J. Reddy

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.

Asymmetric bioreduction of a β-tetralone to its corresponding (S)-alcohol by the yeast Trichosporon capitatum MY 1890

The yeast Trichosporon capitatum MY 1890 was identified by microbial screening as a suitable biocatalyst for the asymmetric bioreduction of 6-bromo-β-tetralone to its corresponding (S)-alcohol (β-tetralol). This β-tetralol is a precursor to the chiral drug candidate MK-0499, a potassium channel blocker targeted for the treatment of ventricular arrhythmias. Process development studies, employing statistical exploratory designs, yielded a 10-fold increase in the rate of bioreduction and improved the (S)-β-tetralol enantiomeric excess from 71% to 99%. The (S)-β-tetralol enantiomeric excess was found to be highly dependent on glucose catabolism by T. capitatum. Elevated enantiomeric excesses were achieved when switching to a glycerol containing medium. Other process parameters such as pH, temperature and medium composition were found to mostly influence the rate of bioreduction. The developed shake flask process was scaled up to laboratory reactors (23-l scale) and supported the production of gram quantities of highly optically pure (S)-β-tetralol.

Conversion of indene to cis-(1S),(2R)-indandiol by mutants of Pseudomonas putida F1

Two mutation and selection methods were used to isolate mutants of Pseudomonas putida F1 which convert indene to cis-(1S),(2R)-indandiol in a toluene-independent fashion. Using soybean or silicone oil as a second phase to deliver indene to the culture, cis-(1S),(2R)-indandiol, cis-(1R),(2S)-indandiol, 1,2-indenediol (or the keto-hydroxy indan tautomer), and the monooxygenation products 1-indenol and 1-indanone were produced from indene as a function of time. Similarly the enantiomeric excess of the cis-(1S),(2R)-indandiol produced also increased with increasing time. In addition, mutants were isolated which produced cis-(1S),(2R)-indandiol of lower optical purity which corresponded to reduced levels of 1,2-indenediol. These data suggest this toluene dioxygenase produces cis-(1S),(2R)-indandiol of low optical purity and that cis-glycol dehydrogenase plays a role in resolving the two cis-1,2-indandiol enantiomers.

Development of a bioconversion process for production of cis-1S,2R-indandiol from indene by recombinant Escherichia coli constructs

Abstract Recombinant Escherichia coli cells expressing the toluene dioxygenase (TDO) genes from Pseudomonas putida convert indene to cis-1S,2R-indandiol, a potentially important intermediate for the chemical synthesis of the HIV-1 protease inhibitor, Crixivan. A bioconversion process was developed through optimization of medium composition and reaction conditions at the shake-flask and 23-l fermentor scales. A cis-1,2-indandiol productivity of approx. 1000 mg/l was achieved with construct TDO123, which represents a 50-fold increase over the initial titer. Varying the bioconversion conditions did not change the enantiomeric excess (e.e.) for the 1S,2R enantiomer from about 30%, suggesting that toluene dioxygenase intrinsically converts indene to 1S,2R- and 1R,2S-indandiols at a ratio of 2:1. Further inclusion of the Pseudomonas dehydrogenase gene in construct D160-1 led to the production of chirally pure cis-1S,2R-indandiol (e.e. > 99%) as a result of the selective degradation of the 1R,2S enantiomer, with the overall yield (650 mg/l) proportionally reduced. A single stage process was developed for D160-1 and scaled up to the 23-l fermentor, achieving a cis-1S,2R-indandiol titer of 1200 mg/l.

Process optimization for large-scale production of TGF-α-PE40 in recombinant Escherichia coli: effect of medium composition and induction timing on protein expression

The effects of medium composition and induction timing on expression of a chimeric fusion protein TGF-α -PE40 (TP-40) in Escherichia coli strain RR1 were examined using a complex medium at several fermentor scales. Two distinctive phases in E. coli catabolism were identified during fermentation based on preferential utilization between protein hydrolysate and glycerol. Maximum specific and volumetric productivities were achieved by inducing the culture when the cells were switching substrate utilization from protein hydrolysate to glycerol. By increasing the yeast extract concentration in the production medium, initiation of the catabolic switch was delayed until high cell mass was achieved. The final titer of TP-40 at the 15-L fermentation scale was doubled from 400 mg L−1 to 850 mg L−1 by increasing the yeast extract concentration from 1% to 4% (w/v) and delaying the time of induction. This fermentation process was rapidly scaled up in 180-L and 800-L fermentors, achieving TP-40 titers of 740 and 950 mg L−1, respectively.

Bioconversion of indene to trans-2S,1S-bromoindanol and 1S,2R-indene oxide by a bromoperoxidase/dehydrogenase preparation from Curvularia protuberata MF5400

Abstract 1S,2R-Indene oxide is the precursor of cis -1S,2R-aminoindanol, a key intermediate for the Merck HIV–1 protease inhibitor, Crixivan®. As an alternative to the challenging chemical synthesis of this chiral epoxide from indene, the biotransformation route using an enzyme catalyst was examined. Approximately 3% of the 400 fungal cultures isolated from high salt environments were found to possess neutral haloperoxidase activities. Subsequent studies revealed that indene conversion by these positive cultures could only be obtained when both hydrogen peroxide and bromide ions were present. The products were generally racemic trans -bromoindanols which upon basification yielded racemic epoxides. Finally, it was found that a crude enzyme preparation from the fungal culture Curvularia protuberata MF5400 converted indene to the chiral 2S,1S-bromoindanol which can be chemically converted to the desired 1S,2R-epoxide through basification or used directly in the asymmetric synthesis of cis -1S,2R-aminoindanol. The bioconversion rate and the enantiomeric excess (ee) achieved with this cell-free system were heavily pH dependent. An initial 1.5-h reaction at pH 7.0 gave ∼10% yield of the chiral bromoindanol or epoxide from indene, and the yield was rapidly improved to >30% for trans -2S,1S-bromoindanol with an ee of 80%. Reaction mechanistic studies revealed that the stereoselectivity observed was apparently due to a specific dehydrogenase activity present in MF5400 which was also found to resolve chemically synthesized racemic trans -2-bromoindanols.

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.

Scale-up studies on a defined medium process for pilot plant production of illicicolin by Gliocladium roseum.

Illicicolin was cultivated at the 600‐L pilot scale for purposes of material generation and process development. The initial medium containing oat flour was difficult operationally as a result of excessive foaming during sterilization, so a new defined medium process (with either glucose or sucrose as the carbon source), developed at the 23‐L scale, was scaled up and improved for pilot scale needs. Pilot scale media development efforts focused on exploring the highest concentration of media (1.0× to 3.0×) that could be cultivated at the pilot scale and not be limited by mixing or oxygen mass transfer. The process was scaled up successfully and peak titers improved 7.5‐fold, from about 200 mg/L in the initial complex medium to 1500 mg/L in the final defined medium.