Whole-cell biotransformation

Poppe L, Novak L (1992) Selective biocatalysis. A synthetic approach. VCH, Weinheim Roberts SM, Wiggins K, Casy G (1992) Preparative biotransformations. Whole cell and isolated enzymes in organic synthesis. WUey, Chichester Flohe L (1979) CIBA Foundation Symposium 65 95... 

Roberts, S. M., Wiggins, K., Casy, G., Phythian, S. Preparative Biotransformations Whole Cell and Isolated Enzymes in Organic Synthesis, John Wiley Sons Chichester, 1992. 

A further important aspect is the feasibility of whole cell biotransformations. Whole cell biotransformations show a lot of advantages as compared to isolated enzymes, such as the improved stability of enzymes. If both, producing and regenerating enzymes, are available in one single strain, no addition of expensive cofactor is necessary because the intracellular cofactor pool can be utilized. Whole cell biotransformations are therefore very promising for technical applications, and making these conversions an intensively studied subject in the last years. The use of recombinant DNA techniques offers many possibilities to create capable systems. This chapter describes the most important whole cell biotransformations developed in the past as well as relevant processes with small-scale and technical application. 

P Zandbergen J van der Linden, J Bmssee, A van der Gen. Preparative Biotransformations. Whole cell and isolated enzymes in organic synthesis. Update 5, 4 5.1. Wiley Looseleaf Publications, Ed. SM Roberts 1995. 

Biotransformations are carried out by either whole cells (microbial, plant, or animal) or by isolated enzymes. Both methods have advantages and disadvantages. In general, multistep transformations, such as hydroxylations of steroids, or the synthesis of amino acids, riboflavin, vitamins, and alkaloids that require the presence of several enzymes and cofactors are carried out by whole cells. Simple one- or two-step transformations, on the other hand, are usually carried out by isolated enzymes. Compared to fermentations, enzymatic reactions have a number of advantages including simple instmmentation reduced side reactions, easy control, and product isolation. 

The term biotransformation or biocatalysis is used for processes in which a starting material (precursor) is converted into the desired product in just one step. This can be done by use either of whole cells or of (partially) purified enzymes. Product examples range from bulk chemicals (such as acrylamide) to fine chemicals and chiral synthons (chiral amines or alcohols, for example). There are several books and reviews dealing with the use of bio transformations either at laboratory or at industrial scales [1, 10-13]. 

In designing a process we have the choice of using the whole organism or specific enzymes isolated from it. As always both options have pro s and cons. Broadly speaking we could say that biosynthetic processes mostly rely on whole cells, whereas biotransformations can be catalysed by whole cells and by enzyme preparations. 

Benzene dioxygenase is a complex enzyme consisting of three protein components, that catalyse the conversion of benzene to benzene cis-dihydrodiol. Give two reasons why this biotransformation should be carried out using whole cells as opposed to using enzyme preparations. 

A new development is the industrial production of L-phenylalanine by converting phenylpyruvic add with pyridoxalphosphate-dependent phenylalanine transaminase (see Figure A8.16). The biotransformation step is complicated by an unfavourable equilibrium and the need for an amino-donor (aspartic add). For a complete conversion of phenylpyruvic add, oxaloacetic add (deamination product of aspartic add) is decarboxylated enzymatically or chemically to pyruvic add. The use of immobilised . coli (covalent attachment and entrapment of whole cells with polyazetidine) is preferred in this process (Figure A8.17). 

The biotransformation should be carried out using whole cells because ... 

Such isolated enzyme approaches for deracemization have a clear disadvantage in that they require two operational manipulations with an intermediate recovery step. A one-pot strategy is offered by employing whole-cell biotransformations with strains containing set(s) of complementary dehydrogenases operating in both biooxidative and bioreductive modes. Trace amounts of the intermediate ketone species can be isolated in several cases. In order to lead to an efficient deracemization... 

Several suitable whole-cell systems have been identified for deracemization biotransformations on a large diversity of substrates, as compiled recently [48]. In particular, heterocyclic alcohols were successfully converted by Sphingomonas [55]. Access to enantiocomplementaiy products was achieved with various strains of Aspergillus [56] or Rhizopus [57]. Biotransformations can even be accomplished with yacon and ginger [58]. Substrate titers were reported up to 8gl for Candida parapsUosis mediated biotransformations [59]. 

Enzyme-mediated chiral sulfoxidation has been reviewed comprehensively in historical context [188-191]. The biotransformation can be mediated by cytochrome P-450 and flavin-dependent MOs, peroxidases, and haloperoxidases. Owing to limited stability and troublesome protein isolation, a majority of biotransformations were reported using whole-cells or crude preparations. In particular, fungi have been identified as valuable sources of such biocatalysts and the catalytic entities have not been fully identified in all cases. 

Cyclic dithioketals and acetals represent another important class of sulfur containing chiral auxiliaries, which are available in chiral form by biooxidation. Biotransformations were performed on a preparative scale using whole-cells (wild type and recombinant) and isolated enzyme. Again, enantiocomplementary oxidation of unsubstituted dithianes (linear and cyclic, R = H) was observed when using and CPMOcomo (Scheme 9.28) [211,212]. Oxygenation of functionalized substrates (R = substituted alkyl) with gave preferably trans... 

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]. 

In contrast to 2,3-dioxygenases, the related ipso/ortho oxygenation of aryl carbox-ylates has received considerable less attention and has hardly been utilized by the synthetic community, so far. Biooxidation of benzoic acid and P-naphthalene carboxylate provide access to corresponding 1,2-dihydroxylated dihydroaryl compounds in excellent stereoselectivity (Scheme 9.35), analogous to TDO- and NDO-mediated ortho/meta oxygenations. Whole-cell-mediated biotransformations were performed with mutant strains of Rahtonia and Pseudomonas and enable access to preparative quantities in >5 gl titers [261,262]. 

Whole-Cell Biotransformation Processes Used in Commercial... 

D-Pantolactone and L-pantolactone are used as chiral intermediates in chemical synthesis, whereas pantoic acid is used as a vitamin B2 complex. All can be obtained from racemic mixtures by consecutive enzymatic hydrolysis and extraction. Subsequently, the desired hydrolysed enantiomer is lactonized, extracted and crystallized (Figure 4.6). The nondesired enantiomer is reracemized and recycled into the plug-flow reactor [33,34]. Herewith, a conversion of 90-95% is reached, meaning that the resolution of racemic mixtures is an alternative to a possible chiral synthesis. The applied y-lactonase from Fusarium oxysporum in the form of resting whole cells immobilized in calcium alginate beads retains more than 90% of its initial activity even after 180 days of continuous use. The biotransformation yielding D-pantolactone in a fixed-bed reactor skips several steps here that are necessary in the chemical resolution. Hence, the illustrated process carried out by Fuji Chemical Industries Co., Ltd is an elegant way for resolution of racemic mixtures. 

Ernst, M., Kaup, B., Mueller, M. et al. (2005) Enantioselective reduction of carbonyl compounds by whole-cell biotransformation, combining a formate dehydrogenase and a (R)-specihc alcohol dehydrogenase. Applied Microbiology and Biotechnology, 66 (6), 629-634. 

Kaup, B., Bringer-Meyer, S. and Sahm, H. (2004) Metabolic engineering of Escherichia coir, construction of an efficient biocatalyst for D-mannitol formation in a whole-cell biotransformation. Applied Microbiology and Biotechnology, 64 (3), 333-339. 

Application of Whole-Cell Biotransformation in the Pharmaceutical Industry... 

Whole-cell biotransformation processes have been successfully applied for commercial production of pharmaceuticals, either as the drug substance itself or as an intermediate for the synthesis of the final drug substance. Some examples of the whole-cell biotransformation processes used by pharmaceutical industry are shown in Table 10.1. The structures of the biotransformation products are shown in Figure 10.1. 

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