System analysis and metabolic engineering of biocatalytic reaction networks : application to indene bioconversion

Abstract

Metabolic engineering is a science concerned with the improvement of cellular properties through the application of recombinant DNA technology. To this point, the field has frequently been limited to methodological issues pertaining to gene introduction, expression and control by way of molecular biological techniques. While such methods are critical for the implementation of strategies for metabolic pathway design and modification, an equally critical activity is the quantitative analysis of metabolic networks and the evaluation of phenotype/genotype relationships. A prominent new opportunity to extend the scope of metabolic engineering has recently emerged to analyze and engineer biocatalytic microorganisms capable of catalyzing stereospecific transformations useful in chiral pharmaceutical manufacturing. This thesis formulates and applies a general framework for the optimization of uncharacterized bioconversion strains (those biocatalysts with little a priori genetic or metabolic data available) comprising five essential steps: (i) Establishment of an experimental system for strain selection and metabolic network analysis, (ii) definition of the bioconversion network and quantification of network fluxes, (iii) target identification, (iv) flux redistribution, and (v) analysis of the modified bioconversion strain to identify areas for further development. In the case described here, a systematic evaluation of the physiology of the soil bacterium Rhodococcus, and analysis of the relative fluxes of an indene bioconversion network,

(cont.) facilitated the construction of a biocatalyst for production of (2R)-indandiol suitable for the manufacturing of the HIV protease inhibitor, indinavir sulfate (Crixivan, Merck and Co., Inc.). The chemical synthesis of (-)-cis-(lS,2R)-l-aminoindan-2-ol [(-)-CAI], a key precursor in CrixivanT manufacturing, may be carried out by the asymmetric epoxidation of indene to (1S,2R)-indan oxide at up to 87% enantiomeric excess by way of hydrolytic kinetic resolution followed by crystallization to purify the (1S,2R)-indan oxide. To circumvent this technically demanding epoxidation, Merck scientists conceptualized a bioconversion process in which indene is oxidized to one of three derivatives that can serve as precursors to (-)-CAI: cis-(1S,2R)-indandiol, trans-(1R,2R)-indandiol, or (1S,2R)-indan oxide. Rhodococcus sp. were isolated that utilize complex networks of enzymatic reactions to convert indene to several oxygenated derivatives of different stereochemistries. In previous work, prolonged cultivation of Rhodococcus sp. I24 in a continuous flow system with a novel indene air delivery 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 that was 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 ...