Genetic Recombination by Spheroplast Fusion of Sterol-transforming Mycobacterium Strains

Abstract

Wall-deficient forms of fast-growing mycobacteria were produced in growth medium containing vancomycin and glycine, and spheroplasts were prepared by lysozyme treatment of wall-deficient cells. Spheroplasts gave rise to recombinants with high frequency (2-6 %) when they were fused using polyethylene glycol 6000. The results demonstrated that in uivo genetic recombination could be used to produce genetically modified Mycobacterium strains with applications in transformation of steroids. Useful intermediates of steroid drug synthesis and new degradation products were obtained from sterols by selected recombinant strains.

Localization of 17β-hydroxysteroid dehydrogenase in Mycobacterium sp. VKM Ac-1815D mutant strain

“…Glycine is known to incorporated into the uridine nucleotide precursors in l-alanine site, inhibit cell wall synthesis. It has been successfully used for the preparation of a wall-deficient form of mycobacteria [19,21].…”

“…Glycine was reported to facilitate further production of mycobacterial spheroplasts with a high yield [19]. After 24 h growth of Mycobacterium sp.…”

Role of plasmid pJL1 of Arthrobacter oxydans 317 in the degradation of β‐sitosterol

“…Though the enzymology of each step of sterol sidechain cleavage is not known, accumulation of intermediary sterol metabolites with intact steroid nucleus and partially cleaved sterol side chain have been reported (KNIGHT et al 1980, HILL et al 1982, JEKKEL et al 1989. Plasmid mediated catabolic degradation of many organic compounds are known t'oday.…”

“…It was proposed that pJLl is involved in the conversion of 3-oxochol-4-en-24-oic acid to AD and in the 9a-hydroxylation of the steroid nucleus but it is not connected with 1,2-dehydrogenation, a critical reaction step in steroid ring opening.Bacterial strains lacking either 9a-hydroxylase or 1,2-dehydrogenase or both are considered important in sterol biotransformation for production of AD and ADD provided that the strains are capable of complete elimination of C-17 alkyl sidechain of the sterol (MARTIN 1977). Though the enzymology of each step of sterol sidechain cleavage is not known, accumulation of intermediary sterol metabolites with intact steroid nucleus and partially cleaved sterol side chain have been reported (KNIGHT et al 1980, HILL et al 1982, JEKKEL et al 1989. Plasmid mediated catabolic degradation of many organic compounds are known t'oday.…”

Generation of Useful Insertionally Blocked Sterol Degradation Pathway Mutants of Fast-Growing Mycobacteria and Cloning, Characterization, and Expression of the Terminal Oxygenase of the 3-Ketosteroid 9α-Hydroxylase in Mycobacterium smegmatis mc 2 155

“…Economic production of these intermediates is an important goal for pharmaceutical companies. So far, mutation-selection and in vivo genetic recombination have been used for strain improvement (11). Here we describe the use of in vitro DNA recombination for this purpose.…”