Highly multiplexed mammalian metabolic engineering with a shotgun approach.

Shotgun genetic engineering enables screening of millions of mammalian pathway combinations, achieving amino acid biosynthesis with tens of kilobases of synthetic DNA.

• Why it matters: Understanding and optimizing mammalian metabolic pathways is crucial for bioproduction and cell therapy, but increasing pathway complexity makes unbiased screening intractable with traditional methods.
• What they did: The authors developed shotgun genetic engineering (SGE), delivering many barcoded small constructs into mammalian cells, allowing each cell to serve as an independent experiment exploring gene content, stoichiometry, and localization, and screening millions of pathway variants.
• The result: SGE identified functional pathways that support growth without valine or isoleucine, favored mitochondrial localization, and generated datasets that enable machine-learning-guided engineering of complex biosynthetic traits.