TL;DR: Researchers reported a method called shotgun genetic engineering that delivers many small barcoded DNA pieces to mammalian cells so each cell tests a different pathway design, and used it to grow cells without supplied valine or isoleucine.

A multiplexed result published on Oct. 6

A research team reported shotgun genetic engineering, abbreviated SGE, in the journal Nature Biotechnology on Oct. 6, 2026.

The paper is titled “Highly multiplexed mammalian metabolic engineering with a shotgun approach,” according to Nature Biotechnology.

The listed authors are Julie Trolle, Sofia Sessa, Aleksandra Wudzinska, Mark Grivainis, Katerina Rincones, Tori Rodrick, Drew R. Jones, David Fenyö, Sudarshan Pinglay and Jef D. Boeke, according to Nature Biotechnology.

The article is open access, according to Nature Biotechnology.

Author affiliations include the Institute for Systems Genetics in the Department of Biochemistry and Molecular Pharmacology at NYU Langone Health, the Department of Genome Sciences at the University of Washington, the Seattle Hub for Synthetic Biology, the Graduate Program in Molecular Engineering at the University of Washington, the Department of Biochemistry and Molecular Pharmacology at NYU Langone Health, the Brotman Baty Institute for Precision Medicine and the Department of Biomedical Engineering at NYU Tandon School of Engineering, according to Nature Biotechnology.

The same date brought companion coverage in Nature Biotechnology, including “Shotgun engineering fires up mammalian metabolic design” and “High-throughput metabolic engineering in mammalian cells,” according to Nature Biotechnology.

The companion piece “Shotgun engineering fires up mammalian metabolic design” was written by Kshitij Rai and Caleb J. Bashor, according to Nature Biotechnology.

Rai and Bashor are associated with the Department of Bioengineering at Rice University, the Graduate Program in Systems, Synthetic and Physical Biology at Rice University, the Department of Biosciences at Rice University and the Rice Synthetic Biology Institute, according to Nature Biotechnology.

The central claim is that SGE screened millions of pathway combinations in mammalian cells, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

The demonstration focused on essential amino acid biosynthesis, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

In Chinese hamster ovary cells, the work achieved near-wild-type growth without valine and enabled isoleucine-free growth, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

The work also enabled valine-free growth in Jurkat cells, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

The companion summary states that the approach enabled discovery of productive metabolic pathways in mammalian cells, including solutions for synthesizing two essential amino acids, according to “Shotgun engineering fires up mammalian metabolic design.”

The research briefing describes SGE as a multiplexed strategy that screens millions of combinatorial solutions in a single experiment, according to “High-throughput metabolic engineering in mammalian cells.”

What shotgun genetic engineering does

SGE exploits the ease of delivering many barcoded small constructs rather than a single large one into mammalian cells, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

Each cell serves as an independent experiment, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

Each cell carries a synthetic pathway that explores gene content, stoichiometry and organellar localization, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

Functional pathways are identified by sequencing barcodes from cells exhibiting the desired phenotype, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

The briefing describes the same logic as using selection strategies that reveal effective designs, according to “High-throughput metabolic engineering in mammalian cells.”

The News & Views description similarly frames the advance as testing random combinations of genes at scale, according to “Shotgun engineering fires up mammalian metabolic design.”

In this design, pathway diversity is sampled in parallel across many cells, rather than by testing pathways in succession, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

The paper contrasts that parallel sampling with a classical design-build-test framework in which pathways are tested in succession, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

The paper presents transcription units, or TUs, as the building blocks being combined, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

Each TU consists of a gene and its regulatory elements, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

Metabolic control is achieved by coordinating the activity of multiple TUs, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

The design space includes promoters, organellar localization signals, enzymatic function, stoichiometry and copy number, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

For a target pathway and host cell type, the paper lists selection of coding sequences, configuration of regulatory elements, intracellular localization of each encoded protein and relative expression levels and stoichiometry as design decisions, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

The paper states that there is little mechanistic understanding to guide those optimal choices, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

The variables create a vast combinatorial design space, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

As pathway complexity increases, so does the size of that combinatorial design space and the size of required DNA constructs, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

That growth renders unbiased screens intractable under conventional approaches, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

Why mammalian cells needed a different screening logic

Mammalian metabolic engineering advances basic biology, bioproduction and cell therapy, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

Synthetic pathways are frequently constructed by transplanting coding sequences from diverse species into orthogonal hosts, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

That transplantation approach endows hosts with new metabolic capabilities, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

Modern DNA synthesis technologies make it possible to incorporate sequences from across the phylogenetic tree or de novo designed sequences, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

The paper says that expanded sequence access vastly expands the functional landscape accessible to mammalian cells, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

Microbial systems have provided notable successes for pathway transplantation, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

Those successes include cell-based alternatives to industrial manufacturing processes, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

They also include stabilization of pharmaceutical supply chains previously dependent on seasonal plant sources, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

They also include capture of atmospheric carbon dioxide to generate industrially valuable biomass, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

Examples of metabolic engineering at pathway scale in mammalian cells remain rare, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

The paper says improved control of mammalian cell function could enhance cell therapies, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

The same potential includes producing increasingly complex biologics, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

It also includes improving scalable manufacture of viral vectors, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

Iteration speed is one reason mammalian engineering has lagged microbial work, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

In fast-growing microbial systems, the paper cites Escherichia coli with a 20-minute doubling time, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

It cites Saccharomyces cerevisiae with a 90-minute doubling time, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

In those systems, design-build-test cycles can rapidly construct, test and iteratively refine pathways until performance is satisfactory, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

Mammalian cells typically require more than 20 hours to double, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

That longer doubling time drastically slows the pace of iteration, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

The limitation becomes more severe as pathway complexity grows, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

It also becomes more severe as multiple functions are integrated into a single cell, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

Amino acids as the test case

The team selected essential amino acid biosynthesis as the engineering target, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

Mammalian cells normally require external supply of essential amino acids, a premise reflected in the selection-based screening design, according to “High-throughput metabolic engineering in mammalian cells” and “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

The SGE screen sampled millions of pathway combinations for that trait, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

The Chinese hamster ovary cell experiments addressed valine independence, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

Those experiments achieved near-wild-type growth without valine, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

The same cell type was also used to pursue isoleucine independence, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

Those experiments enabled isoleucine-free growth in Chinese hamster ovary cells, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

The paper distinguishes the valine result as near-wild-type growth from the isoleucine result as enabled growth, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

A second mammalian context was tested in Jurkat cells, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

In Jurkat cells, the method enabled valine-free growth, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

The two-cell-line outcome is important because it shows the screen was not confined to Chinese hamster ovary cells, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

The two-amino-acid outcome is important because the News & Views piece describes solutions for synthesizing two essential amino acids, according to “Shotgun engineering fires up mammalian metabolic design.”

Mitochondria and large DNA inserts stood out

Functional pathways favored mitochondrial localization, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

That finding concerns where encoded proteins were directed inside the cell, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

Organellar localization was one of the variables explored by each synthetic pathway, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

The enrichment for mitochondrial localization therefore emerged from the screen rather than being imposed as a premise, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

Functional pathways also required integration of tens of kilobases of synthetic DNA, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

That scale was beyond conventional screening, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

The result illustrates why delivering one large construct per design is limiting, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

Instead, SGE delivered barcoded individual TUs at high multiplicity so each cell carried a unique pathway combination, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

Following selection, cells displaying the desired phenotype could be sequenced to reveal the TU combinations that conferred function, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

The emphasis on localization echoes broader metabolic-engineering experience with compartmentalization, including yeast work in which 26 genes were distributed across six subcellular locations for tropane alkaloid biosynthesis, as cited in “High-throughput metabolic engineering in mammalian cells.”

That yeast example is presented in the briefing as context for why location matters, not as part of the new SGE experiments, according to “High-throughput metabolic engineering in mammalian cells.”

Datasets built for machine learning

The resulting datasets support machine-learning-guided decoding and engineering of complex biosynthetic traits in mammalian systems, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

Because SGE links surviving barcodes to pathway composition, the screen produces a record of which combinations conferred the desired function, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

The authors present that record as a resource for decoding design rules, according to “High-throughput metabolic engineering in mammalian cells.”

The briefing frames selection as the mechanism that reveals effective designs from millions of combinatorial solutions, according to “High-throughput metabolic engineering in mammalian cells.”

The paper places the advance in a field where lack of foundational knowledge has limited mammalian pathway design, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

Data-driven design is therefore presented as a response to limited mechanistic guidance, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach” and “High-throughput metabolic engineering in mammalian cells.”

Sequence data from the study were deposited in the Sequence Read Archive under BioProject PRJNA1282762, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

Code and associated materials were made available through Zenodo under record 21874813, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

How this extends earlier valine work

The new paper builds on a 2022 eLife paper titled “Resurrecting essential amino acid biosynthesis in mammalian cells,” according to “High-throughput metabolic engineering in mammalian cells.”

That earlier paper featured work to engineer valine biosynthesis in Chinese hamster ovary cells, according to “High-throughput metabolic engineering in mammalian cells.”

The 2022 paper is listed among the briefing references as the group’s previous work, according to “High-throughput metabolic engineering in mammalian cells.”

The News & Views bibliography likewise cites the 2022 eLife paper alongside the new Nature Biotechnology paper, according to “Shotgun engineering fires up mammalian metabolic design.”

The distinction in 2026 is multiplexing: the briefing describes SGE as screening millions of combinatorial solutions in one experiment, according to “High-throughput metabolic engineering in mammalian cells.”

The advance therefore moves from demonstrating that valine biosynthesis can be engineered in mammalian cells to sampling many designs in parallel and recovering additional solutions, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach” and “High-throughput metabolic engineering in mammalian cells.”

The addition of isoleucine-free growth in Chinese hamster ovary cells marks another extension beyond the earlier valine-focused demonstration, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

The demonstration of valine-free growth in Jurkat cells further extends testing beyond one host line, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

What the study does and does not show

The study demonstrates a screening method and reports the pathway features it recovered, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

It does not present itself as a manufacturing process for biologics or viral vectors, even though those applications motivate mammalian engineering, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

The stated relevance includes basic biology, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

It includes bioproduction, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

It includes cell therapy, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

More specifically, the paper points to complex biologics and scalable viral-vector manufacture as areas that could benefit from improved control of mammalian cells, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

The paper also points to cell therapies as a beneficiary, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

The experimental evidence reported is growth under amino-acid dropout enabled by synthetic pathways, not production of a therapeutic protein or vector, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

The machine-learning contribution is described as support for guided decoding and engineering, not as a validated predictive product, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

For readers tracking synthetic biology, the durable facts are the publication venue and date, the SGE mechanism, the millions-scale screen, the three growth outcomes, the mitochondrial and large-insert observations and the deposited sequence and code records, according to “Highly multiplexed mammalian metabolic engineering with a shotgun approach.”

Sources referenced