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Metabolic engineering of indene bioconversion in Rhodococcus sp
D E Stafford1, K S Yanagimachi, G Stephanopoulos
1Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Advances in Biochemical Engineering/Biotechnology
|January 31, 2002
Summary
Metabolic engineering of Rhodococcus sp. enhanced (2R)-indandiol production. A mutant strain, KY1, doubled yield by lacking a byproduct-forming dioxygenase, paving the way for improved HIV drug precursor synthesis.
Area of Science:
- Biotechnology
- Metabolic Engineering
- Enzymatic Bioreaction Networks
Background:
- Rhodococcus sp. catalyzes indene to (2R)-indandiol, a precursor for the HIV protease inhibitor Crixivan.
- A novel chemostat system was developed for studying Rhodococcus sp. steady-state physiology.
Purpose of the Study:
- To apply metabolic engineering to optimize the bioconversion of indene to (2R)-indandiol.
- To investigate the enzymatic bioreaction network in Rhodococcus sp. for improved yield.
Main Methods:
- Development of a chemostat with a novel indene air delivery system.
- Continuous cultivation leading to mutant strain KY1 evolution.
- Flux analysis using steady-state metabolite balancing and [14C]-tracer labeling.
Main Results:
- Mutant strain KY1 showed a twofold increase in (2R)-indandiol yield compared to the parent strain.
- KY1 lacked toluene-inducible dioxygenase activity, reducing byproduct formation.
- Flux analysis revealed indene is primarily converted to indan oxide, then hydrolyzed to trans- and cis-indandiols.
Conclusions:
- Metabolic engineering strategies can significantly improve (2R)-indandiol production.
- Targeting indan oxide hydrolysis presents a promising route for further yield enhancement.
- The study identified key enzymatic targets for optimizing the synthesis pathway.