Characterization and engineering of 3-ketosteroid-△1-dehydrogenase and 3-ketosteroid-9α-hydroxylase in Mycobacterium neoaurum ATCC 25795 to produce 9α-hydroxy-4-androstene-3,17-dione through the catabolism of sterols.
3-Ketosteroid-△(1)-dehydrogenase (KstD) is a key enzyme involved in the microbial catabolism of sterols. Here, three homologues of KstD were characterized from Mycobacterium neoaurum ATCC 25795, showing distinct substrate preferences and transcriptional responses to steroids. Single deletion of any MN-kstD failed to result in a stable and maximum accumulation of 9-OHAD due to residual KstD activities. To develop stable 9-OHAD producers, all of these MN-KstDs were inactivated, which led to about 6.02g l(-1) of 9-OHAD from 15g l(-1) of phytosterols. However, the product was mixed with 1.55g l(-1) of AD as a major by-product. To transform AD, the oxygenase component of 3-ketosteroid-9α-hydroxylase (KSH), encoded by kshA, was overexpressed. As a result, the yield of 9-OHAD increased to 7.33g l(-1) with less than 0.31g l(-1) of AD and the selectivity of 9-OHAD production was improved to 95-97% among metabolites.
State Key Laboratory of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, China.
Kang
K
Yao
State Key Laboratory of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, China.
Li-Qin
LQ
Xu
State Key Laboratory of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, China. Electronic address: fqwang@ecust.edu.cn.
Feng-Qing
FQ
Wang
State Key Laboratory of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, China. Electronic address: dzhwei@ecust.edu.cn.
Dong-Zhi
DZ
Wei
D000735:Androstenedione / Q000031:analogs & derivatives* / Q000096:biosynthesis; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism; D009161:Mycobacterium / Q000201:enzymology / Q000235:genetics; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism; D010840:Phytosterols / Q000378:metabolism
D001426:Bacterial Proteins; D010840:Phytosterols; D000735:Androstenedione; C076717:9-hydroxy-4-androstene-3,17-dione; D006899:Mixed Function Oxygenases; D010088:Oxidoreductases; C022656:3-oxosteroid delta(1) dehydrogenase
3-Ketosteroid-△(1)-dehydrogenase; 9α-Hydroxy-4-androstene-3,17-dione; Mycobacteria; Sterols