Improvement of 2-phenylethanol production in Saccharomyces cerevisiae by evolutionary and rational metabolic engineering

2-Phenylethanol (2-PE) is a valuable aromatic compound with favorable flavors and good properties, resulting in its widespread application in the cosmetic, food and medical industries. In this study, a mutant strain, AD032, was first obtained by adaptive evolution under 2-PE stress. Then, a fusion protein from the Ehrlich pathway, composed of tyrB from Escherichia coli, kdcA from Lactococcus lactis and ADH2 from Saccharomyces cerevisiae, was constructed and expressed. As a result, 3.14 g/L 2-PE was achieved using L-phenylalanine as a precursor. To further increase 2-PE production, L-glutamate oxidase from Streptomyces overexpression was applied for the first time in our research to improve the supply of α-ketoglutarate in the transamination of 2-PE synthesis. Furthermore, we found that the disruption of the pyruvate decarboxylase encoding gene PDC5 caused an increase in 2-PE production, which has not yet been reported. Finally, assembly of the efficient metabolic modules and process optimization resulted in the strain RM27, which reached 4.02 g/L 2-PE production from 6.7 g/L L-phenylalanine without in situ product recovery. The strain RM27 produced 2-PE (0.8 mol/mol) with L-phenylalanine as a precursor, which was considerably high, and displayed manufacturing potential regarding food safety and process simplification aspects. This study suggests that innovative strategies regarding metabolic modularization provide improved prospects for 2-PE production in food exploitation.

An Electrochemical Evaluation of Novel Ferrocene Derivatives for Glutamate and Liver Biomarker Biosensing

Here, we present an evaluation of two new monosubstituted ferrocene (Fc) derivatives, 3-(1H-pyrrol-1-yl)propanamidoferrocene and 1-hydroxy-2-[2-(thiophen-3-yl)-ethylamino]ethylferrocene, as glutamate oxidase mediators, together with their preparation and characterisation. Taking into consideration the influence of the electronic effects of substituents on the redox potentials of the Fc species, two candidates with pyrrole or thiophene moieties were proposed for investigation. Film studies involved potential sweeping in the presence of pyrrole or 3,4-ethylenedioxythiophene monomers resulting in stable electroactive films with % signal loss upon cycling ranging from 1 to 7.82% and surface coverage (Γ) 0.47–1.15 × 10−9 mol/cm2 for films formed under optimal conditions. Construction of a glutamate oxidase modified electrode resulted in second-generation biosensing with the aid of both cyclic voltammetry and hydrodynamic amperometry, resulting in glutamate sensitivity of 0.86–1.28 μA/mM and Km (app) values over the range 3.67–5.01 mM. A follow-up enzyme assay for liver biomarker γ-glutamyl transpeptidase realised unmediated and mediated measurement establishing reaction and incubation time investigations and a realising response over <100 U/L γ-glutamyl transpeptidase with a sensitivity of 5 nA/UL−1.

A Highly Sensitive Amperometric Glutamate Oxidase Microbiosensor Based on a Reduced Graphene Oxide/Prussian Blue Nanocube/Gold Nanoparticle Composite Film-Modified Pt Electrode

A simple method that relies only on an electrochemical workstation has been investigated to fabricate a highly sensitive glutamate microbiosensor for potential neuroscience applications. In this study, in order to develop the highly sensitive glutamate electrode, a 100 µm platinum wire was modified by the electrochemical deposition of gold nanoparticles, Prussian blue nanocubes, and reduced graphene oxide sheets, which increased the electroactive surface area; and the chitosan layer, which provided a suitable environment to bond the glutamate oxidase. The optimization of the fabrication procedure and analytical conditions is described. The modified electrode was characterized using field emission scanning electron microscopy, impedance spectroscopy, and cyclic voltammetry. The results exhibited its excellent sensitivity for glutamate detection (LOD = 41.33 nM), adequate linearity (50 nM–40 µM), ascendant reproducibility (RSD = 4.44%), and prolonged stability (more than 30 repetitive potential sweeps, two-week lifespan). Because of the important role of glutamate in neurotransmission and brain function, this small-dimension, high-sensitivity glutamate electrode is a promising tool in neuroscience research.