Concise construction of β-branched aromatic D-amino acids via transaminase-catalyzed dynamic kinetic resolution

Domain: chemistry
Published: 2026-03-06
Editorial outcome: major_revision

Abstract
The synthesis of non-canonical amino acids (ncAAs) has recently made tremendous progress in the field of biocatalysis, with enzymes such as transaminases, dehydrogenases, and ammonia lyases being used. Frameworks of β-branched aromatic D-amino acids are crucial for the efficacy of pharmaceuticals, influencing spatial conformation, binding modes with receptors and pharmacokinetic properties. However, there is a scarcity of studies on the efficient synthesis of β-branched D-amino acids, which has significantly hindered the advancement of structure-activity relationship (SAR) studies. Here, we present the asymmetric synthesis of β-branched aromatic D-amino acid analogues catalyzed by PLP-dependent transaminase BsDAAT (from Bacillus sp. strain YM-1), an efficient transamination system that relies on the dynamic kinetic resolution (DKR) process. Through docking analysis and concise enzyme engineering, we successfully obtain various aromatic D-amino acids with β-substitution in high yields (up to 99%), dr ratios (>20:1) and ee values (up to 99%). These β-branched aromatic D-amino acids are then successfully introduced into the modifications of the antineoplastic reagent Lanreotide.

Introduction
D-amino acids (DAAs), enantiomers of the naturally occurring L-amino acids (LAAs), have attracted significant interest in the fields of medicinal chemistry, material science, and biotechnology due to their distinctive biological functions and broad range of applications.1–4 Although they are less prevalent in nature than LAAs, DAAs with biofunctions may not be easily replaced by their enantiomers. For instance, D-serine (D-Ser) and D-aspartic acid (D-Asp) are prevalent in tissues and bodily fluids of vertebrates. Notably, D-Asp is prevalent in the central nervous systems and endocrine peripheral organs of humans and rats.5,6 DAAs also constitute various active natural products and drugs, such as antibiotics, antidiabetics and chemotherapeutic agents. Examples include Nateglinide (D-Phe), Cefadroxil (D-Hpg), Fluvalinate (D-Val), Octreotide (D-Trp, D-Phe) and Lanreotide (D-Napa, D-Trp) (Fig. 1A).7–10 Structure-activity relationship studies have indicated that LAAs with β-modifications exhibit superior pharmacokinetic and pharmacodynamic properties compared with natural LAAs.11,12 However, there has been no investigation on whether DAAs with β-substitutions could also improve the therapeutic efficacy of bioactive molecules.

The emergence of biocatalysis has led to substantial advancements in the asymmetric synthesis of β-substituted L-amino acids, such as lyases,13–15 transaminases,16–18 and artificial photoenzymatic strategies.19,20 For example, a notable study by Renata et al. achieved the stereoselective synthesis of β-substituted L-amino acids via enzyme-catalyzed dynamic kinetic resolution (DKR). Their work detailed the DKR process of ketoacids and selectivity of TtArAT.16 In another innovative research by Merck, the corresponding bulky β-substituted L-amino acid isomers were synthesized through a dynamic equilibrium driven by the key side reaction of the amine donor.17

In recent years, increasing efforts have been devoted to the synthesis of D-amino acids and their derivatives in accordance with the principles of green chemistry and environmentally sustainable solutions.21,22 Effective biocatalysts include transaminases and dehydrogenases, which have the potential to produce DAAs with a single enantiomer from achiral ketoacids (Fig. 1B).23–27 A recent example of the enzymatic synthesis of β-branched D-amino acids was presented by Seebeck et al., who achieved transformation via a cascading process involving transaminases (TAs) and methyltransferases (MTs) (Fig. 1C).28 Multiple aliphatic β-Me L-/D-amino acids were generated with high stereoselectivity under the catalysis of selected MTs and TAs. However, the enzymatic synthesis of aromatic DAAs with β-branches has not been widely demonstrated in published research.

Herein, we report the enzymatic synthesis of β-substituted aromatic D-amino acids via a dynamic kinetic resolution (DKR) process mediated by transaminase BsDAAT. DKR is a powerful enantioselective methodology that converts both enantiomers of a racemic substrate into a single enantiomeric product. In our DKR process of BsDAAT system, two ketoacid enantiomers could be interconverted rapidly and spontaneously, but only fast-reacting (R)-enantiomer could be selected to undergo enzymatic transformation into the β-substituted D-amino acids. This innovative biocatalytic system yields a diverse array of DAAs with two adjacent chiral centers from the appropriate achiral ketoacids with high efficiencies (up to 99% yield) and stereoselectivities (up to >20:1 dr, >99% ee) (Fig. 1D).

Results
Inspired by the capability of enzyme-catalyzed DKR to establish two consecutive chiral centers, we sought to exploit this strategy to constructing aromatic β-substituted D-amino acids. Following recent investigations on the enzymatic synthesis of DAAs, D-phenylalanine aminotransferase (DAAT) from Bacillus sp. YM-1, which exhibits excellent natural activity and stereoselectivity in production of D-Phe, was selected for our study.27,29 Initially, we evaluated the activity of BsDAAT-catalyzed reaction on racemic 2-oxo-3-phenylbutanoic acid (1s) under conditions: PLP (10 mol%), D-Glu (4 equiv.) and 0.6 mol% of the purified BsDAAT-WT enzyme in a KPi buffer solution (100 mM, pH 8.0) containing 1% (v/v) DMSO, at 37 °C for 18 hours. Although the desired product with high stereoselectivity could be identified by TLC and LCMS analysis, only a small amount of product was obtained from a 0.06 mmol-scale reaction (Fig. 2A, Entry 1 and Supplementary Table 2). To evaluate simpler reaction protocols, we explored transamination reactions catalyzed by whole cells or lysates expressing BsDAAT. However, non-stereospecific product 1 was observed, albeit with increased yields. We hypothesized that the non-native substrate 1s was also bio-transformed by the endogenous enzymes in E. coli, such as transaminase and dehydrogenase.27,30–32 This hypothesis was confirmed by the detection of product 1 catalyzed by cultivated E. coli BL21 (DE3) cells without any exogenous enzymes (Fig. 2B, Supplementary Table 4). Consequently, purified enzymes were used in all subsequent studies.

Following the positive reaction results driven by BsDAAT, we optimized the reaction conditions for substrate 1s by screening the alterable elements (Supplementary Table 3). Screening alternative pH levels, temperatures and enzyme or amino donor loadings did not lead to an obvious improvement in reaction efficiency, but the type of amino donor was verified to be the key factor in this reaction. Under the guidance of previous studies, several D-amino donors with different affinities to wild-type BsDAAT were tested, including D-Ala, D-Gln, D-Glu, D-Asp and D-Asn.29,33 However, it was observed that D-Glu and D-Ala with higher specific activity in transamination toward pyruvate and other amino receptors, only provided a 14% yield. In contrast, D-Lys with weak enzyme affinity, showed a significant improvement in compound 1 production (Fig. 2A). This finding was supported by the study on transamination equilibrium, in which the deamination product of lysine was found to convert into the cyclized by-product 6-amino-2-oxohexanoic acid to drive the reaction forward.17 Based on this, a lower equivalent of D-Lys, a suitable temperature and a lower loading of the purified enzyme were found to be more conducive to the production of target compounds. The final conditions after rational optimization were as follows: substrate (1 equiv.), D-Lys (3 equiv.), PLP (10 mol%), purified enzyme (0.6 mol%) in KPi buffer (100 mM, pH 8.0) containing 1% (v/v) DMSO, at 37 °C for 18 hours. Under these conditions, compound 1 could be obtained with excellent yield (>99%) and stereoselectivity (>20:1 dr), as shown in Fig. 2A (entry 5).

With the successful dynamic kinetic amination of the model substrate, we proceeded to conduct comprehensive investigations into the capabilities of BsDAAT-WT. β-Branched α-ketoacids with various substituents were subjected to optimized enzymatic processes. However, most aryl substrates could not be ideally transformed compared with 1s (Fig. 3). Target compounds were effectively synthesized with high stereoselectivity (>20:1 dr) and varied yields for o-substituted substrates with different halogens (2s–5s), except for 6s. When a -CF3 group was incorporated at the m- (12s) or p- (20s) site, substrates were effectively utilized by BsDAAT to yield compounds 12 and 20 with 14:1 and 9:1 dr, respectively. Although substrates with m- (7s–9s) and p- (16s–19s) halogen substitutions could be recognized, a discernible decrease in yields and dr ratios was evident. Exceptional production efficiency was also demonstrated with substrates 13s and 15s, which have potent electron-withdrawing substituents; however, the capacity for chiral control still needs to be improved. Additionally, substrates modified by alkyl groups such as methyl (10s, 14s), isopropyl (22s), tert-butyl (23s), and acetyl (24s), were effectively converted into unnatural amino acids with yields exceeding 60%. The yields declined when methyl group was replaced by a methoxy group (11s and 21s). In addition to single-substituted ketoacids, substrates with other frameworks were tested using BsDAAT-catalyzed system. These included difluorides 25s and 26s, ketobutyric acid 27s, bulky naphthyl-type substrate 28s and heterocyclic compound 29s. However, the results did not meet the expected level. Broad screening revealed that only 30% of products exhibited optimal stereoselectivity (>20:1 dr), while most substrates delivered dr ratios ranging from 5:1 to 20:1. Substrates 6s, 19s, 28s and 29s, in particular, exhibited significant incompatibility with this wild-type enzyme, resulting in modest yields and rare stereoselectivities (Fig. 3).

At this stage, we decided to conduct an enzyme engineering program to achieve universally satisfactory transformation of the desired β-Me-D-Phe analogues. Based on docking analysis of the BsDAAT-WT crystal structure and substrate 1s, 16 amino acids within 5 Å of the binding model were selected for site mutation (Fig. 4A). Focused rational iterative site-specific mutagenesis (FRISM) was applied to evolutionary studies at sites lining the enzyme’s binding pocket.34 Three amino acids with different steric properties were chosen to replace the amino acids individually at the selected residue sites. Alanine (A), leucine (L) and phenylalanine (F) were chosen as small, medium and large amino acids, respectively, and were used to modify the space available in BsDAAT binding pockets. The obtained mutants were then tested in reactions with three substrates: 6s (a special non-working substrate), 17s (representing most substrates) and 28s (a substrate type with greater steric hindrance). This identified the 'hotspots' for manipulating stereoselectivity and reactivity. The results for three substrates and 46 mutants (expression levels of all variants are shown in Supplementary Fig. 7) are presented in Fig. 4C and Supplementary Fig. 7 (mutant T241L was rarely expressed).

Regarding substrates 17s and 28s, both V33L and V33F exhibited greater stereoselectivity. Notably, V33F displayed a significantly higher activity (>20:1 dr for both 17s and 28s) than V33L (Fig. 4 and Supplementary Table 5). Variant V33F was then tested on all the remaining substrates. The stereoselectivity of most products was greatly improved, achieving a ratio of over 20:1 dr, with over ten compounds reaching an isolated yield of over 99%, which distinguishes V33F from other mutants. However, V33F was incompatible with o-substituted substrates (3s–6s). Among the mutants that were positive towards substrate 6s (V33A, T242A/F, S240F/L, and V33G with isolated yield 18%), V33A exhibited superior reaction efficiency and remarkable stereoselectivity (>20:1 dr). Similar results were obtained when V33A was evaluated on substrates 3s–5s, where sustained stereoselectivity was associated with significantly higher yields (Fig. 3, compounds 3–5). V33F and V33A were identified as prospective mutants for substrate scope investigations. Overall, all primary substrates with low dr ratios were favorably accepted by V33F or V33A, exhibiting increased stereoselectivities (>20:1 dr, >99% ee) and isolated yields (Fig. 3, compounds 1–29; Supplementary Fig. 9). The absolute configuration of (2R,3R)-8 was verified by X-ray crystallography, and those of the other products were assigned by analogy.

Owing to successful acceptance of various substituted β-Me ketoacids, larger groups in β-position were investigated within BsDAAT-WT system. However, no products were detected in reactions involving β-Et (30s), β-nPr (31s), β-cPr or β-iPr. Then we attempted a new round of mutations involving S240, T241 and T242 using a minimization strategy (Supplementary Table 6). These three residues were positioned within the β-chain orientation of the binding pocket in BsDAAT (Fig. 5E). The results indicated that substrates with ethyl or n-propyl were not well tolerated by any single-site or double-site mutant containing glycine. However, they could be successfully converted into the desired product by the triple mutant variant S240G/T241G/T242G, albeit with yields of only 4% and 6%, respectively. Excellent stereoselectivity was only demonstrated with compound 31, with a dr ratio of >20:1, whereas compound 30 displayed suboptimal stereocontrol with a dr ratio of 6:1. Only trace amounts of the product from β-cPr ketoacid could be detected by LCMS. The anticipated product from the β-iPr substrate was undetectable in any of the variation reactions, as confirmed by TLC and LCMS. For substrates with larger groups at the β-site, the S240G/T241G/T242G variant, featuring minimized amino acids, demonstrated improved receptivity to substrates 30s and 31s. However, it remained suboptimal for larger side chains, such as cPr and iPr. Further engineering efforts are required to increase the pocket size to enhance the catalytic effectiveness of aromatic D-amino acids with substantial β-chains.

Following our exploration of the scope of aromatic substrates, we sought to investigate the efficacy of BsDAAT on aliphatic substrates. Replacing the phenyl group with a cyclohexyl group dramatically decreased activity, resulting in no derivatization of the α-ketoacid (Fig. 3). However, the extended backbone cyclohexylbutanone acid (32s) could be catalyzed with a low yield of 28% and an original dr ratio of >20:1. When the carbon cycle transitioned from a cyclohexyl group to a cyclopentyl group, only minimal product could be detected by LCMS. Moderate reactivity was noted for other substrates featuring distinct straight or branched chains with reduced steric hindrance (33s–36s), while no chiral selectivity was observed for β-branched groups (1:1–2:1 dr ratios for compounds 33, 34, 35 and 36). Further innovative evolutionary techniques are required for the stereoselective production of D-amino acids from aliphatic α-ketoacids via the BsDAAT system.36 Research addressing the potential of BsDAAT for the production of aliphatic DAAs with a single isomer is currently in progress.

Based on scope results, it could be observed that in biocatalytic system of wild-type BsDAAT, most aromatic substrates bearing electron-withdrawing substituents were converted into the corresponding products with high yields, such as -F (2s, 7s, 16s), -Cl (3s, 17s), and -NO2 (13s, 15s). Hydrophobic alkyl chain-substituted substrates also exhibited favorable reactivity, including -Me (10s, 14s), -iPr (22s), and -tBu (23s). Substrates 11s and 21s showed reduced yields due to the electron-donating effect from -OMe group. However, substrate 6s with o-CF3 showed significantly reduced reactivity compared to 12s and 20s. This diminished activity likely stemed from the combined influence of electronic and steric factors, along with suboptimal enzyme-substrate binding. The -CF3 group in substrate 6s was positioned closer to reaction center, exerting a more potent electron-withdrawing effect. Reduced electron density around reactive site likely weaken the substrate reactivity and interactions between carboxyl groups and nearby residues, leading to a undesired result. Moreover, -CF3 group at the o-position might compress the key regions required for catalytic or positioning residues. The tetrahedral geometry of group -CF3 could generate strong steric repulsion with surrounding residues, preventing the substrate from achieving the optimal alignment necessary for catalysis, resulting in poor reactivity. But substrates with -Br and -I exhibited limited electronic effects. Their approximately spherical atomic shapes, symmetry, and high polarizability enabled a favorable fit within active site. The resulting low steric barrier overcame any minor electronic perturbations, allowing reactions to proceed successfully.

Compared with wild enzyme, mutants V33F and V33A exhibited enhanced reactivity toward most substrates. This improvement was attributed to both higher enzyme-substrate affinity and increased transamination rates (comparison of kinetic parameters was shown in Supplementary Fig. 8). To gain a better understanding of these optimized results, we conducted molecular docking using BsDAAT and substrates 6s and 28s, matching imines PLP-6s and PLP-28s with BsDAAT-WT and BsDAAT-V33F or V33A , respectively (Fig. 5). As shown in Fig. 5A and 5B, V33F exhibited enhanced π-π interaction with the benzene ring within substrate 28s, making it superior for this biocatalysis. Increased π-π interaction may further constrain the conformation of substrates, confining the methyl group to a small region and yielding desirable products with higher stereoselectivity. In contrast, molecular docking between BsDAAT and substrate 6s revealed that BsDAAT with the small-site mutation V33A exhibited greater accommodation for the o-substitution CF3 (Fig. 5C and 5D). When valine was mutated to alanine, the difference in electronic properties of alkyl side chains was so negligible that could not influence the electrostatic potential of active site. Therefore, electronic effects were not the main driving force behind this mutation. With a smaller side chain, alanine provids additional space and greater conformational flexibility, enabling subtle “induced-fit” adjustments of the active site. This enhanced the ability of pocket to accommodate o-substituted substrates with substantial steric bulk. However, minimization of residues does not always enhance the enzyme activity, and optimal catalytic efficiency requires a precise balance. Similar results were not observed when the residue was minimized to glycine (V33G). Although V33G provided maximal space, the excessive flexibility introduced by glycine disrupted the precise preorganization and stability of the active site, leading to a reduction in enzyme’s intrinsic catalytic capability. For other single-point mutants, they either failed to effectively optimized the binding conformation and stereo-control between substrates and enzyme, or disrupted key interactions between residues and substrates, impairing substrates recognition and catalytic efficiency. The phenol hydroxyl group of Y31, the guanidinium group of R98, and the amino group of K145 side chain played critical roles in recognizing and binding carboxyl groups of the substrates. Substitution of these residues disrupted enzyme-substrate recognition and binding, resulting in interrupted reactions. The hydrophobic interactions network among residues S240, T241, T242 and S243 provided sufficient space to accommodate β-substituted substrates but lacked the ability to control methyl conformation, resulting in the poor performance of corresponding mutants. Residues A99, H100, and others likely served only as structural scaffolds but were not directly involved in transamination reactions, explaining the lack of significant change upon mutations.

Beyond these, we explored the relationships between the active pocket and aromatic substrates with larger β-groups and aliphatic substrates. The binding pocket of BsDAAT was deemed too narrow to accommodate the extension of β-chain (aromatic ketoacids), resulting in undesirable reaction outcomes (Fig. 5E and 5F). Although the triple mutant S240G/T241G/T242G offered more space for β-chains, it was still not fully adaptable. For aliphatic ketoacids, insufficient steric hindrance disrupts the critical capacity for chiral control over most aliphatic substrates in our study. For 32s, limited space restricts the free movement of the side chain of 32s, stabilizing the spatial conformation of methyl group and resulting in the formation of a single isomer. However, as shown in Fig. 5H, the spacious active pocket provides sufficient room for free rotation of side chain, ultimately leading to a dr 1:1 mixture of compound 33 isomers. These findings suggest that substrates featuring π-π interaction groups or steric hindrance may be more advantageous for the current transamination system to achieve the expected yields and configurations.

In order to show the usage of the β-branched DAAs, substrates 1s and 28s were synthesized using the BsDAAT system on a 1-mmol scale, yielding 84% and 48%, respectively. Further scale-up reaction of 1s at 1 gram resulted in 55% yield (Supplementary Fig. 11). Both products exhibited exceptional stereoselectivity (>20:1 dr and >99% ee). The products were then applied in the solid-phase peptide synthesis (SPPS) of Lanreotide derivatives, with D-naphthylalanine (D-Napa) being replaced by β-Me-D-Phe and β-Me-D-Napa (Fig. 6). Lanreotide, a somatostatin analogue with antineoplastic properties, possesses a stable octa-membered cyclic structure (D-Napa-L-Cys-L-Tyr-D-Trp-L-Lys-L-Val-L-Cys-L-Thr, disulfide bridge: Cys2-Cys7), containing two essential unnatural amino acids, D-Trp and D-Napa.37,38 With extensive research into structural modifications and variations in drug indications, the synthesis of Lanreotide analogues was performed on Rink Amide AM resin using a HATU/DIEA condensation system. The final cyclic products were obtained via cyclization, deprotection and resin cleavage, in accordance with standard procedures.39 In essence, the various β-Me-D-Phe derivatives in our study all have the potential to act as building blocks in drugs and active natural products, eliciting diverse biological functions.

In summary, we have developed an unprecedentedly efficient biocatalytic approach to the asymmetric synthesis of β-branched aromatic D-amino acids via DKR process. Variants V33F and V33A from the BsDAAT were identified and characterized in the synthesis of β-Me-D-Phe analogues bearing various substituents, leading to good yields and exceptional stereoselectivities (>20:1 dr, >99% ee for 30 examples). This reaction system could be easily scaled up and incorporated to produce Lanreotide derivatives. This efficient, environmentally friendly approach greatly broadens the range of biocatalytic reactions for aromatic D-amino acids with β-chirality. Future research may focus on the directed evolution of BsDAAT to generate β-methyl aliphatic D-amino acids with higher yields and stereoselectivities via the enzymatic DKR process. Furthermore, large-scale production with more efficient protocols, such as a multi-enzyme cascade in a whole-cell system, will be explored to implement β-branched aromatic D-amino acid analogues in industrial manufacturing and the discovery of innovative medications.

Methods
Construction of mutagenesis libraries
The mutant library was constructed using the FRISM strategy for the amino acid residues within the 5 Å range near the BsDAAT active site.

PCR was performed using BsDAAT-WT plasmid (pET-22b) as the template DNA. The reaction (50 μL final volume) contained ddH2O (33 μL), KOD 10X buffer (5 μL), dNTP (5 μL, 2 mM each), forward primers (1.5 μL, 10 μM each), silent reverse primer (1.5 μL, 10 μM each), template plasmid (1.0 μL, 100 ng/μL each), MgSO4 (2.0 μL, 25 μM each), and 1.0 μL of KOD plus. PCR conditions used were 94 °C, 2 min; 30 cycles of (94 °C, 15 sec; 61 °C, 30 sec; 68 °C, 10 min) and final extension at 68 °C, 10 min. To ensure the elimination of the template plasmid, 50 μL of PCR mixture were mixed with 2 μL DpnI (10 U/μL) and incubated overnight at 37 °C. Upon purification of the DpnI-digested product with a GeneJET PCR purification kit (Thermo Scientific, Waltham, MA, USA), an aliquot of 15 μL was used to transform the E. coli BL21 (DE3) competent cells by chemical transformation method. The transformation mixture was incubated with 900 μL of LB medium at 37 °C with shaking and spread on LB-agar plates containing 100 µg/mL carbenicillin. A single colony from a plate was incubated in 5 mL of LB medium at 37 °C overnight, and the plasmid was extracted with an EasyPure® Plasmid MiniPrep Kit (TransGen Biotech Co., Beijing, China). DNA sequencing was performed by Sangon Biotech (Shanghai, China).

Enzymatic synthesis of β-branched aromatic D-amino acids
The suitable α-ketoacids (0.07 mmol) and PLP (10%, 0.007 mmol) were added to potassium phosphate buffer (5 mL, 100 mM, pH 8.0) containing D-Lys (3 equiv., 0.21 mmol). Purified BsDAAT-WT or mutant enzyme (0.6% mmol) was added as a final, and the mixture was incubated at 37°C overnight in a shaking incubator at 280 rpm. Later, the samples were adjusted to pH = 2.0 with 1M HCl, then the mixture was centrifuged at 15,000 ×g and 4 °C for 30 minutes. Supernatant was filtered by a 0.22 μm PES filter and purified by a reverse C18 silica column using acetonitrile/water: 100% water for 15-20 mins, 0% to 16% acetonitrile over 5 minutes, 16% to 70% acetonitrile over 40 minutes, 65% to 95% acetonitrile over 5 minutes under flow rate 5 mL/min. Structures and diastereomeric ratios (dr) of purified compounds were confirmed by 1H NMR and 13C NMR.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The study by Liu et al describes a chemoenzymatic method enabling stereoselective synthesis of a variety of beta-branched d-amino acids. Their approach leverages DAAT, an aminotransferase derived from Bacillus subspecies, which utilizes d-phenylalanine as an amine donor for transformation of beta-branched alpha-keto acids. The wild-type (WT) DAAT exhibited appreciable catalytic activity and selectivity towards keto acids bearing beta-aryl branches, but certain substrates remained recalcitrant under the optimized conditions. To address this, the authors sought a structure-guided FRISM approach, which led to the identification of several mutants with enhanced activity and stereoselectivity over the WT. Finally, the utility of their method was demonstrated by incorporating the stereoselectively synthesized beta-branched d-amino acids as building blocks in cyclic peptides via SPPS.\n\nThe concept of employing aminotransferase-mediated enzymatic reactions for beta-branched amino acid synthesis is not new, as it has been already demonstrated in the cited work (Ref# 39). Nevertheless, this study offers distinct value. Unlike the earlier study, which focused on l-amino acids, Liu et al achieved stereoselective synthesis of beta-branched d-amino acids for the first time through meticulous selection and engineering of DAAT. In addition, the experimental design and data quality are largely robust. Thus, the findings described in this manuscript would attract considerable attentions, particularly, from researchers in synthetic biology and biocatalysis.\n\nThe followings are several points that need to be addressed:\n[Major points]\n1. Although Ref#39 appears to be a foundational study underpinning the current work, its contribution and relevance are not sufficiently addressed. A more substantial discussion of this prior research should be included—either in the introduction or conclusion—with emphasis on the distinctive aspects of the present study that set it apart.\n2. The observed high yield (>50%) is evidently attributable to rapid epimerization at the β-position, which facilitates dynamic kinetic resolution. This raises an intriguing question in the context of aliphatic substrates. For compounds 33, 34, and 36, DAAT was unable to effectively distinguish between the two β-substituents, likely due to their similar steric and electronic profiles. In contrast, compound 32s exhibited the expected diastereoselectivity, albeit with a notably low yield. This suggests that the poor yield may stem from sluggish epimerization, thereby limiting the efficiency of dynamic kinetic resolution. Stereochemical analysis of the unreacted 32s could offer valuable insight into this mechanistic hypothesis.\n[Minor points]\n1. A separate table of contents (TOC) should be included in the Supporting Information (SI), clearly listing Figures, Tables, and Schemes for improved accessibility.\n2. Table S3, entry 7: The designation “G-Asp” appears to be incorrect and should be revised to “D-Asp.”\n3. page 5, line 110: - The phrase “Exceptional production efficacy” would read more naturally as “Exceptional production efficiency.”\n4. Page 5, line 125: The reference to “Fig. 5A” seems to be a mislabeling and should likely be corrected to “Fig. 4A.”\n5. Page 6, line 143: The term “affinity” may not be the most appropriate descriptor in this context. Consider replacing it with “activity” or “catalytic efficiency” to better reflect enzymatic performance.\n6. Page 11, line 265: The phrase “Purified BsDAAT-WT enzyme” implies exclusivity to the wild-type. If the same purification procedure applies to mutant variants, it would be more accurate to revise this to “Purified BsDAAT WT or mutant enzyme.”", "reviewer_2": "The manuscript by Yan and Yang describes the synthesis of beta-chiral D-amino acids from a-ketoacids using a transaminase. This reaction is a dynamic kinetic resolution, where the a-ketoacid racemizes under the reaction conditions. The described method provides products in high yield with excellent levels of diastereoselectivity. The authors conducted a limited protein engineering campaign to increase the yield for more sterically demanding substrates. They used molecular docking to rationalize the substrate preference and the impact of mutations. Finally, the amino acids produced from this enzyme were used to prepare Lanreotide derivatives.\n\nThis work builds directly on two papers published in the past few years. The first is a report by Renata (Ref 39), where they report the synthesis of beta-chiral L-amino acids from a-ketoacids using L-selective threonine aldolases. The second is by Merck (Ref 16), who uses lysine as an amine donor for the reaction reported by Renata. Consequently, the innovation of this paper lies in identifying a transaminase for preparing the opposite enantiomer using the same strategy described by Merck and Renata. Consequently, the innovation of this work is limited, and it would be better suited for a more specialized journal.\n\nAdditional Questions:\n- As the paper is currently written, the references by Renata and Merck are buried. Given that they clearly are the inspiration for this work, they should be explicitly mentioned in the main text.", "reviewer_3": "The manuscript ‘Concise Construction of β-branched aromatic D-amino acids via transaminase-catalyzed dynamic kinetic resolution’ describes an enzymatic approach to synthesise β-branched aromatic D-amino acids with two adjacent chiral centres using a transaminase enzyme D-phenylalanine aminotransferase from Bacillus sp. YM-1, BsDAAT with excellent yields and stereoselectivity. The authors optimized the expression and reaction conditions of the wild-type enzyme and evaluated its performance across a range of alpha-keto acid containing arylated substrates. They further used docking analysis and enzyme engineering to improve upon the stereoselectivity and identified specific mutations by rational mutagenesis experiments that afforded 99% yields, >20:1 dr, and >99% ee for a third of the substrates tested. The method was adapted to synthesise two β-branched D-amino acids on a preparative scale, which were then used in the solid-phase synthesis of two Lanreotide analogues, thereby providing an example of the application of their approach.\n\nThe manuscript describes an approach with future biocatalytic applications for the synthesis of β-branched D-amino acids, an area that is relatively unexplored. They chose a simple but effective rational mutagenesis approach that resulted in markedly improved yields and stereoselectivity after a single round of mutation in most cases. There was a great amount of information summarized in the substrate scope panel (Figure 3) and mutant screening (Figure 4) that showed the breadth of application towards the construction of these β-methyl aryl D-amino acids. The data visualization could potentially be improved in these two figures to more clearly depict the work, however it is all technically sound. Despite this, there have been great achievements within this field in leveraging the same BsDAAT enzyme to construct D-amino acids (current reference 26) or using other PLP enzymology with Dynamic Kinetic Resolution for β-branched L-amino acids (current reference 39). The manuscript in its current form felt similar to both of these published stories in both concept and execution. There is the potential for novelty within this system that could be explored as suggested in the major comments below. Following additional experimentation and rationalising the improvements (or lack thereof) in reactivity and selectivity within this biocatalytic system, we would be happy to reconsider it for publication in Nature Communications following additional revision.\n\nMajor Comments:\n- It would be beneficial to have a more stringent and detailed computational analysis of the enzyme with the docked substrate(s) to explore the interactions of the residues that determine substrate binding, selectivity and positioning. This would help inform the structure/function basis of the enzymatic transformation.\n- There is not a great deal of discussion about the ‘dynamic kinetic resolution’ aspect of this manuscript mentioned within the title. I don’t believe that time was ever used as a variable when exploring this system – incubating racemic β-branched a-keto acid substrates at shorter time points and analyzing assays by NMR (to assess dr), additional insights into this aspect could be achieved.\n- Showing at least one representative example of how NMR was used to determine the dr ratio would be helpful as an SI Figure.\n- Does BsDAAT exclusively require a D-amino acid to serve as an amino donor? What is its tolerance with L-amino acids?\n- Given that the original isolation of BsDAAT was from a thermophilic Bacillus bacteria and its optimal activity temperature is 60 °C (based on reference 28) was there any effort made to run these reactions at elevated temperatures to improve parameters and/or decrease reaction times? Was this used to help improve the reactivity of substrates that gave poor yields with wild type or engineered enzyme? Could this be used to inactivate endogenous E. coli proteins and enable lysates to be used to prepare these β-branched D-amino acids? Some experimentation here would elevate this manuscript and better distinguish it from others in the field.\n- In a similar vein, could BsDAAT (wild type or engineered) be used to resolve racemic and/or scalemic β-branched a-amino acids by just using a PLP cofactor and no amine donor?\n- Including information about the enzyme TTN or kinetic parameters for at least one of the substrates would be helpful in order to support the claims that this is an efficient biocatalytic approach.\n- It appears that electron-withdrawing and electron-donating substituents on the aryl ring influenced the wild type product yields and dr (somewhat) that seemed to be largely overcome in the V33A/F variants. I would presume that there is an electronic component to this in addition to the steric argument presented – can the authors provide additional clarification as to what is influencing some of the more dramatic changes in the substrate scope panels?\n\nMinor comments\n- For the large-scale synthesis, would be helpful to include g scale along with mmol.\n- Inclusion of a mechanism figure in the SI might be helpful.\n- Please include an SDS-PAGE gel for the purified wild type and major mutant assays used for large scale biocatalytic applications.\n- Please include information about the program/tools used for docking analyses in main text and SI figures.\n- Yields should be reported to integer numbers throughout (no decimal places)\n- Lanreotide derivatives should be characterized by MS/MS to confirm the correct peptide sequence", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Dear reviewer 1:\nWe sincerely appreciate your thoughtful and positive assessment of our work, as well as for the constructive comments provided. We are grateful for the recognition of distinct value and robustness of our study, particularly regarding the stereoselective synthesis of β-branched D-amino acids via a chemoenzymatic approach using engineered BsDAAT. We have carefully considered your suggestions and agree that addressing them will further improve the clarity and impact of the manuscript. Accordingly, we have revised the text to more explicitly acknowledge the prior art on enzymatic synthesis of β-branched amino acids (Ref #39), while more clearly articulating the key advances of our study—specifically, the extension of this strategy to D-amino acids through enzyme discovery and engineering.\nNow, all modifications have been implemented with care, and we believe the revised version presents a more balanced and compelling account of our work. We have provided a point-by-point response to the comments along with the corresponding revisions. Thank you again for your valuable feedback.\nFor major points 1:\nThanks for this common suggestion from both you and Reviewer 2. We have incorporated a more detailed discussion in both the introduction and results sections. In introduction section, we now highlight the innovative aspects and significance of the Renata’s work (Ref 39, now is Ref 16). The revised introduction clearly delineates the differences between our study and Ref 16. Subsequently, in the results section, we have emphasized the DKR strategy reported in Ref 16, which served as a key inspiration for our work.\n-Following your suggestions, the revised content has been highlighted in yellow, in page 2-3.\n-Additionally, we have optimized the overall paper structure for better readability, with structural adjustments marked in blue.\nFor major points 2:\nBased on your suggestion, to make a more detailed explanation about the catalyzed activity of BsDAAT on aliphatic substrates, docking models of substrates 32s and 33s with BsDAAT-WT have been added into Figure 5 (shown in Figure 5G and 5H). Undesired results of aliphatic substrates are mainly attributed to the poor steric hindrance, a brief analysis has been added into the main context in page 9-10, line 251-254.\nFor minor points 1:\nThe separate table of contents has been added into the supporting information page 3, including all figures and tables, highlight with yellow.\nFor minor points 2:\n“G-Asp” has been changed to “D-Asp” in Table S3, page 12, highlight with yellow, shown in the figure below.\nFor minor points 3:\n“Exceptional production efficacy” has been changed to “Exceptional production efficiency” in page 5, line 121, highlight with yellow, shown in the figure below.\nFor minor points 4:\n“Fig. 5A” has been changed to “Fig. 4A” in page 5, line 135, highlight with yellow, shown in the figure below.\nFor minor points 5:\n“affinity” has been changed to “activity” in page 6, line 153, highlight with yellow, shown in the figure below.\nFor minor points 6:\n“Purified BsDAAT-WT enzyme” has been changed to “Purified BsDAAT-WT or mutant enzyme” in page 11, line 317-318, highlight with yellow, shown in the figure below.\n\nDear reviewer 2:\nWe sincerely thank you for reviewing our manuscript and providing highly constructive feedback. Driven by a sustained interest in bioactive molecules and enzymatic catalysis, our research group is dedicated to developing enzymatic strategies for the synthesis of non-canonical amino acids, with the long-term goal of constructing a versatile and efficient synthetic toolkit. We believe our present work strikes a balance between genuine innovation and practical applicability, and we are confident it constitutes a meaningful contribution to the field of non-canonical amino acid synthesis and utilization. Based on your comments and our further considerations, we have incorporated revisions into the text as summarized in the following points.\nFor question 1:\nThanks for your comments. We agree that the pioneering work by Renata and Merck established a valuable foundation for synthesizing β-chiral L-amino acids. However, our work distinguishes itself in several key aspects. First, we performed broad substrate profiling coupled with computational and protein engineering efforts, discovering highly active mutants that enabled the efficient synthesis of a diverse library (>30 examples) of β-substituted D-amino acids. Second, our condition optimization was a systematic experimental process. The screening of various parameters, including (but not limited to) amine donors, led to the excellent efficacy under the combination of D-lysine and BsDAAT. We have great respect for the Merck’s study, however, our research focuses on a distinct enzymatic system (D-selectivity) and explores a different structural space (β-substituted D-amino acids). Both studies ultimately share the common goal of providing valuable tools for the efficient and sustainable synthesis of non-natural amino acids. Third, we have completed scale-up verification of the catalytic system, synthesis of peptide derivatives, and in-depth molecular docking analysis. As the first report on the enzymatic synthesis of β-substituted D-amino acids, this work offers both significant innovation and considerable application value. D-amino acids are of broad applicability in pharmaceutical molecules, and the ability to construct target compounds with a single stereoisomer through a one-step process is of significant importance in synthetic chemistry. Furthermore, Nature Communications is an interdisciplinary journal, and our work indeed represents a convergence of chemical synthesis and biotechnology. So, this work provides a robust, scalable route to important D-amino acid building blocks and will be of broad interest in biocatalysis, chemical biology, and pharmaceutical synthesis, well adopt the scope of Nature Communications.\nFor question 2:\nThanks for this suggestion. As mentioned, the work by Renata et al. served as one of the key inspirations for our study, alongside other foundational reports on D-amino acid synthesis. In structuring our introduction, we aimed to maintain a clear focus on the progress and significance of D-amino acids to align with the central theme of our manuscript. The study by Renata is a seminal contribution to enzymatic amino acid synthesis, demonstrating the combination of DKR with biocatalysis to access β-substituted L-amino acids. Therefore, following this common suggestion from you and Reviewer 1, we have now expanded the introduction (and result section) to include a discussion of this important work.\n-Following your suggestions, the revised content has been highlighted in yellow, in page 2-3, line 42-46, line 70-71.\n-Additionally, we have optimized the overall paper structure for better readability, with structural adjustments marked in blue.\nThe subsequent study by Merck represents another excellent advancement, identifying an improved amine donor for specific L-transaminase. In this research, Merck’s study provided a complementary mechanistic explanation for the best reactivity of D-Lys. Now, we expanded the introduction section about their research, and added the citation in result section, in page 2, line 44-46, and in page 4, line 104-107.\n\nDear reviewer 3:\nWe sincerely appreciate your positive feedback on our work. Our work on the enzymatic synthesis of β-chiral D-amino acids is deeply motivated by the broad utility of amino acid scaffolds in drug discovery and the need for sustainable synthetic methods. This work was inspired by Ref 16 (whose first author is the corresponding author L.-C. Yang in this study) and built upon prior foundations from Ref 27. While it can be viewed as a convergence and extension of these preceding studies, its distinct contribution lies in the systematic exploration of β-substituted D-amino acids—a significant and underexplored class. We extensively diversified the substrate scope by testing variants with different steric and electronic properties, performed product derivatization, and conducted molecular docking studies to rationalize selectivity. This comprehensive approach aims to establish a green and efficient platform for non-canonical amino acid synthesis. This project is part of our group's broader vision to build a modular enzymatic toolkit for accessing diverse nonnatural amino acids. While individual methods may seem similar, each addresses unique synthetic challenges and collectively expands the accessible chemical space for biomedical research. We believe the interdisciplinary nature of our work—merging synthetic chemistry and biocatalysis—makes it a strong fit for Nature Communications. We have addressed all points raised in your review and hope the revised manuscript now fully conveys the significance and innovation of our study.\nBased on your suggestions, we have addressed each of your comments point by point. Regarding the data visualization of Figures 4 and 5, we have decided to retain them after careful consideration about the volume of data and the need to clearly present statistical and comparative results. For the other points raised, our specific revisions and responses are provided below.\nFor major point 1:\nThanks for this suggestion. Based on comprehensive considerations, the role of the residues within active site was indirectly demonstrated through the analysis of our scope results and docking. Specific analysis about this suggestion has been added into the main text, highlighted with yellow (page 9, line 239-246), shown below. Other related analysis about scope, mutant and docking have been added to main text, from page 8 to 10, highlight with yellow/blue. We are confident that the discussion section has been significantly improved to be more complete and clearly articulated.\nFor major point 2:\nThanks for this suggestion. We rely on the foundational work in Ref. 16 (whose first author is the corresponding author of the present study), which provides conclusive evidence for a rapid substrate racemization. Renata’s study has demonstrated that one enantiomer of β-Me-α-ketoacid is selectively recognized and converted by the enzyme, while the other enantiomer racemizes and is subsequently consumed, leading to ~100% yield—a hallmark of an efficient DKR. This racemization is a rapid, spontaneous process in solution that is independent and precedes the enzymatic transformation. Enzyme-catalyzed dynamic kinetic resolution is a well-established process reported in many articles. Therefore, we did not replicate this prior characterization extensively, but provided a brief description of the DKR in the revised text (page 3, line 62-66). DKR description in Fig. 1D have been simplified.\nFor major point 3:\nBased on your suggestions, we have refined the structural determination process. The absolute configuration of the product was primarily established by X-ray crystallography, with NMR spectroscopy and optical rotation serving as corroborating evidence. Comparative NMR analysis with the racemic mixture (dr 1: 1) and the (2S,3R)-configured compound revealed that our product exhibited a distinct set of signals, corresponding to the other diastereomer. This indicated that our product and the (2S,3R) compound were a pair of diastereomers, suggesting possible configurations of (2R,3R) or (2S,3S). Furthermore, since BsDAAT is a known enzyme that specifically produces D-amino acids, the configuration was deduced to be (2R,3R). This assignment was consistent with the optical rotation data, which matched values reported in the literature for analogous structures. Ultimately, the (2R,3R) absolute configuration was unambiguously confirmed by X-ray crystallographic analysis.\nIn summary, by comparing the NMR spectra of our product with those of the racemate and an authentic (2S,3R) sample, and supported by optical rotation and X-ray crystallography data, we determined its absolute configuration to be (2R,3R), shown in page 48.\nIn the text, we did not elaborate on how to determine the product configuration. But in the SI section, we added the NMR spectra of racemic mixture of compound 1, and the (2S,3R)-compound spectrum (from Angew. Chem. Int. Ed. 2021, 60, 17680-17685) for comparison and reference. In addition, the supplementary information provides optical rotation data and single-crystal structures, which further confirm the configuration.\nFor major point 4:\nAccording to your suggestion, subsequent experiments revealed that the enzyme is specific for D-amino acid donors, such as D-lysine and D-glutamate, as no activity was observed with L-lysine even after varying the reaction time (detected by TLC). Besides, no product was observed in the reaction system when the amino donor was omitted. These have also been tested in a past study: (J. Bacteriol. 1955, 69, 357–362.)\nLine 1: compound 1; line 2: reaction with no amino donor; line 3: reaction with L-Lys.\nFor major point 5:\nBased on your suggestion, we conducted further investigations. We hypothesized that V33F remains stable at 60°C, while other endogenous enzymes within the cell lysate are susceptible to thermal denaturation at this temperature. To test this, we incubated 30 mL of cell lysate (OD >30, expressed with V33F) at 60°C for 15 minutes, 600 aiming to selectively deactivate background enzymatic activities. Subsequently, PLP, D-Lys, and the substrate 1 were added, and the reaction was allowed to proceed at 60°C overnight (~10 h). However, product 1 was not detected by TLC analysis after either 1 hour of reaction or following an overnight incubation.\nBased on these results, we can only conclude that the V33F variant, along with other proteins in the lysate, is likely not tolerant of the 60°C environment, leading to the absence of detectable product. For amino acid detection, the ninhydrin reagent offers significantly higher sensitivity than LCMS. Since no product was detected by ninhydrin staining, we did not proceed with other analytical methods. In summary, a 60°C reaction condition appears unsuitable for V33F.\nThe TLC analysis (~10 h) is presented below:\nLine 1: compound 1; line 2: reaction under 60°C; line 3: line 1+line 2\nFor major point 6:\nWe interpret this suggestion as exploring the potential of BsDAAT for the kinetic resolution of racemic β‑Me‑D‑phenylalanine. Our reasoning and experimental test are as follows: First, it is established that BsDAAT is strictly D‑specific and does not act on L‑amino acids. Second, our study demonstrates that V33F selectively recognizes the R‑configured β‑methyl group in the substrate. Together, these points suggest BsDAAT could, in theory, distinguish between enantiomers of β‑Me‑D‑phenylalanine. To test this hypothesis, we used enantiopure product 1 as the amine donor and α-keto acid 20s as the acceptor. No formation of 20 was detected by TLC after overnight incubation, showing that BsDAAT cannot catalyze the reverse transamination using 1. This, coupled with the observed near‑quantitative yield (~100%) in the forward reaction—indicating thermodynamic irreversibility—leads us to conclude that BsDAAT is not applicable for resolving racemic β‑Me‑D‑phenylalanine.\nRegarding other amino acids, the fact that BsDAAT utilizes D-lysine but not L-lysine confirms its potential for kinetic resolution of racemic lysine. While the applicability to β-substituted-D-lysines remains an open question for future study, the results presented in our manuscript on aliphatic amino acids suggest that this enzyme may hold greater potential and offer more distinct advantages in the transformation of aromatic substrates.\nTLC of the reaction mixture is presented below:\nLine 1: compound 1; line 2: reaction mixture; line 3: compound 20\nFor major point 7:\nThanks for this suggestion, related experiments about kinetic parameters of wild type enzyme, V33A, and V33F have been performed and the results are shown below (page 18, in SI document), related description has been added into main text (page 9, line 223-225, highlight with yellow).\nFor major point 8:\nThanks for this suggestion, detailed discussion about the scope results combined with docking analysis have been added into the main text, highlighted with yellow (page 8-10, line 201-246).\nFor minor point 1:\nFollowing your advice, the gram-scale synthesis has been successfully completed, shown in SI page 9 and page 20-21 (Fig. S12).\nFor minor point 2:\nFollowing your suggestion, a mechanism illustration about transaminases has been added to SI, shown in page 10-11, Fig. S1.\nFor minor point 3:\nFollowing your suggestion, a figure about the comparison of BsDAAT-WT and BsDAAT-V33F has been added into SI, shown in Figure S6 in page 14.\nFor minor point 4:\nFollowing your suggestion, docking tools and molecule docking assay have been added into the main text (page 10, Figure 5) and SI (page 10), respectively.\nFor minor point 5:\nFollowing your suggestion, yields with decimal places have been replaced by integer numbers in SI (highlighted with yellow), all yields in main text were integer numbers. Extensive changes have been made; please see the Supporting Information for details.\nFor minor point 6:\nTwo Lanreotide derivatives have been analyzed by MS/MS, the detailed results were shown in SI, after the results of their HRMS and HPLC results, in page 89-91 in SI document.\n\nDear reviewer 4:\nWe appreciate your review of our manuscript. We have carefully addressed all the points raised by the reviewers and provided our responses and revisions accordingly for your evaluation. Please see revision, main text and supporting information, all changes have been marked in yellow/blue.\n\nTo all reviewers:\nThanks for all of your positive feedback. We have addressed the remained comments as detailed below, outlining both our responses and the modifications implemented. Please find our revised manuscript for your review.\nSincerely,\nLicheng Yang"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The revised manuscript has fully addressed the concerns raised in my original review. As noted previously, the chemoenzymatic strategy described in this work will be of significant value for the preparation of β-branched D-amino acids. The experimental design and execution are of an exceptionally high standard, and the data presented are comprehensive and robust, fully supporting the authors’ conclusions. I therefore strongly recommend publication of this manuscript in Nature Communications.", "reviewer_2": "The work is of high quality and certainly suitable for publication. While the general concept has been reported previously, the innovation in identifying a d-selective enzyme and studies to characterize this process is novel. I am convinced by the authors arguments and now believe this work is suitable for publication in Nature Communications.", "reviewer_3": "I appreciate the authors’ responsiveness to adding information to their manuscript and response document and find it greatly enhanced. I have a few minor suggestions, however following their consideration believe that this manuscript is now appropriate for publication in Nature Communications.\n\nResponse to major point 2:\nI agree with all of the points mentioned about DKR, especially in relation to this system. I was asking for that same level of rigor to be applied to BsDAAT given its novel application within this article. I find the additional sentences added to the main text appropriate, however would appreciate the inclusion of an experiment to support it for this homologue.\n\nResponse to major point 3:\nThank you for your addition of these NMR spectra, I believe they substantially help illustrate the two different diastereomers. As a minor correction, I believe that the ‘1H NMR spectrum of racemic compound 1’ is inclusive of both diastereomers and the legend should be adjusted to reflect this (1H NMR spectrum of both diastereomers of 1 – or something similar).\n\nThe rest of the revisions are strong additions to the manuscript and I appreciate the authors’ receptiveness to include them.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Dear reviewer 3:\nWe sincerely appreciate your positive feedback on our work. We added a note under the racemic product 1 explaining it as a mixture of two diastereomers. However, the required control experiments to prove DKR mechanism is difficult to carry out. As you could expect, it would take a great labor work to get access the enantiopure substrate and then run those experiments. For the second reason, we believe the results are in agreement with the previous work in Angew. Chem. Int. Ed. 2021, 60, 17680-17685. We hope you can agree with the explanation just by citing the reference of the ACIE paper. If you feel really necessary to repeat the mechanism study, then we have to ask for more time to do it. Thanks and appreciate your understanding.\nFor major point 1:\nThanks for this suggestion. We agree that detailed control experiments would be beneficial to confirm this DKR mechanism. However, as this enzymatic system is very similar with the previous work (Angew. Chem. Int. Ed. 2021, 60, 17680-17685. I, Licheng Yang, was the co-first author in that work, and I carried out all the control reactions.), we believe the racemization mechanism would be the same. The control experiments wouldn’t make a difference. In addition, it would take a great labor work to prove it, including resolving the racemic ketoacid substrate, carrying out the enzymatic reactions, and check the product/recovered SM ee values. In that way, we have to apply for one extra month to carry out all those control experiments. We hope you could agree with the mechanism explanation by citing the reference of the ACIE paper. Thanks and appreciate your understanding.\nFor major point 2:\nThanks for your suggestion. We’ve added a note to explain the mixture of the two diastereomers for the racemic product."}]

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