Brian Heimbuch

Bioconversion of indene to cis (1S,2R) indandiol and trans (1R,2R) indandiol by Rhodococcus species

Abstract cis (1 S ,2 R ) indandiol or trans (1 R ,2 R ) indandiol are both potential precursors to (−)- cis (1 S ,2 R )-1-aminoindan-2-ol, a key chiral synthon for Crixivan ® (Indinavir), a leading HIV protease inhibitor. Enrichment and isolation studies yielded two Rhodococcus sp. strain B 264-1 (MB 5655) and strain I-24 (MA 7205) capable of biotransforming indene to cis (1 S ,2 R ) indandiol and trans (1 R ,2 R ) indandiol respectively. Isolate MB 5655 was found to have a toluene dioxygenase, while isolate MA 7205 was found to harbor both toluene and naphthalene dioxygenases as well as a naphthalene monooxygenase. When scaled up in a 14- l bioreactor, MB 5655 produced up to 2.0 g/ l of cis (1 S ,2 R ) indandiol with an enantiometric excess greater than 99%. MA 7205 cultivated under similar conditions produced up to 1.4 g/ l of trans (1 R ,2 R ) indandiol with an enantiomeric excess greater than 98%. Process development studies yielded titers greater that 4.0 g/ l of cis indandiol for MB 5655. Due to their resistance to indene toxicity and easy cultivation in bioreactors, both Rhodococcus sp. strains appeared as good candidates for future strain engineering and process development work.

Isolation of zoosporogenous actinomycetes from desert soils

We have undertaken a study to estimate the species diversity of zoosporogenous actinomycetes that can be isolated from an arid environment. The study site encompassed an area of approximately 22 000 square kilometers of the Mojave Desert along the California-Nevada border. A series of 29 soil samples was collected along two intersecting transects of approximately 190 and 240 km which traversed a number of distinct ecosystems. A0 horizon soils were collected from the rhizosphere of the predominant vegetation at each sampling site and screened for the target genera using selective isolation techniques: chemoattraction (xylose and γ-collidine) and baiting with hair. Following incubation of primary isolation plates for 28 days at 28°C, all colonies that exhibited filamentous growth, presence of sporangia and/or motile spores upon direct microscopic observation (450 and 1000×) were further characterized by fatty acid analysis (FAME). Most of the isolates fell into three broad clusters that roughly correlated with presumptive genus assignments. Individual isolates could be assigned to 226 FAME biotypes based on chromatographic similarity (≥85%). The dominant species (514/826 isolates) belong to a previously undescribed taxon that morphologically resemblesGeodermatophilus but possesses unique FAME profiles that include at least three novel lipids. The remainder of the isolates were species ofActinoplanes, indeterminate species or vagrant isolates ofStreptomyces.

Actinoplanic acids A and B as novel inhibitors of farnesyl-protein transferase

Actinoplanic acids A and B are macrocyclic polycarboxylic acids that are potent reversible inhibitors of farnesyl-protein transferase. Actinoplanic acids A and B were isolated from Actinoplanes sp. MA 7066 while actinoplanic acid B was isolated from both MA 7066 and Streptomyces sp. MA 7099. Actinoplanic acids A and B are competitive with respect to farnesyl diphosphate and are selective inhibitors of farnesyl-protein transferase because they do not inhibit geranylgeranyl-protein transferase type 1 or squalene synthase. MA 7066 is believed to be a novel species of actinomycetes while MA 7099 is believed to be a novel strain of Streptomyces violaceusniger on the basis of morphological, biochemical and chemotaxonomic characteristics as well as its production of actinoplanic acids.

Microbial hydroxylation of rustmicin (galbonolide A) and galbonolide B, two antifungal products produced by Micromonospora sp.

In order to synthesize derivatives of galbonolide A and B with improved chemical stability and antifungal activity profiles, a panel of microorganisms consisting of various species of actinomycetes and fungi were screened. As a result, an organism, Streptomyces halstedii, was identified, which catalyzed the formation of two polar compounds, one from each of the galbonolides. The synthesis and the relative stability of these compounds were optimized by using washed cells, which had been prepared from the transforming organism, and reaction conditions, which included the usage of MES buffer with pH adjusted to 5.5 and incubation at 27°C. Under conditions thus established, two compounds were isolated and characterized by a combination of UV, mass, and NMR spectroscopic analysis. The data indicate the synthesis of 21-hydroxy derivatives of galbonolides A and B with reduced but still significant anti-fungal activity when compared to the parent galbonolides.