A review on enzyme-catalyzed synthesis of chiral amino acids.

doi:10.12211/2096-8280.2024-015

Abstract

Abstract:

Chiral amino acids represent a crucial class of chiral building blocks with significant value in food, medicine, chemical industry, and agriculture. The market scale of pharmaceuticals, pesticides, food, and chemical industries relying on chiral amino acids is substantial and has been attracting increasing attention. The pursuit of efficient, environmentally friendly, and cost-effective synthesis of chiral amino acids has long been a goal for scientists. Commonly used preparation methods for chiral amino acids fall into four categories: protein hydrolysis, fermentation, chemical synthesis, and enzyme-catalyzed synthesis. Among these, enzyme-catalyzed synthesis has demonstrated great potential due to its mild reaction conditions, high stereo-selectivity, simplicity of steps, and wide application range. In recent years, with the rapid development of bioinformatics, protein engineering, and computational biology, there has been an increasing number of high-performance enzyme preparations developed, leading to a steady increase in the diversity of enzymes and the gradual diversification of catalyzed reactions, further promoting the wide application of enzyme-catalyzed synthesis of chiral amino acids. The enzyme-catalyzed synthesis of chiral amino acids can be categorized into three groups: asymmetric synthesis, deracemization synthesis, and kinetic resolution. Kinetic resolution, due to its theoretical yield of only 50% and low atom economy, is not suitable for industrial applications. In contrast, asymmetric synthesis and deracemization synthesis with theoretical yield of 100% find wider industrial application. This article reviews the application of enzymatic asymmetric synthesis and deracemization synthesis in the synthesis of chiral amino acids. It includes the development and modification of key enzyme such as amino acid dehydrogenase, transaminase, ammonia lyase, aldolase, amino acid oxidase, and amino acid deaminase, as well as their application in the synthesis of high-value chiral amino acids such as phosphinothricin, tert-leucine, and intermediate of sitagliptin. Additionally, it summarizes the main challenges faced in the field of enzymatic synthesis of chiral amino acids, such as the lack of key enzyme components, and low enantioselectivity, narrow substrate spectra, low catalytic activity, poor stability, limited reaction conditions of wild-type enzymes. Finally, it looks ahead to the application of cutting-edge technologies such as automated experimental devices, machine learning, and artificial intelligence in the field of enzyme modification, as well as the development of more efficient and environmentally friendly catalytic processes through reactor design and reaction process control. These endeavors collectively aim to facilitate the broader industrial application of enzymatic synthesis for chiral amino acids.

Key words: High-value chiral chemicals, Chiral amino acids, Asymmetric synthesis, Deracemization synthesis, Protein engineering, Multi-enzymatic cascade

摘要：

手性氨基酸是一类重要的高价值化学品，广泛应用于食品、医药、化工、农药等多个领域。手性氨基酸常用的制备方法可以分为四类，包括化学合成、蛋白质水解、发酵和酶促合成。其中，酶促合成手性氨基酸以其反应条件温和、立体选择性高、步骤简单、应用范围广等优势备受关注。近年来，得益于生物信息学和蛋白质工程等技术的快速发展，大量性能优异的酶制剂被开发，并成功应用于多种手性氨基酸的制备。本文重点综述了酶促不对称合成和去消旋化合成两种路径在手性氨基酸合成中的应用，包括关键酶制剂氨基酸脱氢酶、转氨酶、氨裂解酶、醛缩酶、氨基酸氧化酶、氨基酸脱氨酶等的开发与改造，及其在草铵膦、叔亮氨酸、西格列汀中间体等高价值手性氨基酸合成中的应用。同时，总结了酶促合成手性氨基酸领域面临的主要困境，如关键酶元件缺乏，以及野生酶非对映体选择性低、底物谱窄、催化活性低、稳定性差、反应条件局限等。最后，展望了自动化实验装置、机器学习和人工智能等前沿技术在酶改造领域的应用，以及通过反应器设计和反应过程控制，开发更为高效和环境友好的催化工艺，推动酶促合成手性氨基酸技术更广泛的工业应用。

关键词: 高价值手性化学品, 手性氨基酸, 不对称合成, 去消旋化合成, 蛋白质工程, 多酶级联

CLC Number:

Q814

Ziyuan WANG, Lirong YANG, Jianping WU, Wenlong ZHENG. A review on enzyme-catalyzed synthesis of chiral amino acids.[J]. Synthetic Biology Journal, DOI: 10.12211/2096-8280.2024-015.

王子渊, 杨立荣, 吴坚平, 郑文隆. 酶促合成手性氨基酸的研究进展[J]. 合成生物学, DOI: 10.12211/2096-8280.2024-015.

Figures/Tables 33

Fig. 1 The application of chiral amino acids.

Table 1 Comparison of three common methods for enzyme-catalyzed synthesis of chiral amino acids

方法

Methods

酶制剂

Enzyme

底物

Substrate

立体选择性

Stereosele-ctivity

理论产率

Theoretical yield

原子经济性

Atomic economy

典型案例

Typical examples

Asymmetric synthesis

Amino acid dehydrogenase,

Transaminase,

Ammonia lyase,

Amino mutase,

Aldolase,

Hydroxymethyltransferase,

etc.

Keto acids,

α, β-unsaturated carboxylic acids,

Amino acids and Aldehydes

High

100%

High

L-tert-Leucine ^[11-12]，

(R)-3-Amino-4-(2,4,5-trifluorophenyl)butyric acid^[13]

Racemization synthesis

Amino acid dehydrogenase,

Transaminase,

Ammonia lyase,

Amino mutases, Aldolase,

Hydroxymethyltransferase,

Amino acid oxidase,

Amino acid deaminase,

Amino acid racemase,

etc.

L-Phosphinothricin^[14-15]，

L-Phenylglycine ^[16]

Dynamic kinetic resolution

Amino acid oxidase,

Amino acid deaminase,

Amino acid dehydrogenase,

Amino acid racemase,

etc.

Fig. 2 Asymmetric reductive amination of keto acids by amino acid dehydrogenases (AADH)

Fig. 3 The structure of glutamate dehydrogenase (A) The crystal structure of glutamate dehydrogenase from Bacillus subtilis (PDB ID: 3K92); (B) The single subunit structure of glutamate dehydrogenase from Bacillus subtilis; (C) The "open" and "close" structure of glutamate dehydrogenase

Fig. 4 The catalytic mechanism of amino acid dehydrogenases

Fig. 5 Multi-enzymatic cascade system for synthesizing L-2-aminobutyric acid from L-threonine (TD-Threonine deaminase, LeuDH-Leucine dehydrogenase)

Fig. 6 Asymmetric transfer of amino groups to keto acids by transaminase (TA)

Fig. 7 General catalytic reaction formula for transaminases (TA)

Fig. 8 The crystal structure of transaminase (A) The crystal structure of (S)-transaminase from Bacillus megaterium (PDB ID: 5G09); (B) The crystal structure of (R)-transaminase from Aspergillus fumigatus (PDB ID: 4UUG)

Fig. 9 The catalytic mechanism of transaminase

Fig. 10 Synthesis of chiral α-amino acids by asymmetric transfer of amino groups to keto acids (A) Cascade synthesis of chiral α-amino acids by branched-chain amino acid aminotransferase (BCAT) and ornithine aminotransferase (OrnAT); (B) Cascade synthesis of L-phosphinothricin (L-PPT) by transaminase (TA), glutamate dehydrogenase (GluDH), and alcohol dehydrogenase (ADH); (C) Based on the alanine dehydrogenase (ALD) - formate dehydrogenase (FDH) amine cycle system, cascade synthesis of L-2-aminobutyrate by threonine deaminase (TD) and TA; (D) Chemical methods are coupled with aspartate transaminase (AspAT) to synthesize L-3,4-dimethoxyphenylalanine.

Fig. 11 Synthesis of intermediate of sitagliptin by asymmetric transfer of amino groups to keto acids (TA-transaminase)

Fig. 12 Synthesis of chiral non-α-amino acids by asymmetric transfer of amino groups to keto acids (A) Coupling different transaminases (TA) to synthesize (S)-4-aminopentanoic acid; (B) Cascade synthesis of (S)-β-phenylalanine by nitrilase and ω-TA

Fig. 13 Enantioselective addition of ammonia to α,β-unsaturated acids by ammonia lyase (AL) or amino mutase (AM)

Fig. 14 General catalytic reaction formula for ammonia lyases used for chiral amino acid synthesis (DAL-Aspartate ammonia-lyase, MAL-Methylaspartate ammonia-lyase, PAL-Phenylalanine ammonia-lyase, HAL-Histidine ammonia-lyase, TAL-Tyrosine ammonia-lyase.)

Fig. 15 The catalytic mechanism of transaminases of ammonia-lyase (A) enolate mechanism. (B) MIO electrophile mechanism

Fig. 16 Enantioselective addition of ammonia to α,β-unsaturated acids by phenylalanine ammonia lyase (PAL) (A) Various aromatic L-amino acids; (B) EMA401 intermediate.

Fig. 17 Computational redesign of aspartate ammonia lyase (DAL) for the synthesis of several unnatural amino acids

Fig. 18 Enantioselective addition of ammonia to α,β-unsaturated acids by Methylaspartate ammonia lyases (MAL) (A) Vitamin B5 derivatives (B) L-dopa (ADC-aspartate-α-decarboxylase, CrpG-β-methylaspartate-α-decarboxylase, GAD-glutamate decarboxylase, PS-pantothenate synthetase)

Fig. 19 Aldol condensation of an amino acid to aldehydes by aldolase or hydroxymethyltransferase (HMT)

Fig. 20 General catalytic reaction formula for threonine aldolase (TA)

Fig. 21 The crystal structure of threonine aldolase (A) The crystal structure of L-threonine aldolase from Aeromonas jandaei (PDB ID: 3WGB); (B) The crystal structure of D-threonine aldolase from Achromobacter xylosoxidans (PDB ID: 4V15)

Fig. 22 The catalytic mechanism of threonine aldolase (A) The catalytic mechanism of L-threonine aldolase; (B) The catalytic mechanism of D-threonine aldolase

Fig. 23 Schematic diagram of the path hypothesis. Aldehydes (MTB) attack Cα of aldimine PLP-Gly through the syn path or anti path to form the corresponding configuration of products[92].

Fig. 24 General catalytic reaction formula for Serine hydroxymethyltransferase (SHMT)

Fig. 25 Deracemization synthesis (A) Multi-enzymatic deracemization synthesis; (B) Chemo-enzymatic deracemization synthesis

Fig. 26 General catalytic reaction formula for Amino acid oxidase (AAO)

Fig. 27 Amino acid oxidase are involved in in the deracemization synthesis of chiral amino acids (A) L-phosphinothricin; (B) L-2-aminobutyric acid. (AAO-amino acid oxidase, AADH-amino acid dehydrogenase, CAT-catalase, TA-transaminase)

Fig. 28 General catalytic reaction formula for L-Amino acid deaminase (L-AAD)

Fig. 29 Amino acid deaminase are involved in in the deracemization synthesis of chiral amino acids (AAD-amino acid deaminase, TA-transaminase)

Fig. 30 Amino acid dehydrogenase are involved in in the deracemization synthesis of chiral amino acids (A) D-alanine; (B) L-phenylglycine. (ALADH-alanine dehydrogenase, TA-transaminase, MR-mandelate racemase, DMDH- D-mandelate dehydrogenase, LeuDH-leucine dehydrogenase.)

Fig. 31 Chemo-enzymatic deracemization synthesis of chiral amino acids (A) D-Amino acids; (B) D-Phenylalanine (AAO-amino acid oxidase, PAL-phenylalanine ammonia-lyase, AAD-amino acid deaminase.)

Table 2 Partially applied enzymatic synthetic routes in production

产品 Products	应用 Applications	合成路线 Synthetic routes	酶制剂 Enzyme	参考文献 References
L-phosphinothricin	Broad-spectrum herbicides	Asymmetric reductive amination of keto acids	Glutamate dehydrogenase, Alcohol dehydrogenase/Glucose dehydrogenase/Formate dehydrogenase	[22,28,35]
L-phosphinothricin	Broad-spectrum herbicides	Deracemization synthesis	D-amino acid oxidase, catalase, glutamate dehydrogenase, Alcohol dehydrogenase/Glucose dehydrogenase/Formate dehydrogenase	[14-15]
L-tert-leucine	Intermediate of azanavir, animal feed additive, nutritional fortifier	Asymmetric reductive amination of keto acids	Leucine dehydrogenase，Alcohol dehydrogenase/Glucose dehydrogenase/Formate dehydrogenase	[11-12, 30,32]
L-tert-leucine		Asymmetric transfer of amino groups to keto acids	Transaminase	[62]
L-2-aminobutyric acid	Intermediate of antituberculosis ethambutol and antiepileptic drug levetiracetam	Asymmetric reductive amination of keto acids	Leucine dehydrogenase，threonine deaminase，Glucose dehydrogenase	[33-34]
		Asymmetric transfer of amino groups to keto acids	Transaminase，Glutamate dehydrogenase，Alcohol dehydrogenase	[63]
		Deracemization synthesis	D-amino acid oxidase, ω- Transaminase	[118]
L- phenylglycine	Intermediate of β-lactam antibiotics	Asymmetric reductive amination of keto acids	Amino acid dehydrogenase，Alcohol dehydrogenase/Glucose dehydrogenase/Formate dehydrogenase	[29, 36-37]
L- phenylglycine	Intermediate of β-lactam antibiotics	Deracemization synthesis	Mandelate racemase, D-mandelate dehydrogenase, Leucine dehydrogenase	[16]
(R)-3-amino-4-(2,4,5-trifluorophenyl)butyric acid	Intermediate of siagliptin	Asymmetric transfer of amino groups to keto acids	Transaminase	[13]
L-norvaline	Intermediate of perindopril	Asymmetric transfer of amino groups to keto acids	Transaminase	[62]
(2R, 4S) -ethyl-5-([1,1′- biphenyl]-4-yl) -4- ((tert butoxycarbonyl) amino)-2-methylvaleric acid	Intermediate of sacubitril	Asymmetric transfer of amino groups to keto acids	Transaminase	[53]
L-3,4-dimethoxyphenylalanine	Drug intermediates, chemical sensors, chiral catalysts, etc	Asymmetric transfer of amino groups to keto acids	Transaminase	[64]
(3S)-5-(Benzyloxy)-6-methoxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic Acid	Intermediate of olodanrigan (EMA401)	Enantioselective addition of ammonia to α,β-unsaturated acids	Phenylalanine ammonia-lyase	[80]
(R)-pantothenic acid	Intermediate of antimicrobials against plasmodium falciparum and multidrug-resistant staphylococcus aureus	Enantioselective addition of ammonia to α,β-unsaturated acids	3-methylaspartate ammonia lyase, Aspartate-α-decarboxylase, β-methylaspartate-α-decarboxylase/glutamate decarboxylase, Pantothenate synthetase	[84]
L-syn-p-methylsulfonylphenylserine	Intermediate of flufenicol	Aldol condensation of an amino acid to aldehydes	L-threonine aldolase	[114-116]
β-(2-furyl)serine	Intermediate of furan antibiotic and 2-amino-1-(2-furanyl)ethanol	Aldol condensation of an amino acid to aldehydes	L-threonine aldolase	[117]

Table 2 Partially applied enzymatic synthetic routes in production

产品 Products	应用 Applications	合成路线 Synthetic routes	酶制剂 Enzyme	参考文献 References
L-phosphinothricin	Broad-spectrum herbicides	Asymmetric reductive amination of keto acids	Glutamate dehydrogenase, Alcohol dehydrogenase/Glucose dehydrogenase/Formate dehydrogenase	[22,28,35]
L-phosphinothricin	Broad-spectrum herbicides	Deracemization synthesis	D-amino acid oxidase, catalase, glutamate dehydrogenase, Alcohol dehydrogenase/Glucose dehydrogenase/Formate dehydrogenase	[14-15]
L-tert-leucine	Intermediate of azanavir, animal feed additive, nutritional fortifier	Asymmetric reductive amination of keto acids	Leucine dehydrogenase，Alcohol dehydrogenase/Glucose dehydrogenase/Formate dehydrogenase	[11-12, 30,32]
L-tert-leucine		Asymmetric transfer of amino groups to keto acids	Transaminase	[62]
L-2-aminobutyric acid	Intermediate of antituberculosis ethambutol and antiepileptic drug levetiracetam	Asymmetric reductive amination of keto acids	Leucine dehydrogenase，threonine deaminase，Glucose dehydrogenase	[33-34]
		Asymmetric transfer of amino groups to keto acids	Transaminase，Glutamate dehydrogenase，Alcohol dehydrogenase	[63]
		Deracemization synthesis	D-amino acid oxidase, ω- Transaminase	[118]
L- phenylglycine	Intermediate of β-lactam antibiotics	Asymmetric reductive amination of keto acids	Amino acid dehydrogenase，Alcohol dehydrogenase/Glucose dehydrogenase/Formate dehydrogenase	[29, 36-37]
L- phenylglycine	Intermediate of β-lactam antibiotics	Deracemization synthesis	Mandelate racemase, D-mandelate dehydrogenase, Leucine dehydrogenase	[16]
(R)-3-amino-4-(2,4,5-trifluorophenyl)butyric acid	Intermediate of siagliptin	Asymmetric transfer of amino groups to keto acids	Transaminase	[13]
L-norvaline	Intermediate of perindopril	Asymmetric transfer of amino groups to keto acids	Transaminase	[62]
(2R, 4S) -ethyl-5-([1,1′- biphenyl]-4-yl) -4- ((tert butoxycarbonyl) amino)-2-methylvaleric acid	Intermediate of sacubitril	Asymmetric transfer of amino groups to keto acids	Transaminase	[53]
L-3,4-dimethoxyphenylalanine	Drug intermediates, chemical sensors, chiral catalysts, etc	Asymmetric transfer of amino groups to keto acids	Transaminase	[64]
(3S)-5-(Benzyloxy)-6-methoxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic Acid	Intermediate of olodanrigan (EMA401)	Enantioselective addition of ammonia to α,β-unsaturated acids	Phenylalanine ammonia-lyase	[80]
(R)-pantothenic acid	Intermediate of antimicrobials against plasmodium falciparum and multidrug-resistant staphylococcus aureus	Enantioselective addition of ammonia to α,β-unsaturated acids	3-methylaspartate ammonia lyase, Aspartate-α-decarboxylase, β-methylaspartate-α-decarboxylase/glutamate decarboxylase, Pantothenate synthetase	[84]
L-syn-p-methylsulfonylphenylserine	Intermediate of flufenicol	Aldol condensation of an amino acid to aldehydes	L-threonine aldolase	[114-116]
β-(2-furyl)serine	Intermediate of furan antibiotic and 2-amino-1-(2-furanyl)ethanol	Aldol condensation of an amino acid to aldehydes	L-threonine aldolase	[117]

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