In Vivo Monitoring of Transcriptional Activity During Metabolic Transition Using a Bioluminescent Reporter in Yeast

Summary

This protocol employs a bioluminescent reporter, allowing measurements of transcriptional activity in Saccharomyces eubayanus to monitor the glucose-to-maltose transition, enabling real-time analysis of metabolic adaptations and supporting strain optimization for industrial fermentation under diverse conditions.

Abstract

Sequential sugar consumption, from a preferred sugar source to a less preferred one, represents a critical metabolic adaptation in yeast, which is particularly relevant for survival in fluctuating environments such as those found in beer fermentation. However, sugar transitions are an environmental variable that is challenging to predict and detect, impacting the outcome of beer fermentations. This protocol describes an in vivo system to monitor transcriptional activation associated with the glucose-to-maltose metabolic shift in Saccharomyces eubayanus that applies to different wild Saccharomyces yeast strains.

The system employs an episomal bioluminescent transcriptional reporter for maltose metabolism, focusing on MAL32, since it provides a good readout for metabolic shifts, as studied in S. cerevisiae. For this, yeast strains were transformed with plasmids containing the MAL32 regulatory region from S. eubayanus, controlling the expression of a gene encoding for a destabilized version of firefly luciferase¹, and a hygromycin resistance gene used exclusively during transformation to ensure plasmid acquisition. Following selection, transformed yeast cells can be cultured under non-selective conditions, as the episomal plasmid remains stable in culture conditions for up to 7 days.

This system was validated under a complex sugar environment in microfermentation assays, confirming the effectiveness of the luciferase reporter in informing metabolic transitions. Samples were collected regularly and analyzed with a luminometer, providing continuous insights into yeast responses. While broadly applicable, this protocol is particularly valuable for assessing yeast performance under fermentation conditions, where metabolic changes pose a significant challenge. Additionally, this methodology can be adapted by selecting alternative promoters to explore a broader range of responses to environmental changes, allowing characterization as well as optimization of wild yeast strains for diverse industrial applications.

Introduction

Microorganisms such as yeasts must constantly adapt to dynamic environmental conditions to maintain fitness and survive¹. These adaptations often involve complex gene regulatory circuits integrating multiple extracellular signals to orchestrate precise metabolic responses^2,3. In industrial settings, the efficiency of these metabolic transitions is critical, particularly in fermentation processes where disruptions can lead to suboptimal yields or incomplete fermentations³. A key metabolic challenge to overcome is when cells transition from a preferred to a seconda....

Protocol

1. Construction of episomal reporters

NOTE: We selected a reporter regulatory region based on yeast literature to construct the episomal plasmid for monitoring maltose consumption^6,11,12. The promoter of the candidate reporter gene was defined as the regulatory sequence immediately upstream from the candidate ORF up to the nucleotide flanking the adjacent upstream ORF. This region was amplified from the genomic DNA of S. eubayanus CBS12357^T reference strain¹⁰. This approach ensures the high....

Discussion

This study demonstrates the effectiveness of an episomal bioluminescent reporter for monitoring transcriptional activation in S. eubayanus under metabolic transitions. By employing MAL32 as a transcriptional reporter¹¹, we could track key metabolic transitions in real time, providing a robust framework for understanding strain-specific adaptations. This reporter, selected for their role in maltose metabolism, offers distinct advantages in evaluating metabolic flexibility in yeast.......

Materials

Name	Company	Catalog Number	Comments
Ampicillin, sodium salt	ThermoFisher Scientific	11593027
D-Glucose	Sigma-Aldrich	G8270
DpnI	New England Biolabs	R0176S
EcoRI	New England Biolabs	R0101S
Hygromycine B	Gold Biotechnology	H-270-1
L-Luciferine	Gold Biotechnology	L-127-10
Maltose monohydrate	Sigma-Aldrich	47288
Phusion Plus PCR Master Mix	ThermoFisher Scientific	F631S
Tecan Infinite 200 PRO M	Tecan
Wizard Plus SV Minipreps DNA Purirfication System	Promega	A1330
XhoI	New England Biolabs	R0146S
Zymoprep Yeast Plasmid Miniprep I	Zymo Research	D2001