Serine Integrase Recombinational Assembly (SIRA) for Rapid Optimization of Metabolic Pathways

A major target in Synthetic Biology is to build microbial cell factories for greener and more economical production of chemicals of therapeutic and industrial importance. Building these cell factories requires genetic engineering of metabolic pathways to produce high yields of compounds for large-scale production. However, engineered metabolic pathways require considerable optimization to limit interference with existing metabolic networks, diversion of cellular resources, and accumulation of toxic intermediates which compromise strain performance. DNA assembly is one of the biggest rate-limiting steps in metabolic pathway optimization. Serine Integrase Recombinational Assembly (SIRA) is a site-specific recombination-based DNA assembly method that uses a one-pot reaction for rapid multi-part assembly¹. With SIRA technology, genes and regulatory sequences in the form of linear DNA parts integrate into a landing site on target DNA. The assembly process can easily construct gene assemblies using rational or combinatorial approaches, 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 DNA parts are assembled, SIRA allows targeted addition, deletion, and replacement of genes and DNA elements. This permits much easier optimization of complex genetic systems than was previously possible. This method is currently being applied to enhancing production of therapeutically and industrially important isoprenoids in E. coli.

1.  Colloms SD, Merrick CA, Olorunniji FJ, Stark WM, Smith MCM, Osbourn A, Keasling KJ & Rosser SJ (2013) Rapid metabolic pathway assembly and modification using serine integrase site-specific recombination. Nucleic Acids Res 42: e23