Maintenance of the recombinant plasmid pIJ2 in chemostat cultures of Streptomyces lividans 66 (pIJ2)

Abstract

The maintenance of the recombinant plasmid p I J 2 in chemostat cultures of Streptomyces lividans 66 (~1 5 2 ) was investigated. The presence of tho plasmid coding for a ncomycin phosphotransferase was detected by plating samples from the continuous cultures on nonselective and selective agar medium containing neomycin.The plasmid was lost from the host strain under all conditions tested. However, the kinetics of the plasmid segregation from the chemostat populations were dependent on the growth-limiting substrate of the medium, the dilution rate of the continuous culture and the cultivation ternperature.Size differences between the original plasmid and plasmid DNA isolated from neomycinresistant clones after long-term chemostat cultivation were not observed. In neomycin-sensitive clones no extrachromosomal DNA was found.Recently a substantial literature has accumulated concerning the structural stability and the maintenance of plasmids in their host organisms. Continuous culture techniques proved as a suitable tool in studying these problems (for review DYKHUIZEN and HARTL 1983). Knowledge about stability and maintenance is relevant particularly in the case of vector and recombinant plasmids. For the successful use of a plasmid-containing bacterial strain in large-scale fermentation of a plasmid-coded product it is necessary that the recombinant plasmid is both structurally stablc and maintained at a sufficient copy number.It was, for example, impossible to produce tryptophan in a continuous fermentation

Taxonomy, Physiology, and Natural Products of Actinobacteria

“…A novel set of genes was recently discovered in Streptomyces that control pellet growth, called mat, for mycelial aggregation (387). The genes were discovered 30 years ago via reverse engineering of a strain of S. lividans that was selected for fragmented growth in a chemostat (388). Deletion of the mat genes (SCO2962 and SCO2963 in S. coelicolor) prevented pellet formation and increased both growth rate and enzyme production in S. lividans (387).…”

A novel locus for mycelial aggregation forms a gateway to improved Streptomyces cell factories

“…Because less fit variants wash out over time there is a strong selection for increased growth rates and high substrate uptake rates. The high dilution rate used for the evolution of the S. lividans derivatives PM01 and PM02 30 years ago [ 27 ] resulted in dramatic morphological changes of this streptomycete that normally grows as dense clumps. Instead, the derivatives produced small and open pellets (PM01, selected after 70 cycles) or complete lack of any pellets (PM02, after 100 cycles).…”

“…A regime to improve growth of Streptomyces lividans 66 by selection in a chemostat for over 100 generations resulted in a stable derivative with a non-pelleting phenotype (PM02), and an intermediate mutant forming loose pellets (PM01) [ 27 ]. PM02 was used to study the segregational stability of plasmids in continuous culture with different growth limiting substrates [ 27 , 28 ]. In this work, we identified the mutations accumulated during the generation of S. lividans 66-PM01 and 66-PM02.…”

Directed selection of differentiation mutants of Streptomyces noursei using chemostat cultivation

“…Based on the observation that plasmid DNA is eliminated from E . coli host strains in dependence on cultivation conditions present in the (ROTH et al 1985).I n order t o study segregation of both chromosomal and extrachromosomal DNA of an antibiotic-producing Xtreptomyces strain, we carried out a number of chemostat cultivations. For these experiments we used the strain Streptomyces noursei IMET 3890b NG13 (for reference FRIEDRICH et al 1984) which is the producer of the streptothricin antibiotic nourseothricin.…”

“…Based on the observation that plasmid DNA is eliminated from E . coli host strains in dependence on cultivation conditions present in the chemostat (NOACK et , WOUTERS and VAN ANDEL 1983, JONES and MELLIN~ 1984 we investigated the maintenance of the recombinant plasmid pIJ2 in Streptornyces lividam in parallel experiments (ROTH et al 1985).…”