Whole-cell biocatalysts pathway

In a patent on biological desulfurization [100] of petroleum/coal, only the use of whole cell biocatalysts was claimed. The biocatalysts included microorganisms belonging to the genus Pseudomonas, Flavobacterium, Enterobacter, Aeromonas, Bacillus or Corynebacterium. The desulfurization pathway (sulfur-specific vs. destructive) was not specified. The Japanese patents No. JP2071936C and JP7103379B seem to be equivalent patents. 

To search for an appropriate whole-cell biocatalyst, it is necessary to identify an organism that contains large amounts of the desired enzyme. Equally important, the organism should not contain related pathway enzymes that modify or destroy the product synthesized by the desired enzyme. 

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. 

Production of artemisinin and paclitaxel precursors by engineered whole-cell biocatalysts from glucose. Introduction of biosynthetic genes from Artemisia annua encoding the amorphadiene synthase and amorphadiene oxidase yielded microbial strains that produce arte-misinic acid. Artemisinic acid can be chemically converted into artemisinin, introduction of the Taxus genes encoding taxadiene synthase and taxadiene 5a-hydroxy-lase resulted in E. constrains that produce key paclitaxel intermediates. The biosynthetic pathway for paclitaxel has not been fully elucidated. 

Compared with isolated enzymes, application of whole cells as biocatalysts is usually more economical since there is no protein purification process involved. Whole cells can be used directly in chemical processes, thereby greatly minimizing formulation costs. Whole cells are cheap to produce and no prior knowledge of genetic details is required. Microorganisms have adapted to the natural environment and produce both simple and complex metabolic products from their nutrient sources through complex, integrated pathways. 

For instance, styrene oxide was resolved by whole cells of Aspergillus niger and Beauveria bassiana via two different pathways showing matching enantio- and regioselectivities with excellent results (Scheme 8). Combination of the two biocatalysts employing a deracemization process in a single reactor led to R) phenylethane-l,2-diol as the sole product in 98% ee and 85% isolated yield [58]. 

In contrast to rational approaches, the directed evolution of enzymes is based on the search of useful functionalities in libraries randomly generated and on improvement by suitable and proper selection. The directed evolution combines two powerful and independent technologies methods for the generation of random genetic libraries and strategies for the selection of variant enzymes with the specific capabilities [499-503]. This process can result in biocatalysts with non-natural proprieties, since the proteins are expressed in recombinant cells decoupled from its biological functions and evolved under unusual conditions. One additional advantage is the possibility to tailor not only individual proteins, but also the whole biosynthetic and catabohc pathways [471]. 

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