Cephalosporin C acylase (CCA) is capable of catalyzing cephalosporin C (CPC) to produce 7-aminocephalosporanic acid (7-ACA), an intermediate of semi-synthetic cephalosporins. Inducible expression is usually used for CCA. To improve the efficiency of CCA expression without gene induction, three recombinant strains regulated by constitutive promoters BBa_J23105, PLtetO1, and tac were constructed, respectively. Among them, BBa_J23105 was the best promoter and its mutant libraries were established using saturation mutagenesis. In order to obtain the mutants with enhanced activity, a high-throughput screening method based on flow cytometric sorting techniques was developed by using green fluorescent protein (GFP) as the reporter gene. A series of mutants were screened at 28 °C, 200 rpm, and 24-h culture condition. The study of mutants showed that the enzyme activity, fluorescence intensity, and promoter transcriptional strength were positively correlated. The enzyme activity of the optimal mutant obtained by screening reached 12772 U/L, 3.47 times that of the original strain.

头孢菌素C酰基转移酶（CCA）能够催化头孢菌素C（CPC）产生7-氨基头孢菌素酸（7-ACA），这是半合成头孢菌素的中间体。诱导表达通常用于CCA。为了提高无需基因诱导的CCA表达效率，分别构建了三个由组成型启动子BBa_J23105，PLtetO1和tac调控的重组菌株。其中，BBa_J23105是最好的启动子，其突变库通过饱和诱变建立。为了获得具有增强活性的突变体，以绿色荧光蛋白（GFP）为报告基因，开发了一种基于流式细胞分选技术的高通量筛选方法。在28°C，200 rpm和24 h培养条件下筛选了一系列突变体。突变体的研究表明，酶活性，荧光强度和启动子转录强度呈正相关。通过筛选获得的最佳突变体的酶活性达到12772 U / L，是原始菌株的3.47倍。