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A landing pad system for multicopy gene integration in Issatchenkia orientalis
Zia Fatma1, Shih-I Tan1, Aashutosh Girish Boob1
1Department of Chemical and Biomolecular Engineering, Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, United States.
Metabolic Engineering
|June 21, 2023
Summary
Researchers developed a new CRISPR-based landing pad system for efficient multiplex genome engineering in the yeast Issatchenkia orientalis. This tool enables the integration of multiple genes, improving organic acid production and advancing metabolic engineering efforts.
Area of Science:
- Microbiology
- Synthetic Biology
- Biotechnology
Background:
- Issatchenkia orientalis is a robust non-conventional yeast capable of growth in acidic conditions, making it suitable for organic acid production.
- Recent advancements in genetic tools for I. orientalis, including CRISPR-Cas9, have facilitated metabolic engineering, but efficient multicopy integration for complex pathways remains a challenge.
Purpose of the Study:
- To develop efficient multicopy integration tools for multiplex genome engineering in I. orientalis.
- To create a versatile landing pad system for constructing large, complex metabolic pathways using CRISPR-Cas9.
Main Methods:
- Developed a bioinformatics pipeline to identify and prioritize intergenic loci for genome editing.
- Characterized 47 guide RNAs (gRNAs) in 21 intergenic regions for cutting efficiency, gene cassette integration, cellular fitness, and GFP expression.
- Engineered a landing pad system enabling the integration of multiple genes using a single gRNA and multiple repair templates.
Main Results:
- Demonstrated simultaneous integration of 2 to 5 genes into target loci with >80% efficiency using the landing pad system.
- Successfully improved 5-aminolevulinic acid production by integrating five gene copies in one step.
- Constructed a succinic acid production pathway by integrating five gene expression cassettes, yielding 9 g/L in batch fermentation.
Conclusions:
- The developed single gRNA-mediated CRISPR platform and landing pad system are effective for building complex metabolic pathways in I. orientalis.
- This system significantly enhances multiplex engineering capabilities, offering a valuable tool for metabolic engineering of non-conventional yeasts.
