PolyFab: an efficient in vitro pathway assembly tool for natural product synthesis in Saccharomyces cerevisiae

Saccharomyces cerevisiae is a widely used industrial chassis for the heterologous biosynthesis of natural products. However, achieving high-level expression of heterologous genes and developing efficient metabolic engineering strategies remain major challenges. Here, based on the polycistronic expression system and the Golden Gate assembly, we have developed an efficient in vitro synthetic pathway assembly method named PolyFab. PolyFab enables the seamless and efficient ligation of multiple transcriptional units (TU) in vitro. When combined with the CRISPR/Cas9 gene editing system, these TUs can be integrated into the yeast genome seamlessly with high efficiency. By using PolyFab, we have accomplished the de novo synthesis of the important flavonoid compound apigenin in yeast and carried out overall metabolic engineering optimization of the yeast chassis. Eventually, through shake flask fermentation, the engineered strain produced 165.49 mg/L of apigenin. This work provides an effective tool for rapid pathway construction and multi-gene integration in yeast, facilitating the microbial production of complex natural products.