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
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.