Promoter Regulation of mtx1 in Lysinibacillus sphaericus and Heterologous Production of the Mosquitocidal Protein Mtx1 in Bacillus subtilis

Mtx1 is a mosquitocidal protein that exhibits high toxicity toward Culex species. It is produced during the vegetative phase of Lysinibacillus sphaericus but at very low levels and is rapidly degraded. The low expression appears to result from a weak promoter and a potential regulatory stem-loop structure in the 5′ untranslated region. To investigate this regulation, promoter variants of mtx1 were constructed to disrupt stem-loop formation, and promoter activity was assessed using green fluorescent protein (GFP) as a reporter. Disruption of the inverted repeat resulted in approximately twofold higher fluorescence compared with the wild-type promoter in L. sphaericus 2297, indicating partial derepression of translation. To improve protein stability, Bacillus subtilis WB800N, a protease-deficient host, was employed for heterologous expression. Truncated Mtx1 (tMtx1) was secreted into the culture medium, and no obvious degradation products were detected by Western blot analysis under the conditions tested. Although the overall yield was low and not quantitatively determined, the secreted protein retained biological activity. Larvicidal assays showed elevated mortality in tMtx1-containing culture supernatants, with an estimated LC50 at approximately a 1:83 dilution and detectable activity up to a 1:512 dilution relative to control cultures. These results demonstrate that the upstream inverted repeat contributes to partial repression of mtx1 expression in L. sphaericus and that protease-deficient B. subtilis can be used as a host for producing biologically active tMtx1, although further optimization will be required to improve yield.