Metabolic engineering of Saccharomyces cerevisiae for efficient production of dihydroartemisinic acid.

Artemisinin is a natural antimalarial drug recommended by the World Health Organization. To address the limitations of natural supply, semi-synthetic production provides a promising alternative strategy. Current semi-synthetic routes typically rely on asymmetric heavy metal-involved hydrogenation to convert artemisinic acid (AA) to dihydroartemisinic acid (DHAA). This study aimed to construct an engineered microbial platform capable of directly producing dihydroartemisinic acid to support artemisinin semi-synthesis. The gene AaDbr2, encoding a key enzyme redirecting metabolic flux from artemisinic acid toward dihydroartemisinic acid biosynthesis, was first introduced into an artemisinic acid-producing strain, and promoter strength was optimized to enhance expression, achieving a dihydroartemisinic acid titer of 79.4 mg/L. The aldehyde dehydrogenase ALDH1 catalyzes the oxidation of artemisinic aldehyde and dihydroartemisinic aldehyde, resulting in the formation of AA and DHAA, respectively. The two genes AaDbr2 and ALDH1 were thus fused in the genome and this strategy largely increased DHAA/AA ratio from 0.6 to 4.2. Furthermore, DHAA production was improved through multiple metabolic engineering strategies, including cofactor engineering to enhance NADPH availability, regulation of diauxic growth behavior to balance cellular proliferation and product synthesis, and optimization of the P450 electron transport system. Finally, batch and fed-batch fermentation in a 5 L bioreactor further increased DHAA production, reaching a final titer of 6.8 g/L, which represents the highest reported DHAA titer to date. This study demonstrates the potential of microbial platforms for the sustainable, economical, and scalable production of dihydroartemisinic acid, paving the way for the industrial semi-synthesis of artemisinin.