Effect Of Manipulation Of Glycolysis And Transport System On L-Threonine Production In Escherichia Coli

L-threonine, an essential amino acid, is widely used in the agricultural, pharmaceutical and cosmetics industries.Acetate is the main by-product of Escherichia coli high cell-density culture (HCDC), and has negative effects on bacterial growth and L-threonine production. Additionally, modifications of transport system, decreasing intracellular concentration of L-threonine, could remove feedbacks of the key enzymes in synthesis approach, which will further improve high yield for L-threonine production.Given acetate formation is correlated with the metabolic flux overflow, namely the carbon flux into glycolysis exceeds the TCA cycle, some glycolysis flux was reduced to minimize acetate production by deleting the pfk or/and pyk gene in E. coli THRD, an L-threonine producer. The experimental results revealed that, in THRDΔpykF, THRDΔpykA, THRDΔpfkA and THRDΔpfkB, acetate concentrations decreased to47.80%,19.54%,1.52%and35.39%of THRD, respectively. THRDΔpykF and THRDΔpykA produced11.05%and5.35%more L-threonine, and achieved a10.91%and5.60%higher yield on glucose, respectively (112.57±2.82、106.79±2.80vs.101.37±2.79g/L).While THRDΔpfkA grew slowly and produced less L-threonine, THRDApfkB produced levels of L-threonine and a yield on glucose similar to that of THRD. The dual deletion mutant THRDΔpfkBApykF gave rise to similar L-threonine concentration with THRDApykF but21.35%less acetate than THRDΔpykF. The level of NADPH in THRDΔpfkA cultures was depressed, while all other mutants produced more NADPH than the THRD. These results demonstrated that modification of glycolysis in E. coli THRD reduced acetate production and increased accumulation of L-threonine.Because the production of acetate is relevance with the uptake of glucose, modification of the sugar transport system is also the important strategy to reduce acetate. Gene ptsG, encoding E Ⅱ CBGlc which plays an important role in the sugar transport system, was deleted in TRFC, an L-threonine producer. According to the results of flask-shaking fermentations, TRFCΔptsG gave rise to similar L-threonine concentrations with TRFC, and had22.83%less acetate. Fed-batch fermentation of TRFCAptsG was carried out in5L fermentor. L-threonine production of TRFCΔptsG was about58.30%of TRFC. Our results suggested that deletion of ptsG could effectively reduce acetate accumulation, but be not conducive to L-threonine production.In Escherichia coli deletion of sstT resulted in a significant reduction in L-threonine uptake rates, while overexpression of rhtC enhanced L-threonine export rates. In this work, gene sstT was deleted in the L-threonine producer E. coli TRFC. And TRFCΔsstT showed a4.00%higher L-threonine production than the control strain TRFC in shake flask fed-batch fermentation. Further, rhtC gene was over expressed in TRFCΔsstT. Compared to TRFCΔsstT+pSTV28, TRFCΔssTr+pSTV28-rhtC were found to produce18.16%more L-threonine concentration in shake flask fed-batch fermentation. Finally, the L-threonine concentration and yield on glucose of TRFCAsstT+pSTV28-rhtC were15.33%and16.14%higher than those of TRFC by the5L fed-batch fermentations, respectively. The results suggested that modification of transport system could effectively decrease the intracellular L-threonine concentration and increase excretion of internal synthesized L-threonine, therefore, could be beneficial for L-threonine production.