Mycobacterium fortuitum NRRL B-8 1 19 transformes sterols into 9 alpha-hydroxy-androsta-4-ene-3,17-dione (90H-AD) at high efficieny. Cells strongly aggregate to the surface of the sterol particles forming stable agglomerates ("substrate immobilized cells"). Substrate uptake and product formation were studied as function of the size of the sterol particles. Using particle sizes comparable with the size of the mycobacterial cells ( < 5pm) highest rates were found for both the substrate uptake and the product formation. After mechanical reinforcement of the contact between mycobacterial cells and the sterol substrate by co-grinding of the components, a significantly increased product formation was observed. Associated cells and cell-sterol agglomerates were investigated by freeze-fracture electron microscopy. The micrographs obtained demonstrate that cells of M . fortuitum are growing into the sterol microcrystallite. The uptake of the sterol substrate is assumed to take place via direct contact between cells and the substrate particles. To understand the transport mechanism a model is proposed that includes a flexible multicomponent mesophase (FMCM) which is placed between cells and particles and which mediates the sterol uptake. The putative FMCM is assumed to be composed of mycobacterial glycolipids, extracellular biolipids, synthetical detergents, sterol and water.Many bacterial species of different genera are capable of utilizing hydrophobic sterol substrates as the only carbon and energy sources in a remarkably high transformation rate. There are a large number of commercially operated processes to cleave selectively the side chain of the sterol molecule at position C-27, yielding C,, of C,, steroid intermediates which are important for the manufacture of steroid drugs. Microbial strains mostly used in fermentation processes are members of the genus Mycobacterium. Many reviews dealing with the biotechnology and biochemistry of these processes have been published during the last decade (ARIMA 1980, MARTIN 1977, 1984, SCHOMER and MARTIN 1980, NOMINE 1980, KIESLICH 1980, HESSELINK 1988, CHEN 1985. Enzymatic mechanisms of both the side chain cleavage and the degradation or modification of the steroid ring skeleton are well elucidated (SCHUBERT et al. , FUJUMOTO et a(. 1982, CHEN 1985. However, elementary mechanisms of the uptake of the hydrophobic solid sterol substrate occurring in fermentation processes are not clearly understood.Since more than ten years two basic mechanisms have been discussed: a) the substrate uptake via solubilized (molecular dispersed) or via pseudosolubilized (micellar) structures independent on the reason for solubilization or pseudosolubilization, and b) the substrate uptake caused by the direct contact between the microbial surface and the surface of the solid substrate microcrystallite (GOSHWAMI et al. 1983, CAMEOTRA, et al. 1983, HOSEL 1982, HOSEL et al. 1981.Elementary steps actually taking place seem to be a quite more complex process influenced by the physiological state of the gro...

Investigations concerning sterols were made in one of strain each of nine species of bacteria and one representative of the basidiomyeetes through adsorption chromatography, gas chromatography and mass spectrometry as well as ultraviolet and infrared spectroscopy.Three strains contained sterols in quantities ranging from 0.0004 to 0.01 O i 0 of the dry weight. I n Azotobacter chroococcum six sterols were demonstrated. I n six strains sterols were not detectable. The limit of detectability at present is a t 0.0001 O/ , , sterol related to 100 g dry cells.From the extract of A erobacter cloacae, which contained no sterol, a C,,-hydroxy-ketone was isolated.

Using a special selection procedure, several mutants of Mycobacterium vaccae were isolated which were capable of converting sterols to 9 alpha-hydroxyandrost-4-ene-3,17-dion (9-OH-AD). Two mutants, Mycobacterium vaccae ZIMET 11052 and 11053, respectively, were further investigated. Strains of the species Mycobacterium fortuitum are mainly used for commercially obtaining 9-OH-AD from sterols. In contrast to the species Mycobacterium fortuitum the species Mycobacterium vaccae has not been reported to contain pathogenic strains. This seems an advantage for industrial application. Mutants with the ability of converting sterols to 9 alpha-hydroxysteroids have a defect in the steroid-1-dehydrogenase activity which is, however, only a partial one. The remaining activity may cause an undesirable degradation of the steroid nucleus. The steroid-1-dehydrogenase activity was tested using an assay developed by ATRAT (1986). We confirmed two apparently distinct steroid-1-dehydrogenases in Mycobacterium fortuitum NRRL B-8119 as reported by WOVCHA et al. (1979). One of them has an activity on androst-4-ene-3,17-dion (AD). The activity is increased by induction with sitosterol. The other one is active on 9-OH-AD. But Mycobacterium vaccae does not possess steroid-1-dehydrogenase activity on 9-OH-AD, and the AD specific steroid-1-dehydrogenase is not effected by sitosterol. The consequence is a high level of protection against steroid nucleus degradation yielding an effective accumulation of 9-OH-AD in fermentations with Mycobacterium vaccae mutants.

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Es wird ein zusammenhängender Reaktionsweg für die Umwandlung von gesättigten 17-Ketosteroiden in aromatisierte Steroide bei M. smegmatis beschrieben. Ein prinzipiell gleichartiger oxydativer Abbau von Steroiden wurde auch bei anderen Mycobakterien nachgewiesen. Die strukturellen Merkmale der aromatisierten seco-Verbindung werden im Zusammenhang mit der Carcinogenese diskutiert.