טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentAchmon Yigal
SubjectMolecular and Process Engineering Approaches for
Biotechnological Production of 2-phenylethanol
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Professor Fishman Ayelet
Full Thesis textFull thesis text - English Version


Abstract

The overall goal of this research was to develop new and efficient biotechnological processes for the production of 2-phenylethanol (2-PE), a rose-like fragrance, from L-phenylalanine (L-phe). 

Three different parallel directions were examined in this research:

A process engineering approach: This work describes the use of hydrophobic polymethylmethacrylate (PMMA) microspheres for continuous removal of 2-PE from the fermentation medium by a mechanism of swelling. In shake flask experiments with conditions simulating 2-PE stress, a 10-fold increase in productivity was measured for systems containing 20% (w/v) microspheres. A 1 L fed-batch fermentation with 8% (w/v) of PMMA microspheres resulted in a total 2-PE concentration of 7.05 g/L, from which 5.40 g/L were incorporated inside the resin, implying 76% encapsulation. This ratio of 0.07 g/g of product per resin is among the highest reported to date.

A classical genetic approach: We used classical breeding in order to generate a Saccharomyces cerevisiae strain characterized by high 2-PE tolerance and with enhanced production capabilities. The genetic improvement program was carried by using a novel method called EZSB (Ether Zymolyase Sexual Breeding) toward 2-PE resistance, and screening of 100 random isolates for 2-PE production after 7 and 10 breeding generation. 2-PE productivity increased in fed-batch fermentations from 2.5 to 4.3 g/L in the 7th generation, and further to 5.6 g/L in the 10th generation. This is the most robust yeast strain reported to date.

A molecular engineering approach: In the present work, a novel metabolic pathway has been designed in E. coli to produce 2-PE, using the Rosa hybrida phenylacetaldehyde synthase (PAAS), a pyridoxal 5 ′-phosphate (PLP)-dependent enzyme capable of transforming L-phe into phenylacetaldehyde by decarboxylation and oxidation. To overcome the plant derived enzyme’s insolubility in E. coli, several plasmids and host strains were tested for their expression ability. The desired results were obtained by using the pTYB21 plasmid containing the intein tag from the Saccharomyces cerevisiae VMA1. It was discovered that the whole cell catalyst expressing intein-PAAS activity, is temperature dependent, working well in the range of 25 °C to 30 °C. When the external PLP cofactor was added, the cells produced 0.39 g/L 2-PE directly from L-phe. A biotransformation that was based only on internal PLP synthesis produced 0.34 g/L 2-PE, thus creating for the first time an E. coli strain that can produce 2-PE from L-phe without the need for an exterior cofactor.