Abstract: [Objective] We reconstructed two recombinant plasmids and studied their effects on L-threonine accumulation of Escherichia coli W3110. [Methods] We amplified the threonine operon containing ThrLp promoter, lead peptide thrL, thrA thrB and thrC genes by PCR from E. coli W3110 chromosome and ligated it into the pMD19 T-vector. Site-directed mutation were carried out by gene splicing by overlap extension PCR to release the feedback inhibition of aspartokinase I (thrA). Two recombinant plasmids WYE112 and WYE134 were transformed into E. coli W3110 by electroporation. Fed-batch cultures of E. coli W3110 were carried out in 5-Liter fermentors and the L-threonine concentration was measured by HPLC. [Results] Fed-batch fermentation results showed that E. coli W3110 could accumulate little L-threonine (0.036 ± 0.004 g/L) but recombinant E. coli W3110 harboring the plasmid WYE112 containing a threonine operon exhibited a L-threonine production of 2.590 ± 0.115 g/L. Furthermore, L-threonine production reached 9.223 ± 1.279 g/L when the feedback inhibition of thrA was released. [Conclusion] Overexpression of threonine operon can lead to the accumulation of L-threonine. Further release of feedback inhibition of aspartokinase I can enhance its accumulation.