Identification and engineering of a cellobiose transporter KmStl1p to enhance cellobiose utilization in Kluyveromyces marxianus and Saccharomyces cerevisiae

Abstract

The yeast Kluyveromyces marxianus, a yeast known for its ability to ferment ethanol at high temperatures, can utilize various sugars including cellobiose, lactose and xylose. This study focused on improving cellobiose utilization by identifying and engineering a cellobiose transporter in K. marxianus. To assess cellobiose utilization capabilities, K. marxianus strains were grown in a cellobiose medium. The strains showed various growth levels, for example, the NCYC2791 strain grew well, while the DMKU3-1042 strain did not. This difference provided a basis for identifying a cellobiose transporter. Thirteen transporter candidate genes from the NCYC2791 genome were expressed in DMKU3-1042. As a result, KmSTL1 overexpression enhanced cell growth in a cellobiose medium. In addition, its disruption in NCYC2791 caused growth defects. To confirm its function, KmSTL1 was co-expressed with a β-glucosidase gene in Saccharomyces cerevisiae EBY.VW1000, which only uptake maltose. This engineered strain grew in cellobiose medium, indicating that KmSTL1 encodes a cellobiose transporter. Expression of GFP-fused KmStl1p in K. marxianus revealed that KmStl1p localized on cell membrane under cellobiose conditions, but was degraded in glucose conditions, suggesting that the transporter is regulated by available sugars. By individually disrupting seven α-arrestin genes in K. marxianus, KmRog3p was identified as a major ubiquitination mediator for KmStl1p degradation. Deletion analysis of KmStl1p revealed that its C-terminus is crucial for recognition by KmRog3p. Furthermore, expressing KmStl1p C-terminus mutants enhanced cellobiose assimilation in both K. marxianus and S. cerevisiae. These findings demonstrate that engineering KmStl1p is an effective strategy to improve cellobiose utilization in yeasts.

Keywords: C-terminal engineering; Cellobiose; Degradation; Kluyveromyces marxianus; KmStl1p; Membrane localization; Saccharomyces cerevisiae; Transporter.