Characterization and application of fusidane antibiotic biosynethsis enzyme 3-ketosteroid- Delta super(1)-dehydrogenase in steroid transformation

• Published in: Applied microbiology and biotechnology Vol. 96; no. 1; pp. 133 - 142
• Main Authors: Chen, Miao-Miao, Wang, Feng-Qing, Lin, Liang-Cai, Yao, Kang, Wei, Dong-Zhi
• Format: Journal Article
• Language: English
• Published: 01.01.2012
• Subjects: Aspergillus fumigatus; Escherichia coli; Pichia pastoris
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-011-3855-5

Microbial Delta super( 1 ) -dehydrogenation is one of the most important transformations in the synthesis of steroid hormones. In this study, a 3-ketosteroid- Delta super(1)-dehydrogenase (kstD sub(F)) involved in fusidane antibiotic biosynthesis from Aspergillus fumigatus CICC 40167 was characterized for use in steroid transformation. KstD sub(F) encodes a polypeptide consisting of 637 amino acid residues. It shows 51% amino acid identity with a kstD from Thermomicrobium roseum DSM 5159. Expression of kstD sub(F) in Escherichia coli and Pichia pastoris showed that all kstD sub(F) activity is located in the cytoplasm. This indicates that it is a soluble intracytoplasmic enzyme, unlike most kstDs from bacteria, which are membrane-bound. The expression of kstD sub(F) was performed in P. pastoris, both intracellularly and extracelluarly. The intracellularly expressed protein displayed good activity in steroid transformation, while the extracellularly expressed protein showed nothing. Interestingly, the engineered P. pastoris KM71 (KM71 sub(I)) and GS115 (GS115 sub(I)) showed different transformation activities for 4-androstene-3,17-dione (AD) when kstD sub(F) was expressed intracellularly. Under the same conditions, KM71 sub(I) was found capable of transforming 1.0 g/l AD to 1,4-androstadiene-3,17-dione (ADD), while GS115 sub(I) could transform 1.5 g/l AD to both ADD and boldenone (BD). The production of BD is attributed to a 17 beta -hydroxysteroid dehydrogenase in P. pastoris GS115 sub(I), which catalyzes the reversible reaction between C17-one and C17-alcohol of steroids. The conversion of AD by GS115 sub(I) and KM71 sub(I) may provide alternative means of preparing ADD or BD. In brief, we show here that kstD sub(F) is a promising enzyme in steroid Delta super( 1 ) -dehydrogenation that is propitious to construct genetically engineered steroid-transforming recombinants by heterologous overexpression.