Metabolic engineering has demonstrated the potential to produce high-value specialty chemicals with greater efficiency than traditional chemical synthesis methods. Plasmid-based expression systems enable rapid strain construction and offer good bench-scale production, but when bench-scale technology is transferred to the industrial scale, the requirement of antibiotic selection becomes prohibitively expensive and the reduced stability of plasmid-based systems is problematic for extended production phases during fed-batch fermentations. The scarless genomic integration of an optimized pathway can lead to greater industrial viability by addressing both of the aforementioned issues. Here, we present our progress towards the development of a system which can enable the scarless integration of large constructs onto the E. coli chromosome. We will present a novel method of lambda-Red mediated scarless integration which utilizes Cas9 counterselection and I-SceI mediated in vivo linearization of the donor vector. Four locations on the BL21*(DE3) chromosome were targeted for investigation into the relative expression levels of integrated pathways. We will present proof of principle data utilizing a fluorescent protein, mCherry, as well as a three-step biosynthetic pathway for a high-value small molecule.

Progress towards improving chromosomal integration of large constructs in E. coli

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.