High Eficient Biosynthesis Of L-2-aminobutyric Acid And Rational Engineering Of The Key Enzymes

Unnatural amino acids?UAAs?are fundamental building blocks of modern medicinal chemistry,because their readily functionalized amine and carboxyl groups can be attached to chiral central core of functional molecules along with one or two potentially diverse side chains.L-2-aminobutyric acid?L-2-ABA?has been widely used as a key chiral intermediate for the synthesis of several important drugs such as levetiracetam.However,the chemical synthesis of L-2-aminobutyric acid has complex process and is usually not environmentally friendly.And the enzymatic synthesis of L-2-ABA,including L-threonine deaminase?EC 4.3.1.19,TD?,L-leucine dehydrogenase?EC 1.4.1.9,LeuDH?and enzymes for NADH regeneration,requires the addition of exogenous expensive cofactor and the cultivation of multiple cells which increase the costs.Other problems including the unstable key enzymes,the low activity of enzymes for NADH regeneration and the poor synergy of multi-enzyme conversion are not conducive to industrial production.Therefore,the engineering of key enzymes-TD and LeuDH to get higher catalytic activity and stability,the exploration of high efficiency cofactor regeneration system and the construction of a more coordinated multi-enzyme catalytic system in a single cell,can solve the problems encountered in the microbial synthesis of L-2-ABA.TD is the key enzyme for 2-oxobutyric acid?2-OBA?production using L-threonine,it is allosteric inhibited by L-Ile and not stable,thus we rational design the regulation domain of TD enzyme to relieving its allosteric inhibitition.We truncated the different peptide lengths at the C-terminal regulatory domain?from residues 11 to 193?of TD and obtained the active protein aggregates.The truncated residues Glu480-Gly514 in the C-terminal regulatory domain were confirmed to be an unstable structure by molecular dynamic?MD?simulations.The truncation variants?11,?25,and?35 were not allosterically regulated by L-Ile at all by kinetics analysis.They showed decreased activity but better thermostability than wild-type enzyme.Among the three variants,?25 showed 3.3-fold higher 2-OBA production in the presence of 2 mM L-Ile and about9-fold higher production at 55°C in comparison to the wild type.The protein aggregates resulting from the aggregation of truncated TD dimers by hydrophobic interaction could resist the binding of L-Ile,as determined by calorimetry experiments.According to attenuated total reflection FTIR?ATR-FTIR?measurements,the protein aggregates kept a secondary structure composition similar to that of the wild-type enzyme.Furthermore,the immobilization of protein aggregates was studied for the first time,the highest capacity of immobilized?25 protein aggregates was 1.35±0.07 mg/mg Fe₃O₄ in PBS buffer?pH 7.0?.The effects of ion strength,mild solvents,and nonionic surfactant on the adsorption efficiency showed that the major interaction forces between Fe₃O₄ nanoparticles and protein aggregates were hydrogen bond and ionic exchange interaction forces,while van der Waals forces might also play a role.By nonspecific adsorption of Fe₃O₄ nanoparticles onto protein aggregates,the protein aggregates could be quickly recycled without enzyme leakage using energy-saving magnetic separation.Low efficiency for natural enzymes including LeuDH are one major challenge for UAA production.In the third chapter,rational engineering of Bc LeuDH with a structure-based design approach was studied,higher enzymatic activity and stability towards?-keto acid reduction were achieved by improving the hydrophobic and rigidity of enzymatic substrate entrance tunnel.High catalytic efficiency for variant E116V was associated with the presence of more hydrophobic tunnels that allowed easy substrate diffusion,which was confirmed in absorbance spectroscopy study.For variant T45M/E116V,melting temperature and half-lives of thermal inactivation at 60°C was62.8 ? and 29.2 h,respectively,much higher than 48.4 ? and 3.4 h of wild type.Structural analysis indicated that an additional hydrogen bond in?5 fold was formed in variant T45M,this resulted a more rigid?5 fold which led to better stability.Furthermore,asymmetric synthesis of?-amino acids with coenzyme regeneration revealed higher productivities for variant T45M/E116V.Furthermore,we studied the importance of Asp117 in proton transfer during enzymatic catalysis and hypothesized catalytic chemical mechanism of LeuDH towards?-keto acids reduction.Low efficiency of the using enzymes for NADH regeneration limited the application of many oxidordeuctases,thus more than 10 type of enzymes used for NADH regeneration were cloned and it was found that alcohol dehydrogenase?EC1.1.1.1,ADH?,glycerol dehydrogenase?EC 1.1.1.6,GlyDH?showed a higher efficiency for NADH regeneration and were used for L-2-ABA production.Glucose dehydrogenase?EC 1.1.1.118,GDH?,formate dehydrogenase?EC 1.17.1.9,FDH?,ADH and GlyDH were used to prepare L-2-ABA by enzymatic or whole-cell methods,while no cofactor NAD?H?was added using whole-cell method.The results showed that the yield of L-2-ABA by enzymatic method and whole-cell method using GDH for NADH regeneration was 102±4.7 and 141.6±7.2 g/L,respectively.GlyDH had higher enzyme activity than FDH and ADH,but the yield of L-2-ABA prepared by enzymatic method and whole cell method using GlyDH was only 16.8±2.4 and 43.8±3.9 g/L,respectively,which were much lower than that using FDH or ADH.In addition,L-lactate dehydrogenase?EC 1.1.1.27,LDH?from different sources was studied and we found that Pf LDH had the highest catalytic activity toward pyruvate and 2-ketobutyric acid,the high activity of Pf LDH could be due to its loose structure of substrate-binding region according to the study of crystal structure comparison.Pf LDH was used for the synthesis of?S?-2-hydroxybutyric acid with the help of ADH,the yield was¹46 g/L and the conversion was 98.2±0.6%.The poor coordination of enzymatic reaction in multi-enzyme catalysis often cause the accumulation of intermediate and low efficiency.In the fifth chapter,we optimized the expression of TD enzyme by regulating the intensity of its RBS sequence to coordinate the conversion rate of TD enzyme and Bc LeuDH-coupled FDH enzyme system,then we constructed a single cell with coordinate multi-enzyme coexpression system.It was found that recombinant strain E.coli BL21/pETDuet-R40 was suitable for efficient transformation of L-2-ABA.The yield reached¹86.6 g/L at 22 h and the conversion rate of L-Thr was near 100%.By studying the reusability of cells in whole cell transformation,it was found that intracellular NAD⁺concentration played an important role.The efficient preparation of L-2-ABA using our single cell had already been applied in industry with 20 ton tank.The yield of L-2-ABA using phosphate buffer for transformation was above 145 g/L,the conversion rate was stable at⁸.0 g/?L h?,and the molar yield was stable at⁹5%.