Enhancing tolerance of Kluyveromyces marxianus to lignocellulose-derived inhibitors and its ethanol production from corn cob via overexpression of a nitroreductase gene

Despite its potential as a sustainable feedstock for producing renewable fuels and chemicals, lignocellulosic biomass (LCB) remains largely untapped due to problems associated with its deconstruction and the toxicity of its sugar-rich hydrolysates in microbial conversion processes. Using genetic modification to facilitate inhibitor tolerance, the present study aimed to construct a robust yeast cell factory for cellulosic ethanol bioproduction from non-detoxified LCB. To this effect, two putative nitroreductase genes (KmHBN1 and KmFRM2) hypothesized to be possible mediators of oxidative stress response were separately overexpressed and disrupted in Kluyveromyces marxianus. In contrast to KmFRM2 modified strains (disrupted or overexpressed) with no significant change, the inhibitor tolerance to phenols, furfural, and acetic acid cocktail was improved in YKmHBN1-URA3 strain overexpressing KmHBN1. Although the recombinant KmHBN1 and KmFRM2 had nitroreductase activity, they did not degrade or convert inhibitors. The change of intracellular reactive oxygen species level (ROS) of KmHBN1 disrupted or overexpressed strains indicated that KmHBN1 affected intracellular ROS elimination. Cells with disrupted KmHBN1 failed to respond to ROS created during inhibitor metabolism, and this disruption also led to reduced expression of respiratory chain genes which translates to a decreased inhibitor tolerance. The overexpression of KmHBN1 not only enhanced the ethanol production from glucose in the presence of inhibitors but also improved ethanol productivity (18.75%) from simultaneously-saccharified and co-fermented inhibitor-ladened corn cob. These findings unlock a new pathway for the creation of robust yeast strains to aid in the biorefinery conversion of lignocellulosic feedstock.