Overexpression of key reductases from bakers yeast in Escherichia coli

3 Overexpression of key reductases from baker s yeast in Escherichia coli 

The problem of low enantioselectivity of whole-cell reduction possessing more than two enzymes with opposite enantioselectivities has been solved by overexpressing the individual enzymes in E. coli. Now, 18 key reductases from baker s yeast have been overproduced in E. coli as glutathione S-transferase (GST) fusion proteins. Therefore, rapid identification of synthetically useful biocatalysts is possible. A set of fusion proteins consisting of GST hnked to the N-terminus of putative dehydrogenases produced by baker s yeast was screened for the reduction of various substrates. For example, ethyl 2-oxo-4-phenylbutyrate was reduced 

A representative set of a- and /3-keto esters was also tested as substrates (11 total) for each purified fusion protein (Fig. 10.9(b) and (c)). The stereoselectivities of -keto ester reductions depended on both the identity of the enzyme and the substrate structure, and some reductases yielded both l- and o-alcohols with high stereoselectivities. While a-keto esters were generally reduced with lower enantioselectivities, it was possible to identify pairs of yeast reductases that delivered both enantiomers in optically pure form. 

Furthermore, two enantiocomplementary baker s yeast enzymes were also used for the reduction of an ct-chloro-jS-keto ester to yield precursors for both enantiomers of the N-benzoyl phenylisoserine Taxol side chain (Fig. 10.10). These results demonstrate the power of genomic fusion protein libraries to identify appropriate biocatalysts rapidly and expedite process development.  

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