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L-Serine is widely used in pharmaceutical, food and cosmetic industries, and the direct fermentation to produce L-serine from cheap carbon sources such as glycerol is greatly desired. The production of L-serine by engineered Escherichia coli from glycerol has not been achieved so far. In this study, E. coli was engineered to efficiently produce L-serine from glycerol. To this end, three L-serine deaminase genes were deleted in turn, and all of the deletions caused the maximal accumulation of L-serine at 0.06 g/L. Furthermore, removal of feedback inhibition by L-serine resulted in a titer of 1.1 g/L. Additionally, adaptive laboratory evolution was employed to improve glycerol utilization in combination with the overexpression of the cysteine/acetyl serine transporter gene eamA, leading to 2.36 g/L L-serine (23.6% of the theoretical yield). In 5-L bioreactor, L-serine titer could reach up to 7.53 g/L from glycerol, demonstrating the potential of the established strain and bioprocess. [ABSTRACT FROM AUTHOR]