Synthesis Of D-amino Acid Via Stereoinversion Of L-amino Acid By Multienzyme Cascade

D-Amino acids are important building blocks in the production of pharmaceuticals and fine chemicals,which are commonly used to synthesize?-lactam antibiotics,fertility drugs,anticoagulants,and pesticides.Therefore,it is of great significance to study the biosynthetic methods of D-amino acids.There are currently a number of enzymatic methods to produce D-amino acids,including hydantoinase process,enzymatic resolution of the racemate,enzymatic hydrolysis and enzymatic asymmetric catalysis of prochiral substrates.However,these methods are not suitable for industrial production,because they usually require specific substrates that are generally expensive and commercially unavailable.Therefore,establishing a universal enzymatic method for the general synthesis of D-amino acids from cheap and readily available precursors is challenging.Designing a multi-enzyme cascade biocatalysis route for synthesizing D-amino acids in one-pot from cheap and available starting substrates has the advantages of low catalytic cost,high catalytic efficiency,saving operation preparation steps and resources.Herein,we constructed and optimized a cascade enzymatic system involving oxidative deamination module?L-amino acid deaminase?and reductive amination module?D-amino acid dehydrogenase?for the biocatalytic stereoinversions of cheap and available L-amino acids into D-amino acids.In addition,two kinds of cascade catalytic routes,in vitro and in vivo,were used to gradually improve the catalytic efficiency of the cascade catalytic route and realized the synthesis of a variety of natural and unnatural D-amino acids.The main results are listed as follows:?1?In this study,based on multi-enzyme cascade catalysis,a cascade catalytic route including oxidative deamination module and reductive amination module was designed to catalyze the stereoinversions of L-amino acids into D-amino acids.Then,the substrate and product were used as target orientation,and the enzymes used for constructing the cascade route were determined by gene database mining.The biocatalysts composed of each catalytic module were Pm LAAD?oxidative deamination module,L-amino acid deaminase from Proteus mirabilis?,St DAPDH/H227V?reductive amination module,diaminopimelate dehydrogenase from Symbiobacterium thermophilum?and Bs FDH?cofactor-recycling system,formate dehydrogenase from Burkholderia stabilis?.The catalytic properties of each module were explored to evaluate the optimal catalytic conditions of the selected biocatalysts.The Pm LAAD whole-cell biocatalyst was more active in Tris-HCl buffer?50 m M,p H 7.0-10.0?at45?and the optimum biocatalyst concentration was found to be 80 mg/m L.The specific activity of St DAPDH/H227V toward phenylpyruvic acid was 0.46 U/mg,and the k_cat was1.13 s^-1.The activity of St DAPDH/H227V was higher,when the p H was 9.5 and the temperature was 37-45?.St DAPDH/H227V catalyzed 30 m M of phenylpyruvic acid after 6h,and the conversion was 100%and the ee value of the product was more than 99%.The Bs FDH exhibited higher specific activity of 2.89 U/mg in Tris-HCl buffer?50 m M,p H 7.5?at45?.When the Bs FDH and St DAPDH/H227V were coupled to construct cofactor-recycling system,the NADP⁺can be used to initiate the cofactor regeneration without affecting the catalytic efficiency.Moreover,there was no by-products in the Pm LAAD and St DAPDH/H227V catalytic process,and the reactions always proceeded in the direction of product formation.Therefore,the selected biocatalysts were favorable for use in the biocatalytic stereoinverting cascade system.?2?In order to improve the catalytic efficiency of the cascade catalytic system,the optimal reaction conditions were explored.The optimal catalytic condition of the in vitro cascade catalytic system was 50 m M Tris HCl buffer?p H 9.0?,and the optimal reaction temperature was 30?.When the concentration of Pm LAAD whole cell catalyst was 40mg/m L,the concentration of St DAPDH/H227V was 4.5 mg/m L,and the concentration of Bs FDH was 0.35 mg/m L.The in vitro cascade catalytic system could catalyze the complete stereoinversion of 80 m M L-phenylalanine to D-phenylalanine,and both the conversion and optical purity were close to 100%.In particular,the cascade biocatalytic system also efficiently converted a variety of readily available and inexpensive aromatic and aliphatic L-amino acids into the corresponding D-amino acids,such as L-phenylalanine,L-homophenyalanine,2-chloro-L-phenylalanine,3-chloro-L-phenylalanine,L-glutamic acid,and L-norvaline.?3?In order to promote the soluble expression of Pm LAAD and improve its catalytic efficiency,the soluble expression of membrane protein Pm LAAD was studied.Through the fusion expression with the maltose-binding protein?MBP?tag,the Pm LAAD was actively expressed as a soluble protein.Then the activities of the Pm LAAD cell lysates,MBP-Pm LAAD cell lysates,the MBP-Pm LAAD fusion protein,and the purified Pm LAAD were analyzed.The purified MBP-Pm LAAD fusion protein had a low activity of 0.025?mol/?mg Pm LAAD·min?using L-phenylalanine as a substrate.The activity of the MBP-Pm LAAD cell lysate was 0.17?mol/?mg Pm LAAD·min?,which was 2.2 times higher than the activity of the Pm LAAD cell lysate.Then the catalytic properties of the MBP-Pm LAAD fusion protein was studied.The optimal catalytic conditions were 37?and p H 8.5,and the MBP-Pm LAAD fusion enzyme was relatively stable under the reaction conditions during the reaction for 10 h,even after the reaction reached equilibrium.Moreover,exogenous FAD had no effect on the enzyme activity.After fusion with MBP-tag,the activity of MBP-Pm LAAD crude enzyme was much higher than that of membrane bound Pm LAAD whole cell.Soluble MBP-Pm LAAD cell lysate could catalyze the complete conversion of 100m M substrate L-phenylalanine to phenylpyruvic acid within 6 h.Therefore,the fusion of the MBP-tag not only improved the soluble expression of the Pm LAAD membrane-bound protein,but also increased its catalytic performance.?4?An in vivo cascade catalytic system consisted of Pm LAAD,St DAPDH/H227V,and Bs FDH was designed and constructed.To generate the in vivo cascade system,three strategies were evaluated to regulate enzyme expression levels,including single-plasmid co-expression,double-plasmid co-expression,and double-plasmid MBP-fused co-expression.By comparing the conversion results of different co-expression systems,the double-plasmid MBP-fused co-expression strain,E.coli p ET-21a-MBP-pmlaad/p ET-28a-stdapdh/H227V-bsfdh,exhibiting high catalytic efficiency,was selected.Then the optimum catalytic conditions of the in vivo cascade cell factory were explored.Under optimal conditions,75 mg/m L of E.coli p ET-21a-MBP-pmlaad/p ET-28a-stdapdh/H227V-bsfdh whole-cell biocatalyst asymmetrically catalyzed the stereoconversion of 150 m M L-phenylalanine to D-phenylalanine,with quantitative yields of over 99%ee in 24 h,by the addition of NADP⁺at 0.1-fold substrate concentration and ammonium formate at two-fold substrate concentration.Compared with the previous in vitro cascade system,the substrate concentration increased nearly 1.9 times.In addition,the in vivo cascade cell factory was used to successfully stereoinvert a variety of aromatic and aliphatic L-amino acids,such as L-tyrosine,L-phenylalanine,L-homophenylalanine,2-chloro-L-phenylalanine,3-chloro-L-phenylalanine,4-chloro-L-phenylalanine,L-leucine,L-norvaline,L-glutamic acid,and L-methionine,with a high conversions?45.3%-100%?and optical purity?>99%ee?.?5?The synthesis of D-amino acid was scaled up to 100 m L to further evaluate the feasibility of the in vivo multi enzyme cascade catalytic system.After optimizing the conditionofenlargedreactionsystem,theE.coli p ET-21a-MBP-pmlaad/p ET-28a-stdapdh/H227V-bsfdh whole cell biocatalyst could catalyze200 m M L-phenylalanine in 100 m L enlarged reaction system.After 48 h,both the optical purity and the conversion of the product were close to 100%,and the isolation yield was 91%.Finally,the product was further confirmed to be D-phenylalanine by MS,¹H-NMR,and¹³C-NMR.The identification results showed that the structure of the product was correct,which further proved the accuracy and universal applicability of the in vivo cascade cell factory in this study.