Massive gene duplication on demand accelerates cellobiose utilization in engineered Saccharomyces cerevisiae

The efficient utilization of sugars from cellulosic biomass is essential for the cost effective production of value-added chemicals using microorganisms. Although Saccharomyces cerevisiae is a robust microbial platform widely used in large-scale fermentation for ethanol production from sugar cane and corn starch, as well as in many other industrial processes for the production of value-added chemicals, glucose repression is a major barrier to its efficient fermentation of sugar mixtures. In our previous study, we demonstrated that engineered yeasts co-fermenting xylose and cellobiose, a dimer of glucose, could overcome the bottleneck. To improve ethanol production, we constructed an efficient cellobiose fermentation pathway in S. cerevisiae by integration of genes for cellobiose assimilation (cdt-1 and gh1-1) in the genome and used evolutionary engineering on the engineered strain, using cellobiose as the sole carbon source. At first, the engineered strain showed low ethanol yield and productivity, but the results increased significantly after a series of subcultures. We identified the genetic changes responsible for the improved phenotype: the evolved strain exhibiting the highest cellobiose fermentation rate contained much higher copy numbers of cdt-1 and gh1-1 (9 and 23) with optimized copy ratios of cdt-1 and gh1-1 in the genome when compared to those of the parental strain (1 and 2, respectively). These results indicate that the chromosome rearrangements by tandem repeats of essential genes achieved efficient utilization of cellobiose under the selective pressure. This study presents that flexible gene amplification on demand in yeast might be an efficient strategy for metabolic engineering.