[Construction of recombinant plasmids containing threonine operon and their effects on L-threonine accumulation].

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 pWYE112 and pWYE134 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 pWYE112 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.