绿灰霉素生物合成途径中3-羟基吡啶甲酸起始结构单元的活化特异性

Substrate specificity of 3-hydroxypicolinic acid unit in the viridogriseinbiosynthetic pathway

摘要:

摘要:【目的】鉴定sgvS 是绿灰霉素生物合成起始结构单元3-羟基吡啶甲酸的必需基因;测定绿灰霉素起始结构单元3-羟基吡啶甲酸的活化特异性。【方法】构建sgvS 的基因失活突变株和反式回补菌株，对其发酵产物进行HPLC 分析。并在突变菌株sgvS 的发酵液中分别添加3-羟基吡啶甲酸，吡啶-2-甲酸，3-氯-吡啶-2-甲酸，4-氯-吡啶-2-甲酸，3，5-二氯-吡啶-2-甲酸，烟酸，2-氟-烟酸，2-氯-烟酸，5-氟-烟酸，6-氟-烟酸和环哌啶-2-甲酸，用HPLC 分析其代谢产物的变化。【结果】突变菌株sgvS 丧失了绿灰霉素的生产能力，sgvS 基因反式回补突变株恢复了绿灰霉素的生产能力。向突变菌株sgvS 中添加3-羟基吡啶甲酸也能恢复绿灰霉素的生产，但添加其它上述衍生物均无法恢复绿灰霉素的生产或介导产生新的结构类似物。【结论】sgvS 是绿灰霉素及其骨架起始单元3-羟基吡啶甲酸生物合成的必需基因，SgvD1 对3-羟基吡啶甲酸结构单元的激活存在着严格的底物选择特异性。

Abstract:

Abstract:［Objective］To explore the role of sgvS in the biosynthesis of 3-hydroxypicolinic acid moiety ofviridogrsein，and to analyze the substrate specificity of 3-hydroxypicolinic acid moiety in the viridogrsein biosynthetic pathway.［Methods］Through gene insertion inactivation and in trans complementation strategies，we obtained the gene inactivation mutant sgvS andits complementation mutant ΔsgvS:: sgvS． Meanwhile， we fed 3-hydroxypicolinic acid， picolinic acid， 2-piperidinecarboxylic acid，3-chloropyridinecarboxylic acid，4-chloropyridinecarboxylic acid，3，5-chloropyridinecarboxylic acid，nicotinic acid，2-chloronicotinic acid，2-fluoronicotinic acid acid，5-fluoronicotinic acid and 6-fluoronicotinic acid to the sgvSmutant，respectively．Thefermentation extracts were analyzed by HPLC.［Results］ sgvS mutant abolished the viridogrsein production; viridogrsein production was restored through in trans complementation of sgvS mutant or by feeding 3-hydroxypicolinic acid to the sgvS mutant．No new viridogrsein analogues were observed by feeding other above mentioned 3-hydroxypicolinic acid analogues tothe ΔsgvS mutant.［Conclusion］sgvS is necessary for the biosynthesis of 3- hydroxypicolinic acid moiety．The biosynthetic protein，SgvD1，activates 3-hydroxypicolinic acid，showing strict substrate specificity en route to the viridogrsein biosynthesis.