Abstract: Four genes have been reported to be involved in serine uptake in Escherichia coli, which were sst T, cyc A, sda C and tdc C.In this study, combinatorial deletion of these genes was performed and effects on L-serine production and strain growth were investigated.The result revealed that, in all 11 multi-gene deletion mutants, sst T·sda C double-gene deletion mutant, sst T·sda C·tdc C and sst T·cyc A·sda C triple-gene deletion mutants had higher L-serine production than the original strain and their L-serine production were 271.11, 312.58 and 322.57 mg/L, respectively. Besides, the sst T·cyc A·sda C·tdc C quadruple-gene deletion mutant achieved the highest L-serine production of 450.58 mg/L, which was 6-fold when compared to that of the original strain.In addition, the cell growth of double-and triple-gene deletion mutants were similar or above to that of the original strain.But the cell growth of the quadruple-gene deletion mutant decreased remarkably, whose OD600 was 28% lower than that of the original strain.