Effects Of Trimethylglycine On L-threonine Production In Escherichia Coli

L-threonine is one of the eight essential amino acids of human and animals.It is widely used in medicine,food,feed and other industries.Trimethylglycine can increase enzyme activity,improve membrane permeability and provide methyl groups.Escherichia coli is a production strain of L-threonine,However,it is challenged by high salt concentration in the fermentation broth,in which the continuous addition of neutralizing agents is required to maintain broth pH within an optimal range for the constant accumulation of cell growth,metabolism,and product.In this paper,the effects of trimethylglycine supplementation on key enzymes activity of L-threonine biosynthesis were studied,and transcriptomic analysis and proteomic analysis were performed during the E.coli JLTHR fermentation process.(1)The effect of trimethylglycine on enzyme activity was examined by using glucose solution without trimethylglycine as the control group.Hexokinase activity increased by 19.3%,the enzyme activity of 6-phosphogluconate dehydrogenase increased by 10.8%,phosphofructokinase activity increased by 10.5%,pyruvate dehydrogenase activity increased by 24.3%,phosphoenolpyruvate carboxylase activity increased by 16.3%,glutamate synthase activity increased by 15.6%,aspartokinase activity increased by 13.2%,homoserine dehydrogenase enzyme activity increased by 27.5%,dihydrodipicolinate synthase activity increased by 14.4%,homoserine kinase activity increased by 20.6%,succinyl homoserine synthase activity increased by 12.6%.(2)The effect of trimethylglycine on the gene transcription level was tested in a fermentation experiment in which E.coli JLTHR was treated with glucose solution without trimethylglycine as a control.The results showed that the cell composition and the expression of some genes related to cell membrane synthesis were increased when the fermentation broth was fed with glucose solution containing 1.5 g/L trimethylglycine.The transcription level of the glycolytic pathway,the tricarboxylic acid cycle,and the pentose phosphate pathway appeared to be upregulated overall.In addition,analysis of amino acid biosynthetic pathways showed that the transcript levels of the cysteine and methionine biosynthetic pathways were reduced to some extent,and the transcription levels of glycine,serine,threonine,valine,leucine and isoleucine were increased to some extent.(3)In this experiment,a quantitative method based on iTRAQ was established,a glucose solution without trimethylglycine was used as a control group to detect the effect of trimethylglycine on the expression level of the protein.In the whole analysis,732 proteins were up-regulated and 811 proteins were down-regulated.There were 14 functional categories for the major molecular functions involved in differential proteins,13 functional categories for participating cellular components,and 19 functional categories for participating biological processes.