The algal cell wall is a potent barrier for delivery of transgenes for genetic engineering. Conventional methods developed for higher plant systems are often unable to penetrate or remove algal cell walls owing to their unique physical and chemical properties. Therefore, we developed a simple transformation method for Chlamydomonas reinhardtii using commercially available enzymes. Out of 7 enzymes screened for cell wall disruption, a commercial form of subtilisin (Alcalase) was the most effective at a low concentration (0.3 Anson units/mL). The efficiency was comparable to that of gamete lytic enzyme, a protease commonly used for the genetic transformation of C. reinhardtii. The transformation efficiency of our noninvasive method was similar to that of previous methods using autolysin as a cell wall-degrading enzyme in conjunction with glass bead transformation. Subtilisin showed approximately 35% sequence identity with sporangin, a hatching enzyme of C. reinhardtii, and shared conserved active domains, which may explain the effective cell wall degradation. Our trans-formation method using commercial subtilisin is more reliable and time saving than the conventional method using autolysin released from gametes for cell wall lysis.

中文翻译：

---

使用市售枯草杆菌蛋白酶（Alcalase）去除莱茵衣藻细胞壁的简单方法。

藻细胞壁是传递转基因进行基因工程的有效屏障。为高级植物系统开发的常规方法由于其独特的物理和化学特性，通常无法穿透或去除藻类细胞壁。因此，我们开发了一种使用商业上可获得的酶对莱茵衣藻的简单转化方法。在筛选用于细胞壁破坏的7种酶中，枯草杆菌蛋白酶（Alcalase）的商业形式在低浓度（0.3 Anson单位/ mL）时最有效。效率与配子体分解酶相当，后者是通常用于莱茵衣藻遗传转化的一种蛋白酶。我们的非侵入性方法的转化效率与以前使用自溶素作为细胞壁降解酶并结合玻璃珠转化的方法相似。枯草杆菌蛋白酶显示与孢子囊蛋白（莱茵衣藻的孵化酶）具有大约35％的序列同一性，并且共有保守的活性域，这可以解释有效的细胞壁降解。与使用从配子中释放的自溶素进行细胞壁裂解的常规方法相比，我们使用商业枯草杆菌蛋白酶的转化方法更可靠，更省时。