Abstract: Twin-arginine translocation gene (tatC) of Lysobacter enzymogenes strain OH11 was knocked out by double-cross homologue recombination. Functions of tatC were investigated by comparisons of phenotypes and phenotype-associated gene expressions between wild type and tatC mutant, more attentions were paid on the relationship between tatC and HSAF, an antifungal secondary metabolite produced by L. enzymogenes. Compared with wild-type OH11, mutation of tatC significantly increased the production of HSAF and the transcriptional expression level of pks/nrps, which was a key gene responsible for HSAF biosynthesis. It was observed that the normal clavated cell morphology was changed into ellipticity when disruption of tatC in L. enzymogenes. Furthermore, mutation of tatC remarkably impacted the growth rate in nutrient-limiting broth, biofilm formation and gliding motility. However,MtatC mutation of tatC did not impact the growth rate in nutrient-rich broth, four-type lytic enzyme production, and inhibitory activities against Pythium apanidermatum, Rhizoctonia solani and Fusarium graminearum.