Kodzo Gbewonyo

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.

On-line and in-situ monitoring technology for cell density measurement in microbial and animal cell cultures

Commercially available on-line and in-situ devices for monitoring cell density are reviewed in this article. Principles of operation are described as well as capabilities of these probes in specific measurement applications based on literature reports. Pilot-scale experimental observations from three optical density probes, the Cerex, Monitek and Wedgewood designs, have been included for Escherichia coli fermentations. Requirements for future on-line and in-situ instruments are discussed as well as the impact of current limitations on widespread application.

Interactions of cell morphology and transport processes in the lovastatin fermentation

The cholesterol lowering drug, Lovastatin (Mevacor), acts as an inhibitor of HMGCoA reductase, and is produced from an Aspergillus terreus fermentation.Pilot scale studies were carried out in 800 liter fermenters to determine the effects of cell morphology on the oxygen transport properties of this fermentation. Specifically, parallel fermentations giving (i) filamentous mycelial cells, and (ii) discrete mycelial pellets, were quantitatively characterized in terms of broth viscosity, availability of dissolved oxygen, oxygen uptake rates and the oxygen transfer coefficient under identical operating conditions.The growth phase of the fermentation, was operated using a cascade control strategy which automatically changed the agitation speed with the goal of maintaining dissolved oxygen at 50% saturation. Subsequently stepwise changes were made in agitation speed and aeration rate to evaluate the response of the mass transfer parameters (DO, OUR, and kLa). The results of these experiments indicate considerable potential advantages to the pellet morphology from the standpoint of oxygen transport processes.

Secondary metabolite scale-up to minimize homolog impurity levels

A mutant strain of Streptomyces hygroscopicus was found to produce up to 9.0 units/L of an immunoregulant precursor, immunomycin, with up to 3.5% of a lower homolog impurity under either dual fed-batch or batch conditions. Glycerol and valine were key nutrients influencing productivity and impurity levels. Soybean oil was successfully substituted for glycerol as a carbon source to minimize shot additions to batch culture. The remainder of the production medium was composed largely of defined components with the exception of yeast extract. Valine limitation increased lower homolog formation while decreasing higher homolog formation; excess valine decreased lower homolog formation below 2-3% while increasing higher homolog formation. Higher homolog formation in the presence of valine seemed to be slower than lower homolog formation in the absence of valine. Valine was believed to be the major butyrate precursor; consequently its availability influenced the impurity profile. A preliminary cost analysis suggests that elimination of added valine from the cultivation and replacement of glycerol with soybean oil can result in a 6.6-fold reduction in media costs relative to the original fed-batch process. Copyright 1998 John Wiley & Sons, Inc.

Effects of medium sterilization on the production of zaragozic acids by the fungusLeptodontidium elatius

The production of zaragozic acids by fermentation of the fungusLeptodontidium elatius was examined at the 800-L fermentor scale under two different production medium batch sterilization conditions. Low production-medium heat input (R0=33.4 min) resulted in a 4′-desacetoxy zaragozic acid C:4′-O-desacetyl zaragozic acid C:zaragozic acid C ratio of 0.53:0.60:1.0. At a higher heat input (R0=50.5 min), the ratio shifted to 1.0:0.66:1.0 with a corresponding 26% increase in total zaragozic acid production. This higher total zaragozic acid titer resulted from an increase in the amount of 4′-desacetoxy zaragozic acid C produced while the levels of the other two analogues remained unchanged. Batch sterilization conditions also resulted in differences in growth, carbon substrate consumption, and oxygen uptake rates. The structures of the zaragozic acids produced suggest a precursor/end product relationship. A biosynthetic model describing the synthesis of the three zaragozic acids listed above is postulated and used to explain the effects of production-medium heat input during sterilization.

Bioconversion of avermectin into 27-OH avermectin

SummaryThe bioconversion of avermectin to its 27-hydroxy derivative is achieved withNocardia autotrophica subsp.canberrica. The approach of increasing bioconversion productivity rather than efficiency was adopted in these studies. Process improvement studies focused on the physico-chemical conditions of the fermentation, examined initially at the shake-flask scale. Bioconversion yields were affected by pH, substrate concentration, time of substrate addition, substrate solubilization, carbon to nitrogen ratio, and medium strength. Optimization of these parameters resulted in a 8-fold process improvement. During pre scale-up studies, the sensitivity of this bioconversion to the antifoam employed was demonstrated and lard oil was selected as giving the best results. Additional process changes were required during scale-up efforts in larger vessels, including replacement of the original substrate solvent with dimethylsulfoxide.