[3S02e] 05. Genome Editing Technology and its Applications - Part 3

6:00 PM - 6:15 PM JST(9:00 AM - 9:15 AM UTC)

[3GET-18] Stable, Scalable, and Cost-Effective Microbial Cell Factories: CRISPR-Based Highly Efficient Genomic Integration and a Precision Arabinose Induction System for Industrial Bioproduction.

○Congqiang Zhang¹, Ngoc-Phuong-Thao Nguyen¹, Sudha Shukal¹ (1. Singapore Institute of Food and Biotechnology Innovation (Singapore))

Keywords:

CRISPR-Cas9 genomic integration,Metabolic engineering,Plasmid-free expression,Arabinose induction system,Precision fermentation,Sustainable biosynthesis

Transitioning microbial cell factories from laboratory to industrial scale demands both genomic stability and precise gene regulation. Here, we present two complementary advances addressing these bottlenecks in E. coli. First, a refined CRISPR-Cas9 methodology enables single-step chromosomal integration of large DNA cargos—up to 17.5 kbp in more-challenging-to-engineer BL21 (40% efficiency) and 9.5 kbp in K12 MG1655 (100% efficiency). Systematic screening of ten genomic loci revealed strain-dependent integration "sweet spots" and demonstrated that strain background and insertion site are the dominant determinants of expression, irrespective of medium composition. Applying this approach, we chromosomally installed a 23-gene astaxanthin biosynthetic pathway in BL21, yielding a fully plasmid-free strain producing 426 mg/L astaxanthin in defined glucose medium. Second, we introduce the Arabinose Univariant Control System (AUCS), a tightly regulated, low-burden expression platform that overcomes the key limitations of both PT7lac and PBAD promoters. By constitutively expressing the AraE transporter and disrupting arabinose catabolism, AUCS eliminates carbon catabolite repression and achieves maximal induction at just 3 μM L-arabinose—a >99% reduction in inducer cost. Paired with a customized promoter library, AUCS supports high-yield expression of single proteins, multi-enzyme operons, and complex pathways (>10 genes) with exceptional reproducibility over 36 generations. Together, these tools provide a robust, scalable, and cost-effective foundation for precision fermentation and sustainable biosynthesis of high-value compounds. Zhang, C. and S. Shukal, Arabinose-Inducible Univariant Control System (AUCS) for Microbial Production of Proteins, Enzymes, and Metabolites. ACS Synthetic Biology, 2026. 15(1): p. 171-180. Nguyen, N.-P.-T., L. Ong, and C. Zhang, Systematic study of genomic loci in Escherichia coli B and K12 for genomic integration: application in plasmid-free astaxanthin production. Metabolic Engineering, 2026. 96: p. 154-164.