Characterization of a Fermentation-Deficient Yeast Mutant

Abstract

Kluyveromyces lactis is a species of yeast isolated from dairy processes that is capable of both fermentation and respiration. The behavior of K. lactis during both respiration and fermentation is not well understood and requires further experimentation. This behavior indicates that K. lactis has the ability to determine its mode of energy production whether through respiration, fermentation, or a combination of both. K. lactis contains a single gene encoding for pyruvate decarboxylase, and a strain lacking this gene cannot perform ethanol fermentation. This strain can only respire, however despite being unable to ferment the mutant strain initially grows like the wild-type strain when it is in fermentative conditions and not like the wild-type strain growing in respiratory conditions. This causes an unusual metabolic phenomenon resulting in the export of pyruvate. The main objectives were characterizing metabolism by determining why it excretes the critical central metabolite pyruvate, and whether there are ways to 'fix' its metabolism to help it grow better or more like the wild type. By measuring growth curves of wild-type and mutant strains of K. lactis in various conditions, including a new pH indicator medium and varying 96-well plate volumes, we can better understand each strain’s metabolism. Interestingly, under fermentative conditions, the mutant initially mimics wild-type growth before entering a prolonged second growth phase, approximately 10 hours after the initial growth, accompanied by extracellular pyruvate accumulation and a drop in medium pH from neutral 7 to an acidic 4. Using growth assays and a pH indicator medium in varying oxygenation levels, we characterized the unique double phase growth of the mutant and the corresponding acidification of its environment. Additionally, through the growth assays, we characterized the efficiency of growth within both strains by calculations of their final optical densities at the varying oxygenation levels. Although pyruvate secretion was initially hypothesized to be the cause of acidification, data suggest alternative mechanisms such as proton export via PMA1, a protein that pumps protons out of the cell, causes the acidification. This work deepens our understanding of K. lactis metabolism and lays the groundwork for future genetic and biochemical strategies to restore balanced growth in pdc1Δ strains by investigating the JEN1 gene. Further testing on genetic mutations highlighting JEN1, a known pyruvate/lactate transporter, for their potential ability to improve the growth phenotype of the pdc1Δ strain