Metabolic Engineering Of Corynebacterium Glutamicum For L-leucine Production

As one of the eight essential amino acids in human body,L-leucine is not only the basic unit of protein,but also has special physiological and biological functions,so it is widely used in food,medicine,feed and other fields,and has a good application prospect.At present,L-leucine is mainly synthesized by microbial fermentation in industrial production,and Corynebacterium glutamicum is the most widely used strain.In this experiment,the wild type strain ATCC 13032 of C.glutamicum was used as the starting strain,combined with the relevant theories of metabolic engineering,using genetic engineering methods and molecular biology techniques,in addition,it was used genetic engineering and molecular biology techniques and analyzed by species selection and process control strategies of five words " Enter,Flow,Moderate,Block and Exit".The results are shown as follow:1.L-valine synthetic route is necessary to synthesis of L-leucine,we introduces the production of L-valine bacteria XV acetyl ilvBNXV hydroxy acid synthase encoding mutations and double copy to remove branched chain amino acids?BCAAs?feedback on key enzymes acetyl hydroxyl acid synthase inhibition to reinforce L-valine synthetic approaches,and constructed the strain AL02,the L-valine has a 18 g/L titer after 36 h shake flask fermentation,it is 22.5 times of C.glutamicum ATCC 13032.The introduction of acetohydroxyacid synthase?AHAS?effectively reduces the feedback inhibition of the final product,and the L-leucine synthesis precursors were strongly supplied,which laid a foundation for the molecular modification of L-leucine synthesis pathway in the next step.2.In order to introduce more carbon metabolic flux into the L-leucine synthesis pathway,we enhanced the expression of the L-leucine synthesis pathway gene on the basis of the enhancement of the L-valine synthesis pathway.? The isopropyl malate synthase encoding mutant gene leuACP in L-leucine producing bacteria CP was introduced.In order to relieve the feedback inhibition of L-leucine on the key enzyme isopropylmalate synthase,three gene copies were integrated on the genome to enhance the expression of the enzyme;? We enhanced the expression of isopropylmalate isomerase and isopropylmalate dehydrogenase in the L-leucine synthesis pathway,and the metabolic flux of the L-leucine synthesis pathway were enhanced;?This experiment introduced the leucine dehydrogenase derived from Bacillus subtilis,which enhanced the transamination reaction of L-leucine synthesis and reduced the need for L-leucine synthesis for reducing power NADPH;The constructed L-leucine engineered strain AL10 was fermented in a 36 h shake flask,and the titer of L-leucine reached 11.6 g/L,which was 18.3 times higher than that of the original strain ATCC 13032.The main by-product detected in the fermentation broth is L-valine,indicating that although the L-leucine synthesis pathway has been strengthened,the isopropylmalate synthase activity still needs to be enhanced.3.NADPH is an important reducing force in cells,and L-leucine has a high demand for NADPH.In order to increase the supply of intracellular NADPH,promote the accumulation of L-leucine.In this paper,the pntAB gene encoding pyridine nucleotide hydrogenase in Escherichia coli was introduced and transcribed by promoter PgapA.L-leucine engineering strain AL11 was constructed,the titer of L-leucine reached 12.5 g/L after 36 h of flask fermentation,which was 19.8 times against the original strain.It can be seen that heterologous expression of pyridine nucleotide transhydrogenase derived from E.coli enhanced the synthesis of L-leucine.4.Pyruvic acid is the main precursor for the synthesis of BCAAs.To enhance the intracellular pyruvate supply,in this paper,the threonine dehydrogenase-encoding gene ilvA was knocked out,and the L-isoleucine synthesis pathway was cleaved to construct the L-leucine engineered strain AL12.The appropriate amount of L-isoleucine was replenished in the fermentation medium and fermented in a shake flask for 36 h.The titer of L-leucine of AL12 was 6.3 g/L.L-leucine accumulation decreased compared with AL11 strain.In addition,the growth cycle of AL12 was significantly prolonged,and the total biomass also decreased significantly,indicating that the blocking of the L-isoleucine synthesis pathway affected the normal growth of the cells and the acid production by fermentation.5.The Tricarboxylic Acid Cycle?TCA?pathway is one of the main pathways for carbon catabolism in C.glutamicum.In order to reducing the L-leucine precursor of pyruvic acid consumption and without affecting bacteria under the condition of normal growth,we used the citrate synthase promoter replace for L-leucine producing bacteria PCP₂928 cell-specific promoters of CP,which reduced the flux of carbon source to the TCA cycle at the acetyl-CoA metabolic node,and build the L-leucine AL13 engineering bacteria.After 36 h shake flask fermentation,the titer of L-leucine of AL13 reached 13 g/L,which was 21 times higher than that of the original strain,which indicated that the strategy played a role in weakening the TCA cycle to a certain extent.More carbon sources are introduced into the synthetic pathway of L-leucine.6.In order to further enhance the activity of isopropylmalate synthase,in this paper,we cloned the isopropylmalate synthase encodilg mutant gene leuACP into a plasmid and introduced it into AL13 to enhance the expression of isopropylmalate synthase.and the L-leucine engineered bacteria AL14 was constructed.And after 36 h shake flask fermentation,the titer of L-leucine reached 26 g/L,which was 42.3 times higher than the original strain.7.In order to comprehensively investigate the productivity of L-leucine engineering strain AL14,we made the fed-batch fermentation experiments in the 5 L fermenter.After 48 h of fed-batch fermentation,the titer of L-leucine reached 39.8 g/L,and the yied was 15.8%.It can be seen that this strain has good industrial production potential.