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

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 β-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 .