Optimizing bioconversion pathways through systems analysis and metabolic engineering

Abstract

We demonstrate a general approach for metabolic engineering of biocatalytic systems comprising the uses of a chemostat for strain improvement and radioisotopic tracers for the quantification of pathway fluxes. Flux determination allows the identification of target pathways for modification as validated by subsequent overexpression of the corresponding gene. We demonstrate this method in the indene bioconversion network of Rhodococcus modified for the overproduction of 1,2-indandiol, a key precursor for the AIDS drug Crixivan. C omplex metabolic and bioconversion pathways containing parallel, branching, and͞or reversible reactions can be studied quantitatively under the framework of metabolic engineering, which uses steady-state fluxes as fundamental determinants of cell physiology (1, 2). It is necessary to use these methods to distinguish the relative importance of competing metabolic reactions to guide target selection for the improvement of biological production of secondary metabolites or small molecules important for pharmaceutical and materials applications (3). To date, applications of metabolic engineering have been limited to primarily linear pathways and cases in which the relevant biochemistry and associated genetics are well established. In many cases, efforts focusing on transformation of cells by ad hoc methods have failed where genes are introduced based on conclusions derived in the absence of quantitative analysis of pathways. Consequently, an approach that considers the systemic properties of a bioconversion to identify rational targets is valuable. Such an approach can be based on determination of fluxes in bioconversion networks, which has been a focus of metabolic engineering for the past 10 years.We have developed and applied a general framework for the optimization of bioconversion systems in the context of the directed biocatalytic production of trans-(1R,2R)-indandiol suitable for the synthesis of the HIV protease inhibitor Crixivan (Merck). Chartrain et al. (4) isolated Rhodococcus sp. I24, which possesses the required oxygenase enzyme activities for converting indene to (2R)-indandiol (Fig. 1). The Crixivan chiral precursor (Ϫ),-cis-(1S,2R)-1-aminoindan-2-ol [(Ϫ)-CAI] can then be synthesized from (2R)-indandiol through a Ritter reaction (5, 6). However, besides the desired (2R)-indandiol product, several other side-products are secreted also in a Rhodococcus sp. I24 fermentation that reduce the desired product yield and selectivity. Therefore, it is of interest to modify I24 genetically to eliminate undesirable reactions and enhance the productforming pathway. Because of the poorly characterized nature of I24 genetics a priori, it is imperative that an approach be developed to prioritize network targets for modification in light of the current state of knowledge of the given biological system.The general framework described here is comprised of five essential steps: (i) establishment of an experimental system for strain selection and metabolic network analysis, (ii) definition of the ...

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