YaliCMulti and YaliHMulti: Stable, efficient multi-copy integration tools for engineering Yarrowia lipolytica

Area of Science:

• Biotechnology
• Synthetic Biology
• Microbial Engineering

Background:

• Yarrowia lipolytica is a key industrial yeast for producing recombinant proteins and natural products.
• Current limitations include unstable plasmid expression and difficult gene integration, hindering efficient production.
• Developing robust genetic tools is crucial for industrial applications.

Purpose of the Study:

• To engineer novel vectors and methods for stable, multi-copy genome integration in Yarrowia lipolytica.
• To overcome challenges associated with low-copy plasmids and inefficient gene integration.
• To enhance the efficiency of metabolic engineering strategies in Y. lipolytica.

Main Methods:

• Exploited sequence diversity in retrotransposon LTRs and rDNA sequences for vector construction.
• Developed recyclable, multi-copy site integration vectors using Cre-LoxP system, amino acids, and antibiotic tags.
• Utilized a rapidly degrading selective marker and weak promoter for high-copy number integration.

Main Results:

• Achieved stable integration of long-pathway genes and high levels of gene expression using the green fluorescent protein (HrGFP) reporter.
• Demonstrated successful production of 7.3 g/L L-ergothioneine and 8.3 g/L (2S)-naringenin.
• Attained the highest reported titers for these compounds in Y. lipolytica using the developed tools.

Conclusions:

• Novel multi-copy genome integration strategies offer convenient and effective tools for Y. lipolytica metabolic engineering.
• The developed vectors and methods significantly improve the stability and efficiency of recombinant protein and natural product production.
• These advancements pave the way for broader industrial applications of Y. lipolytica.