Serine Integrase Recombinational Assembly (SIRA) for Rapid Metabolic Pathway Assembly and Optimization

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Christine Merrick, Sean Colloms, Susan Rosser

University of Glasgow, United States

Biosynthetic pathways can be engineered into bacteria allowing the production of chemicals with commercial and therapeutic applications. Challenges, when engineering biosynthetic pathways, include balancing levels of proteins and pathway intermediates, competition with existing pathways, finding suitable enzymes, maintaining host viability and increasing yield. We are developing a novel technology, Serine Integrase Recombinational Assembly (SIRA), using site-specific recombination, to assemble and optimize the expression of biosynthetic pathways rapidly. With SIRA technology, genes and regulatory sequences, in the form of DNA “cassettes”, integrate into a landing pad on the plasmid or genome where the new pathway is being built. The assembly process can easily construct pathways in predefined or random gene orders, include multiple gene variants at all positions, and facilitate varied gene expression levels by incorporating degenerate ribosome binding sites. Using the carotenoid and violacein biosynthetic pathways as model systems, we have shown assembly and optimization of two fully functional pathways in just two days. Once assembled, SIRA allows targeted addition, deletion, and replacement of genes and DNA elements within a pathway.