[Construction of efficient yeast cell factory for kauniolide, a parthenolide derivative]

Kauniolide, a parthenolide derivative, serves as a key biosynthetic intermediate for various guaianolide-type sesquiterpenoids, such as agrabin, lactucin, and lactupicrin. However, its chemical synthesis is often hampered by demanding reaction conditions and high reagent consumption, which severely limit further development and application. To address this, this study employed systematic metabolic engineering strategies to construct an efficient yeast cell factory for kauniolide production. First, the biosynthetic pathway genes for costunolide-the direct precursor of kauniolide(HaGAS, TpGAO, TpCOS, AaCPR)-were heterologously expressed in a lab-engineered yeast chassis strain with high farnesyl pyrophosphate(FPP) production. This initial strain produced costunolide at a titer of 6.1 mg·L~(-1). Subsequently, the co-expression of AaADH1, AaALDH1, and AaCYB5 was implemented to enhance the germacrene A acid synthesis and promote electron transfer, thereby boosting the catalytic efficiency of the key cytochrome P450 enzymes. This modification increased the costunolide titer to 71.5 mg·L~(-1). To optimize the conversion of costunolide to kauniolide, a promoter compatibility screen for the TpKLS gene was conducted. Among the tested promoters(P_(GAL1), P_(TDH3), P_(TEF1), P_(TPI1), P_(sptGAL2), and P_(skGAL2)), the strain harboring the P_(skGAL2)-driven TpKLS construct showed the highest performance, achieving a kauniolide titer of 28.2 mg·L~(-1). Furthermore, to further enhance the pathway flux, the catalytic efficiency of the TpKLS enzyme was improved through semi-rational design coupled with computer-aided design. The best-performing mutant, TpKLS~(V204R), exhibited a 2.7-fold higher catalytic activity compared to the wild-type enzyme. The final engineered strain, when cultivated in shake-flask fermentation, produced costunolide and kauniolide at titers of 35.0 and 71.1 mg·L~(-1), respectively. The kauniolide titer represents the highest level reported to date in a yeast system. In conclusion, this study successfully constructed an efficient yeast cell factory for kauniolide by reconstructing and optimizing its heterologous biosynthetic pathway. It provides a solid technical foundation and a valuable reference for the scalable biosynthesis of kauniolide and the exploration of its downstream derivatives.