Whole biocatalytic reduction

Goldberg, K., Schroer, K., Luetz, S. and Liese, A. (2007) Biocatalytic ketone reduction - a powerful tool for the production of chiral alcohols - part II whole-cell reductions. Applied Microbiology and Biotechnology, 76, 249-255. 

The biocatalytic reduction of carboxylic acids to their respective aldehydes or alcohols is a relatively new biocatalytic process with the potential to replace conventional chemical processes that use toxic metal catalysts and noxious reagents. An enzyme known as carboxylic acid reductase (Car) from Nocardia sp. NRRL 5646 was cloned into Escherichia coli BL21(DE3). This E. coli based biocatalyst grows faster, expresses Car, and produces fewer side products than Nocardia. Although the enzyme itself can be used in small-scale reactions, whole E. coli cells containing Car and the natural cofactors ATP and NADPH, are easily used to reduce a wide range of carboxylic acids, conceivably at any scale. The biocatalytic reduction of vanillic acid to the commercially valuable product vanillin is used to illustrate the ease and efficiency of the recombinant Car E. coli reduction system." A comprehensive overview is given in Reference 6, and experimental details below are taken primarily from Reference 7. 

Fig. 3.41 Whole cell technology for biocatalytic reduction, example adapted from Ref. [117],...

Much higher productivities can be obtained using isolated enzymes or cell extracts [118]. This approach is therefore highly preferred. Because of the importance of whole cell technology for biocatalytic reduction a few examples will be given. However the main part of this chapter will be devoted to industrial examples of bioreduction involving isolated enzymes and cofactor recycling. 

Another example for biocatalytic reduction using glucose as the reductant, is the production of (R)-ethyl-4,4,4-trifluoro-3-hydroxybutanoate by Lonza [130]. It is a building block for pharmaceuticals such as Befloxatone, an anti-depressant monoamine oxidase-A inhibitor from Synthelabo. The process uses whole cells of Escheria coli that contain two plasmids. One carries an aldehyde reductase... 

The future implementation of biocatalytic reductions is particularly bright. It is clear that the cost of the enzymes (both for the reduction itself as well as the cofactor regeneration) is being reduced all the time, as ever more effective technologies for production are established with better expression and enzyme recovery methods. Although several notable recent examples have focused on isolated enzyme routes, it is dear that in some cases whole cells will prove better. However, several requirements for the future also need to be fulfilled ... 

Deracemization via the biocatalytic stereoinversion is usually achieved by employing whole cells. In the case of secondary alcohols, it is believed that microbial stereoinversion occurs by an oxidation-reduction sequence... 

Prochiral aryl and dialkyl ketones are enantioselectively reduced to the corresponding alcohols using whole-cell bioconversions, or an Ir1 amino sulfide catalyst prepared in situ.695 Comparative studies show that the biocatalytic approach is the more suitable for enantioselective reduction of chloro-substituted ketones, whereas reduction of a,/ -unsaturated compounds is better achieved using the Ir1 catalyst. An important step in the total synthesis of brevetoxin B involves hydrogenation of an ester using [Ir(cod)(py) P(cy)3 ]PF6.696... 

Another very recent development in the field of enzymatic domino reactions is a biocatalytic hydrogen-transfer reduction of halo ketones into enantiopure epoxides, which has been developed by Faber, Bornscheuer and Kroutil. Interestingly, the reaction was carried out with whole lyophilized microbial cells at pH ca. 13. Investigations using isolated enzymes were not successful, as they lost their activity under these conditions [26]. 

Enzyme reductions of carbonyl groups have important applications in the synthesis of chiral compounds (as described in Chapter 10). Dehydrogenases are enzymes that catalyse, for example, the reduction of carbonyl groups they require co-factors as their co-substrates. Dehydrogenase-catalysed transformations on a practical scale can be performed with purified enzymes or with whole cells, which avoid the use of added expensive co-factors. Bakers yeast is the whole cell system most often used for the reduction of aldehydes and ketones. Biocatalytic activity can also be used to reduce carbon carbon double bonds. Since the enzymes for this reduction are not commercially available, the majority of these experiments were performed with bakers yeast1 41. 

Unlike the whole-cell system, enzymatic reductions require the addition of a hydride donating cofactor to regenerate the reduced form of the enzyme. Depending on the chosen ADH, the cofactor is usually NADH or NADPH, both of which are prohibitively expensive for use in stoichiometric quantities at scale. Given the criticality of cofactor cost, numerous methods of in situ cofactor regeneration, both chemical and biocatalytic, have been investigated. However, only biocatalytic regeneration has so far proven to be sufficiently selective to provide the cofactor total turnover numbers of at least 10 required in production. 

An example of biocatalytic C=0 bond reduction has also been reported in the literature. The asymmetric reduction of ketones via whole-cell bioconversions and TH was tested by van Leeuwen et al. as complementary approaches to asymmetric... 

The highly selective biocatalytic reactions afford a substantial reduction in waste. The overall isolated yield is greater than 90%, and the product is more than 98% chemically pure with an enantiomeric excess of >99.9%. All three evolved enzymes are highly active and are used at such low loadings that counter-current extraction can be used to minimize solvent volumes. Moreover, the butyl acetate solvent is recycled with an efficiency of 85%.The E factor (kgs waste per kg product) for the overall process is 5.8 if process water is excluded (2.3 for the reduction and 3.5 for the cyanation) [47]. If process water is included, the E factor for the whole process is 18 (6.6 for the reduction and 11.4 for the cyanation). The main contributors to the E factor are solvent losses which accounted for 51% of the waste, sodium gluconate (25%), NaCl and Na2SO4 (combined circa. 22%). The three enzymes and the NADP cofactor account for <1% of the waste. The main waste streams are aqueous and directly biodegradable. 

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