Metabolic Engineering Of Escherichia Coli For L-threonine Production

L-threonine is an important amino acid that can be added in food,medicine,or feed.L-threonine produced by microbial fermentation has an increasing market demand.Escherichia coli became the important strain for L-threonine production.Though the industrial fermentation technology of L-threonine achieved a very high level,there is still significant room to theoretical yield.In this study,metabolic engineering strategies are used to further enhance the production of L-threonine in a threonine-producer TWF001.The main results are as follows:?1?Enhancing the glyoxylate shunt by deleting iclR has been proved useful in increasing the production of L-threonine in TWF001.We had replaced the two IclR-binding boxes and the native promoter of the aceBAK operon with the trc promoter in TWF001,but the L-threonine production of the resulted strain TWF002 decreased sharply.The production of L-threonine increased 26%in strain TWF003 compared to the control TWF001,in which iclR gene was deleted.The two IclR-binding boxes and the native promoter of the aceBAK operon were replaced by the trc promoter in TWF003,resulted in the strain TWF004.To our surprise,L-threonine production in TWF004 increased 27%comparing to the control TWF001.?2?Strengthening the aspartate aminotransferase in TWF003,the production of L-threonine decreased;strengthening the aspartate aminotransferase in TWF004,the production of L-threonine increased.To draw the carbon flow from Krebs cycle to L-threonine biosynthesis,the trc promoter was inserted in the upstream of aspC in TWF003and TWF004,resulting in TWF005 and TWF006,respectively.Although L-threonine production in TWF005 was similar to that in TWF001,TWF006 produced 12.47 g·L^-1L-threonine,which is 34%increase compared to the control TWF001.?3?Three key genes thrA^*,thrB,and thrC in the L-threonine biosynthetic pathway in E.coli were inserted in pFW01,resulting pFW01-thrA^*BC,and overexpressed in above-mentioned mutant strains to further increase L-threonine production.The thrA^*is a thrA mutant in which the 1034^th C has been replaced with T,and it encodes a mutant of aspartate kinase I which is resistant to feedback inhibition.The L-threonine production of TWF002/pFW01-thrA^*BC,TWF003/pFW01-thrA^*BC,TWF004/pFW01-thrA^*BC,TWF005/pFW01-thrA^*BC,TWF006/pFW01-thrA^*BC increased in different degrees comparing to the vector control strain,which proved effective overexpression of the thrA^*BC operon.TWF006/p FW01-thrA^*BC produced the highest yield of L-threonine among all the strains.The L-threonine production of TWF006/pFW01-thrA^*BC increased 58%than the control TWF001.?4?To further increase L-threonine production,the gene asd encoding aspartate semialdehyde dehydrogenase and three genes encoding the L-threonine exporters were overexpressed in TWF006,together with the genes thrA^*BC,resulting in TWF006/pFW01-thrA^*BC-asd,TWF006/pFW01-thrA^*BC-rhtA,TWF006/pFW01-thrA^*BC-rhtC,and TWF006/pFW01-thrA^*BC-thrE.After 36 h fermentation,L-threonine production in TWF006/pFW01-thrA^*BC-asd reached 15.85 g·L^-1,which is 70%increase compared to the control TWF001.?5?The L-threonine production of TWF006/pFW01-thrA^*BC-asd increased than that with TWF001 in fed-batch fermentation.TWF006/pFW01-thrA^*BC-asd produced 95.73 g·L^-1L-threonine after 48 h fed-batch fermentation in a 2-L jar,while TWF001 produced 79.07g·L^-1 L-threonine.The L-threonine production and the overall yield increased 21%and 29%in TWF006/pFW01-thrA^*BC-asd than the control TWF001,respectively.