title: Metabolic engineering of indene bioconversion in Rhodococcus sp.
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. 124. 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 124. Induction studies with both strains indicated that KY1 lacked a toluene-inducible dioxygenase activity present in 124 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.
authors.affiliation: Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: D E
authors.initials: DE
authors.lastname: Stafford
authors.forename: K S
authors.initials: KS
authors.lastname: Yanagimachi
authors.forename: G
authors.initials: G
authors.lastname: Stephanopoulos
mesh_terms: D002384:Catalysis; D004345:Drug Industry; D007192:Indenes / Q000378:metabolism*; D012240:Rhodococcus / Q000378:metabolism*
chemical_list: D007192:Indenes; C093581:indene