Efficiently rewiring microbial metabolism for molecule production lies at the core of industrial metabolic engineering. Combinatorial libraries are useful for directing metabolism towards molecule production; however, their construction is labor-intensive, and their use in iterative strain engineering campaigns is often restricted by site-specific genomic integration. Here we present an automation-friendly framework for generating reusable and modular integration-based combinatorial libraries that can be used repeatedly to build high-performing strains. We apply this approach to engineer the production of betacyanins, a commonly used red food colorant extracted from beetroots, in Saccharomyces cerevisiae. Iterative implementation of combinatorial libraries targeting the betacyanin biosynthesis pathway (design space: ∼25,000), precursors (design space: ∼43,000), and cofactors (design space: ∼26,000) consistently improved pigment production by 1.2-5.7-fold per cycle over seven rounds of engineering. Sequencing of high-performing library isolates from each round revealed unique insights into betacyanin and yeast metabolism, e.g. we found strong evidence implicating the S. cerevisiae cytochrome b5 in heterologous red beet pigment production. Altogether, this study demonstrates a framework for combinatorial library engineering well-suited for accelerating the development of high-performing cell factories for industrial fermentation processes.

• PMID: 40976504
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Journal Article

Authors

Thomsen PT, Gockel P, Vasileiou C, Mohr I, Pastor MC, Borodina I

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