David A. Hopwood

Engineered biosynthesis of novel polyketides

Polyketide synthases (PKSs) are multifunctional enzymes that catalyze the biosynthesis of a huge variety of carbon chains differing in their length and patterns of functionality and cyclization. Many polyketides are valuable therapeutic agents. A Streptomyces host-vector system has been developed for efficient construction and expression of recombinant PKSs. Using this expression system, several novel compounds have been synthesized in vivo in significant quantities. Characterization of these metabolites has provided new insights into key features of actinomycete aromatic PKS specificity. Thus, carbon chain length is dictated, at least in part, by a protein that appears to be distinctive to this family of PKSs, whereas the acyl carrier proteins of different PKSs can be interchanged without affecting product structure. A given ketoreductase can recognize and reduce polyketide chains of different length; this ketoreduction always occurs at the C-9 position. The regiospecificity of the first cyclization of the nascent polyketide chain is either determined by the ketoreductase, or the chain-extending enzymes themselves. However, the regiospecificity of the second cyclization is determined by a distinct cyclase, which can discriminate between substrates of different chain lengths.

pIJ101, a multi-copy broad host-range Streptomyces plasmid: functional analysis and development of DNA cloning vectors.

SummaryStreptomyces lividans ISP 5434 contains four small high copy number plasmids: pIJ101 (8.9 kb), pIJ102 (4.0 kb), pIJ103 (3.9 kb) and pIJ104 (4.9 kb). The three smaller species appear to be naturally occurring deletion variants of pIJ101. pIJ101 and its in vivo and in vitro derivatives were studied after transformation into S. lividans 66.pIJ101 was found to be self-transmissible by conjugation, to elicit “lethal zygosis” and to promote chromosomal recombination at high frequency in both S. lividans 66 and S. coelicolor A3(2). A restriction endonuclease cleavage map of pIJ101 was constructed for 11 endonucleases; sites for five others were lacking. Many variants of pIJ101 were constructed in vitro by inserting DNA fragments determining resistance to neomycin, thiostrepton or viomycin, and having BamHI termini, into MboI or BclI sites on the plasmid, sometimes with deletion of segments of plasmid DNA. The physical maps of these plasmids were related to their phenotypes in respect of lethal zygosis and transfer properties. In vivo recombination tests between pairs of variant plasmids were also done. These physical and genetic studies indicated that determinants of conjugal transfer occupy less than 2.1 kb of the plasmid. A second segment is required for spread of the plasmid within a plasmid-free culture to produce the normal lethal zygosis phenotype: insertion of foreign DNA in this region caused a marked reduction in the diameter of lethal zygosis zones. The minimum replicon was deduced to be 2.1 kb or less in size; adjacent to this region is a 0.5 kb segment which may be required for stable inheritance of the plasmid. The copy number of several derivatives of pIJ101 in S. lividans 66 was between 40 and 300 per chromosome and appeared to vary with the age or physiological state of the culture. pIJ101 derivatives have a wide host range within the genus Streptomyces: 13 out of 18 strains, of diverse species, were successfully transformed.Knowledge of dispensable DNA segments and the availability of restriction sites for the insertion of DNA, deduced from the properties of plasmids carrying the E. coli plasmid pACYC184 introduced at various sites, was used in the construction of several derivatives of pIJ101 suitable as DNA cloning vectors. These were mostly designed to be non-conjugative and to carry pairs of resistance genes for selection. They include a bifunctional shuttle vector for E. coli and Streptomyces; a Streptomyces viomycin resistance gene of this plasmid is expressed in both hosts.

Physical analysis of antibiotic-resistance genes from Streptomyces and their use in vector construction

Restriction endonuclease cleavage maps of five DNA fragments carrying genes for neomycin phosphotransferase and neomycin acetyltransferase (from Streptomyces fradiae), viomycin phosphotransferase (from S. vinaceus), and ribosomal methylases determining resistance to thiostrepton (from S. azureus) and MLS antibiotics (from S. erythreus) are described, together with a map for the SLP1.2 Streptomyces plasmid used to isolate the fragments. Construction of a versatile Streptomyces cloning vector (pIJ61) is reported. pIJ61 carries neomycin phosphotransferase and thiostrepton resistance genes and has unique BamHI and PstI sites which will allow clone recognition by insertional inactivation of neomycin resistance; cloning sites for several other endonucleases are also present. pIJ28, a shuttle vector for Streptomyces and E. coli, carries neomycin resistance and the SLP1.2 and pBR322 replicons.

Physical and genetical characterisation of a second sex factor, SCP2, for Streptomyces coelicolor A3(2)

SummaryCovalently closed circular (ccc) DNA of uniform monomer size (c. 18×106 daltons) and restriction endonuclease cleavage pattern was isolated from strains of S. coelicolor A3(2) of differing constitution in respect of the SCP1 sex factor: SCP1+, SCP1′, SCP1- and NF (integrated SCP1). No such ccc DNA was found in strains of S. lividans 66 or S. parvulus ATCC 12434 whether or not they contained SCP1. These results confirmed that the 18×106 dalton plasmid is not, and does not include, SCP1, which has not so far been isolated by any of a variety of methods.Genetic data served to identify a second sex factor, SCP2, postulated to be present in SCP2+ state in the starting strains and to be capable of mutation to a variant form, SCP2*, with enhanced sex factor activity. From SCP2* strains, SCP2- cultures were isolated, at an average spontaneous frequency of about 0.8%. Crosses of pairs of SCP1- SCP2- strains were almost, but not completely, sterile; thus SCP1 and SCP2 probably contribute nearly all the fertility naturally occurring in the A3(2) strain. The two sex factors share the property of exerting an effect that may be comparable with “lethal zygosis” caused by F in E. coli: it is shown by SCP1-carrying strains against SCP1-, or SCP2* (but not SCP2+) strains against SCP2- and is revealed as a narrow zone of growth inhibition surrounding the plasmid-carrying culture on a background of the appropriate plasmid-negative strain.Genetically defined SCP2- strains lacked the ccc DNA found in SCP2+ and SCP2* strains. Thus this DNA apparently represents the SCP2 sex factor. A preliminary restriction endonuclease cleavage map of SCP2 was constructed, with single sites for EcoRI and HindIII, four sites for SalPI (=PstI) and more than 20 sites for SalGI (SalI).

Structure and deduced function of the granaticin-producing polyketide synthase gene cluster of Streptomyces violaceoruber Tü22.

A 6.5 kb region of DNA from Streptomyces violaceoruber, which contains polyketide synthase (PKS) genes for production of the benzoisochromane quinone moiety of the antibiotic, granaticin, was cloned and sequenced. Of six open reading frames (ORFs) identified, four (ORFs 1‐4) would be transcribed in one direction and two (ORFs 5 and 6) divergently from ORFs 1‐4. ORF1 and ORF2, which show evidence for translation coupling, encode (deduced) gene products which strongly resemble each other and the Escherichia coli fatty acid ketoacyl synthase (condensing enzyme), FabB. We conclude that ORF1 (which contains a characteristic cysteine residue) functions as a condensing enzyme, possibly as part of a heterodimeric protein including the product of ORF2. The predicted ORF3 gene product strikingly resembles acyl carrier proteins (ACPs) of fatty acid synthase (FAS), particularly in the region of the active site motif, while the predicted ORF5 and ORF6 gene products resemble known oxidoreductases, suggesting that they function as reductive steps required during assembly of the granaticin carbon skeleton. Comparison of the deduced ORF4 gene product with available protein databases failed to elucidate its potential function. The overall conclusion is that the granaticin‐producing PKS would consist of at least six separate enzymes involved in carbon chain assembly, thus resembling a Type II, rather than a Type I, FAS.

The Streptomyces plasmid SCP2: its functional analysis and development into useful cloning vectors

Detailed restriction maps of the plasmid SCP2* and its deletion derivative pSCP103 were constructed. DNA fragments carrying hygromycin (Hyg), thiostrepton (Thio) or viomycin-resistance (VioR) determinants were inserted into pSCP103, and various segments were deleted from the resulting plasmids. Changes in plasmid phenotypes associated with these insertions and deletions allowed the localisation and characterisation of plasmid replication, stability, transfer and fertility functions. Several useful cloning vectors were constructed. They are able to maintain large (greater than 30 kb) DNA inserts, with stable inheritance at a low copy number (1-2 per chromosome) and without structural rearrangements, in Streptomyces hosts. The vectors have a broad host range in the genus Streptomyces. One of them (pIJ903) is a shuttle vector for Streptomyces and Escherichia coli.

Physical and genetic characterisation of the gene cluster for the antibiotic actinorhodin inStreptomyces coelicolor A3(2)

SummaryWe determined the physical and transcriptional organisation of the set of previously cloned biosynthetic genes involved in the production of the polyketide antibiotic actinorhodin byStreptomyces coelicolor A3(2). Complementation and mutational cloning analyses (in part using new ϕC31 phage vectors incorporating a transcriptional terminator to block transcription from vector promoters into the cloned DNA) indicate that all the biosynthetic genes, including at least one regulatory (activator) gene, are clustered in a chromosomal region of about 26 kb. The genes are organised in at least four separate transcription units, ranging in size from 1 kb for the class III gene, to a polycistronic transcript of at least 5 kb for the class I, VII and IV genes. Indirect evidence shows that resistance to actinorhodin is also determined by the cloned DNA.

Nucleotide sequence, transcription and deduced function of a gene involved in polyketide antibiotic synthesis in Streptomyces coelicolor

The BamHI fragment containing the actIII gene, from the actinorhodin (Act) biosynthetic gene cluster of Streptomyces coelicolor A3(2), was sequenced. The derived amino acid sequence for the actIII gene shows homology to known oxidoreductases and the actIII product is believed to be responsible for catalysing a beta-keto reductive step during assembly of the Act polyketide chain. High resolution transcript mapping identified the transcription start point at 33 nucleotides upstream of the putative translation start codon. The transcript ends in a large invertedly repeated sequence. In vivo promoter-probe studies suggest that efficient transcription of the actIII gene requires the product of the actII gene.

Genetics of actinorhodin biosynthesis by Streptomyces coelicolor A3(2).

A series of 76 mutants of Streptomyces coelicolor A3(2) specifically blocked in the synthesis of the binaphthoquinone antibiotic actinorhodin were classified into seven phenotypic classes on the basis of antibiotic activity, accumulation of pigmented precursors or shunt products of actinorhodin biosynthesis, and cosynthesis of actinorhodin in pairwise combinations of mutants. The polarity of cosynthetic reactions, and other phenotypic properties, allowed six of the mutant classes to be arranged in the most probable linear sequence of biosynthetic blocks. One member of each mutant class was mapped unambigiguously to the chromosomal linkage map in the short segment between the hisD and guaA loci, suggesting that structural genes for actinorhodin biosynthesis may form an uninterrupted cluster of chromosomal genes.

Mutagenesis by N-methyl-N′-nitro-N-nitrosoguanidine (NTG) in Streptomyces coelicolor

Abstract Mutagenesis was tested by the reversion of several auxotrophic mutations and by the induction of auxotrophs. Mutagenesis at alkaline pH was quantitatively related to the release of an unstable, mutagenic intermediate of NTG decomposition, presumably diazomethane. No evidence was obtained for mutagenesis by undecomposed NTG at pH 5. At very acid pH (2.5), there was weak mutagenesis, associated with high lethality, presumably by a different mechanism from that operating at alkaline pH. Effective mutagenesis in Streptomyces requires a longer and more intense treatment than in some other microbes; suitable conditions are described.