Simultaneously improving xylose fermentation and tolerance to lignocellulosic inhibitors through evolutionary engineering of recombinant Saccharomyces cerevisiae harbouring xylose isomerase

BACKGROUND Yeasts tolerant to toxic inhibitors from steam-pretreated lignocellulose with xylose co-fermentation capability represent an appealing approach for 2nd generation ethanol production. Whereas rational engineering, mutagenesis and evolutionary engineering are established techniques for either improved xylose utilisation or enhancing yeast tolerance, this report focuses on the simultaneous enhancement of these attributes through mutagenesis and evolutionary engineering of Saccharomyces cerevisiae harbouring xylose isomerase in anoxic chemostat culture using non-detoxified pretreatment liquor from triticale straw. RESULTS Following ethyl methanesulfonate (EMS) mutagenesis, Saccharomyces cerevisiae strain D5A⁺ (ATCC 200062 strain platform), harbouring the xylose isomerase (XI) gene for pentose co-fermentation was grown in anoxic chemostat culture for 100 generations at a dilution rate of 0.10 h⁻¹ in a medium consisting of 60% (v/v) non-detoxified hydrolysate liquor from steam-pretreated triticale straw, supplemented with 20 g/L xylose as carbon source. In semi-aerobic batch cultures in the same medium, the isolated strain D5A(+H) exhibited a slightly lower maximum specific growth rate (μ(max) = 0.12 ± 0.01 h⁻¹) than strain TMB3400, with no ethanol production observed by the latter strain. Strain D5A(+H) also exhibited a shorter lag phase (4 h vs. 30 h) and complete removal of HMF, furfural and acetic acid from the fermentation broth within 24 h, reaching an ethanol concentration of 1.54 g/L at a yield (Y(p/s)) of 0.06 g/g xylose and a specific productivity of 2.08 g/gh. Evolutionary engineering profoundly affected the yeast metabolism, given that parental strain D5A+ exhibited an oxidative metabolism on xylose prior to strain development. CONCLUSIONS Physiological adaptations confirm improvements in the resistance to and conversion of inhibitors from pretreatment liquor with simultaneous enhancement of xylose to ethanol fermentation. These data support the sequential application of random mutagenesis followed by continuous culture under simultaneous selective pressure from inhibitors and xylose as primary carbon source.