Supplementary Material for: A Simple Method for Removal of the <b><i>Chlamydomonas reinhardtii</i></b> Cell Wall Using a Commercially Available Subtilisin (Alcalase)

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 <i>Chlamydomonas reinhardtii</i> 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 <i>C. reinhardtii</i>. 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 <i>C. reinhardtii</i>, and shared conserved active domains, which may explain the effective cell wall degradation. Our trans­formation method using commercial subtilisin is more reliable and time saving than the conventional method using autolysin released from gametes for cell wall lysis.

藻类细胞壁是基因工程中转基因递送的强效屏障。针对高等植物开发的常规转化方法，往往难以穿透或去除藻类细胞壁，这源于后者独特的物理与化学特性。为此，我们依托商用酶制剂，开发了一种简便的莱茵衣藻（Chlamydomonas reinhardtii）转化方法。在针对细胞壁降解筛选的7种酶中，商用枯草杆菌蛋白酶（subtilisin）制剂Alcalase在0.3安森单位/毫升的低浓度下效果最优。其转化效率与配子溶酶（gamete lytic enzyme）相当——该蛋白酶是莱茵衣藻遗传转化中常用的工具酶。本研究的非侵入式转化方法，其转化效率与此前采用自溶素（autolysin）作为细胞壁降解酶、结合玻璃珠转化法（glass bead transformation）的经典方法相当。枯草杆菌蛋白酶与莱茵衣藻的孵化酶孢囊素（sporangin）序列同源性约为35%，且二者共享保守的活性结构域，这或可解释其高效降解细胞壁的机制。相较于传统的从配子中提取自溶素以裂解细胞壁的方法，我们采用商用枯草杆菌蛋白酶的转化方案更为可靠且耗时更短。