Improvement of natural isolates of Saccharomyces cerevisiae strains for synthesis of a chiral building block using classic genetics

• Published in: Applied microbiology and biotechnology Vol. 78; no. 4; pp. 659 - 667
• Main Authors: Nir, Netta, Bahalul, Moran, Feingersch, Roi, Katz-Ezov, Tal, Kashi, Yechezkel, Fishman, Ayelet
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Berlin/Heidelberg : Springer-Verlag 01.03.2008; Springer Berlin Heidelberg; Springer; Springer Nature B.V
• Subjects: 4-chloro-3-hydroxybutanoic acid ethyl ester; 4-chloro-acetoacetic acid ethyl ester; Acetoacetates - metabolism; Acids; Applied Genetics and Molecular Biotechnology; Backcross; Biocatalysis; Biocatalysts; Biological and medical sciences; Biosynthesis; Biotechnology; Butyrates - metabolism; Chlorides - pharmacology; Dehydrogenases; Enzymes; Fundamental and applied biological sciences. Psychology; Genetic engineering; Genetics; Genotype & phenotype; Glucose; Heat-Shock Response; Hydrogen peroxide; Hydrogen Peroxide - pharmacology; Industrial Microbiology; introgression; Israel; Laboratories; Life Sciences; Microbial Genetics and Genomics; Microbiology; National parks; Saccharomyces cerevisiae; Saccharomyces cerevisiae - drug effects; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - isolation & purification; Saccharomyces cerevisiae - metabolism; Soil Microbiology; stress tolerance; Studies; Yeast; Israel; 4-chloro-3-hydroxybutanoic acid ethyl ester; 4-chloro-acetoacetic acid ethyl ester; Biocatalysis; Introgression; Stress resistance; Backcross; Ascomycota; Fungi; Resistance; Yeast; Enantioselectivity; Genetic improvement; Chiral compound; Saccharomyces cerevisiae; Optimization; Stress; Biocatalyst
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-008-1344-2

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.