Recombinant whole cells

Metabolic pathways containing dioxygenases in wild-type strains are usually related to detoxification processes upon conversion of aromatic xenobiotics to phenols and catechols, which are more readily excreted. Within such pathways, the intermediate chiral cis-diol is rearomatized by a dihydrodiol-dehydrogenase. While this mild route to catechols is also exploited synthetically [221], the chirality is lost. In the context of asymmetric synthesis, such further biotransformations have to be prevented, which was initially realized by using mutant strains deficient in enzymes responsible for the rearomatization. Today, several dioxygenases with complementary substrate profiles are available, as outlined in Table 9.6. Considering the delicate architecture of these enzyme complexes, recombinant whole-cell-mediated biotransformations are the only option for such conversions. E. coli is preferably used as host and fermentation protocols have been optimized [222,223]. 

Fig. 2. Whole cell biocatalytic reactions for four types of recombinant whole cell systems. Bioconversion reactions were performed in resting cell condition. All data were based on unit cell concentration (1 mg-dry cell weight ml ). Each value and error bar represents the mean of two independent experiments and its standard deviation.

Fig. 3. Lineweaver-Burk plot analyses for four types of recombinant whole cell biocatalytic reactions.

Recombinant Whole-Cell Biocatalysts Overexpressing Catalytic Enzymes... 

The reduction of several ketones, which were transformed by the wild-type lyophilized cells of Rhodococcus ruber DSM 44541 with moderate stereoselectivity, was reinvestigated employing lyophilized cells of Escherichia coli containing the overexpressed alcohol dehydrogenase (ADH- A ) from Rhodococcus ruber DSM 44541. The recombinant whole-cell biocatalyst significantly increased the activity and enantioselectivity [41]. For example, the enantiomeric excess of (R)-2-chloro-l-phenylethanol increased from 43 to >99%. This study clearly demonstrated the advantages of the recombinant whole cell biocatalysts over the wild-type whole cells. 

Groeger, H., Rollmann, C., Chamouleau, F. et al. (2007) Enantioselective reduction of 4-fluoroacetophenone at high substrate concentration using a tailor-made recombinant whole-cell catalyst. Advanced Synthesis and Catalysis, 349 (4-5), 709-712. 

SYNTHESIS OF CATECHOLS BY A RECOMBINANT WHOLE CELL BIOCATALYST... 

An interesting example of the application of recombinant whole-cell biocatalysis is the conversion of 2-hydroxybiphenyl (2-phenylphenol) to 2,3-dihydroxybiphenyl... 

Figure 15.12 Two-liquid-phase-based biooxidation of styrene to styrene oxide with a recombinant whole-cell biocatalyst.

An example of a whole-cell process is the two-step synthesis of an enantiopure epoxide by asymmetric reduction of an a-chloro ketone (Scheme 6.4), catalyzed by recombinant whole cells of an Escherichia coli sp. overexpressing an (R)-KRED from Lactobacillus kefir and GDH from Thermoplasma acidophilum, to the corresponding chlorohydrin, followed by non-enzymatic base-catalyzed ring closure to the epoxide [17]. 

Matsuyama, A. Yamamoto, H. and Kobayashi, Y. Practical Apphcation of Recombinant Whole-cell Biocatalysts for the Manufacturing of Pharmaceutical Intermediates such as Chiral Alcohols. Org. Process Res. Dev. 2002, 6, 558-561. 

G. F. Herrmann, P. Wang, G.-J. Shen, E. Garcia-Juneeda, S. H. Khan, K. L. Malta, and C.-H. Wong, Large-scale production of recombinant a-1,2-mannosyltranferase from E. coli for the study of acceptor specificity and use of the recombinant whole cells in synthesis, J. Org. Chem. 59 6356 (1994). 

For Baeyer-Villiger oxidations of ketones into lactones, recombinant whole-cell catalysts were constructed using the CHMO from A. calcoaceticus and cells of S. cerevisiae [127-129] or E. coli [130-132] as host system. These designer cells were used for example for the biotransformation of bicyclo[3.2.0]-hept-2-ene-6-one [133-136], In order to avoid the substrate and product inhibition, an adsorber... 

Fig. 40 Concept for the two-step synthesis of enantiomerically pure (S)-epoxides out of aliphatic 1-halogenated 2-ketones. The ketone was reduced by a recombinant whole-cell catalyst bearing alcohol dehydrogenase from Lactobacillus kefir (LKADH) and glucose dehydrogenase (GDH) for regeneration of NADPH. Base-induced cyclization of the enantiomerically pure (5)-(3-halohydrin intermediate gave the desired (S)-epoxides in high yield and enantiomeric purity (>99% ee)...

Hydantoinase-Carbamoylase System for t-Amino Acid Synthesis Despite a number of reports of strains with L-selechve hydantoin-hydrolyzing enzymes [38] the commercial application of the hydantoinase process is stiU restricted to the production of D-amino acids. Processes for the production of L-amino acids are Umited by low space-time yields and high biocatalyst costs. Recently, a new generation of an L-hydantoinase process was developed based on a tailor-made recombinant whole cell biocatalyst. Further reduction of biocatalyst cost by use of recombinant Escherichia coli cells overexpressing hydantoinase, carbamoylase, and hydantoin racemase from Arthrohacter sp. DSM 9771 were achieved. To improve the hydan-toin-converting pathway, the level of expression of the different genes was balanced on the basis of their specific activities. The system has been appUed to the preparation of L-methionine the space-time yield is however still Umited [39]. Improvements in the deracemization process from rac-5-substituted hydantoins to L-amino acids still requires a more selective L-hydantoinase. 

Herrmann GF, Wang P, Shen GJ, Garciajunceda E, Khan SH, Malta KL, Wong CH. Large-Scale Production of Recombinant a-l,2-Mannosyltransferase from Escherichia-Coli for the Study of Acceptor Specificity and Use of the Recombinant Whole Cells in Synthesis. J. Org. Chem. 1994 59 6356-6362. 

All the recombinant whole cells producing the different nitrilases were evaluated for the hydrolysis rate on AdN and for its chemo-selectivity (Table 8). 

Herrmann GF, Wang P, Shen G-J, Wong C-H. Recombinant whole cells as catalysts for enzymic synthesis of oligosaccharides and glycopeptides. Angew Chem 1994 106 1346-1347. 

Novella IS, Fargues C, GreviUot G (1994) Improvement of extraction of penicillin acylase by a combined use of chemical methods. Biotechnol Bioeng 44 379-382 Ospina S, Lopez-Mungufa A, GonztQez R et al. (1992) Characterization and use of a penicillin acylase biocatalyst. J. Chem Technol Biotechnol 53 205-214 Ospina SS, Merino E, Ramirez OT et til. (1995) Recombinant whole cell penicillin acylase biocatalyst production, characterization and use in the synthesis and hydrolysis of antibiotics. Biotechnol Lett 17 615-620... 

Verseck, S., Becker, U., Doderer, K., Ofiwald, S., and Wienand, W. (2009) Production of amino adds using wild type and recombinant whole cell catalysts using platform technologies for enhandng production effidency. ACS Symposium series Vol 1009, Asymmetric synthesis and application of a-amino acids. 375-393. 

Buhler, Schmid, and coworkers [36] described the development of a recombinant whole-cell biocatalyst for the direct terminal alkylamino-functionalization of fatty acid methyl esters (e.g., dodecanoic acid methyl ester). The model substrate was dodecanoic acid methyl ester, which was oxidized by an alkane monooxygenase (AlkBGT) from Pseudomonas putida GPol to the corresponding... 

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