Metabolic Engineering Modification And Fermentation Of Corynebacterium Glutamicum For L-threonine Production

L-threonine,one of the essential amino acid species in humans and animals,has a wide range of applications in many aspects,prompting an increasing demand for L-threonine.Therefore,it is very necessary to realize the construction of a high-yield L-threonine strain.Based on the characteristics of L-threonine biosynthesis,based on the selection of the original L-isoleucine glutamicum Ile903?Corynebacterium glutamicum Ile903?,the electroporation scheme for this strain was first established and then adopted.Metabolic engineering and genetic engineering breeding strategies were used to metabolically modify the biosynthesis pathway of L-threonine and over-expression of related genes and to investigate the accumulation of L-threonine.Finally,based on the original fermentation formula,the organic nitrogen source was properly optimized,and the high-density fermentation characteristics of the mutant were verified by fed-batch fermentation of 30L tank.The main research conclusions are summarized as follows:?1?According to the characteristics of the native L-isoleucine C.glutamicum Ile903,an electrotransformation method suitable for the strain was established under the original electroporation protocol:compatible cells were prepared by incubating cells in BMG medium containing 1.0 g/L of Tween 80 and 3.0 g/L of glycine to 1-1.2 of OD_562nm.The electric field intensity was set at 0.8-0.9 kv/mm,immediately after the electroporation,800?L of BMGS containing 0.5 mol/L sorbitol was added,and immediately placed in a water bath at 46°C for 8 min,and the cell modification system was restricted.The resuscitation culture was cultured at 37°C,200 r/min for 1 h,and then resuscitated at30°C,220 r/min for 1-2 h,and then coated with kanamycin resistance BMG tablet.Under this condition,it facilitates the transformation of foreign DNA into the host cell by electric shock.?2?From the perspective of metabolic engineering breeding,the side branch of the L-threonine synthesis pathway is metabolically modified to block or weaken the carbon flux of the metabolic branch,achieve the goal of“throttle”so as to study the yield of L-threonine was changed,and the metabolically engineered strain Ile903-MX was subjected to shake flask fermentation.The results showed that the accumulation of L-threonine was increased,and the yield of L-threonine was 2.36 times higher than that of the control strain Ile903,which was increase from 1.02 g/L to 3.43 g/L.?3?On the basis of“throttle”,the constructed strain Ile903-MX was further“open sourced”,mainly overexpressing the two key enzymes of the L-threonine synthesis pathway,aspC and lysC,and increasing the carbon metabolism of the L-threonine synthesis pathway.To investigate the acidogenic level of the constructed Ile903-MX/pZ190 strain.The results showed that the L-threonine yield was 3.28 times higher than that of the control strain Ile903,which was increased from 0.98 g/L to 4.2g/L.?4?Based on the original fermentation formula,the metabolic modification of Ile903-MX/pZ190 strain was optimized for organic nitrogen source to obtain the optimal nitrogen source combination.Further,30L tank fed batch fermentation was used to verify the results.The fermentation formula produced L-threonine up to 34.6 g/L,and the conversion rate of sugar acid was 31.4%,which was 24.5%and 6.12%higher than that before optimization.