% of a recipient population to maltose utilization. Amylomaltase constituted up to 10% of the protein of cells containing pLS70. A derivative with a deletion, pLS69, appeared to gain a selective advantage by producing less enzyme. A 10-kilobase restriction fragment containing the sul-d gene for sulfonamide resistance was cloned in the presence of the homologous chromosomal gene. De novo establishment of a recombinant plasmid was just as frequent as transformation in an endogenous plasmid. Despite the processing of DNA during uptake in the transformation of S. pneumoniae, recombinant plasmids can be introduced. Models for the reconstruction of recombinant DNA in cells of S. pneumoniae and Bacillus subtilis are considered and compared.