庆大霉素由棘孢小单孢菌（Micromonospora echinospora）产生，包含四个主要组分C1、C1a、C2和C2a，其中C2和C2a为一对C-6’差向异构体，它们具有相同的抑菌活性但C2却表现为无肾毒性和低细胞毒性。棘孢小单孢菌中庆大霉素生物合成基因簇中GenB1和GenB2为磷酸吡哆醛依赖的氨基转移酶，两者具有不同的底物特异性和对映选择性。GenB2为双功能酶，同时具有异构酶活性，催化C2a/C2的异构化反应。本研究课题将通过定点突变技术确定GenB1和GenB2转氨酶活性中心并研究催化机理，从而揭示为什么二者底物特异性和构型选择性不同；阐明GenB2异构酶功能的分子机理，并利用改造后仅保留氨基转移酶活性而失活其异构酶活性的GenB2*，通过组合生物合成技术获得低毒性的庆大霉素C2高产的基因工程菌株。本项目将为全面阐明庆大霉素生物合成机制和研制低毒庆大霉素奠定基础。

Gentamicin produced by Micromonospora echinospora comprises four major compounds, that is, gentamicin C1, C1a, C2, and C2a. C2 and C2a are epimers with the same bactericidal efficacy while C2 exhibits no nephrotoxicity and little cellular toxicity. GenB1 and GenB2 are pyridoxal phosphate (PLP) -linked aminotransferases with different substrate specificity and enantioselectivity. GenB2 is also an epimerase and catalyzed the epimerization of C2a/C2. Firstly, the activity center of GenB1 and GenB2 transaminase will be determined by Site-directed mutagenesis and the catalytic mechanisms will be detected. Secondly, this project is aim to clearly illustrate that the epimerization of gentamicin C2 to gentamicin C2a. Thirdly,GenB2* mutant that inactivation of epimerase but continue to have aminotransferase activity can be used for creating genetically engineered strain that overproduces low toxicity gentamicin C2. This study will determine the biosynthetic pathways of different gentamicin factors, and will lay foundation for combinatorial biosynthesis of low toxicity gentamicin.