Background: 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.

Objective: 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.

Methods: 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.

Results: 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.

Conclusion: 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.

背景：Mtx2 是一种在球形赖氨酸杆菌营养生长过程中产生的杀蚊剂。该蛋白质与其他毒素对蚊子幼虫有协同作用；因此它可用于蚊虫控制配方。Mtx2 作为生物防治剂的开发需要蛋白质表达系统。

目的：本研究旨在建立枯草芽孢杆菌系统以产生 Mtx2 作为分泌蛋白，因为该蛋白含有推定的信号肽。

方法：最初，使用 GFP 作为枯草芽孢杆菌中的报告基因，比较了四种不同的启动子（P43、Pspac、PxylA 和 PyxiE）的强度。随后，结合选定的启动子和 mtx2 测试了六种不同的信号肽（SacB、Epr、AmyE、AprE、LipA 和 Vip3A），以评估枯草芽孢杆菌 WB800（一种细胞外蛋白酶缺陷株）分泌的 Mtx2 水平。

结果：启动子 PyxiE 显示出最高的 GFP 强度，并被选中用于进一步研究。Mtx2 作为信号肽 LipA 和 AmyE 的分泌蛋白成功产生，它对埃及伊蚊表现出杀幼虫活性。

结论：枯草芽孢杆菌已成功开发为产生分泌型 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。