| Summary: | Abstract Background Escherichia coli is an important strain for l-threonine production. Genetic switch is a ubiquitous regulatory tool for gene expression in prokaryotic cells. To sense and regulate intracellular or extracellular chemicals, bacteria evolve a variety of transcription factors. The key enzymes required for l-threonine biosynthesis in E. coli are encoded by the thr operon. The thr operon could coordinate expression of these genes when l-threonine is in short supply in the cell. Results The thrL leader regulatory elements were applied to regulate the expression of genes iclR, arcA, cpxR, gadE, fadR and pykF, while the threonine-activating promoters P cysH , P cysJ and P cysD were applied to regulate the expression of gene aspC, resulting in the increase of l-threonine production in an l-threonine producing E. coli strain TWF001. Firstly, different parts of the regulator thrL were inserted in the iclR regulator region in TWF001, and the best resulting strain TWF063 produced 16.34 g l-threonine from 40 g glucose after 30 h cultivation. Secondly, the gene aspC following different threonine-activating promoters was inserted into the chromosome of TWF063, and the best resulting strain TWF066 produced 17.56 g l-threonine from 40 g glucose after 30 h cultivation. Thirdly, the effect of expression regulation of arcA, cpxR, gadE, pykF and fadR was individually investigated on l-threonine production in TWF001. Finally, using TWF066 as the starting strain, the expression of genes arcA, cpxR, gadE, pykF and fadR was regulated individually or in combination to obtain the best strain for l-threonine production. The resulting strain TWF083, in which the expression of seven genes (iclR, aspC, arcA, cpxR, gadE, pykF, fadR and aspC) was regulated, produced 18.76 g l-threonine from 30 g glucose, 26.50 g l-threonine from 40 g glucose, or 26.93 g l-threonine from 50 g glucose after 30 h cultivation. In 48 h fed-batch fermentation, TWF083 could produce 116.62 g/L l‐threonine with a yield of 0.486 g/g glucose and productivity of 2.43 g/L/h. Conclusion The genetic engineering through the expression regulation of key genes is a better strategy than simple deletion of these genes to improve l-threonine production in E. coli. This strategy has little effect on the intracellular metabolism in the early stage of the growth but could increase l-threonine biosynthesis in the late stage.
|
|---|
