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September 2nd, 2025
Version: 2
University of Potsdam
synthetic biology
bioRxiv

In Vivo Pathway Optimization in Yeast via LoxPsym-Mediated Shuffling of Upstream Activating Sequences

Ruehmkorff, C.Open in Google Scholar•Tafazoli Yazdi, A.Open in Google Scholar•Hochrein, L.Open in Google Scholar

The budding yeast Saccharomyces cerevisiae plays an integral role in the bioeconomy as a powerful host for industrial bio-manufacturing, driving the production of diverse bio-based products. Achieving optimal product yields requires precise fine-tuning of the expression levels of multiple pathway genes, which often relies on cloning-intensive methods. Here, we present PULSE, an in vivo promoter engineering tool based on a streamlined workflow combining FACS-based screening of a randomized DNA library to identify active promoter elements, and their subsequent assembly into synthetic hybrid promoters where each element is flanked by loxPsym sites. Multiple promoter cassettes can be genome-integrated to generate ready-to-use platform strains, allowing users to easily place their genes of interest under the control of PULSE promoters. By activating Cre-mediated recombination, loxPsym-flanked promoter elements can be recombined, effectively bringing the target genes under control of a vast set of promoters spanning a wide range of expression levels in one simple step. Applying PULSE on two heterologous pathways, a threefold increase in {beta}-carotene production and improved xylose utilization by S. cerevisiae was achieved. These results demonstrate the power and efficiency of PULSE as a versatile platform for metabolic engineering, enabling rapid, cloning-free optimization of biosynthetic pathways in vivo.

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