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