Secretory overproduction of Streptomyces cholesterol oxidase by Streptomyces lividans with a multi-copy shuttle vector

Deletion of a 1,2-kb fragment adjoined upstream of the choP-choA operon and subcloning of the operon into a multi-copy shuttle vector composed of pIJ702 and pUC19 in Streptomyces lividans resulted in the overproduction of Streptomyces cholesterol oxidase extracellularly about 70-fold more than that of the original producer, Streptomyces sp. SA-COO. When the cells of S. lividans carrying the plasmid were grown in an appropriate medium, about 90 to 99% of the enzyme produced was secreted into the medium. Increased concentration of glucose in the culture medium resulted in a significant decrease in the production of the enzyme, while elevated peptone concentrations led to a further increase in the production. The level of the overproduction of cholesterol oxidase was dependent on the copy numbers of the plasmids as well as the presence of the sequences derived from pUC19.