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:
1. Process Engineering: A new in situ product removal procedure was employed in Saccharomyces cerevisiae fermentation in order to avoid the 2-PE toxicity issue. Hydrophobic polymethylmethacrylate (PMMA) microspheres of narrow size distribution were used. Fermentation done with the addition of 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.
2. Natural selective breeding of S. cerevisiae: Using genetic breeding of S. cerevisiae strains, a high 2-PE-producing strain was isolated. The final 2-PE concentration obtained by the selected strain, following ten rounds of breeding, was 5.61 g/L in a 2-L fed-batch fermentation, the highest level reported to date by any microorganism.
3. Genetic engineering of Escherichia coli: A new metabolic pathway in E. coli has been designed to produce 2-PE, using the Rosa hybrid phenylacetaldehyde synthase (PAAS). The enzyme is capable of transforming L-phe into phenylacetaldehyde by decarboxylation and oxidation. The desired results were obtained by using the pTYB21 plasmid containing an intein tag. 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.
