In vivo pathway engineering of saccharomyces cerevisiae for enhanced bioproduction

The budding yeast Saccharomyces cerevisiae is a central host in the bioeconomy, supporting the production of diverse bio-based products. Achieving high product yields requires precise control of gene expression of multiple pathway genes, yet current approaches often rely on cloning-intensive methods. In this doctoral work, PULSE, an in vivo promoter engineering tool, was developed to enable rapid and versatile control of transcriptional strength of multiple genes of a metabolic pathway inside the cell. Establishing PULSE required FACS-based isolation of active promoter elements from randomized DNA sequences, followed by their assembly into synthetic hybrid promoters flanked by loxPsym sites, and culminates in the generation of platform strains. Upon induction of Cre recombinase, loxPsym-flanked elements undergo recombination, generating diverse promoter architectures and subjecting target genes to a broad range of expression levels in a single step. This generates fast and cheap yeast libraries derived from a single isogenic platform strain. Application of PULSE to heterologous pathways in S. cerevisiae resulted in an eightfold increase in β-carotene production and enhanced growth under high xylose concentrations. Remarkably, both improvements were achieved over parental strain where all target genes are strongly expressed. To further expand the platform, a red-light-inducible Dre recombinase was developed; however, the associated rox sites were found to hamper transcriptional activity and this system needs further optimization. These results establish PULSE as a powerful and cloning-free platform for metabolic engineering, providing precise and dynamic control of gene expression and accelerating the optimization of biosynthetic pathways in vivo.