Molecular cloning of the whole biosynthetic pathway of a Streptomyces antibiotic and its expression in a heterologous host

Abstract

The application of molecular cloning to antibiotic-producing microorganisms should lead to enhanced antibiotic productivity and to the biosynthesis of novel antibiotics by in vitro interspecific recombination. To allow such approaches, the genes for antibiotic synthesis must be isolated, analysed and perhaps modified. Certain Streptomyces species produce nearly two-thirds of the known natural antibiotics; the recent development of cloning systems in the genus makes it possible to isolate and analyse Streptomyces genes. However, antibiotics are metabolites which require sets of several enzymes for their synthesis and attempts to isolate the corresponding genes have so far yielded clones carrying either individual genes of the set, or only incomplete gene sets. We describe here the isolation of a large continuous segment of Streptomyces coelicolor DNA which apparently carries the complete genetic information required for synthesis of an antibiotic, actinorhodin , from simple primary metabolites. Not only can the cloned DNA 'complement' all available classes of actinorhodin non-producing mutants of S. coelicolor but, on introduction into a different host, Streptomyces parvulus , it directs the synthesis of the antibiotic. The tendency for the genes for antibiotic synthesis to be clustered together on the chromosomes of Streptomyces species and the availability of plasmid vectors which can carry stable inserts of DNA larger than 30 kilobase pairs (kb) and which can be introduced efficiently into Streptomyces protoplasts, suggest that the experiments described have general significance for this area of biotechnology.

Forty years of genetics with Streptomyces: from in vivo through in vitro to in silico

“…The first example, SLP1, was revealed when pocks were found in a lawn of S. Suarez & Chater (1980), Thompson et al (1980Thompson et al ( ) 1981 First report of amplified DNA segments Robinson et al (1981) 1982 Isolation of promoter sequences Bibb & Cohen (1982 First Streptomyces gene sequenced (aph of S. fradiae) Thompson & Gray (1983) Antibiotic-pathway genes cloned Chater & Bruton (1983), Feitelson & Hopwood (1983), Gil & Hopwood (1983 Complete antibiotic pathway (act) cloned Malpartida & Hopwood (1984)  program for gene recognition Bibb et al (1984) Bibb et al (1985), Buttner et al (1987) Linkage of antibiotic-biosynthetic, -regulatory and -resistance genes Chater & Bruton (1985) John Innes Laboratory Manual published Hopwood et al (1985b) 1987 First Streptomyces transposon discovered Chung (1987) 1987\88 Operon structure (gal and gyl) Fornwald et al (1987), Smith & Chater (1988 Translation without Shine-Dalgarno sequences Janssen et al (1989) Conjugation between E. coli and Streptomyces Mazodier et al (1989) lividans after contact with an S. coelicolor strain devoid of SCP1 and SCP2 (Bibb et al, 1981). The pocks yielded a family of SLP1 plasmids of varying size.…”

“…The first antibioticbiosynthetic genes were cloned, by a variety of methods Chater & Bruton (1985). (Chater & Bruton, 1983 ;Feitelson & Hopwood, 1983 ;Gil & Hopwood, 1983), and soon genes for a whole pathway, the act genes for actinorhodin biosynthesis, were isolated and expressed in a different Streptomyces host (Malpartida & Hopwood, 1984). Just as loss of the blue colour of actinorhodin had been instrumental in the isolation and in vivo mapping of antibiotic-pathway genes (Rudd & Hopwood, 1979), so its reacquisition was crucial to the isolation and in vitro mapping of the genes (Malpartida & Hopwood, 1984, 1986.…”

Tautomycin from the bacterium Streptomyces verticillatus

“…Many are polyketides (e.g. [7]) while others are peptides which, like microcystin, may contain both D-and L-amino acids and linkages other than cu,m-peptide bonds (reviewed in [S]). Recently, a polyketide called tautomycin, produced by Streptomyces griseochromogenes and Streptomyces verticillatus, was purified and characterised [9].…”

Pigmented antibiotic production by Streptomyces coelicolor A3(2): kinetics and the influence of nutrients

“…coelicolor synthesizes two chemically distinct pigments which are generally regarded as secondary metabolites : actinorhodin, a diffusible red-blue pH indicator, and undecylprodigiosin, a red cell-wallassociated compound (Rudd & Hopwood, 1980). Genes which determine the synthesis of these two pigments have been identified (Rudd & Hopwood, 1979, 1980Feitelson et al, 1985 ;) and cloned (Feitelson & Hopwood, 1983;Feitelson et al, 1986;Malpartida & Hopwood, 1984. However, the physiological controls which operate on pigment production in S .…”