Improvement of natural isolates of Saccharomyces cerevisiae strains for synthesis of a chiral building block using classic genetics.
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 H(2)O(2)) and thermal stress (44 degrees 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.
Nir
Netta
N
Department of Biotechnology and Food Engineering, Technion-Israel Institute of Technology, Haifa 32000, Israel.
Bahalul
Moran
M
Feingersch
Roi
R
Katz-Ezov
Tal
T
Kashi
Yechezkel
Y
Fishman
Ayelet
A
D000090:Acetoacetates / Q000378:metabolism; D002087:Butyrates / Q000378:metabolism; D002712:Chlorides / Q000494:pharmacology; D018869:Heat-Shock Response; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007218:Industrial Microbiology; D007557:Israel; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D012988:Soil Microbiology
D000090:Acetoacetates; D002087:Butyrates; D002712:Chlorides; C121244:ethyl 4-chloro-3-hydroxybutanoate; D006861:Hydrogen Peroxide; C024840:ethyl acetoacetate; C001599:chlorite