Genomic and metabolic instability during long-term fermentation of an industrial Saccharomyces cerevisiae strain engineered for C5 sugar utilization.

Abstract

The production of bio-based compounds can be impacted by stochastic cellular mechanisms acting at both the genetic and non-genetic levels, leading to instabilities in the performance of the producer organisms. Here, we investigated the long-term genomic and phenotypic stability of an ethanol-producing Saccharomyces cerevisiae strain engineered by chromosomal integration of transgenes allowing xylose and arabinose utilization. First, along a series of batch cultures in bioreactor to reach around 100 generations, we observed that, after a period of phenotypic stability, the strain exhibited instabilities in terms of xylose and arabinose consumption. Long-read sequencing did not reveal major genomic modifications at the population level that could explain such fluctuations. However, we isolated clones that have partly or fully lost the ability to use arabinose due to copy number variations of the integrated transgenes, most probably arising from homologous recombination (HR) events. Based on the cultivation of subpopulations sorted depending on their expression level of RAD52, a gene whose expression is known to be proportional to the recombination rate, we did not detect different genomic and phenotypic stability in the subpopulations. Thus, this work reveals both phenotypic and genomic variations in an industrial yeast strain that could in the long-term lead to the loss of its production performance.