Fengycin is a of cyclic lipopeptide with antifungal, antitumor and adhesion-preventing activities, with great application potential in biological control, medicine and industry. However, the fermentation yield of fengycin is inadequate, which limits its wider application. In this study, key modules for enhanced fatty acid synthesis were cloned singly and in combination, and the suitability of these modules for B. subtilis ATCC 21332 was investigated. We constructed the engineered strain BSA034, which exhibited a 3-fold increase of fengycin titer, which reached 442.51 mg/L. Reverse transcription and quantitative PCR (RT-qPCR) revealed the effect of enhanced branched-chain fatty acid synthesis on the expression level of the fen operon in B. subtilis ATCC 21332, which significantly contributed to the increase of fengycin production. The exogenous supplementation of glutamate further increased the fengycin titer of BSA034 to 657.55 mg/L. Further comparative metabolomic analysis revealed that glutamate mainly promoted the membrane transport of BSA034, which provides a rational basis for further improving the titer of fengycin in the future.