earticle

영어

In bacteria and fungi, the process of carotenoid desaturation step is derived by a single enzyme, the phytoene desaturase (CrtI). Here we constructed beneficial mutants, which produce only neurosporene or lycopene up to 99% as a major product through random mutagenesis and site-directed mutagenesis. Three generations of random mutagenesis was performed and nine positive mutants were isolated and sequenced. Then we determined the effect of each amino acid changes using by site-directed mutagenesis. Through site-directed mutagenesis, we found two critical mutation points, L163P and G511C. To compare the carotenoid biosynthetic pathway of R.spheroides and P.agglomerans, each gene CrtE (GGPP synthase), CrtB (phytoene synthase), was modified to a self-expression module and then reconstructed as a synthetic operon (pACM_ERS_BRS and pACM_EPA_BPA) in Escherichia coli. With constructed two types of synthetic operon, we compared the different effect when expressed with mutant CrtIRS. The comparison result showed that nuerosporene expression rate was better when mutant CrtIRS was introduced into E.coli that harboring pACM_ERS_BRS, while lycopene expression rate was better when it was introduced into E.coli that harboring pACM_EPA_BPA. For better understanding in functional effect of each mutated point, predicted crystal structure modeling of phytoene desaturase from R.spheariodes was performed and analyzed.