ABSTRACT
beta-Sitosterol side chain degradation by Mycobacterium sp. NRRL MB 3683 results in the formation of androstene derivatives and is increased in the presence of glycine. As the sterol transformation is carried out inside the cell, higher product accumulation could indicate faster diffusion of highly hydrophobic substrate through the cell wall permeability barrier. Cell wall preparations were obtained to analyse the effect of glycine on peptidoglycan components. Peptidoglycan is known to be the target for glycine action. In glycine-treated preparations, the molar ratio of diaminopimelic acid:muramic acid, the marker compounds of tetrapeptides and glycan strands respectively, was about 60% lower than in the control. This indicates a possible reduction in cross-linking between peptide units and the destruction of peptidoglycan. Unexpectedly, glycine also caused changes in the relative proportion of mycolic acids to other lipids occurring in the strain used for this study. The enhancement of beta-sitosterol side chain degradation is likely to result from disturbing the integrity of the cell wall components responsible for the permeability barrier in mycobacteria.
Subject(s)
Androstenes/metabolism , Mycobacterium/metabolism , Sitosterols/metabolism , Biomass , Biotransformation/drug effects , Cell Wall/chemistry , Cell Wall/metabolism , Densitometry , Glycine/pharmacology , Mass Spectrometry , Membrane Lipids/metabolism , Mycobacterium/drug effects , Mycobacterium/growth & development , Mycolic Acids/metabolism , Peptidoglycan/drug effects , Peptidoglycan/metabolismABSTRACT
Postselective (directed) mutagenesis was used to create mutants of M. vaccae B3805 showing changed abilities for steroid biotransformations. Three morphological classes of such mutants, differing in colony colour: yellow, white and pink, were selected. Some of them can be helpful in genetic studies of steroid biotransformation. Two mutants were also shown to be hosts for foreign DNA.
Subject(s)
Mutagenesis , Mutation/physiology , Mycobacterium/genetics , Mycobacterium/metabolism , Biotransformation , DNA, Bacterial/analysis , Kinetics , Phenotype , Plasmids/metabolism , Sitosterols/metabolism , Steroid Hydroxylases/metabolism , Steroids/metabolismABSTRACT
Stable mutants showing improved 11-hydroxylation of Substance S were isolated, following treatment with N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and regeneration of uninucleate protoplasts of the appropriate fungal strains. This procedure was especially suitable for obtaining more directed 11 beta-hydroxylation of Substance S with Curvularia lunata IM 2901. Apart from producing cortisol (11 beta-hydroxy-S), the parent strain formed several by-products that significantly lowered the yield of the desired 11 beta-hydroxyderivative. Isolated mutants of this microorganism carried out directed 11 beta-hydroxylation with only a small amount of one of the by-products, which resulted in a much higher yield of cortisol.
