High throughput investigation of vanillin toxicity to yeast using ambr15 microbioreactors and flow cytometry
July 14-18, 2019
Vanillin (4-hydroxy-3-methoxybenzaldehyde) is the main flavoring component of the vanilla bean and synthetic vanilla flavoring agent and biotechnological production in metabolically engineered yeast has been attempted. However, vanillin is also one of the most toxic of the aromatic aldehydes to yeast, which became known both through the metabolic engineering attempts and in its effects on yeast during cellulosic ethanol fermentation. Cellulosic sugars are derived from lignocellulosic biomass following pretreatment; this process generates a range of yeast toxicants and inhibitors of which vanillin is among the most potent. Several reports utilizing transcriptomics and gene knockouts have been undertaken in yeast to determine the mechanism(s) of toxicity of vanillin and these have concluded protein production effects or increased radical oxygen species (ROS) and among others. Here we investigated the mechanism and potency of vanillin toxicity to Saccharomyces cerevisiae yeast using a parallel microbioreactor system and by assessing cell physiology impacts via flow cytometry. An initial screen of vanillin (0.5-10 mM) toward S. cerevisiae BY4171 indicated potent inhibition of yeast growth at concentrations >2 mM. Control yeast strains (empty vector) or those expressing modified Turquoise Fluorescence Protein (mTFP), were used thereafter. Yeast innocula (OD600 ~0.4) were cultured in the Sartorius/TAP Biosystems ambr15 microbioreactor system, 11 ml final volume at 30°C for up to 99 h in the presence of vanillin at concentrations of 2, 4, 6, 8 and 10 mM in batch mode. Microbioreactor dissolved oxygen (DO) and stirring rates were maintained by the control system and automated sampling of the reactors was conducted at specified time intervals. Biomass production (OD600nm) and supernatant vanillin and vanillyl alcohol concentrations were determined. Single cell physiology indicators such as: membrane integrity, ROS generation and mitochondrial membrane potential were measured with the aid of specific fluorescent dyes and TFP levels were measured using a Beckman Coulter Cytoflex flow cytometer.
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Victoria Haritos, Nguyen Ho, Ankita Suri, and Huadong Peng, "High throughput investigation of vanillin toxicity to yeast using ambr15 microbioreactors and flow cytometry" in "Biochemical and Molecular Engineering XXI", Christina Chan, Michigan State University, USA Mattheos Koffas, RPI, USA Steffen Schaffer, Evonik Industries, Germany Rashmi Kshirsagar, Biogen, USA Eds, ECI Symposium Series, (2019). https://dc.engconfintl.org/biochem_xxi/40