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Site-specific Bacterial Chromosome Engineering: ΦC31 Integrase Mediated Cassette Exchange (IMCE)
Published on: March 16, 2012
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Customizable and stable multilocus chromosomal integration: a novel glucose-dependent selection system in
Shuo Zhang1, Tao Ma1, Fu-Hui Zheng1
1MOE Key Laboratory of Evolution and Marine Biodiversity, College of Marine Life Sciences, Ocean University of China, Yushan Road, No. 5, Qingdao, 266003, Shandong, China.
Biotechnology for Biofuels and Bioproducts
|June 17, 2024
Summary
A novel glucose-dependent selection system was developed for non-conventional yeasts, overcoming limitations of antibiotic and auxotrophic methods. This system uses glucose for stable gene integration, enabling efficient industrial bioproduction.
Area of Science:
- Synthetic biology
- Microbial biotechnology
- Genetic engineering
Background:
- Non-conventional yeasts are valuable for microbial bioproduction but current gene editing relies on costly and environmentally concerning antibiotic or auxotrophic selection methods.
- Existing auxotrophic selection systems lack rigorous selection pressure in industrial settings, compromising the stability of engineered metabolic pathways.
- There is a critical need for alternative selection systems compatible with large-scale industrial fermentation of yeasts.
Purpose of the Study:
- To develop a novel, cost-effective, and environmentally friendly glucose-dependent selection system for non-conventional yeasts.
- To enable customizable and stable multilocus chromosomal integration of target genes for enhanced microbial bioproduction.
- To provide a robust tool for genetic manipulation and strain improvement in yeast biorefineries.
Main Methods:
- Developed a glucose-deficient yeast chassis (Δpfk) by knocking out the phosphofructokinase gene (PFK) in *A. melanogenum* P16.
- Constructed chromosomal integration plasmids with a PFK selection marker driven by promoters of varying strengths.
- Utilized the green fluorescent protein (GFP) as a reporter gene to assess transformation efficiency and integration copy number.
Main Results:
- Achieved 100% transformation efficiency with the novel glucose-dependent selection system.
- Demonstrated customizable chromosomal integration copy numbers (2-54) inversely correlated with promoter strength.
- Confirmed stable integration of target genes during successive fermentation, using glucose as a cost-effective selection molecule.
Conclusions:
- The developed glucose-dependent selection system enables customizable and stable multilocus gene integration in non-conventional yeasts.
- This system offers a promising alternative to traditional selection methods, suitable for industrial fermentation applications.
- The PFK-based selection marker shows potential for broad application across various non-conventional yeast strains.
