Evrimsel mühendislik yöntemi ile Rhodobacter sphaeroides'in CoCl 2 stresine dirençli suşlarının eldesi

Rhodobacter sphaeroides is a metabolically diverse organism which is able to fix molecular nitrogen and produce H 2 as side product. It can grow under both oxic and anoxic conditions heterotrophically or photosynthetically. This wide repertoire of metabolism puts this organism in the centre of metabolic regulation and bioremediation studies. R. sphaeroides is used in the production of vitamin B 12 , coenzyme Q10, 5-aminolevulinic acid (ALA), porphyrin, as well as in bioremediation of heavy metals. R. sphaeroides follows different metabolic strategies when exposed to different stress factors. It is able to regulate its photosynthetic machinery sensing O 2 tensions. When partial O 2 pressure is reduced, R. sphaeroides develops invagination in its cellular membrane. Biosynthesis of chromatophores, which are crucial for photosynthesis, takes place in these invaginations. There are three systems involved in regulation of photosynthetic genes: the PrrBA two subunit activation system, the PpsR repressor and the FnrL, anaerobic regulator. The genes are regulated mainly by O 2 levels. R. sphaeroides has two chromosomes and five naturally occurring plasmids. Many of its genes are duplicated between the two chromosomes. These gene duplications generate the genetic diversity which allows the bacteria widen its spectrum of metabolic capabilities. Chromosome I contains 3106 open reading frames whereas chromosome 2 contains 874. It possesses a flagellum similar to that of E. coli which is able to rotate only in clockwise direction. When exposed to heavy metal stress, R. sphaeroides alters fatty acid composition to increase its membrane integrity. The accumulation of heavy metals in the environments imposes new strategies for restoring the original site conditions. Bioremediation, providing high specificity and versatility has received a great attention in this area. In this study, R. sphaeroides was used to obtain a cobalt-resistant strain. Evolutionary engineering approach of metabolic engineering was used to increase the cobalt tolerance of the bacterium, by growing it under gradually increasing stress conditions. This approach being a 'bottom-up' approach does not require extensive information about the metabolism of the organism. The R. sphaeroides strain R-26 was used in this study. Initial cobalt concentration to be used for selection was determined after screening analysis of the reference strain at 11 different cobalt concentrations. According to the screening results, the selection of the populations was started with 0.1 mM cobalt concentration. Cobalt concentrations were increased gradually from 0.1 mM to 15 mM through 64 generations. Survival rates were monitored every 24 hours by measuring OD 535 values of control and stress cultures. At the same time points, cultures were spread on LB plates to confirm that no contamination has occurred. Cultures which were grown on solid medium were examined morphologically. Cultures that produced carotenoids were identified by their characteristic red colour. When the survival rate decreased to 0.2, the selection process was terminated for further analysis. Growth behaviour of the reference strain and the last population obtained by evolutionary engineering were xixcompared in media with and without cobalt. The cultures showed similar growth characteristics when there was no cobalt in the medium. However, there was a significant difference, when cultures were grown in a medium containing 4 mM cobalt. The 64 th population cells were able to maintain their growth rate nearly the same when exposed to 4 mM cobalt, while the reference strain's growth was inhibited. These results indicate the improved resistance of 64 th population towards cobalt.