Abstract

Biomanufacturing uses engineered biological systems to produce valuable compounds from inexpensive materials like waste, reducing reliance on oil- and gas-based chemical production and promoting sustainable manufacturing. While many microbial species are used as hosts, non-conventional yeasts like Kluyveromyces marxianus have gained interest due to unique traits such as rapid growth, thermotolerance, and broad substrate utilization. Here, we investigated transcriptional networks in K. marxianus by engineering transcription factors (TFs) to enhance industrial stress tolerance. We compared two TF upregulation strategies: activation via fusion to viral activation domains (VPR and VP64) and overexpression via fusion to a strong promoter. Overexpression of the model TF KmGcn4p using the KmPDC1 promoter increased HIS3 expression 5-fold and significantly improved growth in the presence of the HIS3 inhibitor 3-AT. Transcriptomic analysis using Nanopore RNA-seq revealed that KmPDC1-driven overexpression of GCN4, HCM1, MSN2, and PDR1 produced differential gene expression across all three TF subclasses. Finally, to identify TFs directing stress tolerance, a high-throughput competition assay workflow was developed to screen barcoded TF libraries. In a library of 10 barcoded TFs, GCN4 and its haplotig allele (GCN4_hap) were found to be the “winner” TFs with increased frequencies of 0.7 and 1.8 (log2) under 70 mM of 3-AT. Future work will apply this pipeline to screen 291 overexpressed K. marxianus TFs under biomanufacturing-relevant stresses to identify TFs that can improve K. marxianus as an industrial bioprocessing strain.