Interactions of Mycobacterium fortuitum with solid sterol substrate particles

Abstract

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...

Methyl-β-cyclodextrin alters growth, activity and cell envelope features of sterol-transforming mycobacteria

“…The heterophase character of cell interaction with solid substrate particles in the presence of MCD was similar to that described for non-MCD medium (Atrat et al, 1991). It is in accordance with the high adhesiveness of mycobacterial cells grown in the presence of MCD.…”

“…However, in order to make the microbial transformations economically attractive, the bioconversion process is carried out at sterol concentrations much higher than the sterol solubility. In this heterogeneous system, the mycobacterial cells sorbed on the surface of sitosterol particles, and sitosterol uptake occurred via direct contact of the cells with this hydrophobic substrate (Atrat et al, 1991).…”

Microbial conversion of sterol‐containing soybean oil production waste

“…No steroid products were detected during the initial 300-350 h. Based on the current concept of the mechanism of mycobacteria interaction with sterol substrates, 9 the following reasons of low scum bioconversion rate were suggested: (a) the presence of components competing with sterols as substrates for oxidation processes; (b) low availability of scum sterols for cell biocatalyst; and (c) spatial restrictions due to scum agglomeration.…”

Biotransformations Utilizing β-Oxidation Cycle Reactions in the Synthesis of Natural Compounds and Medicines

“…The preferred method of enhancing the bioavailability of phytosterols by improving their solubility is surfactant-facilitated emulsification [61,62]. During the transformation process, sterol uptake occurs by direct contact between microbial cells and the phytosterol particles [61,63,64]. Therefore some microorganisms can use poorly water-soluble hydrocarbons as carbon sources, especially if these microorganisms have the following features: lipophilic cell walls, active transporters and membrane-associated enzymes, as well as the capacity to secrete biosurfactants or bioemulsifiers increasing the availability of hydrophobic compounds [65–67].…”

“…Electron microscopy results led to a “flexible multi-component mesophase” model [63] providing a sharp concentration gradient in the mesophase between particles of sterols and the membrane. The model suggests that there should exist channels of proteins, stretching from the cytoplasm to the surface of the cell, capable of sterol binding or transformation, thus enhancing the transport.…”