Combinatorial optimization of gene expression through recombinase-mediated promoter and terminator shuffling in yeast

Charlotte Cautereels, Jolien Smets, Peter Bircham, Dries De Ruysscher, Anna Zimmermann, Peter De Rijk, Jan Steensels, Anton Gorkovskiy, Joleen Masschelein and Kevin J. Verstrepen ()
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Charlotte Cautereels: VIB-KU Leuven Center for Microbiology
Jolien Smets: VIB-KU Leuven Center for Microbiology
Peter Bircham: VIB-KU Leuven Center for Microbiology
Dries De Ruysscher: KU Leuven
Anna Zimmermann: VIB-KU Leuven Center for Microbiology
Peter De Rijk: VIB Center for Molecular Neurology, VIB
Jan Steensels: VIB-KU Leuven Center for Microbiology
Anton Gorkovskiy: VIB-KU Leuven Center for Microbiology
Joleen Masschelein: KU Leuven
Kevin J. Verstrepen: VIB-KU Leuven Center for Microbiology

Nature Communications, 2024, vol. 15, issue 1, 1-17

Abstract: Abstract Microbes are increasingly employed as cell factories to produce biomolecules. This often involves the expression of complex heterologous biosynthesis pathways in host strains. Achieving maximal product yields and avoiding build-up of (toxic) intermediates requires balanced expression of every pathway gene. However, despite progress in metabolic modeling, the optimization of gene expression still heavily relies on trial-and-error. Here, we report an approach for in vivo, multiplexed Gene Expression Modification by LoxPsym-Cre Recombination (GEMbLeR). GEMbLeR exploits orthogonal LoxPsym sites to independently shuffle promoter and terminator modules at distinct genomic loci. This approach facilitates creation of large strain libraries, in which expression of every pathway gene ranges over 120-fold and each strain harbors a unique expression profile. When applied to the biosynthetic pathway of astaxanthin, an industrially relevant antioxidant, a single round of GEMbLeR improved pathway flux and doubled production titers. Together, this shows that GEMbLeR allows rapid and efficient gene expression optimization in heterologous biosynthetic pathways, offering possibilities for enhancing the performance of microbial cell factories.

Date: 2024
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DOI: 10.1038/s41467-024-44997-7

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