Second-generation products: antibiotics.

There has been outstanding progress in gene cloning from microorganisms producing useful antibiotics. Especially noteworthy is the cloning of a gene for cephalosporin biosynthesis that, with increased copy number, helps to overcome a rate-limiting step in biosynthesis. Although this technology is often fraught with unexpected difficulties, not the least of which is gene expression, yield improvement is a practical application with clear financial benefits to the pharmaceutical industry. Although access to cloned genes for antibiotic biosynthesis is becoming common, expression of the cloned genes in a manner such that yield improvement is achieved is a challenge that will be exciting and beneficial in years to come. Heterologous (interspecies) gene expression in antibiotic-producing microorganisms, particularly the actinomycetes, must be successful if hybrid antibiotics are to be produced. Currently, there are no documented examples of heterologous gene cloning to yield a hybrid antibiotic of utility. Even though a hybrid antibiotic structure has been reported as a result of heterologous gene cloning, the antibiotic is not clinically useful, and the two species used in the experiments are closely related. Although technological advances have enabled successful gene cloning, insufficient attention has been given to the issue of gene expression, particularly heterologous gene expression. The need to develop heterologous gene expression systems is urgent. Until this is done, the inability to express cloned antibiotic biosynthesis genes in heterologous hosts could be a barrier to the generation of useful hybrid antibiotics. The potential for a wide array of new antibiotic compounds that could be generated by gene-cloning technology is vast. At least until the limitations of heterologous gene expression are overcome, the best available screening technology (including molecular and immunological screens) and chemistry might best serve as the most prolific means of discovering useful new antibiotics for agriculture and medicine.

pFJ265, a new cloning vehicle for Streptomyces.

A 9.3-kb plasmid, pNM100, was isolated from Streptomyces virginiae (NRRL 15156) and characterized. Streptomyces genes for thiostrepton and neomycin resistance were cloned into pNM100 to yield a small plasmid derivative, pFJ265, that is suitable for Streptomyces gene cloning. pFJ265 is a 9.2-kb nonconjugative plasmid and has a copy number of several hundred per chromosome.

Development of cloning vehicles from the Streptomyces plasmid pFJ103.

A 20-kb plasmid, pFJ103, was isolated from a strain of Streptomyces granuloruber. A restriction endonuclease map of the plasmid was constructed. A Streptomyces gene that specifies resistance to the antibiotic thiostrepton was subcloned into Escherichia coli plasmid pBR322, inserted into pFJ103 and transformed into Streptomyces ambofaciens protoplasts. Two classes of transformants were obtained. One carries the pFJ104 plasmid consisting of the entire pFJ103 with the 1.8-kb thiostrepton resistance gene insert. The other carries the pFJ105 plasmid consisting of the 2.9-kb replicon segment of pFJ103 with the same thiostrepton resistance insert. A gene for neomycin resistance together with the entire E. coli pBR322 plasmid were cloned into pFJ105. The resulting E. coli-Streptomyces bifunctional vector, pFJ123, transformed both E. coli and Streptomyces. The small size of pFJ105, its ease of isolation, and efficient transformation of Streptomyces protoplasts establishes it, and its derivatives, as useful plasmid cloning vehicles for fundamental and applied studies.

ilvU, a locus in Escherichia coli affecting the derepression of isoleucyl-tRNA synthetase and the RPC-5 chromatographic profiles of tRNAIle and tRNAVal.

A mutation in the ilvU locus of Escherichia coli has led to a complex phenotype that included resistance to thiaisoleucine, a loss of derepressibility of isoleucyl tRNA synthetase, and an alteration of the RPC-5 chromatographic profile of the branched-chain aminoacyl-tRNA's. The alterations were manifest in an increase in the amount of Species 2 of both tRNAIle and tRNAVal at the expense of Species 1. A similar alteration, but independent of (and additive to) that caused by the ilvU mutation, was observed upon limitation of either isoleucine or valine. The shift in profile caused by limitation was also independent of the reduced growth rate or the derepression of the isoleucine and valine biosynthetic enzymes that also result from limitation. During chloramphenicol treatment nearly all tRNAIle and tRNAVal formed appears as species 2. Upon recovery from chloramphenicol, Species 2 of both acceptors are converted to Species 1. It is proposed that the ilvU product not only allows derepression of isoleucyl-tRNA synthetase but also retards the conversion of tRNA2Ile to tRNA1Ile and that of tRNA2Val to tRNA1Val. The mutated ilvU loci abolish the derepression and are more efficient in retarding the conversion.