Enhancing xylose fermentation capacity of engineered Saccharomyces cerevisiae by multi-step evolutionary engineering in inhibitor-rich lignocellulose hydrolysate.

Major progress in developing Saccharomyces cerevisiae strains that utilize the pentose sugar xylose has been achieved. However, the high inhibitor content of lignocellulose hydrolysates still hinders efficient xylose fermentation, which remains a major obstacle for commercially viable second-generation bioethanol production. Further improvement of xylose utilization in inhibitor-rich lignocellulose hydrolysates remains highly challenging. In this work, we have developed a robust industrial S. cerevisiae strain able to efficiently ferment xylose in concentrated undetoxified lignocellulose hydrolysates. This was accomplished with novel multi-step evolutionary engineering. First, a tetraploid strain was generated and evolved in xylose-enriched pretreated spruce biomass. The best evolved strain was sporulated to obtain a genetically diverse diploid population. The diploid strains were then screened in industrially relevant conditions. The best performing strain, MDS130, showed superior fermentation performance in three different lignocellulose hydrolysates. In concentrated corncob hydrolysate, with initial cell density of 1 g DW/L, at 35°C, MDS130 completely co-consumed glucose and xylose, producing ± 7% v/v ethanol with a yield of 91% of the maximum theoretical value and an overall productivity of 1.22 g/L/h. MDS130 has been developed from previous industrial yeast strains without applying external mutagenesis, minimizing the risk of negative side-effects on other commercially important properties and maximizing its potential for industrial application.