ABSTRACT

Dihydrokaempferol (DHK) is a natural flavonoid compound that is widely distributed in plants. It has diverse biological activities and significant application value. The extraction of DHK from plants is costly, and this limitation was addressed in the present study via the construction of a highly efficient synthetic strain. This was achieved by modifying Yarrowia lipolytica through multidimensional metabolic engineering. First, the naringenin synthesis pathway was introduced and subsequently optimized by enhancing shikimate pathway flux, introducing the phosphoketolase pathway, and knocking out competitive pathways. The supply of malonyl-CoA was also optimized to increase the naringenin titer. Next, flavonoid 3-hydroxylase (F3H) was screened, and the highefficiency CsF3HM3 mutant was obtained through protein engineering. Finally, a de novo DHK-synthesizing strain was constructed. The DHK titer reached 3.2 g/L following fed-batch fermentation in a 5 L fermenter, confirming Y. lipolytica as a favorable host for flavonoid synthesis, and providing a reference for improving the synthesis of natural products.

KEYWORDS: dihydrokaempferol, naringenin, multidimensional metabolic engineering, Yarrowia lipolytica, flavonoid 3-hydroxylase, substrate channel remodeling