Interspecific protoplast fusion among macrolide-producing streptomycetes.

Abstract

From auxotrophic and idiotrophic mutants of Streptomyces fradiae (tylosin producer) and Streptomyces sp. AM 4900 (pikromycin producer) or Streptomyces narbonensis (narbomycin producer), prototrophic fusants were obtained at a low frequency by the protoplast fusion technique.In the cross of S. fradiae 261-27E (mvcaminose-idiotroph, ilr) and Streptomyces sp. AM 4900 N3-4, (pikronolide-idiotroph, arg), an unstable prototrophic fusant. strain No. 14, produced a macrolide antibiotic which was not produced by the wild type, parent strains, and the productivity was lost within a few times transfer. It was concluded that the fusant ssas not a recombinant, but a heterokaryon.On the other hand, relatively stable fusants were obtained from the cross of S. fradiae TBM (mycaminose-idiotroph, met) and S. narbonensis NA12US3 (narbonolide-idiotroph, his, str) at a frequency of 3.2' ' 10-5 One of the prototrophic fusants produced narbomvcin, which is believed to be due to a result of interspecific recombination.

Production of new hybrid antibiotics, mederrhodins A and B, by a genetically engineered strain

“…These include mutasynthesis (15), in which an unnatural precursor is fed to an idiotroph genetically blocked in antibiotic biosynthesis, and hybrid biosynthesis (10,11,13), in which a wild-type strain is fed with such a precursor in the presence of an enzyme inhibitor which phenotypically blocks the biosynthetic pathway of the recipient strain. Another approach to the production of novel antibiotics is interspecific mating (9,14) or protoplast fusion (4,5). In the published examples of this approach, the origin of the novel compounds detected is ambiguous; however, it is likely that an alteration of a hitherto unexpressed capacity for antibiotic production of one of the strains in the partnership by transferring into it regulatory genetic material from the other is the predominant mechanism.…”

Isolation and characterization of the interspecific fusants from Streptomycetes obtained using a liquid regeneration method

“…Auxotrophic and antibiotic resistance markers have been used to select recombinants. Because each colony grown using the replica method might have consisted of a mixture of heterokaryons and heterogenotes, which have no genetic cross in their chromosomal DNA, 10 more than three successive purifications might have removed any heterokaryotic or heterogenotic colonies. These results suggest that interspecific gene transfer takes place through protoplast fusion and that the fusants obtained are true recombinants.…”

Use of Permanent Wall-Deficient Cells as a System for the Discovery of New-to-Nature Metabolites

“…Protoplasts carrying multiples chromosomes are typically unable to be sustained for long periods of time and are thus unstable. This instability was observed when the mycaminose-producer Streptomyces fradiae was fused with a picronolide-producing Streptomyces strain [19]. The fusant transiently produced a novel macrolide antibiotic that was not formed by either parent that was hypothesized to have been due to a diploid state of the fusant.…”

“…Three potential outcomes are possible after fusion: (i) recombination between chromosomes, (ii) polyploidy where multiple copies of the recombinant genome are present, and (iii) heterokaryosis where both parental genomes are maintained. L-forms could thus overcome the problem of the short recombination time and fusants that only transiently produce a new metabolite due a diploid state prior to cell wall regeneration [19]. The use of L-forms instead allows for maintenance of heterokaryotic genomes in the same cytoplasmic compartment thereby prolonging opportunities for genetic variation to arise through recombination, resulting in novel production phenotypes.…”