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Background  Expression of  d -xylose isomerase having high catalytic activity in  Saccharomyces cerevisiae  ( S. cerevisiae ) is a prerequisite for efficient and economical production of bioethanol from cellulosic biomass. Although previous studies demonstrated functional expression of several xylose isomerases (XI) in  S. cerevisiae , identification of XIs having higher catalytic activity is needed. Here, we report a new strategy to improve xylose fermentation in the  S. cerevisiae  strain IR-2 that involves an evolutionary engineering to select top-performing XIs from eight previously reported XIs derived from various species.    Results  Eight XI genes shown to have good expression in  S. cerevisiae  were introduced into the strain IR-2 having a deletion of  GRE3  and  XKS1  overexpression that allows use of  d -xylose as a carbon source. Each transformant was evaluated under aerobic and micro-aerobic culture conditions. The strain expressing XI from  Lachnoclostridium phytofermentans  ISDg ( Lp XI) had the highest  d -xylose consumption rate after 72 h of micro-aerobic fermentation on  d -glucose and  d -xylose mixed medium. To enhance  Lp XI catalytic activity, we performed random mutagenesis using error-prone polymerase chain reaction (PCR), which yielded two  Lp XI candidates, SS82 and SS92, that showed markedly improved fermentation performance. The  Lp XI genes in these clones carried either T63I or V162A/N303T point mutations. The SS120 strain expressing  Lp XI with the double mutation of T63I/V162A assimilated nearly 85 g/L  d -glucose and 35 g/L  d -xylose to produce 53.3 g/L ethanol in 72 h with an ethanol yield of approximately 0.44 (g/g-input sugars). An in vitro enzyme assay showed that, compared to wild-type, the  Lp XI double mutant in SS120 had a considerably higher  V  max  (0.107 µmol/mg protein/min) and lower  K  m  (37.1 mM).    Conclusions  This study demonstrated that  Lp XI has the highest  d -xylose consumption rate among the XIs expressed in IR-2 under micro-aerobic co-fermentation conditions. A combination of novel mutations (T63I and V162A) significantly improved the enzymatic activity of  Lp XI, indicating that  Lp XI- T63I/V162A  would be a potential construct for highly efficient production of cellulosic ethanol.    Electronic supplementary material  The online version of this article (10.1186/s13068-019-1474-z) contains supplementary material, which is available to authorized users.

biotechnology for biofuels

Molecular evolutionary engineering of xylose isomerase to improve its catalytic activity and performance of micro-aerobic glucose/xylose co-fermentation in Saccharomyces cerevisiae