State Key Laboratory of New Drug and Pharmaceutical Process, Shanghai Institute of Pharmaceutical Industry, 1111 Zhongshan Road (North No. 1), Shanghai, 200437, China.
Curr Microbiol. 2010 Dec;61(6):609-14. doi: 10.1007/s00284-010-9660-z. Epub 2010 May 9.
7-Amino cephalosporanic acid (7-ACA), which is currently obtained by chemical deacylation from cephalosporin C (CPC), is a major intermediate for industrial production of β-lactam antibiotics. 7-ACA can also be produced from CPC by enzymatic route including two-step and one-step procedures. In our research, an ecs gene coding for CPC acylase was synthesized and cloned into pET-28a(+) to construct an E. coli expression plasmid pYG232. E. coli BL21(DE3) bearing pYG232 was induced by IPTG and successfully expressed the recombinant ECS (88.9 kDa). Under the optimal conditions: 0.5 mg/ml purified ECS protein, 5 mg/ml CPC, 100 mM Tris-Cl (pH 9.6), supplement with 7 mM Zn(2+), slightly shaking for 6 h at 25°C, the transformation productivity was 54.4%. Then, ecs was cloned downstream of an A. chrysogenum endogenous promotor, PpcbC, to construct pYG233 for expression in A. chrysogenum. pYG233 was introduced into a CPC high-producer via integrative transformation of protoplasts. Two independent bleomycin-resistant transformants were investigated by PCR, Southern blotting, quantitative RT-PCR, western blotting, and fermentation. Although these two transformants both have one copy of integrated ecs, they showed different expression level of ECS protein and 7-ACA production. When concentration of CaCO(3) was reduced to 50 mM, ZnSO(4) was increased to 7 mM, CuSO(4) was eliminated from the fermentation media, and the pH was adjusted to 8.0 at day 4 during fermentation, 7-ACA production of one of the transformants could reach 1701 μg/ml, indicated that more than 30% of CPC produced by this high-producer have been transformed into 7-ACA directly in vivo. This is the highest 7-ACA production by direct fermentation ever reported.
7-氨基头孢烷酸（7-ACA）目前是通过化学脱酰基作用从头孢菌素 C（CPC）中获得的，是工业生产β-内酰胺抗生素的主要中间体。7-ACA 也可以通过包括两步法和一步法在内的酶法途径从 CPC 中获得。在我们的研究中，合成了编码 CPC 酰化酶的 ecs 基因，并将其克隆到 pET-28a(+)中，构建了大肠杆菌表达质粒 pYG232。携带 pYG232 的大肠杆菌 BL21(DE3) 被 IPTG 诱导，并成功表达了重组 ECS（88.9 kDa）。在最佳条件下：0.5 mg/ml 纯化的 ECS 蛋白、5 mg/ml CPC、100 mM Tris-Cl（pH 9.6）、补充 7 mM Zn(2+)、在 25°C 下轻微摇动 6 小时，转化率为 54.4%。然后，ecs 被克隆到一个 A. chrysogenum 内源性启动子 PpcbC 的下游，构建了用于在 A. chrysogenum 中表达的 pYG233。pYG233 通过原生质体的整合转化被引入到一个 CPC 高产菌中。通过 PCR、Southern 印迹、定量 RT-PCR、western 印迹和发酵对两个独立的博来霉素抗性转化体进行了研究。尽管这两个转化体都有一个整合的 ecs 拷贝，但它们表现出不同的 ECS 蛋白表达水平和 7-ACA 产量。当 CaCO(3)浓度降低到 50 mM、ZnSO(4)增加到 7 mM、发酵培养基中去除 CuSO(4)、并在发酵第 4 天将 pH 调整到 8.0 时，其中一个转化体的 7-ACA 产量可达到 1701 μg/ml，表明该高产菌产生的 CPC 中有超过 30%已直接在体内转化为 7-ACA。这是迄今为止直接发酵生产 7-ACA 的最高产量。
