Bioconversion network

The foremost requirement is the accurate determination of an observable bioreaction network structure that describes indene oxidation in Rhodococcus. Based on product accumulation profiles and induction studies, indene bioconversion networks have been proposed for several isolates [8]. To validate further these networks for our strains and employ them for flux analysis we developed an experimental system that can maintain cells at steady state while allowing accurate metabolite measurements for flux determination. The system comprised a chemostat with a regular feed of hquid medium and separate supply of the indene precursor through a gas phase hne. Indene uptake and metabolite production rates were easily measured in this system, leading to the calculation of the unknown bioconversion fluxes. Additional measurements for system closure and further validation were obtained by using radiolabeled tracers and measuring the products of their oxidation in Rhodococcus cultures [9,10]. 

Fig. 2. Indene bioconversion network in Rhodococcus strain 124 proposed by Chartrain et al. [8]. Indene is converted to the indandiol enantiomers shown through specific oxygenase activities. Dioxygenases produce the cis enantiomers of indandiol while the monooxygenase converts indene to indan oxide. The indandiols are then converted to l-keto-2-hydroxy-indan through the action of dehydrogenase enzymes and a proposed undetectable 1,2-indenediol intermediate...

To improve the quantitative analysis of the indene bioconversion network, an experimental system enabling the accurate measurement of indene metabolites was developed. A multi-phase fermentation system commonly employed when dealing with substrates or products of relatively low solubility was not desirable for this analysis due to uncertainties associated with the partitioning of the in-... 

The indene bioconversion network proposed for Rhodococcus sp. KYI (Fig. 4) using the induction studies with radiolabeled indene can be used to write five independent mass balances to describe six intracellular fluxes (Table 3). This yields an underdetermined system for the fluxes of the KYI network requiring that at least one flux be directly measured to calculate uniquely the remaining network fluxes. It is further desirable to measure directly additional fluxes to generate redundancies that can be used to confirm the structure of the proposed bioreaction network, validate the flux estimates, and help detect gross measurement errors, if present. 

C]-czs-(lS,2i )-Indandiol was used to measure directly the corresponding steady state dehydrogenase flux, Vrdh> in the KYI indene bioconversion network. By measuring the formation of [ C]-l-keto-2-hydroxy-indan associated with the concomitant depletion of [ C]-czs-(lS,2i )-indandiol in steady state cells, the... 

Fig. 4. Indene bioconversion network in Rhodococcus sp. KYI. Metabolite excretion and uptake rates are denoted by r, while intracellular fluxes are written as y...

Table 3. Steady state metabolite balance equations for the KYI indene bioconversion network ...

The indene bioreaction network that catalyzed the bioconversion to (2R)-indanediol was also investigated in Rhodococcus sp. (Stafford, 2001). Metabolic engineering methodology was applied step-by-step ... 

Further steps will be taken in order to implement bioconversion of the nitro group as well as biochemical glucuronidation and glutathionylation of the drug. Since hydrolysis of the amide bond was found to be caused by the action of glutathione, glutathione metabolism will be implemented into the metabolic network as well. 

Figure 39 In vivo NMR at the natural abundance of clarifies the intermediacy of vinorine (16) in the RauvoMa alkaloid biosynthetic network by conversion into sarpagine (5) and into raucaffncine (30) and bioconversion of ajmaline (1) into raumacline (70) (see also Figures 23, 25 and 35).

Coals are complex and heterogeneous polymer networks with aliphatic and ether bridges between aromatic moieties. There are two different bioconversion... 

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