Optimization Of L-Leucine Production Performance Based On Modified Transamination Pathway Of Corynebacterium Glutamicum

In the biosynthetic pathway of L-leucine in Corynebacterium glutamicum,the last step is a transamination reaction catalyzed by branched-chain amino acid aminotransferase(BCAT).However,BCAT simultaneously catalyzes the aminotransfer reaction between three branchedchain amino acids(BCAAs)and the corresponding α-keto acids,resulting in the fact that the production of L-leucine is often accompanied by accumulation of the other two BCAAs,thereby increasing the difficulty of separatation and extraction.Based on the above-mentioned results,we here engineered the L-leucine-producing strain C.glutamicum FA-1 to screen the transaminases which have specific catalytic synthesis activity for L-leucine by metaboic engineering on transamination pathway and investigated its effect on L-leucine.The main results are as follows:(1)In order to investigate the effect of BCAT on the synthesis of L-leucine and L-valine,we eliminated the BCAT activity by deleting the ilv E gene in C.glutamicum FA-1.Compared with the original strain C.glutamicum FA-1,the consumption rate of glucose of the recombinant strain C.glutamicum FA-1 Δilv E was greatly reduced,and the yields of L-leucine and L-valine were reduced by 87.1% and 92.4%,respectively.Moreover,the by-products were increased due to blockade of metabolic pathways.In addition,the recombinant strain showed decreased level of transaminase activity of L-leucine and L-valine by 94.4% and 93.0% due to the inactivation of BCAT,respectively,but the recombinant strain only showed the auxotrophy of L-valine.It was thus speculated that there may be other transaminases in vivo participate in the synthesis of L-leucine to meet the normal growth of the strain.(2)In order to verify that there be other transaminases are involved in the synthesis of Lleucine in vivo,eight transaminase genes derived from C.glutamicum were cloned and expressed in the recombinant strain.The results showed that only the recombinant strain C.glutamicum FA-1 ΔilvE/p EC-XK99E-aspB had the catalytic synthesis activity on L-leucine,and the yield of L-leucine reached 20.81 g·L-1,whereas the production of L-valine did not change.The enzymatic activity on L-leucine of the recombinant strain increased by 537% as compared with C.glutamicum FA-1 Δilv E.The above results confirmed that there is indeed a transaminase which catalyzes L-leucine synthesis except for BCAT in the ilvE-strain.It was speculated that the normal growth of C.glutamicum FA-1 ΔilvE was not dependent on L-leucine may be due to the presence of aspB gene,which catalyzes the synthesis of L-leucine in vivo to meet the growth requirements of the strain without exogenous supplement.(3)To further determine the effect of aspB on L-leucine production,we inactivated the asp B gene based on the ilv E-mutant strain to construct a double mutant C.glutamicum FA-1△ilvE △aspB.The results showed that the double mutant strain C.glutamicum FA-1△ilvE △aspB had no the catalytic activity on L-leucine and exhibited double defects of Laspartate and L-leucine,demonstrating the normal growth of C.glutamicum FA-1△ilvE was independent of L-leucine due to the presence of the asp B gene,which satisfied the need for Lleucine for normal growth of the strain.The inactivation of BCAT and Asp B disturbed the glycolytic pathway and the TCA cycle,resulting in slow growth,metabolism of the cells and increased by-products(e.g.,L-alanine,L-glutamic acid and pyruvate),but the yield o L-valine was not affected.Since the recombinant strain C.glutamicum FA-1△ilvE △aspB eliminated the effect of L-leucine in vivo,it provided a basis for the next experiment to screen specific transaminase.(4)In order to screen for the transaminases with specific synthesis catalytic activity for Lleucine,26 exogenous transaminase genes were cloned and expressed in the double mutant strain.The results showed that the transaminases EctyrB and Ecavt A only showed L-leucine and L-valine synthesis activity,respectively,and the yield of L-leucine by the recombinant strain FA-1△ilvE △aspB/p EC-XK99E-EctyrB was reached 18.55 g·L-1.Moreover,BCATs with different origins had both L-leucine and L-valine synthesis activities,and their catalytic ability was similar,while other transaminases did not detect the activity on L-leucine and/or L-valine.(5)The evolution analysis and sequence alignment of all transaminase proteins were porformed,and the results indicated that all BCATs were in the same branch of the phylogenetic tree and they had closest relationship with each other.Moreover,the presumed enzyme active site and the binding substrate and cofactor of these BCATs were the same.In addition,EctyrB was in a single branch and had distant relationship with other transaminases.While CgaspB was in a different branch with other AspATs,it was found that CgaspB was consistent with the active sites of other AspATs by sequence alignment.What’s more,the residues of CgaspB to bind substrate and cofactor PLP were unique,and the relationship between them were far.Since CgaspB and EctyrB had specific L-leucine catalytic properties,they can be used to construct L-leucine-producing bacteria to optimize the production of L-leucine.