טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentNetta Hirshberg
SubjectUtilization of Saccharomyces cerevisiae for production of
Valuable Chemicals
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Professor Fishman Ayelet
Full Thesis textFull thesis text - English Version


Abstract

Biocatalysis is the execution of chemical reactions using biological systems. Having a broad substrate acceptance and being inexpensive, readily available, fast growing and easy to use, places Saccharomyces cerevisiae among the most popular biocatalysts. This work describes two model reactions targeted for the pharmaceutical and food industries, catalyzed by S. cerevisiae whole-cells or enzymes.

(S)-4-chloro-3-hydroxybutanoic acid ethyl ester ((S)-CHB) is an important chiral building block used in the synthesis of chiral pharmaceuticals which can be produced by S. cerevisiae. However, previous studies using non-modified S. cerevisiae for (S)-CHB production reported a non-satisfactory enantiomeric excess (ee) of 92%. Attempts for overcoming this drawback have shown only partial success or suffer from high complexity.

The objective of this part was to use classic genetic introgression for combining complex multi-component traits from two parents in a single strain. Lately, natural yeast strains, isolated from Mount Carmel were characterized as resistant to environmental stress. Nevertheless, these strains showed relatively low ee values, while a laboratory strain, Y103, exhibited both the highest specific activity (1.88 µmol/min/mg protein) and the highest enantioselectivity of (98%). The enantioselective lab strain was crossed with Ye-519, a multi-stress resistant isolate (93%) to obtain, after selection for both characteristics, a haploid offspring of backcross-1, C242, exhibiting both multi-stress resistance and high enantioselectivity (98%). 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%. Unlike its original parental strains, the new hybrid strain maintained both parameters constant.

2-phenylethanol (PEA) is an aromatic alcohol with a rose-like odor widely used in the fragrance and food industries. Although the vast majority of this commercial compound is produced chemically, increase in consumers' preference for natural flavors, and economical considerations, motivate the design of high PEA-producing microorganisms. S. cerevisiae naturally transforms phenylalanine (L-Phe), to PEA via the "Ehrlich Pathway". However, competitive pathways, and the inhibitory effect of PEA on cell growth, limit the productivity of the bioprocess. Attempts for improving the production yields focused mainly on in-situ product removal (ISPR). The aim of this part was to clone and overexpress in E. coli the three genes responsible for converting L-Phe to PEA as an alternative approach. Cloning of Aro8, Aro10 and Sfa1 separately in E. coli resulted in overexpression of each gene, and a 5-fold, 2.5-fold, and 4-fold increase in initial activity rate of the respective enzymes. Future cloning of those genes as a cluster will form a clone, solely dedicated to PEA production and free of the S. cerevisiae limitations.
The aromatic aminotransferase from S. cerevisiae is expected to have a broad substrate acceptance and therefore may be suitable for industrial purposes. This enzyme was successfully purified by an affinity column and is in the process of structural and biochemical characterization.

In summary, two genetic approaches were used in this work for improving the S. cerevisiae performance as an industrially oriented biocatalyst. The classic genetic introgression technique allowed the combination of two complex traits (enantioselectivity and stress- tolerance) affected by multiple components, while modern genetic engineering showed promising results for the improvement of a well known and defined metabolic pathway.