Bacillus thuringiensis CT-43 produces thuringiensin.The VirD4 protein encoded by the virD4 gene located on the plasmid pBMB0558 of CT-43 is highly homologous to the coupling proteins (T4CPs) of type IV secretion systems.In type IV secretion systems,T4CPs’ basic function is to recruit substrates to the type IV secretion systems for secretion through the translocation channel.A homologous double - crossover vector was constructed through knocking out the virD4 gene in CT-43 and a mutant BMB1122 was obtained.HPLC result showed that the characteristic peak of thuringiensin was not detected in the fermentation supernatant of mutant BMB1122.The LCMS-IT-TOF result showed that the corresponding m/z with molecular weight of proposed precursors in Thu biosynthesis could be detected in intracellular metabolites of mutant BMB1122,consistent with that of CT-43.The knockout of virD4 gene interrupted the secretion of thuringiensin.Presumably,the virD4 was a critical gene for Thu biosynthesis.