Carbon Distribution And Cofactor Generation Facilitate L-threonine Production Of Escherichia Coli

L-threonine,a kind of essential amino acid,has numerous applications in food,pharmaceutical and aquaculture industries.Fermentative L-threonine production from glucose has been achieved in Escherichia coli.However,there are still several limiting factors severely hindering further improvement of L-threonine productivity in industry,such as the conflict between cell growth and production,byproduct accumulation,and insufficient availability of ATP and cofactor.Here,we proposed several strategies to alleviate the above problems in Escherichia coli THRD,an L-threonine-overproducing strain.First,oxaloacetate and ATP levels were increased by heterologous expression of pckA-encoded phosphoenolpyruvate carboxykinase from Mannheiemia succiniciproducens and pycA-encoded pyruvate carboxylase from Lactococcus lactis.The final threonine yield of the constructed strain THPR1 in shake flask fermentation was 1.22 g/L,which was 12.5% higher than THRD yield(1.08 g/L).Secondly,based on the strain THPR1,citA from Aspergillus niger was induced to enhance the activity of citrate synthase in the early stage,and the aspC gene encoding the aspartate aminotransferase was overexpressed at the late fermentation stage to achieve the stepwise carbon distribution between cell growth and L-threonine production.The threonine yield of the constructed strain THPE2 reached 1.62 g/L after 24 h fermentation,which was 49.7% higher than that of THRD.Finally,to compensate for the lack of intracellular reducing power of the strain during fermentation process,the formate dehydrogenase FDH from Candida boidinii was introduced into the strain THPE2 to increase the supply of NADH.In order to promote the transamination reaction in the threonine production pathway,a glutamic acid replenishment system was constructed by overexpressing gdhA,which encoding glutamate dehydrogenase.And the pntAB gene encoding pyridine nucleotide dehydrogenase was overexpressed to regenerate NADPH from NADH.By optimizing the concentration of sodium formate in the fermentation broth,the L-threonine yield of the final strain THPE5 reached 2.18 g/L,which was 1.02 times higher than that in the original strain THRD.The higher threonine producing E.coli strains constructed in this paper will facilitate the further modification and industrial application of threonine strains.At the same time,the strain transformation strategy proposed in this paper also provides a new idea for production of other metabolites in E.coli.