De novo synthesis of plant polyketide plumbagin in yeast: a platform for sustainable production of naphthoquinones

Plumbagin is a natural naphthoquinone with different pharmacological properties and abundantly present in the roots of Plumbago zeylanica L. In spite of its therapeutic anti-cancerous potential, its limited availability from plant sources has slowed down its large scale production. In the present study, we report the heterologous reconstruction of the complete plumbagin biosynthetic pathway in Saccharomyces cerevisiae using six functionally characterized genes from P. zeylanica viz. Polyketide synthase (PKS), Polyketide cyclase, Aldo-keto reductase (AKR1), two cytochrome P450 monooxygenases (CYP81B140 and CYP81B141), and a cytochrome P450 reductase (CPR). Stepwise pathway engineering was performed to evaluate the necessity and sufficiency of individual and combined gene sets. The expression of PKS alone was not able to synthesize measurable products, while the co-expression of PKS, cyclase, and AKR1 (PCA) enabled the biosynthesis of 3-methyl-1,8-naphthalenediol which is a key intermediate and also confirmed by LC-MS/MS. The addition of CYP81B140 and CPR (PCACC) led to the production of 3-methyl-1,8-naphthalenediol and isoshinanolone, and further addition of CYP81B141 (PCACCC) in yeast effectively biosynthesized plumbagin. These results confirm the functional roles of all six genes and demonstrate full pathway reconstruction in yeast from the primary precursors acetyl-CoA and malonyl-CoA. The present study establishes a microbial production platform for plumbagin and provides valuable insights into the biosynthesis of plant-derived naphthoquinones as well as paving the way for the sustainable and scalable production of medicinally important compounds.