Highly multiplexed mammalian metabolic engineering with a shotgun approach.

Shotgun genetic engineering enables screening of millions of mammalian pathway combinations, achieving complex biosynthetic traits with tens of kilobases of synthetic DNA.

• Why it matters: Understanding and optimizing mammalian metabolic pathways is crucial for bioproduction and cell therapy, but the increasing complexity makes traditional unbiased screening infeasible.
• What they did: The approach involves delivering numerous barcoded small constructs into mammalian cells, with each cell acting as an independent experiment to explore gene content, stoichiometry, and localization, followed by barcode sequencing to identify functional pathways.
• The result: This method successfully engineered amino acid biosynthesis pathways, enabling growth without valine or isoleucine in Chinese hamster ovary and Jurkat cells, and provided datasets for machine learning-driven pathway decoding.