Cloning and expression of Mycobacterium aurum carotenogenesis genes in Mycobacterium smegmatis

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Abstract
This report describes the first successful transfer and complete expression of clustered mycobacterial genes controlling a biosynthetic pathway (carotenogenesis) in a homologous system. A genomic library of pigmented Mycobacterium aurum A+ (yellow-orange) DNA was constructed in shuttle vector pHLD-69. The colourless mutant A11 and the brick-red mutant NgR9 derived from M. aurum A+ were electroporated with the plasmid library. Among the transformants, colonies different in colour from the recipient mutants were detected, and were cloned. One of the clones from the transformed A11 mutant had an yellow-orange phenotype, and was designated A11T; one of the clones from the NgR9 (brick-red) mutant had an yellow-orange phenotype and was designated NgR9T. The carotenoid pigments from the A11T and NgR9T clones were analyzed and in both the end product of carotenogenesis in M. aurum (leprotene) was detected. A11T and NgR9T harboured the same recombinant plasmid (pC1) containing a 11-kb M. aurum fragment. pC1 was used to transform the colourless Mycobacterium smegmatis MC2-155 strain. All the transformants were pigmented. A colony (MC2-T) was arbitrarily chosen and leprotene was detected. It was therefore concluded that M. aurum genes involved involved in carotenogenesis had been cloned, and were expressed not only in M. aurum mutants, but also in M. smegmatis.

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