Bioconversion of indene to cis (1S,2R) indandiol and trans (1R,2R) indandiol by Rhodococcus species

Chartrain, Michel; Jackey, Barbara A.; Taylor, Colleen; Sandford, Vanessa; Gbewonyo, Kodzo; Lister, Leonard M.; DiMichele, Lisa; Hirsch, C. F.; Heimbuch, Brian K.; Maxwell, Carrie; Pascoe, Deborah; Buckland, Barry C.; Greasham, Randolph

doi:10.1016/s0922-338x(99)80005-1

Cited by 58 publications

(49 citation statements)

References 23 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%

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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%

“…The enantiomeric mixture of cis-indandiol produced by I24 (also observed by Chartrain, et al in ref. 4) indicates that cross-induction is occurring between at least two dioxygenases in I24. This crossinduction does not occur in KY1 because of its altered or deleted toluene-inducible dioxygenase gene.…”

mentioning

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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“…Although Rhodococcus species utilize naphthalene as their sole carbon and energy source, [82][83][84][85] to the best of our knowledge, the PAH catabolic genes from these have not been characterized until recently. Larkin et al 86) reported the puriˆcation and characteristics of a novel ISP of naphthalene dioxygenase from Rhodococcus sp.…”

Section: )mentioning

confidence: 99%

Genetics of Polycyclic Aromatic Hydrocarbon Metabolism in Diverse Aerobic Bacteria

Habe

Omori

2003

Bioscience, Biotechnology, and Biochemistry

387

220

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“…Due to such constraints, many biotransformations performed in single phases are susceptible to low yields that render the systems economically unattractive. To improve productivities, specifically in batch mode, an assortment of approaches has been suggested to reduce the toxic effect of substrate/product, the most popular of which has been the addition of an immiscible second phase (Chartrain et al, 1998;Ereldinger et al, 2000;Husken et al, 2001b).…”

Section: Introductionmentioning

confidence: 99%

Solvent selection for enhanced bioproduction of 3‐methylcatechol in a two‐phase partitioning bioreactor

Prpich

Daugulis

2006

Biotech & Bioengineering

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The biotransformation of toluene to 3-methycatechol (3MC) via Pseudomonas putida MC2 was used as a model system for the development of a biphasic process offering enhanced overall volumetric productivity. Three factors were investigated for the identification of an appropriate organic solvent and they included solvent toxicity, bioavailability of the solvent as well as solvent affinity for 3MC. The critical log P (log P(crit)) of the biocatalyst was found to be 3.1 and log P values were used to predict a solvent's toxicity. The presence of various functional groups of candidate solvents were used to predict the absorption of 3MC and it was found that solvents possessing polarity showed an affinity towards 3MC. Bis (2-ethylhexyl) sebecate was selected for use in the biphasic system as it fulfilled all selection criteria. A two-phase biotransformation with BES and a 50% phase volume ratio, achieved an overall volumetric productivity of 440 mg 3MC/L-h, which was an improvement by a factor of approximately 4 over previously operated systems. Additional work focused on reducing the toluene feed in order to minimize possible toxicity and decrease loss of substrate (toluene), a result of volatilization. Toluene losses were reduced by a factor of 4, compared to previously operated systems, without suffering an appreciable loss in overall volumetric productivity.

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Bioconversion of indene to cis (1S,2R) indandiol and trans (1R,2R) indandiol by Rhodococcus species

Cited by 58 publications

References 23 publications

Optimizing bioconversion pathways through systems analysis and metabolic engineering

Optimizing bioconversion pathways through systems analysis and metabolic engineering

Genetics of Polycyclic Aromatic Hydrocarbon Metabolism in Diverse Aerobic Bacteria

Solvent selection for enhanced bioproduction of 3‐methylcatechol in a two‐phase partitioning bioreactor

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