Industrial fermentation processes strive for high robustness to ensure optimal and consistent performance. Medium components, fermentation products, and physical perturbations may cause stress and lower performance. Cellular stress elicits a range of responses, whose extracellular manifestations have been extensively studied; whereas intracellular aspects remain poorly known due to lack of tools for real-time monitoring. Genetically encoded biosensors have emerged as promising tools and have been used to improve microbial productivity and tolerance toward industrially relevant stresses. Here, fluorescent biosensors able to sense the yeast intracellular environment (pH, ATP levels, oxidative stress, glycolytic flux, and ribosome production) were implemented into a versatile and easy-to-use toolbox. Marker-free and efficient genome integration at a conserved site on chromosome X of Saccharomyces cerevisiae strains and a commercial Saccharomyces boulardii strain was developed. Moreover, multiple biosensors were used to simultaneously monitor different intracellular parameters in a single cell. Even when combined together, the biosensors did not significantly affect key physiological parameters, such as specific growth rate and product yields. Activation and response of each biosensor and their interconnection were assessed using an advanced micro-cultivation system. Finally, the toolbox was used to screen cell behavior in a synthetic lignocellulosic hydrolysate that mimicked harsh industrial substrates, revealing differences in the oxidative stress response between laboratory (CEN.PK113-7D) and industrial (Ethanol Red) S. cerevisiae strains. In summary, the toolbox will allow both the exploration of yeast diversity and physiological responses in natural and complex industrial conditions, as well as the possibility to monitor production processes.
In this study, the three-step build-transform-assess
toolbox for
real-time monitoring of the yeast intracellular environment has been
expanded and upgraded to the two-module ScEnSor (S. cerevisiae Engineering + Biosensor) Kit. The Biosensor
Module includes eight fluorescent reporters for the intracellular
environment; three of them (unfolded protein response, pyruvate metabolism,
and ethanol consumption) were newly implemented to complement the
original five. The Genome-Integration Module comprises a set of backbone
plasmids for the assembly of 1–6 transcriptional units (each
consisting of promoter, coding sequence, and terminator) for efficient
marker-free single-locus genome integration (in HO and/or X2 loci).
Altogether, the ScEnSor Kit enables rapid and easy
construction of strains with new transcriptional units as well as
high-throughput investigation of the yeast intracellular environment.
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