Fine-tuned synthetic transcription factors for production of 3'-phosphoadenosine-5'-phosphosulfate in yeast

Technologies developed over the past decade have made Saccharomyces cerevisiae a promising platform for producing various natural products. Balancing multi-enzyme expression, while maintaining robust microbial growth, remains a limiting factor for engineering long biosynthetic pathways in yeast.

Here, we improved the transcriptional capacity of our previously developed synthetic transcription factors (synTFs) derived from the plant JUB1 DNA-binding domain, whose expression is controlled by an β-D-1-thiogalactopyranoside (IPTG)-inducible promoter. To this end, at cysteine positions within surface-exposed loop regions of a JUB1-derived DNA-binding scaffold, we introduced a short peptide to enhance loop flexibility while providing local stability and orientation. The generated synTFs, so-called JUB1-X synTFs, varying in strength, have been successfully used to improve the production of 3'-phosphoadenosine 5'-phosphosulfate (PAPS), a universal sulfate donor necessary for the synthesis of therapeutic glycosaminoglycans and sulfolipids, in yeast. Using the generated yeast strain, in simple batch culture, PAPS accumulation of 21.4 ± 5.8 mg g⁻¹ cdw was achieved after only 5 h of inducing the expression of JUB1-X synTFs.

The design principle demonstrated here provides a generalizable strategy to fine-tune other synTFs derived from heterologous DNA-binding domains, expanding the regulatory capabilities of existing synTF collections. Together, this work offers a modular, scalable approach to constructing high-performance gene circuits and supports the development of yeast cell factories for the synthesis of complex natural products.