Regulation of steroid 16α‐hydroxylation in Streptomyces olivoviridis

Abstract

The steroid 16a-hydroxylase of Streptomyces olivoviridis was shown to be constitutive, but synthetized only in small amounts in the absence of cortisol. The induction of the enzyme with the steroidal substrate resulted in a distinct increase in t h e 16a-hydroxylation rate. This increase in activity, however, could be demonstrated only in later phases of culturing, after the growth of the mycelium was completed. The possible mechanisms controlling the activity of the enzyme during t h e growth phase of t h e culture are discussed.The ability of certain streptomycetes to 16 a-hydroxylate the steroid nucleus was first described by PERLMAN et al. in In previous work less attention was paid to the mechanisms of regulation of steroidhydroxylase activity. In our present paper more detailed studies on the relationship between cell growth, intracellular metabolic pool constituents, and the 16 a-hydroxylation activity were carried out. Materials and methodsMicroorganism: A streptomycete strain, isolated from t h e local soil on grounds of its ability for 16a-hydroxylation of C-21 steroids was used. The choice was dictated by t h e practically onedirectional transformation of hydrocortisone. The strain was identified as Streptomyces olivoviridis at t h e Department of Bacteriology of the State Institute of Hygiene, Warsaw.Media: The stock culture was kept on slants of Bacto-Potato-Dextrose-Agar (DIFCO) a t 4 "C and reinoculated once a month. For conidia germination a medium (designated as medium 1 a ) of the following composition was used (g/l): corn steep liquor 50% -20, sucrose -30, (NH,),SO, --2, CaCO, -7.Growth and bioconversion were investigated on medium D of following composition (g/l) : soluble starch -40, corn steep liquor 5076 -20, CaCO, -5 (MARTINKOVA and DYR 1965). The media were sterilized at 117 "C for 30 min. After sterilization the media were adjusted to p H = 6.7.Reagents : Hydrocortisone (SCHERING) was the steroid substrate. The product of hydroxylation was compared with 1%-hydroxyhydro-cortisone (POLFA). The substrate was dissolved in N,Ndimethylformamide (KOCH-LIGHT Lab.). Sugar and amino acids were purchased from MERCK and CALBIOCHEM, respectively.Cultivation and bioconversion: Conidia were washed off from a 14-day culture on potato slants and t h e suspension was transferred to 20 ml of medium l a in 200-ml-Erlenmeyer flasks. Incu-