Metabolic Engineering of Indene Bioconversion in Rhodococcus sp.

Stafford, Daniel; Yanagimachi, Kurt S.; Stephanopoulos, Gregory

doi:10.1007/3-540-45300-8_5

Cited by 12 publications

(15 citation statements)

References 16 publications

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“…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.…”

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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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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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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“…The transformation of indene by microorganism expressing dioxygenases has also been reported in the literature. A number of products including indene oxide, cis-(1R,2S)-indandiol, cis-(1S,2R)-indandiol, transindandiol, and 1-indenone have been reported [14][15][16][17][18][19]. Substitution of the carbon of indene with the nitrogen of indole resulted in a 2.3-fold decrease in the rate of substrate consumption ( Table 1).…”

Section: Resultsmentioning

confidence: 99%

“…While the product of styrene consumption, styrene oxide, did not appear due to the presence of other enzymes in P. putida CA-3, known to metabolise styrene to central metabolites [8][9][10], analysis of the biotransformation medium for indene revealed the accumulation of a single product 2-indanone from indene ( Fig. A number of products including indene oxide, cis-(1R,2S)-indandiol, cis-(1S,2R)-indandiol, transindandiol, and 1-indenone have been reported [14][15][16][17][18][19]. All of the indene transformed appeared as 2-indanone ( Fig.…”

Section: Growth Of P Putida Ca-3 With Various Alkenesmentioning

confidence: 99%

Aromatic and aliphatic hydrocarbon consumption and transformation by the styrene degrading strainPseudomonas putidaCA-3

Dunn

Curtin

O'Riordan

et al. 2005

FEMS Microbiology Letters

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Pseudomonas putida CA-3 is capable of consuming a number of aromatic and aliphatic hydrocarbons. With the exception of styrene none of the alkenes tested are capable of supporting the growth of P. putida CA-3 as sole sources of carbon and energy. The highest rate of alkene consumption was observed with styrene as the substrate. A 6.5- and 15.5-fold lower rate of substrate consumption was observed with indene and indole with the concomitant formation of 2-indanone and indigo, respectively. The presence of a sulphur (benzothiopene) or oxygen (benzofuran) in the cyclopentene ring resulted in further decreases in the rate of substrate consumption by whole cells of P. putida CA-3. P. putida CA-3 is incapable of consuming benzene and consumes toluene at a low rate. No detectable products were observed in supernatants of cultures incubated with benzothiopene, benzofuran or toluene. The aliphatic alkenes 1-octene and 1,7-octadiene were both consumed by whole cells of P. putida CA-3 at a rate equivalent to indene consumption. The consumption of (R) styrene oxide was 1.7- and 1.25-fold higher than that of the S isomer and the racemic mix, respectively. The rate of racemic indene oxide, 1,2-epoxyoctane and 1,2-epoxy-7-octene consumption was lower than their equivalent alkene and 55-, 11.8-, and 27.5-fold lower than the rate of racemic styrene oxide consumption. A transposon mutant incapable of growth with styrene or styrene oxide failed to transform indole to indigo. The ratio of styrene utilisation relative to other substrates changes in the mutant strain compared to the wild-type strain, e.g., Indene biotransformation by mutant AF5 is 1.9-fold higher than styrene consumption compared to the wild-type strain CA-3 where the rate of styrene consumption is 6.7-fold higher than indene consumption. This trend is also observed for other alkenes and epoxides.

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“…Rhodococcus 124 exhibits both monooxygenase as well as dioxygenase activity (Chartrain et al, 1998). Figure 1 shows the currently understood indene bioconversion pathway for Rhodococcus 124 (Stafford et al, 2001). The biotransformation of indene to cis-(1S,2R) indandiol is characterized by low yields with the production of various undesirable monooxygenation by-products such as 1-indenol and 1-indanone.…”

Section: Introductionmentioning

confidence: 99%

Application of multi‐parameter flow cytometry using fluorescent probes to study substrate toxicity in the indene bioconversion

Amanullah¹,

Hewitt²,

Nienow³

et al. 2002

Biotech & Bioengineering

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The bioconversion of indene to cis-(1S,2R) indandiol, a potential key intermediate in the synthesis of Merck's HIV protease inhibitor, CRIXIVAN trade mark, can be achieved using a Rhodococcus strain. This study using Rhodococcus I24 reports on the application of multiparameter flow cytometry for the measurement of cell physiological properties based on cytoplasmic membrane (CM) integrity and membrane depolarization as indicators of toxic effects of the substrate, indene. Quantification of intact polarized CM, intact depolarized CM and permeabilized CM of a large population of bacterial cells has been conducted using specific intracellular and membrane-binding fluorescent stains. Measurements of oxygen uptake rate (OUR) and optical density (OD) as indicators of metabolic activity and biomass growth, respectively, were also made. Indene concentrations of up to 0.25 g/L (0.037 g indene/g dry cell weight) did not significantly (<5% compared to control) affect cell light-scattering properties, intact CM, membrane polarization, respiratory activity, or biomass growth. Between this value and 1.5 g/L (0.221 g indene/g dry cell weight), the changes in intact CM, respiratory activity and biomass growth were relatively insignificant (<5% compared to control), although dissipation of the membrane potential of a significant proportion of the cell population occurred at 0.50 g/L (0.074 g indene/g dry cell weight). At 2.5 g/L (0.368 g indene/g dry cell weight) there was a significant increase in the dead cell population, accompanied by changes in the extracellular cationic concentrations and substantial decrease in respiratory activity. The primary effect of indene toxicity was the disruption of the proton motive force across the cytoplasmic membrane which drives the formation of ATP. The disruption of the proton motive force may have been due to the measured changes in proton permeability across the membrane. In addition, indene may have directly inhibited the membrane-bound enzymes related to respiratory activity. The overall consequence of this was reduced respiratory activity and biomass growth. The cell physiological properties measured via flow cytometry are important for understanding the effects of toxicity at the cellular level which neither measurements of biomass growth or indandiol formation rates can provide since both are cell averaged measurements. The technique described here can also be used as a generic tool for measuring cell membrane properties in response to toxicity of other indene-resistant strains that may be possible to use as recombinant hosts to perform the biotransformation of indene. This study has demonstrated that flow cytometry is a powerful tool for the measurement of cell physiological properties to assess solvent toxicity on whole cell biocatalysts.

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Metabolic Engineering of Indene Bioconversion in Rhodococcus sp.

Cited by 12 publications

References 16 publications

Optimizing bioconversion pathways through systems analysis and metabolic engineering

Optimizing bioconversion pathways through systems analysis and metabolic engineering

Aromatic and aliphatic hydrocarbon consumption and transformation by the styrene degrading strainPseudomonas putidaCA-3

Application of multi‐parameter flow cytometry using fluorescent probes to study substrate toxicity in the indene bioconversion

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