Construction of Cell-Free Synthesis System and Synthetic Precursor of Azadirachtin A

【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