| Title | Overexpression of the sprD Gene Encoding Streptomyces griseus Protease D Stimulates Actinorhodin Production in Streptomyces lividans |
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| Author | Si-Sun Choi, Won-Jae Chi, Jae Hag Lee 1 , Sang-Soon Kang 2 , Byeong Chul Jeong, and Soon-Kwang Hong * |
| Address | Department of Biological Science, Myongji University, Yongin 449-728, Korea; 1 Seoil College, Seoul 131-208, Korea; 2 Division of Science Education, C |
| Bibliography | Journal of Microbiology, 39(4),305-313, 2001, |
| DOI | |
| Key Words | sprD, S. griseus, protease, actinorhodin, S. lividans |
| Abstract | The sprD gene encoding Streptomyces griseus protease D (SGPD), a chymotrypsin-like protease, was cloned from Streptomyces griseus IFO13350 and sequenced. Most of the amino-acid sequence deduced from the nucleotide sequence is identical to that of Streptomyces griseus IMRU3499 except that one amino acid has been deleted and Trp369 has been substituted into Cys369 in the SGPD from S. griseus IFO13350 without affecting the protease activity. The sprD gene was overexpressed in Streptomyces lividans TK24 as a heterologous host. Various media with different compositions were also used to maximize the productivity of SGPD in the heterologous host. The SGPD productivity was best when the transformant of S. lividans TK24 was cultivated in R2YE medium. The relative chymotrypsin activity of the culture broth measured with an artificial chromogenic substrate, N-succinyl-ala-ala-pro-phe-[rho]-nitroanilide, was 16 units/ml. A high level of SGPD was also produced in YEME and SAAM media but it was relatively lower than in R2YE medium, and negligible amounts of SGPD were produced in GYE, GAE and Benedict media. The growth of S. lividans reached the maximum level of cell mass at days 3 and 4 of the culture, but SGPD production started in the stationary phase of cell growth and kept increasing till the 10th day of culture in R2YE and YEME medium, but in GYE media the productivity reached maximum level at 8 days of cultivation. The introduction of the sprD gene into S. lividans TK24 triggered biosynthesis of the pigmented antibiotic, actinorhodin, which implies some protease may play a very important role in secondary-metabolite formation in Streptomyces. |
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