Engineering a carbon source-responsive promoter for improved biosynthesis in the non-conventional yeast Kluyveromyces marxianus

Many desired biobased chemicals exhibit a range of toxicity to microbial cell factories, making industry-level biomanufacturing more challenging. Separating microbial growth and production phases is known to be beneficial for improving production of toxic products. Here, we developed a novel synthetic carbon-responsive promoter for use in the rapidly growing, stress-tolerant yeast Kluyveromyces marxianus, by fusing carbon-source responsive elements of the native ICL1 promoter to the strong S. cerevisiae TDH3 or native NC1 promoter cores. Two hybrids, P_IT350 and P_IN450, were validated via EGFP fluorescence and demonstrated exceptional strength, partial repression during growth, and late phase activation in glucose- and lactose-based medium, respectively. Expressing the Gerbera hybrida 2-pyrone synthase (2-PS) for synthesis of the polyketide triacetic acid lactone (TAL) under the control of P_IN450 increased TAL more than 50% relative to the native NC1 promoter, and additional promoter engineering further increased TAL titer to 1.39 g/L in tube culture. Expression of the Penicillium griseofulvum 6-methylsalicylic acid synthase (6-MSAS) under the control of P_IN450 resulted in a 6.6-fold increase in 6-MSA titer to 1.09 g/L and a simultaneous 1.5-fold increase in cell growth. Finally, we used P_IN450 to express the Pseudomonas savastanoi IaaM and IaaH proteins and the Salvia pomifera sabinene synthase protein to improve production of the auxin hormone indole-3-acetic acid and the monoterpene sabinene, respectively, both extremely toxic to yeast. The development of carbon-responsive promoters adds to the synthetic biology toolbox and available metabolic engineering strategies for K. marxianus, allowing greater control over heterologous protein expression and improved production of toxic metabolites.