Construction and fermentation of L-threonine-producing recombinant Escherichia coli

Abstract:

[Background] Escherichia coli is often used to produce threonine because of its excellent growth performance and clear genetic background. [Objective] In order to construct a high-yield strain of threonine, the nonessential genes of the E. coli THR in threonine biosynthetic pathway were knocked out while the key enzymes essential for threonine synthesis were heterologously expressed. [Methods] The lysC, pfkB and sstT of E. coli THR were deleted by using the FLP/FRT recombinant enzyme system. In addition, the recombinant plasmid with the genes lysCfbr and thrE from Corynebacterium glutamicum and the gene gapC from Clostridium acetobutylicum was constructed. Then this plasmid was introduced into different host strains. [Results] The target strain E. coli THR3 as obtained from the parental strain E. coli THR by deleting its threonine synthesis pathway: aspartate kinase III-coding gene lysC and phospho fructose kinase II-coding gene pfkB as well as the threonine absorption protein-coding gene sstT. The threonine production of strain E. coli THR3 as up to 75.64±0.35 g/L, was 9.9% higher than that of the parental stain E. coli THR (68.7 g/L). In addition, heterogonous expression of threonine secretory transporter-coding gene thrE and aspartic kinase-coding lysCfbr from C. glutamicum as well as heterologous expression of NADP+ dependent glyceraldehyde-3-phosphate dehydrogenase coding gapC from Clostridium acetobutylicum were beneficial to increase the threonine production. The recombinant strain E. coli THR6 could produce 105.3±0.5 g/L of threonine with a biomass of 18.26 g DCW/L and the threonine yield (g/g) from glucose increased by 43.20% and the acid production capacity per unit increased to 5.76 g/g DCW in fed-batch culture accumulation. [Conclusion] Knocking out a gene alone or modifying a pathway does not allow large amounts of threonine to be synthesized and accumulated. Co-transformation of multiple metabolic pathways is the most effective way to construct threonine engineered strains.