Substrate-Specific Engineering Of Amino Acid Dehydrogenase And Its Synthesis Of Noncanonical Amino Acids

Noncanonical amino acids are a class of amino acids that are not naturally incorporated into proteins during translation.They have a wide range of application values in the fields of biomedicine and biotechnology.For example,L-tert-leucine is an important intermediate in the synthesis of anti-tumor inhibitors,anti-HIV protease inhibitors and other drugs.Some non-α-amino acids（such asγ-aminovaleric acid）can participate in the construction of active peptides with special functions.In addition,with the development of the strategy of incorporating non-encoded amino acids into proteins,the application scope of noncanonical amino acids has been further expanded.noncanonical amino acids can participate in the construction of artificial enzymes that are more in line with industrial needs.Therefore,the development of diverse noncanonical amino acid synthesis strategies has important scientific significance and application value.Biocatalytic synthesis of chiral amino acids has the characteristics of mild reaction conditions,environmental friendliness and high optical purity of products.The enzymatic asymmetric synthesis of chiral amino acids currently developed mainly includes four pathways,namely aldol condensation of an amino acid to aldehydes,asymmetric transfer of an amino group to keto acids,enantioselectivity addition of ammonia toα,β-unsaturated acids,and asymmetric reductive amination of keto acids.Among them,the asymmetric reductive amination pathway of keto acids catalyzed by amino acid dehydrogenases（such as leucine dehydrogenase,phenylalanine dehydrogenase）can directly use cheap ammonia as the amino donor with high atom utilization,and the optical purity of the product can often exceed 99%e.e.,which has been widely used in the synthesis of high value-added non-standard amino acids.However,the products catalyzed by these enzymes are mainlyα-amino acids,and few amino acid dehydrogenases for synthesizing non-α-amino acids have been developed.Herein,we successfully improved the catalytic efficiency of leucine dehydrogenase from Bacillus cereus（Bc LeuDH）to synthesize L-tert-leucine,and successfully constructed an engineered glutamate dehydrogenase(Ec Glu DH^K116Q/N348M)that can synthesize（R）-4-aminovaleric acid asymmetrically.The main results are listed as follows:（1）Using the physicochemical characteristics of NAD（P）H and 2,4-dinitrophenylhydrazine（DNPH）,we constructed two high-throughput screening methods（DNPH assay and NAD（P）H assay）for the detection of asymmetric reductive amination activity of amino acid dehydrogenases and their effectiveness was evaluated.Experiments show that DNPH assay is suitable for the rapid detection of carbonyl compounds（keto acids,ketone alcohols,ketones）in this study.This method is cheap and fast,and is suitable for the screening of relatively large mutant libraries;the NAD（P）H assay requires the addition of expensive reducing coenzyme,which is not as economical as the DNPH detection method,but the false positive rate of this method is only 0.3%,which is less than half of the DNPH detection method.The complementary advantages of the two high-throughput screening methods provide favorable conditions for the subsequent substrate specificity engineering of amino acid dehydrogenases.（2）Comprehensive screening by two high-throughput screening methods,four dominant mutants（E116V,D126E,E24V/E116V and E24A/D126E）with improved catalytic efficiency to trimethylpyruvate were identified.Among them,the k_cat/K_m of the best mutant E24V/E116V for trimethylpyruvate and NADH were 5.3 times and 3 times that of wild-type Bc LeuDH,respectively.The mutant E24V/E116V showed better affinity for NADH,and its K_m was nearly0.3 of that of wild-type Bc LeuDH.Under the condition of 0.05 m M NAD⁺,the mutant E24V/E116V completed the complete conversion of 0.57 M trimethylpyruvate in 75 min,and the specific space-time conversion rate and TTN reached 22.8 mmol·h^-1·L^-1·g^-1and 11400,respectively.In addition,these mutation sites have reference significance for the improvement of the activity of amine dehydrogenase constructed based on Bc LeuDH.（3）In order to create an engineered amino acid dehydrogenase that can catalyze non-αamino acids,two substrate-specific engineering schemes for amino acid dehydrogenases were designed in this study.Scheme 1:Convert the specific recognition of the substrate backbone of the enzyme from the carboxyl group（COOH）to COOHCH2-to construct an amino acid dehydrogenase that can catalyzeβ-amino acids.Scheme 2:While changing the specific recognition of the substrate main chain of the enzyme from a carboxyl group to a methyl group,the substrate side chain specific recognition of the enzyme was modified to make the engineered enzyme catalytically active for substrates with carboxyl side chains,thereby constructing an engineered amino acid dehydrogenase capable of asymmetric synthesis of non-α-amino acids.Based on two important residues（K70 and N263）of Bc LeuDH anchoring the carboxyl group of the substrate backbone,a two-site combinatorial saturation mutant library was constructed,and the mutant library was screened with 3-amino-5-methylhexanoic acid and 2-pentanone as substrates,respectively.The corresponding engineered amine dehydrogenase（Bc-LAm DH-M₀,K70S/N263L）was successfully constructed,and the specific recognition of its substrate backbone was changed from the original carboxyl group to the methyl group.On the basis of Bc-LAm DH-M₀,an iterative saturation mutation was used to superimpose the dominant mutation residues（24,116,126）,and a mutant Bc-LAm DH-M₂（E24V/E116V）with improved catalytic activity was constructed.The catalytic activity of Bc-LAm DH-M₂to 2-pentanone was2.2 times that of Bc-LAm DH-M₀,and the ee value of the product（R）-2-aminopentane was more than 99%.In the detection of the substrate spectrum,it was found that the catalytic activity of the dominant mutant Bc-LAm DH-M₂ for aliphatic ketones and alicyclic ketones has been widely improved.In the molecular engineering of the specific recognition of the substrate side chain of Bc-LAm DH-M₂,an engineered enzyme with catalytic activity for 4-oxopentanoic acid（a substrate for the introduction of a carboxyl group into a side chain）has not been obtained,which may be due to the strong hydrophobicity of the substrate pocket of LeuDH.（4）In this study,glutamate dehydrogenase（Glu DH）with a hydrophilic substrate pocket was selected to implement the Scheme 2,trying to construct an engineered amino acid dehydrogenase that can catalyze non-α-amino acids.In order to select suitable enzymes for substrate-specific engineering,this study performed substrate spectrum of three Glu DHs（Cg Glu DH from Corynebacterium glutamicum,Cs Glu DH from Clostridium symbiosum and Ec Glu DH from Escherichia coli）with crystal structures.Compared with Cg Glu DH and Cs Glu DH,Ec Glu DH not only has higher catalytic activity for the substrates 2-ketoglutarate(50.55 U·mg^-1)and oxaloacetate(0.45 U·mg^-1),but even has detectable catalytic activity for L-homoserine(0.004 U·mg^-1)with a hydroxyl group at the end of the side chain.In order to improve the catalytic activity of Ec Glu DH for oxaloacetate,the specific recognition of the substrate side chain of Ec Glu DH was modified in this study.The k_catand k_cat/K_mof the optimal mutant K92V obtained on oxaloacetate was 9.7-fold and 40-fold higher than that of Ec Glu DH,respectively.（5）In order to construct an engineered amino acid dehydrogenase that can catalyze the synthesis of non-α-amino acids,this study confirmed two key residues of the carboxyl group of the backbone of the Ec Glu DH anchor substrate through the crystal structure analysis and sequence alignment of Glu DH.Based on these two sites,an“small-intelligent”combinatorial saturation mutation library was constructed,and the optimal mutant Ec Glu DH^K116Q/N348Mwas successfully obtained by screening the activity of the substrate levulinic acid,and its catalytic activity to levulinic acid could reach 108.6 m U·mg^-1.Ec Glu DH^{K116Q/N348M/K92V} produced weak activity against（R）-3-aminobutyric acid(0.23 m U·mg^-1).In conversion experiments under optimal reaction conditions（1 m M NADP⁺,p H 8 and 45°C）,the coenzyme recycling system constructed with Ec Glu DH^K116Q/N348M and formate dehydrogenase converted 0.4 M levulinic acid by 97%within 11 h,generating the corresponding product（R）-4-aminopentanoic acid with>99%ee.