Enzymatic cascade systems for D-amino acid synthesis: progress and perspectives


D-amino acids, different from the ubiquitous L-amino acids, are recognized as the “unnatural” amino acids. The applications of D-amino acids have drawn increasing interest from researchers in recent years, and D-amino acids are widely used in various industries, including for food products, pharmaceuticals, and agricultural chemicals. Inspired by the prevalent applications, many synthetic methods for D-amino acids have been developed, which are mainly divided into chemical synthetic methods and biosynthetic methods. Chemical synthesis of D-amino acids has a variety of disadvantages such as multiple reaction steps, low yields, low reaction rates, and difficulties in product extraction. Thus, biosynthetic methods utilizing enzymes are attracting increasing attention because they are more energy-saving and environmentally friendly compared to traditional chemical synthesis. Among all enzymatic methods, multi-enzymatic cascade catalytic methods have significant advantages, such as lower costs, no need for intermediate separation, and higher catalytic efficiency, which is ascribed to the spatial proximity of biocatalysts. In this review, advances in multi-enzyme cascade catalytic systems as well as chemo-enzymatic approaches to synthesize D-amino acids are discussed.


D-amino acids are considered as “unnatural” amino acids because they do not engage in natural protein synthesis as L-amino acids do. In the past, D-amino acids did not receive as much attention from researchers as their L-enantiomers did and were considered rare in nature. However, with the development of analytical technologies and research methods, D-amino acids have gradually been found to occur in nature [1,2,3,4]. For example, peptidoglycan in the cell wall of bacteria contains D-amino acids [5]. D-methionine and D-leucine are produced by specific enzymes in the stationary phase in Vibrio cholerae, while Bacillus subtilis produces D-tyrosine and D-phenylalanine. D-amino acids may regulate peptidoglycan synthesis and participate in controlling cell wall assembly and modification [2]. In an analysis of 43 species of marine invertebrates in eight phyla, D-amino acids were found in 18 species in six phyla (42%) [6]. In plants, free D-amino acid levels are approximately 0.2–8% relative to the corresponding L-amino acids [7]. In the brain and retina of mammals, metabolic processes of D-serine, such as biosynthesis, extracellular release, uptake, and degradation, occur [8].

In contrast to their sparse existence in nature, D-amino acids are widely used in the pharmaceutical, agricultural, food, and cosmetics industries [9,10,11]. A variety of D-amino acids have been used in the production of ampicillin and amoxicillin (antibiotics), nateglinide (antidiabetic), tadalafil (erectile dysfunction), and fluvalinate (pesticide) [12]. D-lysine is used as an analog of luteinizing hormone-releasing hormone [13] and as a drug carrier in the form of polylysine [14]. D-arylalanines are useful intermediates in the synthesis of β-lactam antibiotics, small peptide hormones, and pesticides [15]. D-phenylglycine and D-para-hydroxyphenylglycine are building blocks for semi-synthesizing cephalosporins and penicillins [11]. Many foods contain D-amino acids, and humans consume on average more than 100 mg of D-amino acids every day [3]. The global market for D-amino acids was estimated at US$ 178.3 Million in 2020 and is forecasted to reach US$ 227.7 Million by 2027 (Global Industry Analysts, Inc. https://www.strategyr.com/market-report-d-amino-acids-forecasts-global-industry-analysts-inc.asp).

Chiral amino acids are produced via the following three main strategies: biological fermentation by microorganisms, kinetic resolution of racemates, and asymmetric synthesis of prochiral materials [16]. The current methods for synthesizing D-amino acids can be divided into the following two categories: chemical synthesis and biocatalytic methods. The D-amino acids produced by chemical synthesis are mainly obtained by chiral asymmetric resolution of D, L-amino acid racemates or by catalyzing chiral or prochiral compounds. However, there are many shortcomings in the chemical synthesis of D-amino acids, such as multiple reaction steps, low yields, low reaction rates, and difficulties in product extraction. In contrast, the biocatalytic method is mild, efficient, energy-saving, and environmentally friendly, which is why it is widely applied to the production of D-amino acids.

The enzymes for D-amino acid production mainly include hydantoinase, L-amino acid oxidase (LAAO), D-amino acid transferase (DAAT), D-amino acid dehydrogenase (DAADH), D-amide hydrolase, and D-ammonia lyase. For example, D-stereospecific amide hydrolases, such as N-acyl-d-amino acid amidohydrolase, D-amino acid amidase, D-aminopeptidase, and alkaline D-peptidase, can be used for kinetic resolution of racemic amino acid amides to produce D-amino acids because these hydrolases only react with the D-enantiomer, but the theoretical yield is merely 50% [17]. The D-amino acid amidase from Arabidopsis SV3 (Ochrobactrum anthropi SV3), which was isolated and characterized in 1989, can act on D-amino acid amides with aromatic or hydrophobic side chains, such as D-phenylalanine amide, D-tyrosine amide, and D-serine amide [18]. LAAO from Rhodococcus sp. AIU Z-35-1 is used to produce a variety of D-amino acids, such as D-glutamic acid, D-arginine, and D-homoserine, because of its wide substrate specificity. However, the theoretical yield is only 50% for most [19].

Although a variety of D-amino acids have been successfully synthesized with these enzymes, some of the enzymes mentioned above cannot produce D-amino acids by themselves or their catalytic activities are low. Hence, the multi-enzyme cascade catalytic reaction for D-amino acid production has received increasing attention from researchers.

Multi-enzymatic cascade reactions are defined as the combination of several enzymatic transformations in concurrent one-pot processes [20]. Compared with traditional single-step catalysis, multi-enzyme cascade reactions have many obvious advantages. The coupling of multiple enzymes improves the catalytic efficiency, eliminates the cumbersome separation and purification steps for intermediates, and directly reduces the cost of conversion. Consequently, a higher yield can be obtained [21, 22]. Multi-enzyme cascade systems have been widely applied in the synthesis of industrial products [23]. N-acetyl- D-neuraminic acid (Neu5Ac) is the starting material to produce anti-influenza virus agent Zanamivir and has great commercial value [24]. It has been successfully synthesized by multi-enzyme reactions with epimerases and aldolases, and the immobilization techniques make the multi-enzyme processes possible for large-scale production [20]. Chiral α-hydroxy ketones, important building blocks in fine chemical and pharmaceutical industries, have also been synthesized by one-pot cascades consisting of alcohol oxidase, benzaldehyde lyase and catalase as well [25].

In recent years, research regarding enzymatic cascade reactions has become increasingly popular. More researchers have chosen to couple two or more enzymes, or even combine chemical catalysts with biocatalysts for the production of targets [26,27,28]. There are many examples of using multi-enzyme systems to synthesize D-amino acids, since the catalytic properties and advantages of various enzymes can be combined to produce D-amino acids with higher efficiency and stereoselectivity [15, 29,30,31].

In this review, advances in multi-enzyme cascade catalytic systems and chemo-enzymatic approaches for the synthesis of D-amino acids are discussed. The methods for producing D-amino acids are classified into the following four categories: chemo-enzymatic approaches, two-enzyme catalytic systems, three-enzyme catalytic systems, and four-enzyme catalytic systems. Each category is addressed and discussed in detail in the current examples. The research prospects of relevant enzyme discovery and reaction route design are also discussed.

Chemo-enzymatic cascade systems

Chemo-enzymatic approaches involve a combination of chemical and biological catalysts. In the past, chemical synthesis was the most commonly used approach for obtaining target products. However, there are circumstances in which chemical catalysts cannot synthesize the target product independently; therefore, biological catalysts are recruited to assist with the catalytic reactions. In recent years, chemo-enzymatic approaches have been utilized to produce D-amino acids.

Chemo-enzymatic cascades involving ammonia lyase

Ammonia lyases are defined as carbon–nitrogen lyases that release ammonia and an unsaturated or cyclic derivative. They typically catalyze the cleavage of the C–N bond of α-amino acids [32]. Ammonia lyases can be classified into various types according to their structure and functions, among which phenylalanine ammonia lyase (PAL) catalyzes the reversible elimination of ammonia from the corresponding aromatic L-phenylalanine to produce cinnamic acid (CA) [33, 34]. PAL is widely employed with ammonia to produce amino acids. Substituted phenylalanines, including (S)-m-methoxyphenylalanine, (S)-p-bromophenylalanine, and (S)-m-(trifluoromethyl) phenylalanine, were successfully obtained through the variant I460V of PAL by Tork et al. [35]. PAL was also immobilized on different supports to catalyze the production of D-amino acids [36,37,38]. For example, PAL from Petroselinum crispum was immobilized in a novel microfluidic device (Magne-Chip) that comprises microliter volume reaction cells filled with PAL-coated magnetic nanoparticles [37]. PAL from Rhodotorula glutinis JN-1 was immobilized on a modified mesoporous silica support (MCM-41-NH-GA), and D-phenylalanine was produced in a scaled-up recirculating packed-bed reactor with a productivity of 7.2 g L−1 h−1 [38].

Ahmed et al. developed and optimized a chemo-enzymatic approach to synthesize a series of N-protected non-natural L- and D-biarylalanine derivatives (Fig. 1). The two following effective biocatalytic strategies were proposed to obtain L- and D-4-bromophenylalanine: hydroamination of 4-bromocinnamic acid and reductive amination of bromophenylpyruvate [39]. Hydroamination of 4-bromocinnamic acid was catalyzed by PAL without any cofactors. After testing several different sources of PAL, AvPAL, a PAL from the cyanobacterium Anabaena variabilis, was found to have the best catalytic activity. It was then mutated to further increase the conversion rate, and L-bromophenylalanine was obtained with > 99% enantiomeric excess (e.e.) D-Bromophenylalanine was obtained by the second catalytic strategy, where DAADH was utilized to catalyze the reductive amination of 4-bromophenylpyruvate, coupling the glucose/glucose dehydrogenase (GDH) system to regenerate the cofactor NADPH. In this strategy, 4-bromophenylpyruvate was completely converted into D-bromophenylalanine with > 99% e.e. Next, a palladium catalyst was employed to protect the amino group and conduct Suzuki–Miyaura coupling with arylboronic acid, and the target product L- and D-biarylalanine derivatives were obtained with high yield and optical purity under mild aqueous conditions.

Fig. 1

Two strategies for L- and D-4-bromophenylalanine synthesis by chemo-enzymatic methods. AvPAL refers to phenylalanine ammonia lyase from the cyanobacterium Anabaena variabilis; DAADH refers to D-amino acid dehydrogenase; GDH refers to glucose dehydrogenase

PAL was also coupled with the chemical catalyst NH3:BH3 to produce D-amino acids. Parmeggiani et al. designed a novel one-pot multi-enzyme method to synthesize substituted D-phenylalanine from a cheap substrate, CA [40]. This PAL-mediated synthesis of D-phenylalanine is both economical and low-cost. This cascade catalysis process consists of the two following steps: the amination step catalyzed by PAL and the deracemization step catalyzed by a chemical enzymatic method. In this chemo-enzymatic method, L-amino acid deaminase (LAAD) is used for deracemization, and the chemical reagent is NH3:BH3, which is more compatible with enzymes. PAL undergoes a hydroxylamination reaction to catalyze the substrate into D,L-phenylalanine. LAAD then stereoselectively oxidizes L-phenylalanine into amino acids, which are then chemically reduced to L- or D-phenylalanine. In this process, D-phenylalanine is accumulated (Fig. 2).

Fig. 2

Chemo-enzymatic synthesis of D-phenylalanine derivatives involving phenylalanine ammonia lyase and L-amino acid deaminase with reducing agent NH3:BH3. PAL refers to phenylalanine ammonia lyase; LAAD refers to L-amino acid deaminase

A previous study also developed a facile high-throughput solid-phase screening method based on the intensity of color to identify PAL mutants with a higher conversion rate. D-amino acid oxidase and horseradish peroxidase were utilized to convert colorless 3,3′-diaminobenzidine to a polymeric brown dye, the color intensity of which scales up with the amount of hydrogen peroxide generated by D-phenylalanine oxidation. The best mutants were applied to the cascade reaction system to convert a series of CAs into optically pure D-phenylalanine derivatives with a maximum yield of 80%.

Zhu et al. also constructed a similar one-pot two-enzyme catalytic system to synthesize D-arylalanine [15]. This system mainly involves two enzymes; AvPAL and LAAD, from Proteus mirabilis. Wild-type PAL with low stereoselectivity can catalyze the amination of substituted trans-CA (t-CA) to generate a racemic mixture of D,L-arylalanine. The Asn residue in the active site of wild-type AvPAL is mutated and successfully increases the stereoselectivity towards D-arylalanine by 2.3 fold, but L-arylalanine remains in the products of t-CA amination. Therefore, L-arylalanine is converted to D-arylalanine through stereoselective oxidation by LAAD and non-selective reduction by the borohydride compound NH3:BH3. The optimal t-CA conversion rate reaches 82% with an optical purity of D-phenylalanine e.e.D > 99%. In addition, the conversion rate and optical purity of the catalytic system vary according to the position and electronic properties of the CA substituents. Meta-substituted t-CA and t-CA with strong electron-withdrawing groups (such as nitro-) on the benzene ring show higher overall conversion rates. For example, the yield of m-nitro-D-phenylalanine reaches 96%, and the optical purity exceeds 99%.

The catalytic route involving PAL and LAAD has also been employed by Parmeggiani et al., who proposed four routes combining chemical catalysts and enzymes to produce D-(2,4,5-trifluorophenyl) alanine, a key precursor of the antidiabetic sitagliptin (Fig. 3) [41]. For example, of the proposed reaction routes, the intermediate keto acid was obtained by standard Erlenmeyer-Plöchl synthesis involving the chemical reagents N-acetylglycine, acetic anhydride, and anhydrous sodium acetate and then transformed into the product through reductive amination catalyzed by DAADH with GDH or DAAT with D-aspartate oxidase. Another route resembled the synthetic strategy using PAL and LAAD, as mentioned above.

Fig. 3

Four chemo-enzymatic routes from corresponding trifluorobenzaldehyde to D-(2,4,5-trifluorophenyl) alanine consisting of chemical catalytic steps and enzymatic catalytic step. The biocatalysts used here include D-amino acid dehydrogenase (DAADH), glucose dehydrogenase (GDH), D-amino acid transferase (DAAT), L-amino acid deaminase (LAAD), D-aspartate oxidase (DDO), and ene-reductase (ERED)

Chemo-enzymatic cascades involving L-amino acid oxidase

The widely used chemical reductant NH3:BH3 has also been coupled with other kinds of biocatalysts, such as LAAO, to produce D-amino acids. LAAO and LAAD are flavoenzymes containing non-covalently bound flavin adenine dinucleotide (FAD), which catalyzes the stereospecific oxidative deamination of L-amino acids into α-keto acids, ammonia, and hydrogen peroxide [42]. Nakano et al. found a potential ancestral variant of LAAO (AncLAAO) in Pseudoalteromonas piscicida and successfully expressed it in Escherichia coli [43]. This novel LAAO has a significantly broad substrate selectivity. While coupling with the chemical reductant NH3:BH3, 16 kinds of racemates of D,L-amino acids can be completely converted into D-amino acids, including nine D,L-phenylalanine derivatives and six D,L-tryptophan derivatives (Fig. 4).

Fig. 4

Deracemization reaction cascades employing L-amino acid oxidase (LAAO) and chemical reductant NH3:BH3 to obtain enantiomerically pure D-amino acids

A similar chemoenzymatic catalytic strategy was employed by Schnepel et al. who synthesized substituted D-tryptophan derivatives through a specific LAAO (RebO) from the actinomycete Lechevalieria aerocolonigenes [44]. In the presence of the reductive agent NH3:BH3, halotryptophan was produced by RebO and halogenase. The conversion rate and e.e. of 5- and 7-bromotryptophan reached 90% and 92%, respectively. Another similar situation was introduced by Alexandre et al. to produce a range of D-amino acids by deracemization of D,L-amino acids, among which optically pure D-leucine was produced with a 98% yield [45].

Although many chemo-enzymatic approaches have successfully produced D-amino acids, these approaches are limited by chemical catalysts that are not economical or environmentally friendly. Furthermore, the enzymes may be unstable while coexisting with the chemical catalysts; thus, multi-enzyme catalytic systems have more developing potential compared with these chemical enzymatic methods.

Two-enzyme cascade catalytic systems

Two-enzyme cascade catalytic systems are the most widely reported multi-enzyme systems for D-amino acid production. The two-enzyme systems are discussed in two parts: racemate resolution and asymmetric synthesis. Most examples discussed here involve co-enzyme recycling systems because the catalyzing enzymes are generally cofactor-dependent.

L-amino acid dehydrogenase and NADH oxidase (NOX) for the resolution of racemates

The first example of a two-enzyme system aims to produce D-tert-leucine. Owing to the bulky and hydrophobic tert-butyl side chain, tert-leucine has been increasingly used in the synthesis of biologically active compounds and chiral auxiliaries in recent years [46]. L-tert-leucine is successfully obtained using amino acid dehydrogenase. However, this method of reductive amination is not suitable for the synthesis of D-tert-leucine because there is no corresponding D-specific leucine dehydrogenase (LeuDH). A new two-enzyme catalytic approach has been reported that has resulted in breakthroughs to the effective production of D-tert-leucine. L-LeuDH was employed to oxidatively resolve a racemic mixture of D,L-tert leucine [47]. Since the reaction catalyzed by this NAD+-dependent dehydrogenase is reversible, it was coupled with the NAD+ regeneration reaction catalyzed by NOX from Lactobacillus brevis. In this case, NAD+-dependent dehydrogenase can not only catalyze predominantly the reaction of oxidative deamination but NAD+ supply is also guaranteed. The NAD+ regeneration step is an efficient and irreversible reaction. With this enzymatic catalytic system, L-tert-leucine is completely oxidized, and D-tert-leucine (e.e. > 99%) is successfully prepared (Fig. 5).

Fig. 5

Selective oxidation of racemic tert-leucine to produce D-tert-leucine catalyzed by leucine dehydrogenase (LeuDH) with NAD+ regenerated by NADH oxidase (NOX)

Succinylase and racemase for the resolution of racemates

A one-pot method for D-amino acid production was constructed using two enzymes for the dynamic kinetic resolution of N-succinyl-amino acids [48]. These two enzymes are D-succinylases (DSAs) from Cupriavidus sp. P4-10-C and N-succinyl-amino acid racemase (NSAR) from Geobacillus stearothermophilus NCA1503. Both DSA and NSAR have broad substrate specificity, and D-succinyl-amino acid is the natural substrate of D-acyl-amino acid racemase. Therefore, DSA and NSAR have strong potential for producing D-amino acids through kinetic resolution. The N-succinyl-D,L-amino acids are enantioselectively hydrolyzed to N-succinyl-D-amino acids by DSA, and the residual N-succinyl-L-amino acids are racemized back to N-succinyl-D,L-amino acids.

In addition, the amount of DSA affects the optical purity of the generated D-amino acids. A smaller amount of DSA tends to generate products with higher optical purity. The highest optical purity was obtained in a 1 mL reaction system with 1.5 U of NSAR and 100 mM of D-succinyl-phenylalanine substrate. After 20 h of reaction at 40 °C, the product D-phenylalanine reached an e.e. of 91.6% and a conversion rate of 82.4%. Even though the two enzymes have the lowest activity on N-succinyl-D,L-tryptophan, 100 mM of N-succinyl-D,L-tryptophan was successfully resolved into D-tryptophan (conversion rate 81.8%, e.e. 94.7%). This enzymatic method is expected to be applied to the industrial production of various D-amino acids (Fig. 6) [12, 48].

Fig. 6

Dynamic kinetic resolution of N-succinyl-D,L-amino acids to produce D-phenylalanine by D-succinylase (DSA) and N-succinyl-amino acid racemase (NSAR)

Amino acid racemase and decarboxylase for the resolution of racemates

Since many enzymes that have been reported to directly transfer D,L-amino acids to D-amino acids are rarely active towards lysine [49], Wang et al. developed a strategy that employs enzymes that selectively degrade L-lysine from a mixture of D,L-lysine [30]. Because of the overcapacity of L-lysine in industry today wherein the annual output exceeds two million tons, L-lysine is promising as a starting material to produce D-amino acids. This method involves two enzymes, lysine racemase and decarboxylase. First, the substrate L-lysine is racemized by lysine racemase to generate D,L-lysine. The L-lysine in D,L-lysine is selectively degraded into cadaverine by lysine decarboxylase, which has the highest catalytic activity in the form of crude enzymes compared with permeabilized cells and resting whole cells. In this cyclic process, D-lysine accumulates in a stepwise manner. The reaction temperature, pH, metal ion additives, and pyridoxal 5′-phosphate (PLP) content in the cascade catalytic system have been studied and further optimized. After 1 h of the racemization reaction and 0.5 h of the decarboxylation reaction, 750.7 mM of D-lysine is generated (substrate L-lysine concentration be 1710 mM). The D-lysine yield reaches 48.8% with e.e. > 99% (Fig. 7).

Fig. 7

Two-enzyme catalytic cascade of lysine racemase and lysine decarboxylase for the production of enantiomerically pure D-lysine

D-amino acid dehydrogenase and glucose dehydrogenase for asymmetric synthesis

DAADH is an NADPH-dependent oxidoreductase that catalyzes the asymmetric reductive amination of α-keto acids to the corresponding D-amino acids. Due to its low abundance and membrane-bound characteristics, natural DAADH has not been reported to synthesize D-amino acids through its reductive amination process [50,51,52]. Due to the weak ability of natural DAADH to catalyze the production of D-amino acids, many researchers have made mutations and modifications to refine it. For example, Novick modified the meso-2,6-D-diaminopimelic acid dehydrogenase (DAPDH) from Corynebacterium glutamicum by protein engineering methods and obtained a highly stereoselective DAADH [50]. By combining rational and random mutations, after three rounds of mutagenesis, a BC621 variant with D155G/Q151L/R196M/T170I/H245N substitutions was generated. This variant can be used to produce D-amino acids in the presence of the cofactor NADPH and ammonia. In this case, the corresponding α-keto acid was reduced to generate a variety of D-amino acids. The variant enzyme exhibits broad substrate specificity and has been successfully used to produce various D-amino acids with e.e. > 95%. Various D-amino acids, such as D-alanine, D-valine, and D-lysine, were successfully synthesized through meso-DAPDH by Gao et al. [53]. To expand the substrate-binding pocket of meso-DAPDH from Symbiobacterium thermophilum to allow bulky substrates, such as 2-keto acids, site saturation mutagenesis was carried out on four residues (Phe146, Thr171, Arg181, and His227) [52]. Recently, Hayashi et al. generated a single D94A mutant using site-directed mutagenesis [54]. The catalytic activity toward various D-amino acids, such as D-phenylalanine, D-leucine, D-norleucine, D-methionine, D-isoleucine, and D-tryptophan, was consequently improved, among which D-phenylalanine had the highest activity of 5.33 μmol min−1 mg−1 at 50 °C. Cheng et al. ulteriorly engineered this biocatalyst by introducing the double mutation W121L/H227I, whose activity toward sterically bulkier 2-keto acids was significantly enhanced [55]. Among all mutants, the specific activity of a single mutant H227V was 35.1 times higher than that of the wild-type enzymes.

In addition to DAADH, a two-enzyme system involving DAPDH and GDH for cofactor recycling produces D-branched chain amino acids. The function of D-branched chain amino acids has attracted increasing attention from researchers in recent years owing to their significant biological activity. For example, D-isoleucine is related to the growth of Matsutake mushrooms [56], D-leucine causes the disassembly of Bacillus subtilis biofilms, and D,L-[1-13C] isoleucine can be used to elucidate the biosynthetic pathway of alkaloids in Penicillium sp. EPF-6 [57]. Motivated by the multiple applications of D-branchedchain amino acids, Hironaga et al. developed a two-enzyme catalytic system that enantioselectively synthesizes D-branchedchain amino acids [58]. The two enzymes in this catalytic system are thermostable DAADH [59] and thermostable GDH. GDH together with D-glucose and NADP+, was used to regenerate the cofactor NADPH. The optimized system successfully converted 2-oxo-4-methylpentanoic acid to D-leucine, and the yield and optical purity were both higher than 99% (Fig. 8). In addition, the activity of the thermostable DAADH was increased by removing its C-terminal His tag, and higher activity towards D-phenylalanine, D-norleucine, and D-methionine was achieved by Asp94Ala substitution [51]. Although this system was successfully applied to synthesize D-leucine and has the potential to synthesize a series of other D-amino acids, there remain the following few shortcomings: the yield of D-leucine is influenced by the concentration of NADP+ and NADP+ may be unstable at a relatively higher pH (10.5) of this system.

Fig. 8

The DAADH/GDH system for the synthesis of enantioselective D-branched amino acids. DAADH refers to D-amino acid dehydrogenase; GDH refers to glucose dehydrogenase

L-methionine γ-lyase and transaminase for asymmetric synthesis

Amino transferases are also prevalent enzymes in the production of D-amino acids. Aminotransferase, or transaminase (TA), belonging to fold types I and IV of PLP-dependent enzymes, can catalyze the transamination from a suitable amine donor to a carbonyl acceptor with the aid of the cofactor PLP. PLP-dependent TAs are classified into α-TAs and ω-TAs according to the substrate they catalyze [60]. For example, five types of D-amino acids (D-alanine, D-homoalanine, D-fluoroalanine, D-serine, and D-norvaline) were synthesized by ω-TAs with > 99.7% e.e. using racemic α-methylbenzylamine as an amino donor [61]. A variety of keto- acids, including α-ketobutyrate, glyoxylate, indole-3-pyruvate, α-ketovalerate, 3-methyl-2-ketobutyrate, and 4-hydroxyphenyl-pyruvate, can be transferred to the corresponding D-amino acids by D-amino acid transaminase from Lactobacillus salivarius [62]. Due to the small substrate-binding pocket of ω-TAs, Han et al. introduced mutations to the ω-TA from Ochrobactrum anthropi and generated the variant L57A, the activity of which toward substrates with bulkier substituents (up to n-butyl substituent) was remarkably improved [63]. L- and D-norvaline (e.e. > 99%) were obtained by this variant with conversion rates of 99.3% and 51.6%, respectively.

Silva et al. used a two-step enzymatic method to convert the cheap and easily available natural amino acid L-methionine into both enantiomers of homoalanine [29]. L-methionine was first catalyzed into 2-oxobutyrate by L-methionine γ-lyase (METase) from Fusobacterium nucleatum. Amino acid aminotransferases from different sources were then used to generate L- or D-homoalanine. Among them, D-homoalanine was obtained through D-amino acid aminotransferase (DAAT) from Bacillus sp. using D-alanine as an amino donor. D-homoalanine was obtained with a yield of 69% and an optical purity of 90% (Fig. 9).

Fig. 9

The two-step enzymatic method to convert L-methionine into D-enantiomers of homoalanine. METase refers to L-methionine γ-lyase, DAAT refers to D-amino acid aminotransferase

Three-enzyme cascade catalytic systems

Although many two-enzyme catalytic systems have successfully produced D-amino acids, there are still conditions in which two enzymes are not sufficient to generate D-amino acids, or the substrates for some two-enzyme systems are expensive. Three-enzyme cascade catalytic systems are complementary to two-enzyme systems.

L-amino acid deaminase, D-amino acid dehydrogenase, and a cofactor recycling system

This three-enzyme cascade uses L-amino acids as the starting material and employs a cofactor recycling system. LAAD and DAADH are responsible for converting L-amino acids to their corresponding D-amino acids without the need to add intermediate keto acids. The most commonly used biocatalysts for cofactor regeneration are GDH and formate dehydrogenase (FDH).

This strategy was employed by Parmeggiani et al. to construct a three-enzyme system to produce D-arylalanines [64]. This is a pragmatic and effective method involving mainly DAADH and GDH to catalyze the reductive amination of keto acids to produce D-arylalanines. The authors first used LAAD to deaminate L-arylalanine and converted it to the corresponding keto acid. LAAD, which is different to the FAD-containing flavoenzyme LAAO, catalyzes the deamination of L-amino acids to the corresponding α-keto acids without producing hydrogen peroxide [65,66,67]. Then, the keto acid is converted into the corresponding D-arylalanine through the DAADH/GDH system (Fig. 10). The conversion rate of the one-pot two-step strategy is 95%. This strategy also shows impressive enantioselectivity (98%) and yield on a variety of substrates on the preparative scale. In this method, an engineered whole-cell catalyst and an enantiomeric complementary deaminase are facilely combined to mediate the production of D-phenylalanine derivatives. This one-pot biocatalytic method provides a new and effective strategy for the biocatalytic production of D-amino acids.

Fig. 10

One-pot two-step cascade for the synthesis of D-arylalanine by stereo-conversion. LAAD refers to L-amino acid deaminase; DAADH refers to D-amino acid dehydrogenase; GDH refers to glucose dehydrogenase

Zhang et al. also successfully synthesized optically pure D-phenylalanine from the corresponding L-enantiomer through a three-enzyme cascade catalytic system [68]. In addition to LAAD and DAPDH, which first convert L-phenylalanine to the intermediate phenylpyruvate and then to D-phenylalanine, FDH was employed to catalyze the regeneration of the cofactor NADPH. D-phenylalanine was produced in quantitative yield with > 99% e.e. (Fig. 11).

Fig. 11

One-pot three enzyme catalytic system consisting of oxidative deamination by LAAD, reductive amination by DAPDH, and a cofactor recycling system by FDH. LAAD refers to L-amino acid deaminase; DAPDH refers to meso-diaminopimelate dehydrogenase; FDH refers to formate dehydrogenase

L-amino acid dehydrogenase, transaminase, and NADH oxidase

Another example involving a cofactor recycling system is a one-pot multi-enzyme catalytic system that deracemizes a racemic mixture of aliphatic amino acids to produce D-amino acids [69]. This catalytic system consists of the three following enzymes: L-alanine dehydrogenase (AlaDH), D-selective ω-TA, and NOX. This cascade system primarily undergoes two reactions; (1) the stereoinversion of L-amino acids to D-enantiomers catalyzed by L-AlaDH and ω-TA, and (2) the regeneration of the cofactor NAD+ by NOX (Fig. 12). D-alanine was obtained with a yield of 95% ultimately with a reaction system comprising 100 mM of isopropylamine and 1 mM of NAD+ after 24 h. The optical purity exceeded 99%. This method is efficient and economical because racemic amino acids can be easily prepared by the Strecker synthesis method without the need for expensive intermediate keto acids.

Fig. 12

Deracemization cascade by AlaDH, ω-TA, and NOX to produce D-amino acids. L-AlaDH refers to L-alanine dehydrogenase; ω-TA refers to D-selective ω-transaminase; NOX refers to NADH oxidase

Hydantoin racemase, hydantoinase, and D-carbamoyl hydrolase

Hydantoinase is a conventional catalyst for the production of D-amino acids. The enzymatic catalysis process of hydantoinase coupled with D-carbamyl hydrolase and hydantoin racemase uses 5-substituted D,L-hydantoin as a starting material, which can be obtained by chemical methods. The catalytic process consists of the following two steps: the racemate 5-substituted D,L-hydantoin is generated by hydantoin racemase; 5-substituted D,L-hydantoin is hydrolyzed by D-hydantoinase to the corresponding D-N-carbamoyl amino acids, and then undergoes decarbamylation by D-carbamoyl hydrolase to generate the corresponding D-amino acid. D-phenylglycine, D-p-hydroxyphenylglycine, D-tryptophan, D-phenylalanine, D-valine, D-alanine, and D-methionine were produced by this method [9]. Chen et al. successfully converted 50 g L−1 of D,L-5-isopropylhydantoin into D-valine within 48 h through the hydantoinase process, with a conversion rate of 88% [70] (Fig. 13). This hydantoinase process has also been employed to produce D-tryptophan [71]. Hydantoin racemase from Arthrobacter aurescens and D-hydantoinase from Agrobacterium tumefaciens were coupled with D-carbamoylase from Arthrobacter crystallopoietes, and the dynamic resolution resulted in D-tryptophan with a 99.4% yield and e.e. > 99.9%. Although hydantoinase has been successfully applied to synthesize several D-amino acids, its catalytic activity and substrate range need to be further improved.

Fig. 13

The hydantoin process to produce D-valine with hydantoin racemase, D-hydantoinase, and D-carbamoylase

Tryptophan synthase, L-amino acid deaminase, and aminotransferase

LAAD and aminotransferase were applied in a two-enzyme system to synthesize D-phenylalanine and D-phenylalanine derivatives containing electron-donating or -withdrawing substituents [31]. The aminotransferase variant, T242G, was employed in this cascade. D-phenylalanine was generated with a conversion rate of 99% and e.e. > 99%, and D-phenylalanine derivatives were also obtained with good yield (> 94%) and optical purity (> 98%).

The DAAT was further mutated to increase the binding affinity towards D-tryptophan, which has a bulky indole side chain [72]. The resultant variant DAAT-V33G/T242G with a 35-fold improvement in kcat/Km towards D-tryptophan compared with the wild-type was coupled with tryptophan synthase from Salmonella enterica and LAAD from Proteus myxofaciens to produce D-tryptophan from indole. In a larger reaction scale (200 mL), optically pure D-tryptophan was produced with a yield of 66%. A wide range of D-tryptophan derivatives was also obtained through this three-enzyme procedure (Fig. 14).

Fig. 14

Three-enzyme system consisting of tryptophan synthase (TrpS), L-amino acid deaminase (LAAD), and the engineered D-amino acid transferase (DAAT) for D-tryptophan synthesis

Four-enzyme cascade catalytic systems

Transaminase, amino acid racemase, amino acid dehydrogenase, and formate dehydrogenase

Nakajima et al. developed an effective four-enzyme catalytic system to produce various D-amino acids with the addition of corresponding α-ketoglutarate and ammonia. A variety of D-amino acids were successfully obtained with > 80% yield [73]. The four enzymes utilized in this cascade system are DAAT, glutamate racemase, glutamate dehydrogenase, and FDH. In this multi-enzyme system, glutamate dehydrogenase and glutamate racemase undergo a coupling reaction to regenerate D-glutamate in the presence of ammonia, α-ketoglutarate, and cofactor NADH. Glutamate is then used as an amino donor together with the corresponding α-ketoglutarate to enantioselectively synthesize D-amino acids with DAAT. FDH is responsible for the regeneration of the co-enzyme NADH. Since D-glutamate can be regenerated through the cyclic system of glutamate dehydrogenase and glutamate racemase, only a small amount of D-glutamate is needed to trigger the system (Fig. 15).

Fig. 15

Four-enzyme catalytic system combining α-ketoglutarate and ammonia to produce D-amino acids. The four enzymes used here are formate dehydrogenase, glutamate dehydrogenase, D-amino acid transferase, and glutamate racemase

Although the reactions catalyzed by DAAT, glutamate racemase, and glutamate dehydrogenase are all reversible, the reaction catalyzed by FDH trends towards oxidative decarboxylation, which converts the formation of formate to CO2 and generates NADH. This multi-enzyme system will still proceed towards the generation of D-amino acids.

D-valine was first successfully synthesized through this multi-enzyme system with an optical purity of 100%. D-valine, D-alanine, D-leucine, D-methionine, and D-aspartic acid were also produced with molar yields > 80% after the optimization of this system, although the overall output was low and the highest production of D-valine was only 450 μM.

Due to the instability of glutamate racemase, a multi-enzyme system was further developed by assembling L-AlaDH and alanine racemase to replace glutamate dehydrogenase and glutamate racemase [74]. D-glutamic acid, D-leucine, D-norleucine, and D-methionine were synthesized with a high conversion rate and optical purity through this new multi-enzyme system. However, the yield of D-phenylalanine and D-tyrosine is less than 50% even though the optical purity of these two products is nearly 100% because D-phenylalanine and D-tyrosine are poor substrates for DAAT, but the yield can be increased by adding excess DAAT.

Bae et al. also studied a multi-enzyme system composed of glutamate racemase, thermostable DAAT, glutamate dehydrogenase, and FDH. The system was optimized in terms of substrate concentration, reaction conditions, and feeding methods. D-phenylalanine and D-tyrosine were successfully produced from the corresponding α-keto acids, even though these two D-amino acids are not ideal substrates for DAAT. Their research has improved the catalytic capacity of this multi-enzyme system to synthesize aromatic D-amino acids with a conversion rate as high as 100% [75]. In addition, the authors developed a thermostable glutamate racemase from Bacillus thermophilus SK-1. After 35 h of reaction, the multi-enzyme system produced 58 g L−1 optically pure D-phenylalanine from equimolar amounts of phenylpyruvate [76].

Conclusion and perspective

D-amino acids have a variety of applications in the pharmaceutical, agricultural, food, and cosmetics industries and have attracted increasing attention in recent years. In this review, various multi-enzyme cascade systems for D-amino acid synthesis are introduced and discussed. Compared to traditional chemical synthesis, multi-enzyme systems have significant advantages, including circumventing the separation of intermediates, avoiding the accumulation of toxic intermediates, reaching higher yields, and reducing operational workload. In addition, multi-enzyme cascade systems are more economical and environmentally friendly. Thus, multi-enzyme systems would be more feasible for the industrial production of D-amino acids.

Although multi-enzyme cascade systems are promising to produce D-amino acids, there remain several shortcomings of multi-enzyme systems for D-amino acids production at an industrial scale. Since all the enzymes in the catalytic cascade system are in the same environment, the optimum reaction conditions of the enzymes should be compatible. Besides, enzymes are vulnerable to the changes of temperature, pH, metal ion, and other factors, which limits the application of multi-enzyme systems in the industrial production of D-amino acids.

To further improve the applicability of the multi-enzyme system, effective technologies for novel enzyme discovery still need to be developed. Since enzymes are vulnerable to environmental conditions, exploiting new technologies to enhance the stability of enzymes under unfavorable reaction conditions remains necessary. For example, the stability, catalytic activity, and substrate selectivity of biocatalysts should be improved, and the substrate spectrum should be broadened via protein engineering methods.

Designing new multi-enzyme cascade routes requires careful consideration. Some multi-enzyme routes may have major reaction steps to drive the entire system or rate-limiting steps that restrict the conversion rate, which should be taken into consideration. Furthermore, the enzymes in a cascade reaction system can be assembled in a confined environment, especially using the designed nanomaterial facilitating substance and electron transfer involved in the reactions for D-amino acid production, such as inorganic materials, nanoparticles, polymersomes, and bio-based scaffolds, including DNA scaffolds (DNA strips, tiles, bundles, or cages) and protein scaffolds (protein–protein interactions and peptide-protein complexes). Multi-enzyme assembled systems can spatially organize enzyme-mediated reaction steps and provide intermediate channeling to improve the catalytic performance of the entire cascade system. With the development of bio- and reaction-compatible materials, multi-enzyme co-immobilization and self-assembly would be promising for the improvement of enzymatic cascade systems.


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Financial supports from the National Natural Science Foundation of China (NSFC) (No. 31872891), the 111 Project (No. 111-2-06), the High-End Foreign Experts Recruitment Program (No. G20190010083), the National Program for Support of Top-Notch Young Professionals, the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, Top-Notch Academic Programs Project of Jiangsu Higher Education Institutions, the Jiangsu Province “Collaborative Innovation Center for Advanced Industrial Fermentation” Industry Development Program, the Program for the Key Laboratory of Enzymes of Suqian (No. M201803), and the National First-Class Discipline Program of Light Industry Technology and Engineering (No. LITE2018-09) are greatly appreciated.


Financial supports from the National Natural Science Foundation of China (NSFC) (No. 31872891), the 111 Project (No. 111-2-06), the High-End Foreign Experts Recruitment Program (No. G20190010083), the National Program for Support of Top-Notch Young Professionals, the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, Top-Notch Academic Programs Project of Jiangsu Higher Education Institutions, the Jiangsu Province “Collaborative Innovation Center for Advanced Industrial Fermentation” Industry Development Program, the Program for the Key Laboratory of Enzymes of Suqian (No. M201803), and the National First-Class Discipline Program of Light Industry Technology and Engineering (No. LITE2018-09) are greatly appreciated.

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Fan, A., Li, J., Yu, Y. et al. Enzymatic cascade systems for D-amino acid synthesis: progress and perspectives. Syst Microbiol and Biomanuf (2021). https://doi.org/10.1007/s43393-021-00037-9

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  • D-amino acid
  • Multi-enzyme system
  • Cascade reaction
  • Racemates resolution
  • Asymmetric synthesis