Improvement of natural isolates of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains for synthesis of a chiral building block using classic genetics |
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Authors: | Netta Nir Moran Bahalul Roi Feingersch Tal Katz-Ezov Yechezkel Kashi Ayelet Fishman |
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Institution: | (1) Department of Biotechnology and Food Engineering, Technion-Israel Institute of Technology, Haifa, 32000, Israel |
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Abstract: | The asymmetric bio-reduction of 4-chloro-acetoacetic-acid-ethyl-ester to the pharmaceutical building block (S)-4-chloro-3-hydroxybutanoate-ethyl-ester requires the utilization of an enantioselective robust biocatalyst. Some of the
natural Saccharomyces cerevisiae strains, isolated from Mount Carmel National Park in Israel, were characterized as resistant to environmental stress. Nevertheless,
these strains showed relatively low enantiomeric-excess (ee), while a laboratory strain, Y103, exhibited a selectivity of
98% ee. The enantioselective lab strain was crossed with the multi-stress resistant environmental isolate (93% ee) followed
by backcross with Y103, to subsequently obtain a haploid offspring of backcross-1, exhibiting both high multi-stress resistance
and high enantioselectivity (98% ee). Introducing osmotic (1 M NaCl), oxidative (0.6 mM H2O2) and thermal stress (44°C) to growing cultures of the enantioselective parent, resulted in a decrease of 24–32% in specific
activity, while the enantioselectivity of the stress-resistant parent decreased by 4–12% ee. Unlike its original parental
strains, the new strain maintained constant specific activity and enantioselectivity when introduced to the various stress
factors. This work shows that the classic introgression method, can serve as a viable approach for creating a robust enantioselective
biocatalyst, designed for industrial production of chiral compounds. |
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Keywords: | Saccharomyces cerevisiae 4-chloro-3-hydroxybutanoic acid ethyl ester 4-chloro-acetoacetic acid ethyl ester Backcross Stress resistance Biocatalysis Introgression |
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