Abstract
We have applied the methodology of metabolic engineering in the investigation of the enzymatic bioreaction network in Rhodococcus sp. that catalyzes the bioconversion of indene to (2R)-indandiol suitable for the synthesis of cis-1-amino-2-indanol, a precursor of the HIV protease inhibitor, Crixivan. A chemostat with a novel indene air delivery system was developed to facilitate the study of steady state physiology of Rhodococcus sp. I24. Prolonged cultivation of this organism in a continuous flow system led to the evolution of a mutant strain, designated KY1, with improved bioconversion properties, in particular a twofold increase in yield of (2R)-indandiol relative to I24. Induction studies with both strains indicated that KY1 lacked a toluene-inducible dioxygenase activity present in I24 and responsible for the formation of undesired byproducts. Flux analysis of indene bioconversion in KY1 performed using steady state metabolite balancing and labeling with [14C]-tracers revealed that at least 94% of the indene is oxidized by a monooxygenase to indan oxide that is subsequently hydrolyzed to trans-(1R,2R)-indandiol and cis-(1S,2R)-indandiol. This analysis identified several targets in KY1 for increasing (2R)-indandiol product yield. Most promising among them is the selective hydrolysis of indan oxide to trans-(1R,2R)-indandiol through expression of an epoxide hydrolase or modification of culture conditions.
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Stafford, D.E., Yanagimachi, K.S., Stephanopoulos, G. (2001). Metabolic Engineering of Indene Bioconversion in Rhodococcus sp.. In: Nielsen, J., et al. Metabolic Engineering. Advances in Biochemical Engineering/Biotechnology, vol 73. Springer, Berlin, Heidelberg. https://doi.org/10.1007/3-540-45300-8_5
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DOI: https://doi.org/10.1007/3-540-45300-8_5
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