Work

Reusable and modular combinatorial libraries for iterative metabolic engineering of Saccharomyces cerevisiae

Year: 2025

Type: article

Abstract: 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 . • Framework for reusable and modular combinatorial library engineering in yeast • Iterative libraries consistently improved betacyanin production 1.2-5.7-fold per cycle over seven rounds of engineering • Best-performing strain secreted 217.5 mg/L betanin and 4.3 mg/L isobetanin in shake flasks • Enrichment of cytochrome b5 implicates its role in CYP76AD activity and betacyanin production

Source: Metabolic Engineering

DOI: 10.1016/j.ymben.2025.09.006

Authors: Philip Tinggaard Thomsen, Peter Gockel, Christina Vasileiou, Ingrid Mohr, Marc Cernuda Pastor, Irina Borodina

Institutions: Novo Nordisk Foundation, Technical University of Denmark

Language: English

Topic: Microbial Metabolic Engineering and Bioproduction

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

SDG: Responsible consumption and production

Open Access status: hybrid

Estimated APC paid: $4,780