Accelerated proliferation of Kluyveromyces marxianus on glucose and xylose: Insights from adaptive laboratory evolution and MAPK/cell cycle genetic variations

Achieving cost-effective production of lignocellulosic biochemicals hinges on yeast's ability to utilize xylose efficiently. While Kluyveromyces marxianus is a natural xylose-fermenting yeast, its fermentation efficiency remains inadequate, particularly in conversion yield. This study aims to increase the efficiency of xylose utilization by employing adaptive laboratory evolution (ALE) to generate robust strains for single-cell proteins. After 20 batches of serial passaging culture, we successfully obtained an evolved strain, K. marxianus ZHY-3. ZHY-3 was cultured in a synthetic medium with xylose as the only carbon source in shake flasks. Compared with the wild-type ZHY-1, the specific growth rate increased by 3.5-fold, and the xylose yield coefficient increased by 78.6%. In addition, the specific growth rate of ZHY-3 in the glucose synthesis medium has also increased. In order to clarify its potential mechanism, we conducted a whole-genome analysis, and the results showed that in ZHY-3, phenotype-related single nucleotide polymorphisms (SNP) and insertion/deletion variations (InDel) exist in the mitogen-activated protein kinase (MAPK) signaling pathway and cell cycle process regulation related genes. These findings provide a potential strategic target for future metabolic engineering and show that ZHY-3 is a new candidate strain capable of utilizing lignocellulose biomass. • ALE significantly improved K. marxianus growth on glucose and xylose. • Specific growth rate on xylose increased by 3.5-fold after 20 passages. • Evolved strain ZHY-3 showed 78.6% higher xylose yield that wild-type. • WGS identified key variations in MAPK and cell cycle regulation pathways. • Potential attenuation of Hog1-mediated arrest may promote cell proliferation.