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영어

Rapamycin is a macrocylic polyketide with immunosuppressive, antifungal, and anticancer activity. The production level of rapamycin in Streptomyces hygroscopicus ATCC29253 was enhanced by classical mutagenesis and metabolic engineering approach. This study describes the development of a novel highthroughput method for rapid screening of rapamycin-producing strains using agar diffusion method and metabolic engineering strategy to enhanced rapamycin production. Rapamycin production by a mutant strain UV2-2 induced by Ultraviolet (UV) mutagenesis was improved by approximately 3.2-fold compared to that of wild-type strain. The comparative analysis of gene expression and intracellular acyl-CoA pool between wild-type and the UV2-2 strains revealed that the increased production of rapamycin in UV2-2 was due to the prolonged expression of rapamycin biosynthetic genes but the low concentration of intracellular methylmalonyl-CoA was limiting the rapamycin biosynthesis in both strains. In an attempt to promote intracellular pool of methylmalonyl-CoA, propionyl-CoA carboxylase pathway was introduced into the mutant strain UV2-2 along with supplementation of methyloleate, resulting in an additional 1.7-fold improvement of rapamycin titers. These results demonstrate that the combined approach involving traditional mutagenesis and metabolic engineering can be successfully applied for the rapamycin-producing strain.