Microbial conversion of mevinolin.

About 3000 microorganisms (bacteria, Actinomyces, Zygomyces, Deuteromyces) were screened for their capacity to convert mevinolin. Absidia coerulea IDR 705 was found to produce two hydroxylated derivatives of mevinolin, 2 and 3. Compound 2 is a new transformation product while compound 3 was described as a chemical modification product of mevinolin. By combination of spectroscopic techniques, the structures of 2 and 3 were identified with beta,delta-dihydroxy-7-(1,2-dihydro-2-hydroxymethyl-6-methyl-naphthal en-1-yl)-heptanoic acid delta-lactone and beta,delta-dihydroxy-7-[1,2,3,5,6,7,8,8a-octahydro-3,5-dihydroxy-2, 6-dimethyl-8-(2-methyl-butyryloxy)-naphthalen-1-yl]-hepta noi c acid delta-lactone, respectively. The inhibitory effects of the two derivatives on the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase were similar to that of mevinolin.

Microbial transformation of beta-sitosterol and stigmasterol into 26-oxygenated derivatives.

The genetically modified Mycobacterium sp. BCS 396 strain has been used to transform sterols with stigmastane side chain in order to obtain 26-oxidized metabolites. beta-Sitosterol (I) was transformed to 4-stigmasten-3-one (II), 26-hydroxy-4-stigmasten-3-one (III), and 3-oxo-4-stigmasten-26-oic acid (IV), while stigmasterol (V) was converted to 4,22-stigmastadien-3-one (VI), 6 beta-hydroxy-4,22-stigmastadien-3-one (VII), 26-hydroxy-4,22-stigmastadien-3-one (VIII), 3-oxo-4,22-stigmastadien-26-oic acid methyl ester (IX), and 3-oxo-1,4,22-stigmastatrien-26-oic acid methyl ester (X) with that strain. In both beta-sitosterol and stigmasterol, 26-oxidation generates the R-configuration on C-25.

Novel 26-oxygenated products in microbial degradation of ergosterol.

In order to investigate the effect of the different stereochemistry of C-24 on the microbial C-26 oxidation of sterol side-chain the genetically modified Mycobacterium sp. BCS 396 strain was used to transform erogsterol. Ergosterol was converted to 3-oxo-4,22-ergostadien-26-oic acid methyl ester, 3-oxo-1,4,22-ergostatrien-26-oic acid methyl ester, and 3-oxo-1,4,22-ergostatrien-26-oic acid, the structures of which have been determined by IR, 1H NMR, 13C NMR, and mass spectroscopy. The X-ray structure of 3-oxo-4,22-ergostadien-26-oic acid methyl ester revealed that oxidation at C-26 of the ergostane side-chain generates a chiral center with S-configuration at C-25 as a result of chiral induction of the C-24 center.

Development of genetic systems for the mycobacteria.

Requisite to a detailed understanding of the molecular basis of bacterial pathogenesis is a genetic system which allows for the transfer, mutation, and expression of specific genes. Genetic analysis of mycobacteria has been exceedingly difficult since the mycobacteria grow slowly and no natural efficient method of gene transfer within the pathogenic has thus far been found. Using a molecular genetic approach, we have developed both the vectors and the methodology for efficient gene transfer in the mycobacteria. Initially, a novel of type of mycobacteriophage vector was developed, termed a shuttle phasmid. This hybrid shuttle vector replicates in Escherichia coli as a plasmid and in mycobacteria as a phage, capable of introducing foreign DNA into a wide variety of mycobacterial species. A set of shuttle phasmids, constructed from a temperate mycobacteriophage, retained their ability to lysogenize their mycobacterial hosts and could thus introduce foreign DNA stably into mycobacterial cells. An E. coli gene conferring kanamycin-resistance was cloned into these vectors and shown to express in the mycobacteria, thus providing the first selectable marker gene for subsequent genetic studies. Using kanamycin-resistance gene as a selection, the M. fortuitum plasmid pAL5000 replicon, and electroporation; a plasmid transformation system has been developed for both M. smegmatis and BCG. We now plan to use these phage and plasmid systems to analyze, genetically, the virulence attributes of the pathogenic mycobacteria. In addition, by introducing and expressing foreign antigens in BCG, we hope to develop a novel recombinant multi-vaccine vehicle capable of conferring immunity to a variety of bacterial, viral, and parasitic pathogens.

Lysogeny and transformation in mycobacteria: stable expression of foreign genes.

Requisite to a detailed understanding of the molecular basis of bacterial pathogenesis is a genetic system that allows for the transfer, mutation, and expression of specific genes. Because of the continuing importance of tuberculosis and leprosy worldwide, we initiated studies to develop a genetic system in mycobacteria and here report the use of two complementary strategies to introduce and express selectable genetic markers. First, an Escherichia coli cosmid was inserted into the temperate mycobacteriophage L1, generating shuttle phasmids replicating as plasmids in E. coli and phage capable of lysogenizing the mycobacterial host. These temperate shuttle phasmids form turbid plaques on Mycobacterium smegmatis and, upon lysogenization, confer resistance to superinfection and integrate within the mycobacterial chromosome. When an L1 shuttle phasmid containing a cloned gene conferring kanamycin resistance in E. coli was introduced into M. smegmatis, stable kanamycin-resistant colonies--i.e., lysogens--were obtained. Second, to develop a plasmid transformation system in mycobacteria, M. fortuitum/E. coli hybrid plasmids containing mycobacterial and E. coli replicons and a kanamycin-resistance gene were constructed. When introduced into M. smegmatis or BCG (Mycobacterium tuberculosis typus bovinus var. Bacille-Calmette-Guérin) by electroporation, these shuttle plasmids conferred stable kanamycin resistance upon transformants. These systems should facilitate genetic analyses of mycobacterial pathogenesis and the development of recombinant mycobacterial vaccines.