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

• Published in: Biotechnology and bioengineering Vol. 81; no. 4; pp. 405 - 420
• Main Authors: Amanullah, A., Hewitt, C. J., Nienow, A. W., Lee, C., Chartrain, M., Buckland, B. C., Drew, S. W., Woodley, J. M.
• Format: Journal Article
• Language: English
• Published: New York Wiley Subscription Services, Inc., A Wiley Company 20-02-2003; Wiley
• Subjects: and by-product toxicity; Biological and medical sciences; Bioreactors; Biotechnology; Catalysis; Cell Division - drug effects; Cell Membrane - drug effects; Cell Membrane - metabolism; Cell Membrane - pathology; Dose-Response Relationship, Drug; Escherichia coli - cytology; Escherichia coli - drug effects; Escherichia coli - growth & development; Escherichia coli - metabolism; flow cytometry; Flow Cytometry - methods; Fundamental and applied biological sciences. Psychology; HIV protease inhibitor; indene bioconversion; Indenes - metabolism; Indenes - toxicity; Membrane Potentials - drug effects; Methods. Procedures. Technologies; Others; Oxygen - metabolism; product; Pseudomonas putida - cytology; Pseudomonas putida - drug effects; Pseudomonas putida - growth & development; Pseudomonas putida - metabolism; Rhodococcus - cytology; Rhodococcus - drug effects; Rhodococcus - growth & development; Rhodococcus - metabolism; Sensitivity and Specificity; Species Specificity; strain selection; substrate; substrate, product, and by‐product toxicity; Various methods and equipments; Pseudomonadales; Flow cytometry; Toxicity; strain selection; HIV protease inhibitor; Rhodococcus; Biotransformation; Enantiomer; Bacteria; Pseudomonadaceae; Pseudomonas aeruginosa; Antiviral; Indene derivatives; indene bioconversion; Quantitative analysis; Enzyme; Retroviridae; Enzyme inhibitor; Fermentation; Lentivirus; Virus; Substrate; substrate, product, and by-product toxicity; Peptidases; Hydrolases; Actinomycetes; Inhibitor; Human immunodeficiency virus
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.10479

The bionconversion of indene to cis‐(1S,2R)‐indandiol, a potential key intermediate in the synthesis of Merck's HIV protease inhibitor, CRIXIVAN™, 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. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 405–420, 2003.