Production of Natural 2-Phenylethanol from Glucose or Glycerol with CoupledEscherichia coliStrains Expressingl-Phenylalanine Biosynthesis Pathway and Artificial Biocascades

Abstract

2-Phenylethanol (2-PE) is a fragrance widely used in food and cosmetics. Natural 2-PE is preferred in these applications but with high price and very limited availability. Microbial synthesis from glucose or glycerol is an attractive way to produce natural 2-PE, but suffers from low product titer. Escherichia coli NST74-Phe-Sty was engineered to coexpress the L-phenylalanine (L-Phe) biosynthesis pathway and enzyme cascade of L-Phe to 2-PE, producing only 0−0.17 g/L 2-PE from glucose at 22−37 °C due to the incompatibility of the temperature for enzyme expression and activity. Enhanced production of 2-PE (8.4−9.1 g/L) from glucose or glycerol was achieved by coupling of E. coli NST74-Phe expressing the L-Phe biosynthesis pathway for L-Phe production at the optimal temperature of 37 °C with E. coli T7-Sty expressing enzyme cascades of L-Phe to 2-PE at the optimal expression temperature of 22 °C and the optimal biotransformation temperature of 30 °C. The 2-PE titer is 4.7-fold higher than the best reported 2-PE concentration produced from glucose. The coupled strains approach could be generally applicable for enhancing microbial production of useful chemicals from sugars or glycerol.

Sensing, Uptake and Catabolism of L-Phenylalanine During 2-Phenylethanol Biosynthesis via the Ehrlich Pathway in Saccharomyces cerevisiae

“…Interestingly, hydrolysed corn stover or molasses can be used as a carbon source to produce 2-PE by nonconventional yeast or Bacillus licheniformis, with higher 2-PE concentrations of 3.67 g/L and 4.41 g/L, respectively (Mierzejewska et al, 2019;Zhan et al, 2020). To reduce the cost, in current studies, Escherichia coli has been developed for the biotransformation of L-Phe, glucose, or glycerol to 2-PE with resting cells, leading to a higher 2-PE concentration (9.1 g/L) from glucose or glycerol (Liu et al, 2018;Lukito et al, 2019;Sekar et al, 2019).…”

CRISPR-mediated multigene integration enables Shikimate pathway refactoring for enhanced 2-phenylethanol biosynthesis in Kluyveromyces marxianus

“…2-PE is a valuable compound for a variety of different applications including next-generation biofuels and flavor and fragrance additives for food, beverages, perfumes, and personal care products. As a potential alternative to chemical synthesis and isolation from natural sources, bioproduction from engineered yeast and bacteria has been investigated by a number of researchers [ 17 – 19 , 49 ]. One of these works was able to increase 2-PE production in K. marxianus to upward of 1.1 g/L by growth selection in the presence of a toxic l -phenylalanine paralog, p -fluorophenylalanine [ 18 ].…”

Fluorescent enzyme-coupled activity assay for phenylalanine ammonia-lyases

“…FDC1 from Saccharomyces cerevisiae has already been shown to transform a wide range of phenylacrylate analogues 42 , 43 supporting the applicability of the assay for activity screens towards various non-natural substrates of interest. Nonetheless, the sequential use of PAL and FDC1 reactions in genetically engineered whole cells was also demonstrated within the biosynthesis of styrene, 1,2-ethanediols or other valuable products 44 – 46 . The 1,3-dipolar cycloaddition reaction between an alkene and a tetrazole represents an attractive method of fluorophore-forming bioorthogonal chemistry, with various diaryltetrazoles shown to be highly sensitive fluoroprobes for the detection of alkenes 47 – 49 .…”