A modular single- and dual-gene expression toolkit for Kluyveromyces marxianus

Kluyveromyces marxianus is a fast-growing, food-grade yeast with broad substrate utilization, however, its limited expression tools hinder its synthetic biology applications. Here, we developed single- and dual-gene expression systems based on screened elements, including a promoter pENO and a bidirectional promoter pHTX. These systems enabled stable expression across multiple K. marxianus strains, with expression levels affected by genetic element combinations, carbon source, and culture time. Using the single-gene system with a short intergenic sequence (IGG) for bicistronic expression, the production of leghemoglobin from Vigna angularis (VaHB) reached 30.4 mg/L using galactose as carbon source. In contrast, the dual-gene system achieved 48.4 mg/L of VaHB with the co-expression of HEM1, enhancing heme biosynthesis using glucose as a carbon source. Additionally, Simian Virus 40 (SV40) nuclear localization signals (NLS) directed fluorescent proteins to the nucleus, enabling subcellular targeting. This toolkit supports efficient and context-responsive gene expression in K. marxianus, facilitating its development as a versatile microbial chassis for industrial protein production and synthetic biology applications.