National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathum Thani 12120, Thailand.
Protein Pept Lett. 2021;28(9):1054-1060. doi: 10.2174/0929866528666210616103337.
Mtx2 is a mosquitocidal toxin produced during the vegetative growth of Lysinibacillus sphaericus. The protein shows synergism with other toxins against mosquito larvae; hence it could be used in mosquito control formulations. The protein expression system is needed for Mtx2 development as a biocontrol agent.
This study aimed to set up a Bacillus subtilis system to produce Mtx2 as a secreted protein since the protein contains a putative signal peptide.
Initially, four different promoters (P43, Pspac, PxylA, and PyxiE) were compared for their strength using GFP as a reporter in B. subtilis. Subsequently, six different signal peptides (SacB, Epr, AmyE, AprE, LipA, and Vip3A) were tested in conjunction with the selected promoter and mtx2 to evaluate levels of Mtx2 secreted by B. subtilis WB800, an extracellular protease-deficient strain.
The promoter PyxiE showed the highest GFP intensity and was selected for further study. Mtx2 was successfully produced as a secreted protein from signal peptides LipA and AmyE, and it exhibited larvicidal activity against Aedes aegypti.
B. subtilis was successfully developed as a host for the production of secreted Mtx2, and the protein retained its larvicidal activity. Although the Mtx2 production level still needs improvement, the constructed plasmids could be used to produce other soluble proteins.
Mtx2 是一种苏云金芽孢杆菌在营养生长阶段产生的杀蚊毒素。该蛋白与其他毒素对蚊虫幼虫有协同作用；因此，它可以用于蚊虫防治制剂。需要有 Mtx2 蛋白表达系统来开发它作为一种生物防治剂。
本研究旨在建立枯草芽孢杆菌系统来生产 Mtx2 作为一种分泌蛋白，因为该蛋白含有一个假定的信号肽。
最初，使用 GFP 作为报告蛋白比较了四个不同的启动子（P43、Pspac、PxylA 和 PyxiE）在枯草芽孢杆菌中的强度。随后，测试了六个不同的信号肽（SacB、Epr、AmyE、AprE、LipA 和 Vip3A）与选定的启动子和 mtx2 结合，以评估枯草芽孢杆菌 WB800（一种缺乏细胞外蛋白酶的菌株）分泌的 Mtx2 水平。
启动子 PyxiE 显示出最高的 GFP 强度，被选为进一步研究的对象。Mtx2 成功地从 LipA 和 AmyE 信号肽作为分泌蛋白产生，并表现出对埃及伊蚊的杀幼虫活性。
成功地将枯草芽孢杆菌开发为生产分泌型 Mtx2 的宿主，并且该蛋白保留了其杀幼虫活性。尽管 Mtx2 的生产水平仍有待提高，但构建的质粒可用于生产其他可溶性蛋白。
