Effects of soybean oil and alanine on rifamycin SV biosynthesis in 250 mL flask were studied in this paper.When 3 mL/L soybean oil and 0.8 g/L alanine was added into the medium at 72 h,rifamycin SV production was increased by 5.3% and 6.9% over the control,respectively.Response surface methodology was used to study the interaction between soybean oil and alanine.Result showed that their optimal amount was 2.57 mL/L and 0.89 g/L,respectively.Under the optimal conditions,rifamycin SV production reached 5179 U/mL that was 12.3% higher than control.The results of organic acids analysis demonstrated that utilization rate of a-ketoglutarate and citriate was 0.86 mg/(L·h) and 0.77 mg/(L·h),respectively,which was 115.7% and 23.3% higher than that of control in the fermentation process from 72 h to120 h.Meanwhile,succinate concentration reached 1.01 g/L that was 16.1% higher than the control.The result indicated that the conversion efficiency of a-ketoglutarate and citrate to succinate in the TCA cycle was greatly improved with the addition of oil and alanine,which led to enhanced synthesis of succinyl-CoA and methylmalony-CoA and the facilitation of rifamycin SV biosynthesis.