Metabolic engineering of escherichia coli for the production of L-threonine

Abstract

A genetically defined L-threonine production strain of Escherichia coli was constructed by rational metabolic engineering. Feedback inhibition of aspartokinase I and III by L-threonine and L-lysine, respectively, were removed by site-directed mutagenesis and the native promoter containing the transcriptional attenuator leader regions of the thrABC operon was replaced with the tac promoter. The lysA and metA genes were deleted to make more precursors available for L-threonine. The point mutation in the structural gene of ilvA and deletion of tdh gene were carried out to prevent the threonine degradation. This engineered TH07 strain harboring a plasmid overexpressing the threonine operon showed a yield of 0.146 g L-threonine per g glucose. Engineering of central carbon metabolism, especially anaplerotic pathways, was performed based on comparative transcriptome analysis with the aid of in silico simulation approach. Promoter replacement of the native ppc promoter with a trc promoter and deletion of iclR gene for activating glyoxylate shunt enzymes led to the enhanced production of L-threonine by 51%. From the transcriptome profiling, further improvement was achieved by deletion of tdcC gene which encoding an anaerobic threonine transporter and amplification of three kinds of threonine exporters. The resulted TH27C strain (TH07 $\Delta iclR$ Pppc::Ptrc tdcC::$Cm^R$) overexpressing the $thrA^{S345P} BC$, rhtC, rhtA, and rhtB was able to produce 11.8 g/l L-threonine from 30 g/l glucose in batch culture, resulting in a impressively high yield of 0.393 g L-threonine per g glucose. Finally, based on in silico simulation approach, an improved strain more applicable to industrial fermentation process was developed by replacing a native acs promoter with the strong trc promoter, threreby decreasing the acetate accumulation during fermentation. With fed-batch fermentation, 82.4 g/l of L-threonine was produced from 280 g/l of glucose after 50 hours cultivation. Thes...