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Harnessing Candida tenuis and Pichia stipitis in whole‐cell bioreductions of o ‐chloroacetophenone: Stereoselectivity, cell activity, in situ substrate supply and product removal
Author(s) -
Gruber Christoph,
Krahulec Stefan,
Nidetzky Bernd,
Kratzer Regina
Publication year - 2013
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.201200322
Subject(s) - pichia stipitis , stereoselectivity , yeast , substrate (aquarium) , in situ , biology , chemistry , biochemistry , catalysis , saccharomyces cerevisiae , organic chemistry , ecology
Generally, recombinant and native microorganisms can be employed as whole‐cell catalysts. The application of native hosts, however, shortens the process development time by avoiding multiple steps of strain construction. Herein, we studied the NAD(P)H‐dependent reduction of o ‐chloroacetophenone by isolated xylose reductases and their native hosts Candida tenuis and Pichia stipitis. The natural hosts were benchmarked against Escherichia coli strains co‐expressing xylose reductase and a dehydrogenase for co‐enzyme recycling. Xylose‐grown cells of C. tenuis and P. stipitis displayed specific o ‐chloroacetophenone reductase activities of 366 and 90 U g CDW –1 , respectively, in the cell‐free extracts. Fresh biomass was employed in batch reductions of 100 mM o ‐chloroacetophenone using glucose as co‐substrate. Reaction stops at a product concentration of about 15 mM, which suggests sensitivity of the catalyst towards the formed product. In situ substrate supply and product removal by the addition of 40% hexane increased catalyst stability. Optimisation of the aqueous phase led to a ( S )‐1‐(2‐chlorophenyl)ethanol concentration of 71 mM (ee > 99.9%) obtained with 44 g CDW L –1 of C. tenuis. The final difference in productivities between native C. tenuis and recombinant E. coli was < 1.7‐fold. The optically pure product is a required key intermediate in the synthesis of a new class of chemotherapeutic substances (polo‐like kinase 1 inhibitors).