Lin Xuemei, Lambertz Jan, Dahlmann Tim A, Nowaczyk Marc M, König Burghard, Kück Ulrich
Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum, 44780 Bochum, Germany.
Plant Biochemistry, Ruhr-University Bochum, 44780 Bochum, Germany.
J Fungi (Basel). 2022 Apr 26;8(5):450. doi: 10.3390/jof8050450.
The pharmaceutical industry has developed various highly effective semi-synthetic cephalosporins, which are generated by modifying the side chains of the core molecule 7-aminocephalosporanic acid (7-ACA). In industrial productions, the 7-ACA nucleus is obtained in vitro from cephalosporin C (CPC) by chemical or enzymatic processes, which are waste intensive and associated with high production costs. Here, we used a transgenic in vivo approach to express bacterial genes for cephalosporin C acylase (CCA) in the CPC producer . Western blot and mass spectrometry analyses verified that the heterologous enzymes are processed into α- and β-subunits in the fungal cell. Extensive HPLC analysis detected substrates and products of CCAs in both fungal mycelia and culture supernatants, with the highest amount of 7-ACA found in the latter. Using different incubation times, temperatures, and pH values, we explored the optimal conditions for the active bacterial acylase to convert CPC into 7-ACA in the culture supernatant. We calculated that the best transgenic fungal strains exhibit a one-step conversion rate of the bacterial acylase of 30%. Our findings can be considered a remarkable contribution to supporting future pharmaceutical manufacturing processes with reduced production costs.
制药行业已经开发出了多种高效的半合成头孢菌素，这些头孢菌素是通过修饰核心分子7-氨基头孢烷酸（7-ACA）的侧链而产生的。在工业生产中，7-ACA核是通过化学或酶促过程从头孢菌素C（CPC）体外获得的，这些过程浪费资源且生产成本高昂。在此，我们采用了一种体内转基因方法，在CPC产生菌中表达头孢菌素C酰基转移酶（CCA）的细菌基因。蛋白质免疫印迹和质谱分析证实，异源酶在真菌细胞中被加工成α亚基和β亚基。广泛的高效液相色谱分析在真菌菌丝体和培养上清液中均检测到了CCA的底物和产物，其中培养上清液中7-ACA的含量最高。我们通过使用不同的孵育时间、温度和pH值，探索了活性细菌酰基转移酶在培养上清液中将CPC转化为7-ACA的最佳条件。我们计算得出，最佳转基因真菌菌株中细菌酰基转移酶的一步转化率为30%。我们的研究结果可被视为对支持未来降低生产成本的制药制造工艺做出的重大贡献。
