Abstract
Hyoscyamine and scopolamine are two pharmaceutical tropane alkaloids (TAs) whose biosynthesis is dependent on tropinone and littorine. The biosynthesis process of tropinone and littorine was elucidated only very recently, with the discoveries of four novel TAs biosynthesis genes, namely type III polyketide synthase ( PYKS ), tropinone synthase ( CYP82M3 ), phenyllactate UDP-glycosyltransferase ( UGT1 ), and littorine synthase ( LS ). Yet their values in engineering TAs production remain unknown. In this study, their roles in engineering TAs’ production were studied via gene overexpression methods applied to root cultures of Atropa belladonna . PYKS or CYP82M3 overexpressed separately did not or slightly affected the biosynthesis of tropine, and had no effect on the accumulation of TAs. Co-overexpression of PYKS and CYP82M3 led to much higher levels of tropinone and tropine than those produced by the overexpression of either PYKS or CYP82M3 . Overexpression of either UGT1 or LS significantly elevated the production of littorine, hyoscyamine, anisodamine, and scopolamine. Higher levels of these TAs were produced when UGT1 and LS were overexpressed together. Finally, when the four TA biosynthesis genes were overexpressed together, the highest production of TAs was obtained, indicating that synergistic effect did significantly augment TAs’ production. The effects overexpressing PYKS, CYP82M3, PYKS+CYP82M3, UGT1, LS, and UGT1+LS on the biosynthesis of tropane alkaloids in root cultures of Atropa belladonna . ↑, increased production; ↓, decreased production; -, no alteration. • Co-overexpressing PYKS and CYM82M3 increased tropinone and tropine contents. • Co-overexpressing UGT1 and LS increased hyoscyamine and scopolamine contents. • Four genes co-overexpressed, the highest levels of tropane alkaloids were obtained. • Tropinone and littorine biosynthesis are two rate-limiting steps.
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