Microbial Conversion of Indene to Indandiol: A Key Intermediate in the Synthesis of CRIXIVAN

Buckland, Barry C.; Drew, Stephen W.; Connors, Neal; Chartrain, Michel; Lee, Chanyong; Salmon, Peter; Gbewonyo, Kodzo; Zhou, Weichang; Gailliot, Pat; Singhvi, Rahul; Olewinski, R.; Sun, Wei; Reddy, J.; Zhang, Jinyou; Jackey, Barbara A.; Taylor, Colleen; Göklen, Kent E.; Junker, Beth; Greasham, Randolph

doi:10.1006/mben.1998.0107

Cited by 70 publications

(62 citation statements)

References 22 publications

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“…Further optimization of bioconversion conditions is likely to improve the product titer obtained in this study. In particular, the use of a two-phase system similar to that used previously should minimize exposure of the cells to the possibly toxic and͞or inhibitory indene metabolites (4,7,20).…”

Section: Discussionmentioning

confidence: 99%

Optimizing bioconversion pathways through systems analysis and metabolic engineering

Stafford

Yanagimachi

Lessard

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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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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Section: Discussionmentioning

confidence: 99%

Optimizing bioconversion pathways through systems analysis and metabolic engineering

Stafford

Yanagimachi

Lessard

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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“…It has been noted that the chiral products of the dioxygenase reaction can be converted in a variety of synthetic reactions to advanced intermediates for natural-product synthesis (8,15). Toluene dioxygenase (TDO) from Pseudomonas putida readily dihydroxylates aromatic carbocycles with one or two small hydrophobic substituents (6,33).…”

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confidence: 99%

Laboratory Evolution of Toluene Dioxygenase To Accept 4-Picoline as a Substrate

Sakamoto

Joern

Arisawa

et al. 2001

Appl Environ Microbiol

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We are using directed evolution to extend the range of dioxygenase-catalyzed biotransformations to include substrates that are either poorly accepted or not accepted at all by the naturally occurring enzymes. Here we report on the oxidation of a heterocyclic substrate, 4-picoline, by toluene dioxygenase (TDO) and improvement of the enzyme's activity by laboratory evolution. The biotransformation of 4-picoline proceeds at only ϳ4.5% of the rate of the natural reaction on toluene. Random mutagenesis, saturation mutagenesis, and screening directly for product formation using a modified Gibbs assay generated mutant TDO 3-B38, in which the wild-type stop codon was replaced with a codon encoding threonine. Escherichia coli-expressed TDO 3-B38 exhibited 5.6 times higher activity toward 4-picoline and ϳ20% more activity towards toluene than wild-type TDO. The product of the biotransformation of 4-picoline is 3-hydroxy-4-picoline; no cis-diols of 4-picoline were observed.Dioxygenase enzymes involved in the catabolism of aromatic hydrocarbons by soil microorganisms nicely illustrate nature's ability to adapt to different carbon sources through evolution of the substrate specificity of the biodegradation enzymes (35). The biodegradation pathway often begins with the dioxygenasecatalyzed regio-and enantio-specific introduction of molecular oxygen into aromatic compounds to form the corresponding arene cis-diols, with consumption of NADH (13, 36) ( Fig. 1).It has been noted that the chiral products of the dioxygenase reaction can be converted in a variety of synthetic reactions to advanced intermediates for natural-product synthesis (8, 15). Toluene dioxygenase (TDO) from Pseudomonas putida readily dihydroxylates aromatic carbocycles with one or two small hydrophobic substituents (6, 33). Activity towards much larger, fused-ring substrates or substrates with polar or bulky substituents is considerably reduced or nonexistent (33).We are attempting to extend the utility of dioxygenase-catalyzed biotransformations by engineering the catalysts to accept a wider range of substrates, including small heterocyclic compounds and polar-substituted carbocyclic aromatics. N-Heterocyclic compounds are useful for the synthesis of biologically active compounds; recent reports describe the regioand/or stereo-controlled biooxidation of N-heterocycles (20,26). Bicyclic compounds such as quinolines and benzofurans are accepted by TDO, with oxygen insertion occurring primarily in the carbocylic rings (3-5).To date, reactions on single-ring heterocycles have not been reported. We have found that TDO catalyzes the oxygenation of 4-picoline (4-methylpyridine), although at a much slower rate than on its preferred substrate toluene.A strategy of DNA shuffling to create libraries of hybrid genes and screening has been used with some success to extend the substrate range of dioxygenases that degrade environmental pollutants such as polychlorinated biphenyls (7, 21). An alternative approach is to fine tune a single gene by accumulating beneficial mutat...

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“…Its chemical synthesis proceeds through epoxidation of indene to indan oxide at ee 87%, followed by two steps to afford 85 in greater than 99% ee. Two alternative processes currently exist: biotransformations of indene (86) either through the transformation of indandiol (88) at >95% ee followed by its conversion to 85, or through from indan oxide (87) at high enantio-purity followed by its conversion to 85 (Buckland, 1999). In both, cis-(1S, 2R)-indandiol (88) or trans-(1R, 2R)-indandiol (89) are potential precursors to 85.…”

Section: Sources Of Chiral Drugsmentioning

confidence: 99%