Measurement of strain-dependent toxicity in the indene bioconversion using multiparameter flow cytometry.

The bionconversion of indene to cis-(1S,2R)-indandiol, a potential key intermediate in the synthesis of Merck's HIV protease inhibitor, CRIXIVAN trade mark, can be achieved using Rhodococcus, Pseudomonas putida, and Escherichia coli strains. This study reports on the application of multiparameter flow cytometry for the measurement of cytoplasmic membrane integrity and membrane depolarization as indicators of toxic effects of the substrate, product, and by-products using each of these strains. Measurements of oxygen uptake rate (OUR) and optical density (OD) as indicators of metabolic activity and biomass growth, respectively, were also made. Measurements of the cytoplasmic membrane potential, cell viability, and respiratory activity provided a sensitive set of parameters to assess toxicity in the indene bioconversion and provided the basis for process improvements and strain selection. The toxic concentrations of the substrate, product, and by-products for each strain have been determined. The results show that it is possible to accumulate cis-(1S,2R)-indandiol and cis-1-amino-2-indanol up to 20 g/L without significant negative effects on cell physiology using any of the strains tested. The Gram-negative P. putida (421-5 and GM 730) and E. coli strains were more resistant to indene and the isolated chemicals of the biotransformation than the Gram-positive Rhodoccoccus I24 strain, possibly due to the presence of the outer membrane and efflux pump mechanisms. P. putida GM 730 and the E. coli TDO 123 strains responded similarly to toxic effects, and the E. coli TDO 123 strain was more resistant than the P. putida 421-5 strain. In addition to the recommendations for strain selection, the identified targets for bioprocess improvement include a combination of genetic as well as process engineering approaches.

Application of multi-parameter flow cytometry using fluorescent probes to study substrate toxicity in the indene bioconversion.

The bioconversion of indene to cis-(1S,2R) indandiol, a potential key intermediate in the synthesis of Merck's HIV protease inhibitor, CRIXIVAN trade mark, can be achieved using a Rhodococcus strain. This study using Rhodococcus I24 reports on the application of multiparameter flow cytometry for the measurement of cell physiological properties based on cytoplasmic membrane (CM) integrity and membrane depolarization as indicators of toxic effects of the substrate, indene. Quantification of intact polarized CM, intact depolarized CM and permeabilized CM of a large population of bacterial cells has been conducted using specific intracellular and membrane-binding fluorescent stains. Measurements of oxygen uptake rate (OUR) and optical density (OD) as indicators of metabolic activity and biomass growth, respectively, were also made. Indene concentrations of up to 0.25 g/L (0.037 g indene/g dry cell weight) did not significantly (<5% compared to control) affect cell light-scattering properties, intact CM, membrane polarization, respiratory activity, or biomass growth. Between this value and 1.5 g/L (0.221 g indene/g dry cell weight), the changes in intact CM, respiratory activity and biomass growth were relatively insignificant (<5% compared to control), although dissipation of the membrane potential of a significant proportion of the cell population occurred at 0.50 g/L (0.074 g indene/g dry cell weight). At 2.5 g/L (0.368 g indene/g dry cell weight) there was a significant increase in the dead cell population, accompanied by changes in the extracellular cationic concentrations and substantial decrease in respiratory activity. The primary effect of indene toxicity was the disruption of the proton motive force across the cytoplasmic membrane which drives the formation of ATP. The disruption of the proton motive force may have been due to the measured changes in proton permeability across the membrane. In addition, indene may have directly inhibited the membrane-bound enzymes related to respiratory activity. The overall consequence of this was reduced respiratory activity and biomass growth. The cell physiological properties measured via flow cytometry are important for understanding the effects of toxicity at the cellular level which neither measurements of biomass growth or indandiol formation rates can provide since both are cell averaged measurements. The technique described here can also be used as a generic tool for measuring cell membrane properties in response to toxicity of other indene-resistant strains that may be possible to use as recombinant hosts to perform the biotransformation of indene. This study has demonstrated that flow cytometry is a powerful tool for the measurement of cell physiological properties to assess solvent toxicity on whole cell biocatalysts.

Directed evolution of toluene dioxygenase from Pseudomonas putida for improved selectivity toward cis-indandiol during indene bioconversion.

Toluene dioxygenase (TDO) from Pseudomonas putida F1 converts indene to a mixture of cis-indandiol (racemic), 1-indenol, and 1-indanone. The desired product, cis-(1S,2R)-indandiol, is a potential key intermediate in the chemical synthesis of indinavir sulfate (Crixivan), Merck's HIV-1 protease inhibitor for the treatment of AIDS. To reduce the undesirable byproducts 1-indenol and 1-indanone formed during indene bioconversion, the recombinant TDO expressed in Escherichia coli was evolved by directed evolution using the error-prone polymerase chain reaction (epPCR) method. High-throughput fluorometric and spectrophotometric assays were developed for rapid screening of the mutant libraries in a 96-well format. Mutants with reduced 1-indenol by-product formation were identified, and the individual indene bioconversion product profiles of the selected mutants were confirmed by HPLC. Changes in the amino acid sequence of the mutant enzymes were identified by analyzing the nucleotide sequence of the genes. A mutant with the most desirable product profile from each library, defined as the most reduced 1-indenol concentration and with the highest cis-(1S,2R)-indandiol enantiomeric excess, was used to perform each subsequent round of mutagenesis. After three rounds of mutagenesis and screening, mutant 1C4-3G was identified to have a threefold reduction in 1-indenol formation over the wild type (20% vs 60% of total products) and a 40% increase of product (cis-indandiol) yield.

Gene therapy and metabolic engineering.

Gene therapy involves the introduction of normal, healthy genes into cells to correct the underlying cause of a wide variety of inherited and acquired diseases. Future progress in developing effective clinical protocols involving gene therapy for the treatment of cellular dysfunction associated with disease may incorporate metabolic engineering. Metabolic engineering can be applied to gene therapy for the successful identification of disease genes; elucidation of disease pathways; development of safe and efficient gene-delivery systems; and regulation and control of gene expression. Cystic fibrosis, cancer, and diabetes are reviewed as examples of diseases where gene therapy approaches are being studied.

Biocatalysis for pharmaceuticals--status and prospects for a key technology.

In reviewing how biocatalysis can be applied to improve chiral synthesis for pharmaceuticals it becomes clear that there will be many opportunities using a simple enzyme system but that many of the more useful applications will require the whole cell because of the requirement for cofactors. An assessment is made of the opportunities to apply metabolic engineering to construct de novo metabolic pathways for the biosynthesis of useful advanced intermediates and a conceptual example is provided for the biosynthesis of cis-aminoindanol. We predict that in the future novel pathways will be assembled for a one-step biosynthesis of many semisynthetic natural products.

The enzymatic transformation of water-insoluble reactants in nonaqueous solvents. Conversion of cholesterol to cholest-4-ene-3-one by a Nocardia sp. Reprinted from Biotechnology and Bioengineering, Vol. XVII, Pages 815-826 (1975).

The rapid conversion of cholesterol to cholestenone by Nocardia in the presence of high proportions of water-immiscible solvent has been demonstrated. At high agitator speeds, the reaction rate was not limited by the rates of transfer of oxygen or cholesterol to the microorganisms. Using 100 g of thawed cells in 200 ml of carbon tetrachloride containing 16% (w/v) cholesterol, at 20 degrees C cholestenone was formed at 7 g/hr. Cells could be separated easily from the organic solvent and reused. After 7 runs (69 hr) the reaction rate had fallen only to half the value for the first run.

Production of cis-1,2-dihydroxy-3-methylcyclohexa-3,5-diene (toluene cis glycol) by Rhodococcus sp. MA 7249.

An attractive method for producing cis-1,2-dihydroxy-3-methylcyclohexa-3,5-diene (toluene cis glycol) was developed employing a cis dihydrodiol dehydrogenase "deficient" strain of Rhodococcus (MA 7249). The toluene cis glycol produced was found to have optical rotations of [alpha]D25 = +25.8 (c 0.45, CH3OH) and +72.8 (c 0.42, CHCl3) which indicated an absolute configuration of (1S,2R) when compared with previously published values. When cultivated in laboratory fermentor in the presence of toluene vapors, MA 7249 reached a toluene cis glycol concentration up to 18 g/l in 110 h. Culture MA 7249 also accumulated cis (1S,2R) dihydrodiols from dihydronaphthalene, biphenyl, chlorobenzene, and styrene.

Metabolic engineering and directed evolution for the production of pharmaceuticals.

The tools of metabolic and enzyme engineering have been well developed in academic laboratories and are now being applied for the optimization of biocatalysts used in the production of a wide range of pharmaceutically important molecules. Engineered microorganisms with a diverse set of modified or non-native enzyme activities are being used both to generate novel products and to provide improved processes for the manufacture of established products, such as in the production of precursors, intermediates, and complete compounds of importance to the pharmaceutical industry, including polyketides, nonribosomal peptides, steroids, vitamins, and unnatural amino acids. The use of directed evolution has rapidly emerged to be the method of choice for the development and selection of mutated enzymes with improved properties. A variety of such methods have been used to alter the activity, stability and availability of an array of enzymes. The industrial practice of these technologies at large scale is, however, in its infancy and stands as an exciting challenge for process scientists today.

Microbial conversion of indene to indandiol: a key intermediate in the synthesis of CRIXIVAN.

Indene is oxidized to mixtures of cis- and trans-indandiols and related metabolites by Pseudomonas putida and Rhodococcus sp. isolates. Indene metabolism is consistent with monooxygenase and dioxygenase activity. P. putida resolves enantiomeric mixtures of cis-1,2-indandiol by further selective oxidation of the 1R, 2S-enantiomer yielding high enantiomeric purity of cis-(1S, 2R)-indandiol, a potential intermediate in the synthesis of indinavir sulfate (CRIXIVAN), a protease inhibitor used in the treatment of AIDS. Molecular cloning of P. putida toluene dioxygenase in Escherichia coli confirmed the requirement for the dihydrodiol dehydrogenase in resolving racemic mixtures of cis-indandiol. Rhodococcus sp. isolates convert indene to cis-(1S, 2R)-indandiol at high initial enantiomeric excess and one isolate also produces trans-(1R, 2R)-indandiol, suggesting the presence of monooxygenase activity. Scale up and optimization of the bioconversions to these key synthons for chiral synthesis of potential intermediates for commercial manufacture of indinavir sulfate are described.

Process optimization for large-scale production of TGF-alpha-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-alpha-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.

Fed-batch culture of recombinant NS0 myeloma cells with high monoclonal antibody production.

An amplified NS0 cell line transfected with a vector expressing a humanized monoclonal antibody (MAb) against CD-18 and glutamine synthetase (GS) was cultivated in a 1.5 L fed-batch culture using a serum-free, glutamine-free medium. Concentrated solutions of key nutrient components were fed periodically using a simple feeding control strategy. Feeding amounts were adjusted daily based on the integral of viable cell concentration over time (IVC) and assumed constant specific nutrient consumption rates or yields to maintain concentrations of the key nutrient components around their initial levels. On-line oxygen uptake rate (OUR) measurement was used to aid empirically the adjustment of the feeding time points and amounts by inferring time points of nutrient depletion. Through effective nutritional control, both cell growth phase and culture lifetime were prolonged significantly, resulting in a maximal viable cell concentration of 6.6 x 10(9) cells/L and a final IVC of 1.6 x 10(12) cells-h/L at 672 h. The final MAb concentration reached more than 2.7 g/L. In this fed-batch culture, cellular metabolism shifts were repeatedly observed. Accompanying the culture phase transition from the exponential growth to the stationary phase, lactate, which was produced in the exponential growth phase, became consumed. The time point at which this metabolism shift occurred corresponded to that of rapid decrease of OUR, which most likely was caused by nutrient depletion. This transition coincided with the onset of ammonia, glutamate and glutamine accumulation. With removal of the nutrient depletion by increasing the daily nutrient feeding amount, OUR recovered and viable cell concentration increased, while cell metabolism shifted again. Instead of consumption, lactate became produced again. These results suggest close relationships among nutrient depletion, cell metabolism transition, and cell death. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 783-792, 1997.

A fluid dynamic study using a simulated viscous, shear thinning broth of the retrofitting of large agitated bioreactors.

Studies were conducted(1) in 19-m(3) fermentors (14-m(3) working volume) using four Rushton turbines, four Prochem Maxflo Ts, and three Lightnin' A315s and the results in water have been reported earlier. Here, a 1.7 wt/vol% Xanthan solution has been used as the working fluid, simulating viscous broths to give Reynolds numbers (Re) between 1800 and 4500. As predicted from small-scale studies, the power numbers at these values of Re were similar to those in water. The K factor (the ratio of power draw under aerated conditions compared to non-aerated) was the same as in water at the higher values of Re, but at the lower values it fell more rapidly with increasing aeration rate and to a lower value than in water. At all times, K was higher than with Rushton turbines. Vibration characteristics were also measured. Under aerated conditions, the fermentors vibrated with an amplitude 75% to 100% less than in water due to viscous damping. With increasing air flow, the amplitude increased steadily due to the presence of very large and rapidly rising bubbles in such fluids to give values 2.5 to 3 times those in water. Nevertheless, these mechanical problems can be overcome, allowing such agitators to be used successfully in high viscosity mycelial fermentations.

Development of a defined medium fermentation process for physostigmine production by Streptomyces griseofuscus.

Physostigmine is a plant alkaloid of great interest as a therapeutic candidate for the treatment of Alzheimer's disease. Fortunately, this compound is also produced by Streptomyces griseofuscus NRRL 5324 during submerged cultivation. A fermentation process that used chemically defined medium was therefore developed for its production. By means of statistical experimentation, the physostigmine titer was quickly increased from 20 mg/l to 520 mg/l with a culture growth of 19 gl dry cell weight on the shake-flask scale. Further medium optimization resulted in a yield of 790 mg/l in a 23-1 bioreactor using a batch process. A titer of 880 mg/l was attained during scale-up in a 800-1 fermentor by employing a nutrient-feeding strategy. This production represents a 44-fold increase over the yield from the initial process in shake-flasks. The defined-medium fermentation broth was very amenable to downstream processing.

Large scale production of recombinant mouse and rat growth hormone by fed-batch GS-NSO cell cultures.

Investigations of biological effects of prolonged elevation of growth hormone in animals such as mice and rats require large amounts of mouse and rat growth hormone (GH) materials. As an alternative to scarce and expensive pituitary derived materials, both mouse and rat GH were expressed in NSO murine myeloma cells transfected with a vector containing the glutamine synthetase (GS) gene and two copies of mouse or rat GH cDNA. For optimal expression, the mouse GH vector also contained sequences for targeting integration by homologous recombination. Fed-batch culture processes for such clones were developed using a serum-free, glutamine-free medium and scaled up to 250 L production scale reactors. Concentrated solutions of proteins, amino acids and glucose were fed periodically to extend cell growth and culture lifetime, which led to an increase in the maximum viable cell concentration to 3.5×10(9) cells/L and an up to 10 fold increase in final mouse and rat rGH titers in comparison with batch cultures. For successful scale up, similar culture environmental conditions were maintained at different scales, and specific issues in large scale reactors such as balancing oxygen supply and carbon dioxide removal, were addressed. Very similar cell growth and protein productivity were obtained in the fed-batch cultures at different scales and in different production runs. The final mouse and rat rGH titers were approximately 580 and 240 mg/L, respectively. During fed-batch cultures, the cell growth stage transition was accompanied by a change in cellular metabolism. The specific glucose consumption rate decreased significantly after the transition from the growth to stationary stage, while lactate was produced in the exponential growth stage and became consumed in the stationary stage. This was roughly coincident with the beginning of ammonia and glutamate accumulation at the entry of cells into the stationary stage as the result of a reduced glutamine consumption and periodic nutrient additions.

Microfiltration of recombinant yeast cells using a rotating disk dynamic filtration system.

To develop a highly efficient cell harvest step under time constraint, a novel rotating disk dynamic filtration system was studied on the laboratory scale (0.147-ft.(2) nylon membrane) for concentrating recombinant yeast cells containing an intracellular product. The existing cross-flow microfiltration method yielded pseudo-steady state flux values below 25 LMH (L/m(2). h) even at low membrane loadings (10 L/ft.(2)). By creating high shear rates (up to 120,000(-1)) on the membrane surface using a rotating solid disk, this dynamic filter has demonstrated dramatically improved performance, presumably due to minimal cake buildup and reduced membrane fouling. Among the many factors investigated, disk rotating speed, which determines shear rates and flow patterns, was found to be the most important adjustable parameter. Our experimental results have shown that the flux increases with disk rotating speed, increases with transmembrane pressure at higher cell concentrations, and can be sustained at high levels under constant flux mode. At a certain membrane loading level, there was a critical speed below which it behaved similarly to a flat sheet system with equivalent shear. Average flux greater than 200 LMH has been demonstrated at 37-L/ft.(2) loading at maximum speed to complete sixfold concentration and 15-volume diafiltration for less than 100 min. An order of magnitude improvement over the crossflow microfiltration control was projected for large scale production. This superior performance, however, would be achieved at the expense of additional power input and heat dissipation, especially when cell concentration reaches above 80 g dry cell weight (DCW)/L. Although a positive linear relationship between power input and dynamic flux at a certain concentration factor has been established, high cell density associated with high viscosity impacted adversely on effective average shear rates and, eventually, severe membrane fouling, rather than cake formation, would limit the performance of this novel system.

Pilot-scale harvest of recombinant yeast employing microfiltration: a case study.

In order to develop a cost-effective recovery process for an intracellular product, crossflow microfiltration was studied for the harvest of a recombinant yeast under severe time constraint. It was required to process yeast broth in a short period of time to minimize the risk for product degradation. Preliminary microfiltration studies employing flat sheet membranes showed high throughout with initial fluxes on the order of water fluxes (> 1000 LMH, regime I, < 2 min), followed by a rapid decay towards a low pseudo-steady state flux (20 LMH, regime II, > 2 min). Exploitation of these high fluxes and control of their eventual decline were crucial in establishing a rapid crossflow filtration process. The effect of several parameters, such as initial cell concentration, shear rate, transmembrane pressure, membrane pore size and medium composition on filtration performance were investigated to better understand the flux decline mechanisms. We found that the major contributor to flux decay was reversible fouling by the cake formation on the membrane surface. Within the operating boundaries of our microfiltration system, large-pore membrane (0.65 micron) was much more desirable for harvesting our yeast (10 microns size) without cell leakage than smaller pore ones (0.22 micron and 0.45 micron). Among adjustable operating parameters, feed flow rate (i.e., shear rate) exerted significant impact on average flux, whereas manipulation of transmembrane pressure afforded little improvement. Although initial cell concentration affected adversely the permeation rates, growth medium components, especially soy-peptone, was deemed pivotal in determining the characteristics of cell cake, thus controlling yeast microfiltration.

Use of comparative reasoning tools for evaluation of the effect of sterilization conditions on fermentations to produce acidic fibroblast growth factor.

The effects of various medium sterilization conditions on fermentations of a recombinant, acidic fibroblast growth factor (aFGF) producing Escherichia coli have been studied. Changes in the medium resulting from sterilization were monitored by pH and absorption spectra. This simple experiment provided excellent data for the demonstration of the usefulness of comparative reasoning tools in order to evaluate the effect of sterilization on fermentation performance. The time profiles of the main parameters (e.g., carbon dioxide evolution rate, dissolved oxygen, pH, and aFGF productivity) were simplified into piecewise contiguous linear segments, each of which was sequentially numbered. The length, position, and slope of each tine were then characterized. Application of the comparative reasoning tools confirmed that separate sterilization of the glucose was necessary for the success of the process, despite adding to the cost and complexity. The comparative data analysis also showed that scaleup with longer sterilization holding and cooling times would not be detrimental to aFGF production. (c) 1995 John Wiley & Sons, Inc.