Abstract: [Objective] Azadirachtin is an active limonoids and its biosynthetic pathway is still poorly understood. Azadirachtin A biosynthesis in cell-free synthesis system from Azadirachta indica leaf was investigated,and its preparation and reaction conditions were optimized. Some selected synthetic precursor of azadirachtin A was screened according to the relative output of azadirachtin A. The azadirachtin A biosynthesis pathway in vitro was discussed, and it would provide a foundation for the construction of azadirachtin allobiosynthesis platform and the realization of azadirachtin allobiosynthesis.[Method] The cell-free synthesis system of azadirachtin A was constructed and optimized through completely randomized and orthogonal experimental design, including buffer solution type, concentration, pH, extraction time and solid-liquid ratio. In addition, the reaction system was optimized, reaction termination reagents and stabilizers, reaction temperature, time, substrate concentration and cofactor. 2,3-oxidosqualene, lanosterol, butyrospermol, euphol, desacetylsalannin, nimbin and salannin were used to study the synthetic precursor of azadirachtin A.[Result] The optimal preparation process of azadirachtin A cell-free synthesis system was that fresh leaves were extracted for 1 h with 200 mmol·L^-1 Tris-HCl buffer (pH7.0) and the solid-liquid ratio was 1:20(g·mL^-1). The optimized cell-free synthesis conditions of azadirachtin A were as follows:Incubations were done in 2 mL centrifuge tubes in a total volume of 800 μL, containing 300 μL of the enzyme preparation diluted in 200 mmol·L^-1 Tris-HCl (pH7.0), 400 μL cofactor mixture (1 mmol·L^-1 Mg²⁺, 1 mmol·L^-1 Mn²⁺, 0.1 mmol·L^-1 ATP, 0.1 mmol·L^-1 NADPH⁺ and 5 mmol·L^-1 ascorbic acid), and 100 μL of 250 μmol·L^-1 substrate. After 60 min incubation at 30℃, the reaction was stopped by the addition of 200 μL acetic acid. The synthesis of azadirachtin A was promoted differently by 2,3-oxidosqualene, butyrospermol, euphol, desacetylsalannin, nimbin and salannin, while lanosterol inhibited the synthesis of azadirachtin A.[Conclusion] Lanosterol was not the precursor of azadirachtin A. It was further verified that 2,3-oxidosqualene and butyrospermol were precursors of azadirachtin A. Furthermore, euphol, nimbin, desacetylsalannin and salannin could be the precursors of azadirachtin A, and salannin was located in the downstream of the synthesis pathway.

Key words: azadirachtin A, cell-free system, precursor screen, biosynthetic pathway