Engineered Saccharomyces cerevisiae strain for improved xylose utilization with a three-plasmid SUMO yeast expression...

Area of Science:

• Biotechnology
• Synthetic Biology
• Molecular Biology

Background:

• Developing efficient protein production systems in Saccharomyces cerevisiae is crucial for various biotechnological applications.
• Simultaneous expression of multiple proteins can be challenging due to vector compatibility and selectable marker limitations.

Purpose of the Study:

• To develop a versatile, multi-plasmid yeast expression system for high-level soluble protein production.
• To integrate this system into an automated platform for strain engineering and screening.
• To engineer Saccharomyces cerevisiae for enhanced xylose utilization.

Main Methods:

• Utilized a three-plasmid system with portable small ubiquitin-like modifier (SUMO) vectors and endogenous yeast protease Ulp1.
• Employed PCR assembly and amino acid scanning mutagenesis for gene cloning and library generation.
• Integrated the expression system onto a fully automated, plasmid-based robotic platform for strain screening.

Main Results:

• Achieved simultaneous high-level expression of three different proteins using distinct selectable markers (URA, TRP, LEU).
• Engineered yeast strains expressing xylose isomerase (XI) and Yersinia pestis xylulokinase demonstrated improved growth on xylose.
• The presence of lycotoxin-1 (Lyt-1) did not impede the enhanced xylose utilization.

Conclusions:

• The developed SUMO-based multi-plasmid system facilitates efficient co-expression of multiple proteins in Saccharomyces cerevisiae.
• This system, integrated with automation, enables rapid screening of engineered strains for improved metabolic functions.
• The engineered yeast strains show promise for enhanced industrial applications involving xylose fermentation.