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Measurement of strain-dependent toxicity in the indene bioconversion using multiparameter flow cytometry.
A Amanullah1, C J Hewitt, A W Nienow
1Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Torrington Place, London W1CE 7JE, United Kingdom. ashraf_amanullah@merck.com
Biotechnology and Bioengineering
|December 20, 2002
Summary
Multiparameter flow cytometry assessed toxicity in microbial bioconversion of indene to cis-(1S,2R)-indandiol. Gram-negative bacteria showed higher resistance, enabling high product accumulation up to 20 g/L.
Area of Science:
- Biotechnology
- Microbial Biocatalysis
- Process Optimization
Background:
- The bioconversion of indene to cis-(1S,2R)-indandiol is crucial for synthesizing HIV protease inhibitors like CRIXIVAN.
- Assessing microbial toxicity is vital for optimizing bioprocesses and selecting robust strains.
Purpose of the Study:
- To apply multiparameter flow cytometry to evaluate the toxicity of indene bioconversion on microbial strains.
- To determine toxic concentrations of substrate, product, and by-products for Rhodococcus, Pseudomonas putida, and Escherichia coli.
- To identify optimal strains and bioprocess improvements for high-yield cis-(1S,2R)-indandiol production.
Main Methods:
- Multiparameter flow cytometry was used to measure cytoplasmic membrane integrity and depolarization.
- Oxygen uptake rate (OUR) and optical density (OD) were monitored for metabolic activity and biomass growth.
- Cell viability and respiratory activity were assessed to determine toxic effects.
Main Results:
- Gram-negative P. putida and E. coli strains exhibited greater resistance to indene and related chemicals than Gram-positive Rhodococcus.
- cis-(1S,2R)-indandiol and cis-1-amino-2-indanol could be accumulated up to 20 g/L without significant adverse effects on cell physiology.
- E. coli TDO 123 demonstrated higher resistance than P. putida 421-5, with P. putida GM 730 and E. coli TDO 123 showing similar responses.
Conclusions:
- Multiparameter flow cytometry provides sensitive parameters for assessing toxicity in bioconversion processes.
- Strain selection based on toxicity resistance, particularly Gram-negative bacteria like P. putida GM 730 and E. coli TDO 123, is recommended for process improvement.
- Bioprocess optimization can be achieved through a combination of genetic engineering and process engineering approaches.