Influence of prefoldin subunit 4 on the tolerance of Kluyveromyces marxianus to lignocellulosic biomass-derived inhibitors.

ABSTRACT

BACKGROUND: Kluyveromyces marxianus is a potentially excellent host for microbial cell factories using lignocellulosic biomass, due to its thermotolerance, high growth rate, and wide substrate spectrum. However, its tolerance to inhibitors derived from lignocellulosic biomass pretreatment needs to be improved. The prefoldin complex assists the folding of cytoskeleton which relates to the stress tolerance, moreover, several subunits of prefoldin have been verified to be involved in gene expression regulation. With the presence of inhibitors, the expression of a gene coding the subunit 4 of prefoldin (KmPFD4), a possible transcription factor, was significantly changed. Therefore, KmPFD4 was selected to evaluate its functions in inhibitors tolerance. RESULTS: In this study, the disruption of the prefoldin subunit 4 gene (KmPFD4) led to increased concentration of intracellular reactive oxygen species (ROS) and disturbed the assembly of actin and tubulin in the presence of inhibitors, resulting in reduced inhibitor tolerance. Nuclear localization of KmPFD4 indicated that it could regulate gene expression. Transcriptomic analysis showed that upregulated gene expression related to ROS elimination, ATP production, and NAD+ synthesis, which is a response to the presence of inhibitors, disappeared in KmPFD4-disrupted cells. Thus, KmPFD4 impacts inhibitor tolerance by maintaining integration of the cytoskeleton and directly or indirectly affecting the expression of genes in response to inhibitors. Finally, overexpression of KmPFD4 enhanced ethanol fermentation with a 46.27% improvement in productivity in presence of the inhibitors. CONCLUSION: This study demonstrated that KmPFD4 plays a positive role in the inhibitor tolerance and can be applied for the development of inhibitor-tolerant platform strains.