Bioreduction whole-cell

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... 

Katz, M., Frejd, T., Hahn-Haegerdal, B. and Gorwa-Grauslund, M.F. (2003) Efficient anaerobic whole cell stereoselective bioreduction with recombinant Saccharomyces cerevisiae. Biotechnology and Bioengineering, 84 (5), 573-582. 

Whereas several anti-cholestemic drugs are produced wholly by fermentation, the side chain of several others is accessible through enzymatic synthesis. (3R,5S)-Dihydroxyhexanoate, a key intermediate of fluvastatin, is accessible by reduction of the diketo acid, either by bioreduction with whole cells (85% yield, 97% e.e.), or cell extracts (72% yield, 98.5% e.e.), or, for syn-(3h,5.S)-dihydroxy-6-Cl-hexanoate, regioselective and (ft)-specific reduction with ADH to yield (5S)-6-chloro-3-ketohexanoate. 

The side chain of such statins, the (3i ,5S)-dihydroxyhexanoates, is a key intermediate and are accessible by reduction of the diketo acids. One way is bioreduction with whole cells (Patel, 1993) with glycerol-grown suspensions of Adnetobacter calcoaceticus SC 13876, yields of 85% and 97% e.e. were achieved for the benzyloxy-(3P,5S)-derivative. If cell extracts were used and NAD+, glucose, and glucose dehydrogenase supplied, both intermediate monohydroxy (in the 3- and 5-position) compounds were produced before the desired dihydroxyhexanoate, and the overall reaction resulted in 72% yield and 98.5% e.e. (Patel, 2001). 

What are the bottelnecks for bioreduction The drawbacks of a bioreduction process involving whole cells of microoganisms can be summarized i) Microbial strains possessing both carbonyl reductase activity and cofactor (NAD(P)H)-regenerating activity are necessary to obtain a highmolar yield, because a stoichiometric amount of cofactor is required for substrate reduc-... 

Hall, M., Hauer, B., Stuermer, R., Kroutil, W., and Faber, K. 2006. Asymmetric whole-cell bioreduction of an oc,P-unsaturated aldehyde (citral) competing prim-alcohol dehydrogenase and C-C lyase activities. Tetrahedron Asymm., 17, 3058-3062. 

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. 

Scheme4.10 Whole-cell bioreduction using a two phase system.

Fig. 11 Biotechnological approaches for the production of y-valerolactone (y-VL) from levulrnic acid. The key step is the bioreduction of levulinic acid 4-hydroxyvalerate (4-VL), which subsequently can lactonize to y-VL. (a) Biotransformation using Pseudomonas putida whole-cell overexpression of thioesterase tesB and paraoxonase I PONl (Martin et al. 2010). (b) A new variant of 3-hydroxybutyrate dehydrogenase 3HBDH from Alcaligenes faecalis catalyzes the bioreduction of levulinic acid to 4-hydroxyvalerate (4-HV) (Yeon et al. 2013). (c) Chemoenzymatic route using carbonyl reductase from Candida parapsilosis CPCR2 and lipase B CAL-B from Candida antarctica (Gotz et al. 2013)...

As already observed for the tetrasubstituted C=C double bonds of substituted cinnamaldehydes, the bioreduction of a,p-dimethyl nitrostyrenes was also shown to be efficient albeit poorly diastereoselective, in the case of both whole cells and with isolated ene reductases [53,100]. 

As mentioned in the case of enals, whole-cell systems often show undesired side activities in this case ester hydrolysis took place quantitatively in the reaction medium (due to the presence of hydrolases, such as proteases and lipases) and the optically pure chloro acids were obtained. The bioreduction of the same compounds has recently been revised, employing isolated ene reductases OYEl-3, to prevent hydrolysis and demonstrate conclusively that the reduction in yeast cells is due to the action of ene reductases and the methylesters are accepted substrates [118]. (H)-enantiomers are obtained from ( )-stereoisomers of the starting materials, and the opposite (S)-enantiomers are obtained from the (Z)-isomers (isomerism-based strategy. Figure 3.2a), though with lower ee values. The saturated products obtained (either esters or acids) can be employed as synthones in a large variety of applications (e.g., (S)-98 is the key precursor of the natural antibiotic armentomycin). 

In this chapter we would like to highlight some recent examples, from 2006 onward, regarding the employment of whole-cell systems or (partially) purified enzymes to obtain, through bioreduction, biologically relevant (family of) compounds or intermediates, focusing on the enzymatic preparation and the cofactor regeneration system employed (Scheme 4.1). This chapter has been divided depending on the chemical structure of the substrates. Thus, Section 4.2 will provide recent examples of bioreductions over a- or P-keto esters (Section 4.2.1), diketones (Section 4.2.2), halo... 

Toxic substrates and products to whole-cell biocatalysts. Finally, in whole-cell format, the substrate and/or product of the bioreduction can be toxic to the cells, preventing cofactor regeneration. Such irreversible loss of regeneration capacity is, of course, catastrophic for the process. In principle, this can be overcome by maintaining a low substrate concentration, but this will ultimately prevent a sufficiently high product concentration for an effective process. In some cases, dependent upon the water-solubility (and if the substrate is a liquid), it may be possible to feed the substrate, such that a low concentration is provided to the cells in the reactor, but at the end of the reaction a high product concentration is achieved. However, in nearly all cases at the required concentration for an... 

For the aforementioned reasons the use of isolated enzymes is often preferred due to reduction in side reactions and higher productivities (see Ref. [12] for a review of this topic). However this brings other challenges such as the need for effective cofactor regeneration. The choice between enzyme and whole-cell biocatalysts is complex and requires more work in the future to establish a clearer strategy to help the process design and implementation of bioreductions. 

The enantio- and diastereoselective bioreduction of 2-oxocycloalkanecarboni-triles 10 lead to the cis relative configuration of (3-hydroxy nitriles 10a [17]. These reductions were accomplished by whole cells of the yeast Saccharomyces montanus (Scheme 12.5). The presence of the acidic a-hydrogen is very important since (3-keto nitriles that are fully substituted in the a-position do not racemize under the bioreduction conditions. The products were transformed into optically active 2-amino and 2-aminomethyl cycloalkanols. 

A few years later, the same research group demonstrated the bioreduction of a-chloro-P keto esters 29-33. The production of at least two of the four possible a-chlorO P-hydroxy ester diastereomers with high stereoselectivities, by using whole cells of an E. coli strain overexpressing a single yeast reductase identified from screening studies, was successfully accomplished (Scheme 12.16) [33]. 

Scheme 12.19 Stereoselective synthesis of the chiral chlofibrate analog 39b by whole-cell bioreduction.

Bioreduction of a,p-unsaturated ketones and aldehydes by non-conventional yeast (NCY) whole-cells. Bioresour. Technd., 102, 3993-3998. 

Prior to the widespread awdlabdity of recombiant carbonyl reductases enzymes, the use of microbial reductions using either actively growing or dormant cells was commonplace Bakers yeast in particular, was a readily available source of stereoselective carbonyl reductases enzymes. Even with the widespread knowledge of the power of recombinant CRED biocatalysts, the literature is still rife with wild-type whole-cell microbial reductions. The reductions presented have advanced well beyond the early Bakers yeast reduction and have an apphcation even today. When the whole-cell fermentation is developed and finely tuned, high titers of product alcohol are possible and Scheme 6.4 shows m example of a keto-amide 12 bioreduction performing at 100 g/L with more than 98% ee with multi-kg isolation [12]. The bioprocess was performed over 8 days at pH 7 using the yeast Candida sorbophila. 

This section gives the organic chemist examples of how to perform the bioreductions in the laboratory using both wild-type whole cell and recombinant systems. 

Whole-cell miaobial bioreduction involves both whole cell preparation followed by incubation with ketone substrate [13]. 

Enzymes with different stereochemical preferences for 3-oxo esters and 2-alkyl 3-0X0 esters have been isolated [57,62,63,68]. They are NADPH-dependent enzymes and are able to catalyze the reduction of oxo esters of different type. However, they are not available for enzyme-catalyzed reaction in substitution of the whole-cell catalyst. Synthetic applications make use of whole-cell biocatalysts. Valuable intermediates in synthesis are keto esters possessing additional functionality. Thus 5 -4-chloro-3-hydroxybutanoic acid 33 (Scheme 12) has been obtained by reduction with suspended cells from cultures of G. candidum. The compound is the intermediate in the synthesis of the cholesterol antagonist 34. In the biotransformation process a reaction yield of 95% and optical purity of 96% were obtained at 10 g/L. The optical purity was increased to 99% by heat treatment of cell suspensions prior to conducting the bioreduction [69]. 

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