[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.


CONCLUSIONS
Overexpression of threonine operon can lead to the accumulation of L-threonine. Further release of feedback inhibition of aspartokinase I can enhance its accumulation.
Zhang
Xue
X
College of Biological Engineering, Tianjin University of Science & Technology, Tianjin 300457, China. meixiabe@yahoo.com.cn
Yan
Ji'ai
J
Yu
Lei
L
Zhang
Guoqiang
G
Zhang
Yun
Y
Chen
Ning
N
Wen
Tingyi
T
D001222:Aspartate Kinase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005285:Fermentation; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors; D009154:Mutation; D009876:Operon; D017853:Phosphotransferases (Alcohol Group Acceptor); D010957:Plasmids / Q000235:genetics*; D013912:Threonine / Q000096:biosynthesis / Q000235:genetics*
D013912:Threonine; D017853:Phosphotransferases (Alcohol Group Acceptor); D001222:Aspartate Kinase