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

Our research or main research focuses on microbial diversity, phylogenomics, and fermentation applications, emphasizing in yeast diversity, adaptations, and specifically in industrial application of yeast biology. Specifically going to that applications, we study fermentation efficiency, flavor production, and stress tolerance in yeast. So, our most recent advances are related to yeast phylogenomics, particularly from Patagonia.

Then we use this to generate novel lager yeast for beer fermentation, but we're also using novel yeast for low alcohol beer. Altogether we use different omic techniques and experimental evolution to develop this novel yeast for biogenological applications. We are currently using different techniques like genome sequencing, long-read genome sequencing, and also transcriptomics.

Then altogether, we use this information to make some experimental evolution of the different strains that we have, and then we try to validate some of the genomic changes using CRISPR techniques or different yeast transformation techniques, and in that way we get into the molecular details of yeast adaptation to fermentative environments. So, we have two big challenges. One is to generate novel lager yeast with efficient beer fermentation, and another one is all of the opposite.

We want to obtain novel strains, particularly from Patagonia, that are able to generate low alcohol beer. I would highlight two main results from our lab. One of them is that we identify an out of Patagonia origin of the mother of the lager yeast, Saccharomyces eubayanus.

And now we have also generated dozens of novel lager hybrids for beer fermentation. I think those are the two main findings of our research. To begin, pick a sample from a transformed yeast strain patch and inoculate it into a yeast peptone medium containing 5%glucose and 200 micromolar hygromycin.

Incubate the culture at 25 degrees Celsius without shaking for 24 hours. Refresh the cultures in fresh medium at a 1 to 10 dilution before incubating for another 24 hours. Wash the cells with sugar-free yeast peptone medium, then inoculate them into the test media at a 1 to 10 dilution.

Supplement the test media with luciferin to a final concentration of 3 millimolar, and with 200 micromolar hygromycin to maintain plasmid stability. For mixed sugar growth testing, use a glucose-maltose matrix with increasing glucose and maltose concentrations in yeast peptone medium. Dispense 200 microliters of culture into each well of a 96 well plate.

Now use a luminometer plate reader to measure luminescence and optical density at 620 nanometers every 30 minutes for 72 hours. Set the integration time to one second and disable attenuation to ensure continuous reporter activity and cell density monitoring. For fermentation sampling and luminescence monitoring, first dissolve malt extract in water to prepare malt extract medium.

Pre-culture transformed yeast strains in 5 milliliters of YPD medium at 20 degrees Celsius with agitation at 200 RPM for 24 hours. Transfer the pre-culture to 50 milliliters of malt extract medium, then incubate at 20 degrees Celsius with agitation at 200 rotations per minute for another 24 hours. Centrifuge the culture at 21, 380 G for one minute at room temperature to harvest the cells.

Then resuspend the cells in 12 degrees plate of malt extract medium in triplicates for 50 milliliter microfermentations. Supplement the medium with 0.3 milligrams per liter zinc chloride to enhance fermentation performance. Calculate the inoculum volume to ensure consistent cell densities across replicates.

Inoculate the prepared medium with the calculated amount of inoculum. Then place the cultures at 20 degrees Celsius without shaking for 14 days. Monitor fermentation progress by recording the carbon dioxide lost daily.

Weigh the vessels each day to measure cumulative weight loss as an indicator of carbon dioxide production. For luminescence monitoring, periodically sample 200 microliters from each microfermentation. Add luciferin to achieve a final concentration of 3 millimolar.

Measure luminescence and optical density at 620 nanometers using a luminometer plate reader without attenuation and one second of integration time. The the episomal reporter plasmid pRS426-PMAL32-LUC-HphMX was successfully constructed with the PMAL32 promoter luciferase ORF, and hphMX cassette. Differential luciferase activity was observed among the three transformed strains under varying glucose and maltose concentrations.

CBS12357T and CL467.1 showed activation without glucose while QC18 required above 1%glucose concentration for activation. Under microfermentation conditions, all three transformed strains demonstrated strong luminescence peaking at different times. Luminescence was absent in wild type strains.