Secretory overproduction of Arthrobacter simplex 3-ketosteroid delta 1-dehydrogenase by Streptomyces lividans with a multi-copy shuttle vector.
The gene for 3-ketosteroid delta 1-dehydrogenase (ksdD) of Arthrobacter simplex was expressed in Streptomyces lividans and the secreted enzyme was overproduced by using a multi-copy shuttle vector composed of pIJ702 and pUC19. Deletional analysis of the recombinant plasmid showed that the entire coding sequence of the ksdD gene was located within a 7-kb segment of the chromosomal DNA obtained from the enzyme-producing strain of A. simplex. When S. lividans carrying the recombinant plasmid was grown in an appropriate medium, the cells produced about 100-fold more 3-ketosteroid delta 1-dehydrogenase than the original strain. Although the percentage of enzyme secreted was changed during cultivation, a maximum 55% of the enzyme was secreted into the cultured medium of S. lividans, while A. simplex did not produce the enzyme extracellularly. Secretory overproduction of 3-ketosteroid delta 1-dehydrogenase in S. lividans was also identified by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and on native gel, and the enzyme reaction was confirmed by reverse-phase HPLC using 4-androstene-3,17-dione as a substrate.
Choi
K P
KP
Department of Biotechnology, Faculty of Engineering, Osaka University, Japan.
Molnár
I
I
Murooka
Y
Y
D000730:Androstadienes / Q000378:metabolism; D000735:Androstenedione / Q000378:metabolism; D001173:Arthrobacter / Q000201:enzymology / Q000235:genetics*; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D017077:Culture Media, Conditioned / Q000737:chemistry; D004269:DNA, Bacterial / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D010088:Oxidoreductases / Q000096:biosynthesis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D017384:Sequence Deletion; D013302:Streptomyces / Q000378:metabolism*
D000730:Androstadienes; D001426:Bacterial Proteins; D017077:Culture Media, Conditioned; D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; C006573:1,4-androstadiene-3,17-dione; D000735:Androstenedione; D010088:Oxidoreductases; C022656:3-oxosteroid delta(1) dehydrogenase