Genetic recombination through protoplast fusion in Streptomyces

Abstract

MEMBERS of the genus Streptomyces yield over 60% of known antibiotics¹, including more than 70 commercial products², as well as at least one very important enzyme (glucose isomerase). Members of the related genera Nocardia and Micromonospora produce some further valuable antibiotics (rifamycins, gentamicins). Recombination through conjugation is widespread in these actinomycetes³ but only to a very limited extent has it been applied to strain improvement. The main reason for its neglect has probably been the necessity to introduce selectable (for example drug resistance) or more generally counter-selectable (auxotrophic) markers into the strains to be crossed in order to identify recombinants occurring at frequencies of 10⁻⁶ or lower among predominantly asexual progeny. The marking of strains is time-consuming; moreover it may induce deleterious mutations along with the markers, and these will usually go undetected since auxotrophic mutations themselves often depress antibiotic yield. In certain cases, recombination occurs so rarely that single markers in each parent cannot be used to distinguish it from reverse mutation⁴, while in some strains no recombination has been detected³. The availability of a simple generally applicable procedure to recombine actinomycete strains at high frequency would therefore greatly facilitate the routine use of recombination in strain improvement. We report here such a procedure for Streptomyces which depends on polyethylene glycol (PEG)-induced protoplast fusion and regeneration. Recombination frequencies achieved by this technique are so high that selectable markers could be dispensed with, at least in certain kinds of strain improvement programmes.