Naslov
Genetic engineering in S. rimosus and its effects on DNA rearrangements

Autori
Lesnik, Urska ; Fujs, Stefan ; Raspor, Peter ; Hranueli, Daslav ; Hunter, S. Iain ; Petković, Hrvoje

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
Book of Abstracts / Vaclav Paces - Prag : Czech Academy of Sciences, 2006, 102-103 (P171)

Skup
10th International Symposium on the Genetics of Industrial Microorganisms

Mjesto i datum
Prag, Češka Republika, 24.06.2006. - 28.06.2006

Vrsta sudjelovanja
Poster

Vrsta recenzije
Nije recenziran

Ključne riječi
Streptomyces rimosus; oxytetracycline biosynthesis; gene-cluster; DNA rearrangements; genetic instability

Sažetak
Many phenotypic characteristics that are associated with differentiation and secondary metabolism are genetically unstable, including aerial mycelium formation, pigmentation, sporulation, resistance to antibiotics, and/or antibiotic production. Genetic instability in Streptomyces is a very common phenomenon with spontaneous mutants occurring at frequencies higher than 10-3. S. rimosus, the oxytetracycline (OTC) producer, is not an exception. However, higher frequencies (up to 100%) of instability at the genome level are induced by genetic modification of the otc genes of S rimosus strains, causing the frequency of occurrence of gene rearrangements to be close to 100%. The studies on the genetics of OTC biosynthesis in S. rimosus have been severely hampered by genetic rearrangements. Therefore, we have considered various approaches to avoid or at least reduce this problem. The transfer of entire otc cluster into the genetically amenable host S. lividans reduced the gene rearrangement almost entirely. Unfortunately, the yield of the OTC produced was reduced up to 100-fold. The compound(s) produced by the genetically engineered S. lividans strain produced only a trace amount of the desired products thus making the compound purification and structure determination work extremely difficult. Despite the observed gene instability induced by genetic manipulations, the over-producing S. rimosus strains seemed to be the best system to study OTC biosynthesis. In our attempt to reduce gene instability, we have employed various gene disruption and replacement strategies using streptomycete replicative vectors, E. coli non-replicative vectors ("suicide disruption") and linearised double and single strand constructs. The frequency of plasmid DNA transformation (integration) and the DNA rearrangement patterns varied according to the method applied. This work has allowed us to asses the best approach to reduce the gene instability in order to isolate the desired mutants.

Izvorni jezik
Engleski

Znanstvena područja
Biotehnologija

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