Promoter engineering by shuffling Upstream activating sequences via LoxPsym Supported Evolution

We developed a promoter engineering tool that enables simultaneous, dynamic and cloning-free adjustment of the expression levels of multiple genes in vivo based on the Cre-loxP system. In our system, each pathway gene is regulated by a distinct synthetic hybrid promoter, composed of a core element and unique combinations of upstream activating sequences (UAS-elements) flanked by loxPsym sites. Cre activity alters the order, orientation and number of UAS-elements and thus the strength of the promoter. In this way, we were able to generate promoter cassettes comparably strong to the native TDH3 promoter. Remarkably, starting from an isogenic yeast colony, we were able to generate a diverse yeast culture showing different expression levels post-recombination, independent of cloning and transformation efficiency.