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Background  To effectively convert lignocellulosic feedstocks to bio-ethanol anaerobic growth on xylose constitutes an essential trait that  Saccharomyces cerevisiae  strains normally do not adopt through the selective integration of a xylose assimilation route as the rate of ATP-formation is below energy requirements for cell maintenance ( m  ATP ). To enable cell growth extensive evolutionary and/or elaborate rational engineering is required. However the number of available strains meeting demands for process integration are limited. In this work evolutionary engineering in just two stages coupled to strain selection under strict anaerobic conditions was carried out with BP10001 as progenitor. BP10001 is an efficient ( Y  ethanol  = 0.35 g/g) but slow ( q  ethanol  = 0.05 ± 0.01 g/g BM /h) xylose-metabolizing recombinant strain of  Saccharomyces cerevisiae  that expresses an optimized yeast-type xylose assimilation pathway.    Results  BP10001 was adapted in 5 generations to anaerobic growth on xylose by prolonged incubation for 91 days in sealed flasks. Resultant strain IBB10A02 displayed a specific growth rate  μ  of 0.025 ± 0.002 h -1  but produced large amounts of glycerol and xylitol. In addition growth was strongly impaired at pH below 6.0 and in the presence of weak acids. Using sequential batch selection and IBB10A02 as basis, IBB10B05 was evolved (56 generations). IBB10B05 was capable of fast ( μ  = 0.056 ± 0.003 h -1 ;  q  ethanol  = 0.28 ± 0.04 g/g BM /h), efficient ( Y  ethanol  = 0.35 ± 0.02 g/g), robust and balanced fermentation of xylose. Importantly, IBB10A02 and IBB10B05 displayed a stable phenotype. Unlike BP10001 both strains displayed an unprecedented biphasic formation of glycerol and xylitol along the fermentation time. Transition from a glycerol- to a xylitol-dominated growth phase, probably controlled by CO 2 /HCO 3  - , was accompanied by a 2.3-fold increase of  m  ATP  while  Y  ATP  (= 87 ± 7 mmol ATP /g BM ) remained unaffected. As long as glycerol constituted the main by-product energetics of anaerobic growth on xylose and glucose were almost identical.    Conclusions  In just 61 generation IBB10B05, displaying ~530% improved strain fitness, was evolved from BP10001. Its excellent xylose fermentation properties under industrial relevant conditions were proven and rendered it competitive. Based on detailed analysis of growth energetics we showed that  m  ATP  was predominantly determined by the type of polyol formed rather than, as previously assumed, substrate-specific.

Stepwise metabolic adaption from pure metabolization to balanced anaerobic growth on xylose explored for recombinant Saccharomyces cerevisiae