A Tri-enzyme Cascade For Production Of L-2-aminobutyrate From L-threonine

L-2-aminobutyrate（L-ABA）is a very important non-natural amino acid,which isa key chiral intermediate in synthesizing a variety of drugs,such as anti-tuberculosis drugs ethambutol and anti-epileptic drugs levoetiracetam and Boisetan,etc.Therefore,several effective synthesis methods have been developedto meet the growing market demand for L-ABA.The synthesis of L-ABA can be divided into the chemical method and the enzymatic method.The chemical process usually has the disadvantages of low optical purity,harsh reaction conditions and tedious steps.Enzymatic preparation is mainly based on L-threonine deaminase（TD）,L-leucine dehydrogenase（LDH）and a tri-enzyme cascade transformation system providing the regeneration of cofactor NADH to produce L-ABA.However,this method also has some problems,such as low catalytic activity of key enzymes and poor coordination of multi-enzyme cascade paths,which is not able to industrial production.Therefore,in this study,protein engineering was carried out for the key enzyme LDH to make it have higher catalytic activity,and a more stable and cooperative cascade catalytic system was constructed,which could effectively solve the problems in the process of enzymatic preparation of L-ABA.（1）Design,construction and verification of cascade reaction pathway for L-ABA production.First,the pathase was screened based on literature research:L-threonine deaminase from C.glutamicum,leucine dehydrogenase from B.thuringiensis and formate dehydrogenase from C.boidinii were selected according to specific enzyme activity as the target enzymes of the cascade reaction.Subsequently,the cascaded reaction pathway was constructed in vitro,and the product L-ABA was identified by high performanceliquid chromatography（HPLC）and mass spectrometry（MS）to verify the feasibility of the pathway.Finally,by measuring the catalytic activities of the three enzymes in vitro,the low catalytic activity of BtLDH was determined to be the rate-limiting enzyme of the cascade reaction.（2）Rational design can improve the catalytic activity of BtLDH.Based on crystal structure and reduction amination mechanism analysis,protein engineering of BtLDH was carried out to enhance the catalytic activity of BtLDH.Compared with the wild type,the enzyme activity of BtLDH^{M3（A262S/P150M/V296C）}was increased by 21.7 times and k_cat/K_mby 10.6times.Protein structure comparison and MD simulation showed that:（ⅰ）BtLDH^M3shortened the hydride transfer distance between C4H on the NADH nicotinamide ring and the Cαof substrate 2-OBA;（ⅱ）BtLDH^M3increased the proportion of proteins in closed conformation by limiting the transition from closed conformation to open conformation,and further improved the catalytic efficiency of reduced amination.（3）In vivo three enzyme cascade production of L-ABA.First,the activity ratio of the three enzymes was optimized in vitro,and the optimal ratio was controlled at 0.4:1:1.In order to optimize the activity ratios of these three enzymes in vivo,E.coli 02 with the highest production of L-ABA was obtained by carrier optimization strategy.The optimal mutant BtLDH^M3was constructed into E.coli 02 to obtain a new strain E.coli 02-1,which basically realized the equilibrium of the three-enzyme cascade reaction in vitro.The optimal transformation conditions for E.coli 02-1 conversion of L-threonine to L-ABA were determined as follows:the optimal molar ratio of L-threonine to ammonium formate was1:1.5,20 g·L^-1whole cell catalyst,the conversion temperature was 37℃,and the optimal pH was 8.0.When the reaction was amplified to 5 L fermenter,160 g·L^-1L-threonine could be converted into 130.2 g·L^-1L-ABA within 12 h,with the conversion rate reaching 95.0%and ee>99%.