Nguyen, N.-P.-T., Ong, L., & Zhang, C. (2026). Systematic study of genomic loci in Escherichia coli B and K12 for genomic integration: application in plasmid-free astaxanthin production. Metabolic Engineering, 96, 154–164. https://doi.org/10.1016/j.ymben.2026.03.013
Abstract:
Escherichia coli is a major workhorse for industrial biotechnology, yet reliance on plasmids is hindered by intrinsic instability. While genomic integration offers superior stability, it remains challenging for integrating large DNA fragments, particularly in difficult-to-engineer B strains (e.g., BL21) compared to K12 strains like MG1655. Moreover, a systematic study comparing genomic expression across B and K12 strains and different loci is absent. To address these issues, we firstly refined a CRISPR methodology that achieved unprecedented integration efficiencies in both strains: 40% for single-step insertion of a 17.5-kbp fragment in BL21 and 100% for a 9.5-kbp fragment in MG1655. Using this updated workflow, we rationally screened and compared 10 genomic loci in BL21 and MG1655 across varying media. The results demonstrated that host strain and genomic locus selection are more critical for expression than carbon source or medium composition. Although BL21 generally exhibits higher gene expression than MG1655, expression at certain loci can be very low. Therefore, we highlight the need for greater caution in locus selection for BL21 due to its higher variability. We leverage our validated loci for the scarless and marker-less integration of the astaxanthin synthesis pathway (23 genes, including multi-copy key genes, crtY, crtZ, crtW, crtE, crtB, and crtI) into the BL21 chromosome. The resulting plasmid-free strain produced 426 mg/L astaxanthin in glucose defined medium, showcasing a robust and generalizable strategy for complex pathway integration and optimization in challenging host strains.
License type:
Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)
Funding Info:
This research is supported by core funding from: BMRC Central Research Fund (CRF) Applied/Translational Research
Grant Reference no. : SIF-GF21-X101
This research is supported by core funding from: Central Research Fund
Grant Reference no. : SIF-GF25-0370
This research / project is supported by the Agency for Science, Technology and Research (A*STAR) - Career Development Fund
Grant Reference no. : C233312033
This research / project is supported by the Agency for Science, Technology and Research (A*STAR) - Singapore-Australia Bilateral Programme on Innovations in Food for Sustainability
Grant Reference no. : R24I4IR001