Objectives To improve the S-adenosylmethionine (SAM) production in methionine-free medium, effects of deleting genes of SAM decarboxylase (speD) and homoserine kinase (thrB) on SAM titers were investigated, and the SAM synthetase gene (SAM2) was also overexpressed. Results InB.amyloliquefaciensHSAM2, deletingspeDto block the SAM utilization pathway significantly reduced the SAM titer. After knockout ofthrBto block the branched pathway, the resulted mutant HSAM4 produced 143.93 mg/L SAM, increasing by 42% than HSAM2. Further plasmid-based expression ofSAM2improved the SAM titer to 226.92 mg/L, and final optimization of key fermentation parameters resulted in the maximum SAM titer of 412.01 mg/L in flasks batch fermentation. Conclusions DeletingthrBand overexpressingSAM2gene were efficient for enhanced SAM production inB. amyloliquefaciens. The maximum SAM titer in flasks batch fermentation was much higher than that of previous reports.