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.

In Situ Product Recovery of Bio-Based Industrial Platform Chemicals: A Guideline to Solvent Selection

In situ product recovery (ISPR), in the form of an extractive fermentation process, can increase productivity and product titers in the sustainable production of platform chemicals. To establish a guideline for the development of industrially relevant production processes for such bio-based compounds, a wide screening was performed, mapping the potential of an extensive range of solvents and solvent mixtures. Besides solvent biocompatibility with Saccharomyces cerevisiae, distribution coefficients of three organic acids (protocatechuic acid, adipic acid and para-aminobenzoic acid) and four fragrance compounds (2-phenylethanol, geraniol, trans-cinnamaldehyde and β-ionone) were determined. While for highly hydrophobic fragrance compounds, multiple pure solvents were identified that were able to extract more than 98%, reactive extraction mixtures were proven effective for more challenging compounds including organic acids and hydrophilic alcohols. For example, a reactive mixture consisting of 12.5% of the extractant CYTOP 503 in canola oil was found to be biocompatible and showed superior extraction efficiency for the challenging compounds as compared to any biocompatible single solvent. This mapping of biocompatible solvents and solvent mixtures for the extraction of various classes of industrial platform chemicals can be a tremendous step forward in the development of extractive fermentations.

Enhanced 2-phenylethanol production by newly isolated Meyerozyma sp. strain YLG18 and characterization of its synthetic pathways

Background2-Phenylethanol (2-PE) is an aromatic alcohol which has been widely used in cosmetics, perfume and food industries owning to its delicate rose scent. The newly isolated yeast Meyerozyma guilliermondii YLG18 was able to tolerate high exogenous 2-PE and produce 2-PE with two different pathways.ResultsA unique Meyerozyma sp. strain YLG18 was obtained in this study, which was capable of tolerating 4.0 g/L exogenous 2-PE. Response surface methodology (RSM) was implemented to improve the maximum 2-PE production. At optimized conditions: temperature, 24.7℃; initial glucose, 63.54 g/L; initial L-phe, 10.70 g/L, 2-PE production was increased to 2.55 g/L, leading to 104% increase compared to the pre-optimized one. In situ product recovery (ISPR) could further help improve the final 2-PE production to 3.20 g/L with fatty acid methyl ester as the extractant, representing the highest 2-PE production by using Meyerozyma sp.. Furthermore, genes involved in 2-PE synthesis were identified and their expression levels between Shikimate pathway and Ehrlich pathway were compared. Based on the genomic and transcriptional analysis, a penta-functional enzyme AroM in Shikimate pathway and an aspartate aminotransferase (AAT) with the potential to convert phenylalanine into phenylpyruvate in Ehrlich pathway were identified.ConclusionsThese findings would help broaden our knowledge and add to the pool of known 2-PE generating microbes and genes. Moreover, this study describes a potential, new 2-PE producer that lays foundation for the industrial-scale production of 2-PE and its derivatives in the future. Key words: 2-phenylethanol; Meyerozyma guilliermondii; RSM; ISPR; gene analysis