A microbial platform for selective biosynthesis of monoterpene esters

Domain: physics
Published: 2026-04-09
Editorial outcome: minor_revision

Abstract
Reconstructing the precise biosynthesis of structurally complex natural esters, such as monoterpene esters, in engineered microbes remains a major challenge, owing to the limited repertoire of highly selective alcohol acyltransferases and the lack of compatible pathway modularity. Here, we establish a dual-substrate microbial platform to profile the activities of alcohol acyltransferase (AAT) to synthesize three distinct classes of monoterpene esters: monoterpenyl esters, monoterpenoate esters, and monoterpenyl monoterpenoate esters, enabling access to both natural and non-natural monoterpene ester biosynthetic pathways. Through structure-guided critical residue engineering and dual-substrate molar ratio tuning, we achieve selective biosynthesis of >C2 acyl-CoA-derived monoterpene esters, despite competing intracellular acetyl-CoA. Coculture engineering further redistributed metabolic fluxes between acyl-CoA and alcohol precursors, yielding 11.50 g/L linalyl acetate and 3.16 g/L geranyl butyrate in 1-L bioreactor. This study expands the biosynthetic space of monoterpene esters and provides a versatile strategy to control AAT selectivity, offering a plug-and-play, scalable framework for ester biomanufacturing.

Introduction
Natural esters, primarily recognized as either volatile compounds responsible for fruity and floral aromas or as acylated derivatives of complex natural products, are ubiquitous in flowers, traditional fermented foods, and medicinal plants (e.g., rose1, 2, wine3, Baijiu4, Strychnos nux-vomica5, etc). Since the term "ester" was first introduced in 1848 by German chemist Leopold Gmelin to describe the condensation product of acids and alcohols6, natural esters have garnered extensive attention due to their structural diversity, sensory importance, and biosynthetic complexity. Their entire biosynthesis involves intricate modular biosynthetic pathways distributed across microbial and plant cellular systems4, 7. Engineering microbes to synthesize these esters presents considerable challenges, particularly in achieving controlled and efficient biosynthetic pathways. A primary hurdle is the unclear specificity and selectivity of enzymes at critical metabolic junctions8. Natural ester intracellular biosynthesis is predominantly mediated by alcohol acyltransferases (AATs), which catalyze the esterification of acyl-CoAs and alcohols9. However, the inherent broad substrate promiscuities of AATs for both donor dimensions typically form multiple ester products instead of the desired singular compound in engineered microbial or natural systems10. Furthermore, intracellular ester biosynthesis relies heavily on the availability of dual substrates—acyl-CoAs and alcohols—whose availability is regulated by primary or secondary metabolic networks11, 12, such as glycolysis, citric acid cycle, fatty acid biosynthesis/degradation, amino acid biosynthesis/degradation, mevalonate pathway, polyketide biosynthesis, etc. Typically, the imbalance or limitation in precursor supply from these upstream biosynthetic pathways, whether in nature or engineered hosts, can critically hinder AAT-driven ester formation, thereby reducing product formation specificity and efficiency.

Over the past two decades, advancements in metabolic engineering have significantly facilitated microbial design, enabling AAT-mediated biosynthesis of structurally diverse esters using engineered microbes consuming glucose, glycerol, or ethanol13-16. Ester biosynthesis approaches in engineered microbes have involved esterification of short-chain fatty alcohols (SCFAlcs) with various acyl-CoAs by AATs17, optimization of enzyme specificity through substrate-tunnel engineering to reduce intermediate-derived by-product formation7, and coculture strategies to minimize cellular metabolic burdens and substrate promiscuity by dissecting the entire biosynthetic pathway and temporally cascade inoculations18. Despite these progressions, precision ester biosynthesis in engineered microbes remains constrained by insufficiently characterized biocatalytic modules of AATs and the incompatibility of upstream metabolic pathways17, 19-21.

Monoterpene esters (MEs), a highly valuable class of natural products extensively applied as flavors, fragrances, food additives, cosmetic ingredients, advanced biofuels, insecticides, etc (Supplementary Tables 1-2, Supplementary Data 1)9, 22-26, exemplify these constraints in microbial biosynthesis. Structurally, MEs originate primarily from the esterifying reactions between monoterpenols (MTs) and acyl donors, or between alcohols and monoterpenoic acid (MTa)-derived acyl-CoAs (Fig. 1). However, current engineered microbial systems produce a narrow range of MEs, primarily derived from acetyl-CoA27, including geranyl acetate (GAC)28, neryl acetate29, and perilla acetate30. Controlling the specificity of ME biosynthesis critically depends on the characterization and development of highly selective AATs capable of utilizing C>2 acyl-CoAs amid high intracellular acetyl-CoA concentrations. Moreover, as both monoterpenes and acyl-CoAs share acetyl-CoA as a common precursor, finely tuning the molar ratio and compatibility of metabolic fluxes toward alcohol and acyl-CoA biosynthesis is essential to achieve high specificity and titer of ME in engineered microbes.

Here, we develop a dual-substrate microbial platform to profile the catalytic activities of AATs in engineered Escherichia coli, synthesizing three distinct classes of MEs: Monoterpenyl Esters (MTEs), Monoterpenoate Esters (MTaEs), and Monoterpenyl Monoterpenoate Esters (MtMtaEs) (Fig. 1 and Supplementary Fig. 1). By bioconverting MTs or MTas individually with their pairing substrates, we generate 64 MTEs, 3 MTaEs, and 4 MtMtaEs, a type of underexplored monoterpene dimeric esters with four isoprene units. This comprehensive profiling enables the identification of AAT exhibiting either broad or highly specific dual-substrate acceptance patterns for both alcohols and acyl-CoAs. Structure-guided engineering of the critical residue in AATs under adjusted acyl-CoA-to-alcohol molar ratios efficiently mitigated substrate specificity imbalances, in particular between acetyl-CoA and butyryl-CoA, allowing the precise biosynthesis of a variety of MEs and aromatic esters with >99% selectivity. Leveraging a modular coculture strategy to enhance dual-substrate availability and compatibility, we demonstrate the de novo biosynthesis of 7 MTEs using an E. coli-E. coli consortium with MT as the bridging molecule, achieving a titer exceeding 11 g/L for one MTE in a 1-liter bioreactor. This study highlights the advantages of AAT-mediated ME biosynthesis and provides a versatile framework for selective ester biosynthesis in engineered microbes, significantly enhancing the diversity, substrate specificity, and pathway modularity.

Results
Biosynthesis of monoterpenoate esters

To explore the diversity of MTE biosynthesis using various sourced AATs, the acid donor should be available as CoA-activated molecules (Fig. 2a). We selected a butyrate-acetoacetate CoA-transferase from Clostridium acetobutylicum (CaBACT) for activating short-chain fatty acids (SCFAs) into acyl-CoAs31. The selection of the 14 AATs from bacteria, yeast, and plant was based on the previously reported studies, indicating their enzymatic activity in forming acetate-based MTEs7, 19, 32, 33. The phylogenetic analysis reveals the evolutionary divergence and conservation among the amino acid sequences of AATs (Fig. 2b and 2c, Supplementary Fig. 2). The close clustering of LiAAT4, FvAAT, and FaAAT in the phylogenetic tree suggests that these AATs may share a similar dual-substrate specificity. Using engineered E. coli strains expressing individual AATs with or without CaBACT, we conducted esterification reactions by feeding selected MTs and carboxylic acids. The resulting MTEs were captured into the organic phase of culture during fermentation and analyzed by GC-MS (Supplementary Fig. 3). Characteristic ester bond fragmentation patterns were observed, with monoterpenols such as geraniol, nerol, and lavandulol forming base ions at m/z = 69 and linalool at m/z = 93 due to its distinct allylic rearrangement (Supplementary Table 3)34. Characteristic m/z fragments (43, 57, 71, 85) derived from C2-C5 SCFAs enabled unambiguous MTE identification, even without NIST MS references (Supplementary Data 2). Four representative AATs, including FvAAT, FaAAT, LiAAT4, and ScAAT1, were characterized for their activities with acetyl-CoA, propionyl-CoA, and valeryl-CoA. FvAAT, FaAAT, and LiAAT4 displayed broad catalytic activity, capable of accepting C2-C5 acyl donors and diverse MTs (Fig. 2d, Supplementary Figs. 4-25). Among all the validated AATs, LiAAT4 and ScAAT1 showed catalytic activity toward primary-to-tertiary MTs (Fig. 2d).

To activate aromatic acids into their respective acyl-CoAs, we constructed the plasmids harboring the genes coding for an individual AAT with an ATP-dependent fatty/aromatic acid-CoA ligase (ACSL) from Thauera terpenica (TtLACSL)35, Pseudomonas aeruginosa PAO1 (PaLACSL)36, or ATP-dependent benzoate-CoA ligase from Hypericum (HzBCL)37. A preliminary experiment revealed that only TtLACSL exhibited benzoate-CoA activation activity when benzoate and geraniol were co-fed to the strains co-expressing individual ACSL or BCL with FaAAT. We then constructed strains with plasmids co-expressing TtLACSL with various AATs for further evaluation. In addition to FvAAT, FaAAT, LiAAT4, and ScAAT1, we validated three benzyl alcohol O-benzoyltransferases (BABTs) to assess their activities on aromatic acid-derived MTE formation. PhBABT was previously reported to esterify aromatic acid-derived acyl-CoAs with linalool and geraniol38. Identified via BLAST with ≥70% sequence identity to PhBABT in NCBI protein database, we included two additional BABTs (Fig. 2c, Supplementary Fig. 2). The evaluation indicated that FvAAT, FaAAT, and PmBABT produced the corresponding benzoate MTEs (Fig. 2d, Supplementary Figs. 4-25). FvAAT and FaAAT exhibited a wide range of substrate specificity for various MTs for benzoyl-CoA-derived MTEs, which are widely used in the flavor, fragrance, and cosmetic industries, where they contribute sweet, floral, and fruity notes39. PmBABT showed better catalytic activity for the accessed MTs and could exclusively generate benzoate MTEs (e.g., lavandulyl benzoate, geranyl benzoate, neryl benzoate, and perillyl benzoate, Fig. 2d, Supplementary Figs. 7, 9, 11, and 13). Building on these findings, we profiled the capacity of FvAAT and FaAAT to form phenylacetyl-CoA- and phenylpropionyl-CoA-derived MTEs. The identification shows that FaAAT possesses broad catalytic activities for esterifying aromatic acid-derived acyl-CoA with multiple MTs (Fig. 2d, Supplementary Figs. 4-25).

Biosynthesis of monoterpenoate and monoterpenyl monoterpenoate ester

Primary MTs, in plant or microbe, would undergo oxidation to form corresponding MTas, typically mediated by two sequential steps conduct using alcohol dehydrogenases40, 41. We hypothesized that the activation of MTa with HS-CoA by TtLACSL would possibly form monoterpenoyl-CoA, which can be subsequently esterified with SCFAlcs into MTaE (Fig. 3a). This design enables the expansion of microbial ME diversity by leveraging AAT promiscuity towards underexplored dual-substrate reactions. We evaluated the activities of FvAAT, FaAAT, LiAAT4, ScAAT1, ScEEB1, ScEHT1-1, ScEHT1-2, PmBABT, MsBABT, and PhBABT, to form MTaTE (Fig. 3b, the last three were not shown in the figure as no product was detected). By pairing two MTas with three SCFAlcs for the bioconversion, the analysis of the formed compounds in the organic phase using GC-MS showed that (S)-(-)-perillic acid was successfully activated with HS-CoA by TtLACSL and esterified by FaAAT, LiAAT4, and ScEHT1-2 into three respective MTaEs, namely ethyl (S)-(-)-perillate, propyl (S)-(-)-perillate, and butyl (S)-(-)-perillate (Fig. 3b-d). The fragmentation of these MTaEs during ionization would generate a base peak of (S)-(-)-perillic acid with an m/z = 121 (Supplementary Table 4), and characterization of these bioconversion-produced MTaEs was verified by mass spectrometry using the NMR-confirmed, chemically synthesized standards as references (Supplementary Figs. 26-28). As such, incorporating the MTaEs biosynthesized here into the ME family demonstrates the capacity to harness the promiscuity of plant-derived AATs for diversifying natural and non-natural ME biosynthesis.

The conceptual visualization of the dual-substrate ME biosynthesis inspired us to explore the biosynthesis of the third type of MEs (Fig. 1). Theoretically, monoterpenoyl-CoA activated from MTa and HS-CoA could undergo esterification by AATs using either structurally homo- or hetero-type MTs as alcohol donors, resulting in the formation of MtMtaEs (Fig. 3e). Although MtMtaE consists of four isoprene units and shares the same carbon count (C20) with canonical diterpenes, they are structurally distinct as they are not synthesized via the geranylgeranyl pyrophosphate pathway. We paired and co-fed 11 MTs with two MTas into the medium to explore the possibility of MtMtaE formation by FvAAT, FaAAT, LiAAT4, ScAAT1, ScEHT1-2, PmBABET, MsBABT, and PhBABT (Fig. 3f, the last three were not shown in the figure as no product was detected). The analysis of the compounds formed in the organic phase using GC-MS revealed that both geranic acid and (S)-(-)-perillic acid could be activated by TtLACSL to their CoA molecules, which were esterified by FaAAT into four respective MtMtaEs (Fig. 3g-i). The base peaks of geranic acid and (S)-(-)-perillic acid would be m/z =69 and 121, respectively (Supplementary Table 4). The characterization of these bioconversion-produced MtMtaEs was verified by mass spectrometry with the NMR-confirmed, chemically synthesized standards as references (Supplementary Figs. 29-31). MtMtaEs represent a unique class of monoterpene dimers with the molecular formula C20H30O2, previously detected in atmospheric secondary organic aerosols42, yet their biosynthetic origins in living organisms remain largely unexplored. The dual-substrate microbial platform accelerates the discovery and characterization of MtMtaEs, offering a viable method for biosynthesizing monoterpene dimeric esters. Importantly, the conversion of volatile and membrane-permeable monoterpenes into bulkier MtMtaEs alters their physicochemical properties, reducing volatility while favoring sequestration in subcellular storage compartments such as lipid droplets or peroxisomes. Such compartmentalization not only possibly alleviates product toxicity but also establishes a stable reservoir that supports more efficient and sustainable monoterpene biosynthesis.

Reprogramming AAT selectivity via single-residue mutagenesis and dual-substrate ratio

We performed molecular docking of the AlphaFold2-predicted FaAAT, LiAAT4, FvAAT, and PmBABT in complex with acyl-CoAs, revealing distinct differences in active-site organization that underlie their distinct substrate preferences (Supplementary Fig. 32). Although these AATs share a conserved acyl-CoA binding framework coordinated by key catalytic residues (e.g., His and Asp), the composition and geometry of the acyl-binding pocket vary substantially. FaAAT and FvAAT, which preferentially accept short-chain acyl donors, possess relatively narrow and hydrophobic acyl pockets shaped by residues such as Ala/Val and Phe/Leu, forming a compact environment around acetyl-CoA (Supplementary Fig. 32a-b). In contrast, LiAAT4 exhibits a more restricted cavity due to substitution by bulkier residues (e.g., Tyr), whereas PmBABT displays a distinct adaptation for aromatic substrates, with benzoyl-CoA stabilized by expanded hydrophobic contacts and additional polar interactions (Supplementary Fig. 32c-d). Consistent with these pocket-level differences, substrate tunnel architectures across these AATs differ in length, bottleneck radius, and residue composition (Supplementary Fig. 33). FaAAT and FvAAT feature extended tunnels that can accommodate elongated aliphatic substrates, whereas PmBABT exhibits shorter and more constrained pathways. These global tunnel features rationalize broad differences in substrate scope among AATs; however, they primarily govern substrate accessibility rather than fine discrimination between structurally similar acyl donors. In particular, the precise selectivity of FaAAT toward acetyl- versus butyryl-CoA cannot be explained by tunnel geometry alone, implicating localized interactions and dynamic effects near the catalytic center.

Accordingly, we focused on FaAAT to further investigate how ligand-binding residues govern acetyl-CoA versus butyryl-CoA selectivity using geraniol as a fixed alcohol donor. Molecular docking using the AlphaFold2-predicted FaAAT suggested that acyl-CoA binds within a shared pocket adjacent to the catalytic residue His157, with 303Phe, 162Gly, 250His, 378Ser, and 380Trp located in close proximity to the acyl-CoA moiety (Supplementary Fig. 34). Among them, 303Phe was proposed as a key residue proximal to 157His that influences the relative binding geometry of C2 and C4 acyl-CoAs to geraniol (Fig. 4a-b, Supplementary Fig. 34a). To test this hypothesis, structure-guided NDT codon-based saturation mutagenesis (N = A/C/G/T, D = A/G/T, T = thymine) was performed. Several variants exhibited selectively enhanced on butyryl-CoA over acetyl-CoA (Supplementary Fig. 34b-f). Replacement of 303Phe with small side-chain residues (e.g., Gly, Ser, and Asn) significantly reduced GA formation without causing a substantial decrease in geranyl butyrate (GB) titer. Additional substitutions at G162, H250, S378, and W380 on F303GFaAAT did not yield further improvements in selectivity or product titer (Supplementary Fig. 35), indicating that, for short-chain acyl-CoA discrimination in FaAAT, double-site tunnel reshaping is less effective than localized residue engineering near the catalytic center, likely as excessive tunnel perturbation compromises productive acyl-CoA positioning.

Phylogenetic analysis revealed substantial diversity among plant-derived AATs; however, a conserved aromatic or bulky aliphatic residue (Tyr, Phe, or Ile) is frequently observed at position 303 (Fig. 4c, Supplementary Fig. 36). Furthermore, mutagenesis at position 303 in FaAAT resulted in substrate-dependent changes in the production profiles of MTaEs and MtMtaEs (Supplementary Fig. 37), indicating that this residue influences acyl-donor preference beyond the geraniol-based system.

Representative poses of acetyl-CoA-geraniol and butyryl-CoA-geraniol complexes generated from the AlphaFold2-predicted F303GFaAAT revealed altered substrate orientations and contact patterns in the vicinity of the active site after mutation (Fig. 4d-e). However, because molecular docking provides only static and non-unique binding hypotheses, we then examined mutation-induced effects using pre-reaction state (PRS) molecular dynamics (MD) simulations. These analyses revealed that the F303G mutation does not uniformly shorten substrate contact distances but instead redistributes dynamic interaction patterns within the acyl-CoA thioester environment, particularly involving 284Asn and 378Ser, in a substrate-dependent manner (Supplementary Fig. 38). Moreover, comparative perturbation response scanning molecular dynamics (PRS-MD) analyses revealed that homologous mutations in closely related FaAAT2 can modulate distinct reaction coordinates. In FaAAT, the F303G mutation primarily reshapes thioester torsional conformations, whereas in FaAAT2 with an isoleucine at the corresponding position, the analogous I303G mutation in FaAAT2 alters the Bürgi-Dunitz attack geometry. This divergence indicates that mutation-induced selectivity arises from enzyme-specific reorganization of reactive conformational ensembles rather than static distance changes alone (Supplementary Fig. 39).

The intracellular availability of acyl-CoA and MT affects the apparent specificity of dual-substrate esterification catalyzed by AATs, which can be modulated by varying the molar ratio of the substrates. To confirm this, we profiled the formations of GB and GA at varying dual-substrate molar ratios, both wtFaAAT and F303GFaAAT exhibited high exponential curve fitting confidence for GA and GB formation (R2>0.99, Fig. 4f, Supplementary Data 3, No. 31-34). The selectivity for GLB formation was improved at all tested dual-substrate molar ratios (Fig. 4g). The strain harboring F303GFaAAT achieved a GB selectivity of 98.95% under a 4:1 butyrate-to-geraniol molar ratio, representing a 3.12-fold (p < 0.001) increase relative to wtFaAAT under a 1:4 ratio; even under 1:4 butyrate-to-geraniol molar ratio, F303GFaAAT still exhibited a 2.21-fold improvement in GB selectivity compared with wtFaAAT (p < 0.001; Fig. 4g). Consistent with this trend, the other two beneficial substitutions at position 303 of FaAAT also showed improved GB selectivity under higher butyrate-to-geraniol ratios (Supplementary Fig. 34g-i).

To elucidate the enzymatic basis underlying these mutation-enhanced selectivity effects, we next characterized the intrinsic catalytic properties of wtFaAAT and F303GFaAAT in vitro. The F303G mutation in FaAAT resulted in no detectable product in the acetyl-CoA/geraniol reaction, indicating a loss of enzyme catalytic activity toward acetyl-CoA upon introduction of the F303G mutation under the tested conditions in vitro (Fig. 4h, Supplementary Figs. 40-41). In contrast, for the butyryl-CoA/geraniol reaction, quantitative kinetic analysis was feasible. The Km value showed no significant change upon mutation (1.04-fold difference relative to the wild type, p = 0.66), indicating that substrate binding affinity for butyryl-CoA was largely preserved. However, the catalytic efficiency (kcat/Km) was significantly reduced to 0.35-fold of wtFaAAT (p < 0.001) (Fig. 4h, Supplementary Fig. 40). These kinetics are consistent with mutation-induced destabilization of catalytically productive acetyl-CoA conformations, arising from altered local hydrogen-bonding networks involving 284Asn and 378Ser, while largely preserving butyryl-CoA binding (Supplementary Fig. 42).

Despite the favorable shift in acyl-donor preference conferred by the F303G mutation in FaAAT, the overall catalytic performance of FaAAT remained constrained under cellular conditions, prompting us to examine whether enzyme solubility represents an additional limiting factor. We observed poor soluble expression of both wtFaAAT and F303GFaAAT (Supplementary Fig. 40a), which likely caused the reduced catalytic activity for GB formation (Fig. 4f and 4i). Phylogenetic analysis and pairwise sequence alignment revealed that FaAAT2, identified from the NCBI protein database (Supplementary Fig. 40b-c), contains an Ile at position 303 and differs from FaAAT by five amino acid residues (Supplementary Fig. 36), showing improved solubility compared with FaAAT (Supplementary Fig. 40d). Profiling GB and GA production by the strains expressing wtFaAAT2 and I303GFaAAT2 varying molar ratios of butyrate-to-geraniol generated high-confidence exponential curve fitting (Supplementary Fig. 43, R²>0.98, Supplementary Data 3, No. 39-42) and a GB titer (Fig. 4i) approaching the theoretical yield (1.45 g/L, Supplementary Fig. 44). The GB selectivity achieved by the strain harboring I303GFaAAT2 (97.62%) was slightly lower than that of F303GFaAAT under a 4:1 molar ratio of butyrate-to-geraniol, but still represented a 4.22-fold increase compared to wtFaAAT2 under 1:4 molar ratio of butyrate-to-geraniol (p < 0.001) (Fig. 4j). The enhanced GB selectivity observed with I303GFaAAT2 was consistent with altered catalytic behavior, as confirmed by steady-state kinetic assays (Fig. 4k, Supplementary Fig. 40e-f), which suggests that, in FaAAT2, the apparent acyl-CoA selectivity is governed by a combined contribution of catalytic turnover, enzyme solubility, and global active-site interaction networks (Supplementary Fig. 45), rather than being solely dictated by local acyl-CoA binding energetics.

To place these enzyme determinants within a unified catalytic framework, we next examined the intrinsic reaction mechanism shared by AAT and its variants. The kinetic data are consistent with a sequential bi-bi mechanism for AAT and its variants under the tested conditions, in which geraniol and acyl-CoA form a catalytically competent ternary complex (Fig. 4I, Supplementary Fig. 46-47). To quantitatively address the catalytic mechanism, we have performed ab initio QM calculations on an enzyme model that encompasses the core reaction center (157His in FaAAT, geraniol, and CoA thioester, Supplementary Fig. 48). This model confirms the proposed mechanism: 157His facilitates geraniol deprotonation followed by nucleophilic attack on the thioester carbonyl, with a rate-determining barrier of 17.6 kcal/mol (ΔHǂ). Notably, the subsequent proton transfer from the imidazolium to thiolate is essentially barrierless, and the overall reaction is slightly exergonic (ΔG = -3.1 kcal/mol). The computed energy profile aligns well with the mechanistic scheme in Fig. 4I, suggesting the critical role of pre-organized active-site architecture in transition-state stabilization. Building on this mechanistic understanding, to validate the combined strategy of single-residue engineering and dual-substrate ratio control for precision ester biosynthesis, we characterized more substrate pairings: butyryl-CoA with nerol, butanol, and benzyl alcohol; geraniol with propionyl-CoA and benzoyl-CoA; and benzoyl-CoA with benzoyl-CoA. Exponential fittings of the product titers on the selected dual-substrate molar ratio showed high confidence (average R²>0.98 across all dual-substrate pairings (Supplementary Data 3, No. 1-30, 47-72). Modulating molar ratios of acyl-CoA-to-alcohol from 4:1 to 1:4 with varied wtAAT and its mutants significantly enhanced selectivity by 3.71-fold (neryl butyrate, p < 0.001), 1.94-fold (butyl butyrate, p < 0.001), 3.19-fold (benzyl butyrate, p < 0.001), 2.10-fold (geranyl propionate, p < 0.001), 60.13-fold (geranyl benzoate, p < 0.001), and 1.37-fold (benzyl benzoate, p < 0.001) (Supplementary Figs. 49-54), outperforming the reported AATs under comparable conditions (Supplementary Fig. 44). These results support that dual-substrate molar ratio modulation adjusts the apparent AAT selectivity, while critical residue engineering alters the AAT’s intrinsic specificity, both of which aided precision ester biosynthesis.

Coculture to de novo monoterpenoate ester biosynthesis

Connecting isopentenyl diphosphate (IPP) plus dimethylallyl diphosphate (DMAPP) or only two DMAPPs generates various prenyl diphosphate (PDSP) by PDP synthase (PDPS). Subsequent releasing diphosphate from PDP by MT synthase (MTS) would form corresponding MTs (Supplementary Fig. 55a)24. To establish de novo MT biosynthetic route from glycerol in E. coli, six representative MTs were selected—linalool, geraniol, α-terpineol, borneol, nerol, and lavandulol—covering primary, secondary, and tertiary MTs. The entire biosynthetic pathway was modularly assembled with four units: an upstream mevalonate (MVA) module, a downstream MVA module, a PDPS module, and an MTS module. Each module operon-related gene was placed under the control of two inducible promoters, enabling fine-tuned flux regulation (Supplementary Fig. 56-61). Systematic metabolic engineering revealed several key strategies to enhance MT production: increasing acetyl-CoA availability through the ethanol utilization pathway (EUP)43, 44, optimizing gene expression across modules to balance pathway flux, and reducing by-product formation via targeted gene deletions (Supplementary Fig. 55b-g, Supplementary Figs. 56-61). For GPP-derived MTs, changing MTSs in the downstream biosynthetic pathway resulted in evident differences in product titers (Supplementary Fig. 55b-e). Under optimized strain and induction conditions, the titers of linalool, geraniol, α-terpineol, borneol, and nerol reached 3.33 g/L, 1.18 g/L, 198.98 mg/L, 3.84 mg/L, and 224.42 mg/L, respectively. The titer of linalool achieved was 16.74-fold higher (p < 0.001) than that of α-terpineol. This disparity is attributable to differences among MTSs in both catalytic efficiency toward GPP and effective expression levels24, resulting in varied production titers of MTs. Besides, protein engineering of MTS to enhance their activities was therefore critical for improving MT biosynthesis. In lavandulol biosynthesis, we screened a panel of E. coli endogenous phosphatases and identified EcRdgB as the most active enzyme toward lavandulyl pyrophosphate. We further performed structure-guided multiple-round single-site mutagenesis to enhance its catalytic efficiency, obtaining the engineered variant F118D-H182CEcRdgB, which significantly improved the hydrolysis of lavandulyl pyrophosphate and consequently increased the lavandulol titer by 7.64-fold (p < 0.001) (Supplementary Fig. 61).

Inspired by the modularity of acyl-CoA and MT biosynthetic pathways in nature, we constructed a MT-producing strain and an ester-producing strain for coculture engineering (Supplementary Fig. 62a). This strategy simplifies the genetic manipulation cycle and alleviates metabolic competition for shared precursors such as acetyl-CoA45. By combining the above MT-producing strains with an esterification module containing AATs with defined substrate specificity, we produced natural linalyl acetate (LA) and GB via microbial biosynthesis. LA and GB have high industrial relevance: LA is the dominant high-value fragrance component in lavender essential oil and is widely used in perfumery, while GB is a commercially important fruity/floral flavor compound used in food, beverage, and fragrance formulations27, 46. Besides, their biosynthetic precursors—linalool and geraniol—are among the monoterpene alcohols with the higher achievable microbial titers in this study and other extensive studies, enabling a representative demonstration of MTEs biosynthesis. For acetate MTE biosynthesis using a single strain, introducing an additional plasmid overexpressing LiAAT4 with an inducible promoter into the linalool-producing strain would be achieved. However, even under optimized induction conditions, the highest titer of LA reached 0.058 g/L by the single-strain mode (Fig. 5a-b, Supplementary Fig. 63). We cocultured the linalool-producing strain with the ester-producing strain containing LiAAT4 (Fig. 5c) and optimized the critical parameters. Optimization of inoculation ratio at the induction stage, coupled with EUP module integration into the ester-producing strain, substantially enhanced LA production (Supplementary Fig. 63), achieving a titer of 3.22 g/L, representing a 55.2-fold improvement over monoculture systems (p < 0.001) (Fig. 5b). Next, to construct a single-strain system for GB production, we transformed the third plasmid with the genes coding the enzymes of butyryl-CoA biosynthetic pathway47 and wtFaAAT into the geraniol-producing strain (Fig. 5d, Supplementary Fig. 64). However, this three-plasmid-containing strain did not give a detectable titer of GB after optimizing the induction conditions for pathway balancing (Fig. 5e). We subsequently cocultured a geraniol-producing strain and a strain with butyryl-CoA biosynthetic pathway and wtFaAAT, which produced 0.077 g/L of GB (Fig. 5e-f). Because wtFaAAT provided a consistent baseline for optimizing coculture conditions, we subsequently replaced it with F303GFaAAT and I303GFaAAT2 in the ester-producing strain, which increased the GB titer to 0.32 g/L with a selectivity of 97.81% (Fig. 5e, Supplementary Fig. 64). We further expanded the product profile to biosynthesize acetate and butyrate MTEPs from glycerol using coculture strategy, and achieved the titers of lavandulyl acetate, neryl acetate, bornyl acetate, lavandulyl butyrate, and neryl butyrate to 94.49, 197.12, 2.40, 8.72, and 65.72 mg/L (Fig. 5g-k, Supplementary Fig. 62b-f).

To scale up the coculture process for ME biosynthesis, we ran fed-batch fermentation in a 1-liter bioreactor. A titer of 11.50 g/L LA was obtained, with 0.11 g/L linalool residing after 96h fermentation (Fig. 5l). Chiral column analysis of the fermenter samples confirmed the stereoselective biosynthesis of R-(–)-LA in enantiopure form, in contrast to chemically synthesized racemic LA (RS-mixture, Supplementary Fig. 62g). The GB-producing coculture reached a titer of 3.16 g/L, with 0.033 g/L GA and 0.046 g/L geraniol accumulated after 83 h, corresponding to a GB selectivity of 98.97% (Fig. 5m). Importantly, GB was obtained as an isomerically pure compound, whereas chemically synthesized standards consisted of mixed isomers including neryl butyrate (Supplementary Fig. 62h). In both cases, the target esters were captured in situ by an overlaying organic phase, which enabled efficient product partitioning from the aqueous broth and visual accumulation of ester-rich layers for further downstream purification (Fig. 5n and 5o). These results highlight the scalability, specificity, and stereochemical fidelity of the coculture strategy for de novo biosynthesis of MEs.

Methods
Strains, chemicals, and culture media

The primers and genes used in this study were synthesized at Sangon Biotech Shanghai Co., Ltd., China and Jiutian Gene Technology (Tianjin) Co., Ltd, China. The sequence information of primers and genes are listed in Supplementary Data 4 and Supplementary Data 5. E. coli DH5α (TSC-C01-100, Beijing Tsingke Biotechnology Co., Ltd, China) was used for general cloning purposes. E. coli MG1655(DE3) ∆endA ∆recA (https://www.addgene.org/37854/) was used for fermentation. E. coli BL21(DE3) (TSC-E01, Beijing Tsingke Biotechnology Co., Ltd, China) was used for protein purification. All the chemicals used in this study are listed in Supplementary Data 6. For genome editing of E. coli MG1655(DE3) ΔendA ΔrecA, a two-plasmid CRISSPR-Cas9 system was used56, and the pTarget plasmids employed are detailed in Supplementary Data 7 and 8. Colony PCR was used to evaluate the correctness of the genes deleted at the selected locus. Fermentation process for ME formation identification was carried out in M2 medium (pH 7.0), consisting of 10 g/L glycerol, 26.6 g/L K2HPO4, 8 g/L (NH4)2HPO4, 12 g/L tryptone (LP0042B, Oxoid, Thermo Fisher Scientific Inc., USA), 24 g/L yeast extract (LP0021B, Oxoid, Thermo Fisher Scientific Inc., USA), and appropriate antibiotics.

Plasmid construction and transformation

The construction of the plasmids in this study was based on the GT DNA assembly standard57 when designing overlapping sequences. The assembly of all the PCR-obtained DNA fragments was done using the homologous recombination enzyme (2 × ClonExpress Mix, KAO-201, Toro Bioscience, China) and carried out with an equimolar amount of each DNA fragment in a PCR instrument at 50 °C for 15~30 mins. The standard heat-shock protocol was used to transform the assembled mixture into E. coli DH5α. The culture plate was incubated overnight at 37 °C until the colonies reached an appropriate size, after which they were harvested for subsequent colony PCR verification. The colonies containing the corrected plasmid assembly were inoculated into 5 mL LB medium and used to extract the plasmids for Sanger sequencing. All the plasmids constructed are listed in Supplementary Data 7 and 8. The standard electroporation protocol transformed the sequencing-confirmed plasmid into E. coli MG1655(DE3) ∆endA ∆recA or other derivative strains. All the strains constructed and used in this study are listed in Supplementary Data 9.

Bioconversion of paired MT with acid into ME

The expression plasmids carrying individual AAT (Supplementary Data 7 and 8) were transformed into E. coli MG1655(DE3) ∆endA ∆recA. A single colony of the resulting strain was inoculated into 1 mL LB medium (ampicillin 50 μg/mL) and incubated overnight as the seed culture. Next, 2% (v/v) of seed culture was inoculated into 2 mL M2 medium with 50 μg/mL ampicillin into 2 mL of culture medium in a 15 mL high-borosilicate glass test tube (15 × 150 mm, Product No. 120307005, Changde Beckman Biotechnology Co., Ltd, China). Upon reaching OD600 of 1.0, isopropyl-β-D-thiogalactopyranoside (IPTG, 0.1 mM), the pairing substrates of acid and alcohol were added. For MTE biosynthesis, 20 μL of acid substrate (100 g/L aqueous stocks) was added at a final concentration of ~1 g/L. MTs dissolved in dodecane or hexadecane (6.67 g/L) were added into the culture with 300 μL (15% v/v) to a final concentration of 1 g/L. For MTaE biosynthesis, geranic acid or (S)-(-)-perillic acid (500 g/L in dimethyl sulfoxide, DMSO) was added with 4 μL to a final concentration of ~1 g/L, followed by adding 20 μL of alcohol substrate (ethanol, propanol, butanol) in ultrapure water (100 g/L) into the culture. Subsequently, 300 μL of dodecane overlay was supplemented for in situ product extraction during fermentation. For MtMtaE biosynthesis, MTa and MT were paired and supplemented at the same final concentration as specified above. The cultures were incubated at 30 °C, 250 rpm for 30 h. After fermentation, the upper organic phase was collected after centrifugation at 16,000 g for 5 min, and 10 μL of the upper phase was mixed with 190 μL ethyl acetate for GC-MS analysis.

Bioconversion of paired substrates into other esters

To biosynthesize butyl butyrate, benzyl benzoate, and benzyl butyrate, the expression plasmids carrying individual AAT (Supplementary Data 7 and 8) were transformed into E. coli MG1655(DE3) ∆endA ∆recA. The culture processes are as described in “Bioconversion of paired MT with acid into ME”). The concentration of butanol or benzyl alcohol was 1 g/L (100 g/L aqueous stock), and butyrate or benzoate was added into the culture with a molar ratio of acid-to-alcohol of 2:1 for screening strains containing mutants of AAT. For the dual-substrate molar ratio test, the alcohol was still fixed at 1 g/L, and the acid substrate was added in the ratios of 4:1, 2:1, 1:1, 1:2, and 1:4. For benzyl benzoate, the concentration of benzyl alcohol was reduced to 0.3 g/L to avoid cellular toxicity when evaluating the dual-substrate molar ratio.

Metabolite analysis

GC-MS analysis: A 2 μL aliquot of the above-obtained mixtures of organic solvents with ethyl acetate was used for GC-MS analysis. GC-MS was performed using a system equipped with HP-5MS Capillary Column (30 m × 0.25 mm × 0.25 μm, Agilent Technologies, CA, USA) in a split ratio 1:10. The oven temperature program was as follows: initial hold at 50 °C for 1 min, ramp to 100 °C at 5 °C/min (5 min hold), then to 200 °C at 5 °C/min (5 min hold), followed by a 5 °C/min ramp to 300 °C (5 min hold). The MS source and quadrupole were maintained at 230 °C and 150 °C, respectively, with a scan range of 40-500 m/z. When exporting the extracted ion chromatograms (EIC) based on the information provided in Supplementary Table 3-4 and Supplementary Data 2, fixed offsets were applied to the Y-axis to distinguish the chromatographic traces of different samples for clearer visualization, and the X-axis range was constrained to focus on the regions of interest. It should be noted that these adjustments were made solely to optimize the graphical presentation, and all original raw data remain complete and unaltered, which are fully provided in Supplementary Data 10. GC-MS spectra exported from the instrument software (Agilent MassHunter Workstation Software, Version B.07.00). For each spectrum, the exported table (CSV format, Supplementary Data 11) was parsed by skipping the first two metadata/header lines and extracting the m/z and ion-count columns. Peak intensities were normalized to relative intensity (%) by setting the base peak to 100 (intensity/max intensity × 100). The normalized spectra were then rendered as mass spectra with uniform peak-line width, and the 15 most intense ions were annotated by their m/z values.

GC analysis: GC analysis was conducted using a GC9720Plus (Zhejiang Fuli Analytical Instruments Co., Ltd, China) or Agilent 8860 (Agilent Technologies, CA, USA) equipped with an HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm, Agilent Technologies, CA, USA) or Chiral GC capillary column, Rt-βDEXse (30 m × 0.32 mm × 0.25 μm, Restek Corporation, PA, USA). Post-fermentation, 20 μL of the organic phase sample was mixed with 80 μL of ethyl acetate for GC analysis. A 2 μL aliquot of the mixture of organic solvents with ethyl acetate was injected into the GC for analysis. For the HP-5MS capillary column, the oven temperature program was initiated with an isothermal hold at 50 °C for 2 min, followed by a controlled temperature ramp at a rate of 15 °C/min up to 250 °C, where it was maintained for an additional 7 min to ensure optimal separation. For the Chiral GC capillary column, the oven temperature was programmed as follows: held at 50 °C for 1 min, increased to 100 °C at a rate of 10 °C/min and held for 1 min, then raised to 120 °C at a rate of 0.5 °C/min and held for 1 min, and finally increased to 230 °C at a rate of 20 °C/min and held for 2 min.

HPLC analysis: High-performance liquid chromatography (HPLC) analysis was conducted using an LC-20A system (Shimadzu, Japan) equipped with an Aminex HPX-87H Column (300 × 7.8 mm, Bio-Rad Laboratories, Inc., USA). To quantify glycerol, ethanol, and other fermentation metabolites, 1 mL of cell culture supernatant was collected from centrifuging (16,000 g, 5 min) at each sampling time point and filtered. A 10 μL of the obtained samples was injected for analysis. The separation was performed under isocratic conditions with a mobile phase of 5 mM H2SO4 aqueous solution at a flow rate of 0.7 mL/min. The column temperature was maintained at 40 °C, and analytes were detected using a refractive index detector. Commercial standards of glycerol and acetate were employed to establish calibration curves for accurate concentration determination.

Sequence alignment and phylogenetic tree analysis of AATs

Protein sequences of AATs (Supplementary Data 5) were aligned using ClustalW with default gap penalties (gap opening: 10.0; extension: 0.10 for pairwise and 0.20 for multiple alignments). Divergent sequences were delayed using a 30% cutoff. Phylogenetic analysis was conducted using MEGA software employing the Maximum Likelihood method58. The Jones-Taylor-Thornton model was selected for amino acid substitution59, with uniform rates among sites and inclusion of all sites in the analysis. The initial tree was automatically generated using the BioNJ method. The ML heuristic method applied was Nearest-Neighbor Interchange, and the analysis was run using four computational threads. The resulting aligned FASTA file containing 14 AATs was submitted to the ESPript 3.0 server (https://espript.ibcp.fr/ESPript/ESPript/) for structure-informed alignment annotation60. A corresponding PDB file of FvAAT was uploaded to map secondary structure elements onto the alignment. Besides, to create pairwise alignment with each two AAT protein sequences, the FASTA file containing 14 AATs (Supplementary Data 5) was used for sequence similarity calculation in the Biopython pairwise2 module with the globalxx scoring scheme in Python 3.1261. The similarity score between each pair of protein sequences was normalized by the length of the shorter sequence and expressed as a percentage. A similarity matrix was constructed and visualized as a heatmap using the Seaborn and Matplotlib libraries in Python 3.12.

Structure modeling

Protein structural models of AATs and their mutants were generated using AlphaFold62 (https://alphafold.ebi.ac.uk/) or SWISS-MODEL63 (https://swissmodel.expasy.org/), depending on sequence availability and prior structural annotation. For AAT structures not available in the AlphaFold database, homology models were constructed using the SWISS-MODEL server. Protein sequences were submitted to the SWISS-MODEL workspace, and the top-ranked template based on sequence identity and coverage was selected for model generation. All resulting models were confirmed as monomers unless otherwise indicated. Template entries and sequence identity (%) between query proteins and their corresponding structural templates are summarized in Supplementary Table 5. The modeled structures were used for active site residue mapping, enzyme-ligand docking, and tunnel analysis.

Molecular docking

Molecular docking of AATs with selected ligands was performed using AutoDock Vina v1.1.264. Three-dimensional ligand structures were retrieved from the PubChem database (https://pubchem.ncbi.nlm.nih.gov) and converted to PDB format using Open Babel65. Ligand and protein preparation, including the addition of Gasteiger charges and polar hydrogen atoms, was conducted using AutoDock Tools v1.5.666. Docking was carried out between each AAT model and its corresponding ligand, with docking grids centered around the putative active sites of dual substrates based on the results of tunnel analysis. All docking parameters, including grid box size, center coordinates, and exhaustiveness values are available via Zenodo67.

AAT substrate tunnel analysis

Dual-substrate access tunnels of AATs were analyzed using the CAVER 3.0.3 PyMOL plugin (integrated in PyMOL 3.1.0, Schrödinger)68, 69. The analysis was performed on the representative AAT PDB structures. The starting point for tunnel calculation was defined as the N3 atom of the catalytically relevant histidine in the active site. The following parameters were used for tunnel detection: probe radius of 0.9 Å, shell radius of 3.0 Å, and shell depth of 4.0 Å. For tunnel clustering and prioritization, default settings were applied. All tunnels were visually inspected and rendered using PyMOL. Key surrounding residues were annotated for each identified tunnel, and comparative analysis was performed across different mutants to assess changes in tunnel geometry and accessibility. The tunnel analysis pse files are available via Zenodo67.

Synthesis of chemical standards

The chemical standards of MTaE and MtMtaE were synthesized as follows70: (1) to synthesize ethyl, propyl, and butyl (S)-(-)-perillate, a mixture of perillic acid (1.52 g) and potassium carbonate (1.93 g) was dissolved in 20 mL of dimethylformamide (DMF). Under stirring at room temperature, a solution of iodoethane/iodopropane/iodobutane (1.87/2.04/2.21 g) in DMF was added dropwise. After completion, the reaction mixture was heated to 40 °C and stirred for 5 h. The mixture was then quenched with saturated brine (10 mL), and the aqueous layer was extracted with ethyl acetate. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to afford the target compounds as colorless liquids. (2) to synthesize perillyl generate, the reaction was carried out under a nitrogen atmosphere. Iodine (3.81 g) was dissolved in 150 mL of dry dichloroethane at room temperature (25 °C), followed by sequential addition of PPh3 (3.94g), imidazole (2.25 g), and geranic acid (1.85 g). The mixture was stirred for 15 min before adding Gd(OTf)3 (0.06 g) and heating to 45~50 °C for 30 min. Subsequently, perillyl alcohol (1.52 g) was added, and the reaction temperature was maintained for 2~3 h until completion was confirmed by thin-layer chromatography (petroleum ether:ethyl acetate=20:1, v/v). The workup involved quenching with 1 M sodium bicarbonate solution (100 mL) with 10 min of stirring. After phase separation, the aqueous layer was further extracted with dichloroethane (50 mL). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to afford the target compounds as oily liquid. (3) to synthesize geranyl generate and geranyl (S)-(-)-perillate, under a nitrogen atmosphere at room temperature (25 °C), iodine (3.81 g) was dissolved in 150 mL of anhydrous dichloroethane, followed by the addition of PPh3 (3.94 g), imidazole (2.25 g), and geranic acid (1.85 g)/(S)-(-)-perillic acid (1.83 g). The mixture was stirred for an additional 15 min. Then Gd(OTf)3 (0.06 g) was introduced, and the temperature was raised to 45~50 °C with continuous stirring for 30 min. Geraniol (1.54 g) was added, and the reaction was maintained for 2~3 h until completion, as monitored by thin-layer chromatography (petroleum ether: ethyl acetate=20:1, v/v). The reaction mixture was quenched with 1M sodium bicarbonate solution (100 mL) and stirred for 10 min. After phase separation, the aqueous layer was further extracted with dichloroethane (50 mL). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to afford the target compounds as oily liquid. All the structures of the products were confirmed by ¹H NMR (Bruker 400 MHz, CDCl3). The spectra were processed and analyzed using MestReNova 14.0.

Construction of NDT-based mutagenesis library and screening

Based on the analysis of molecular docking of AATs with ligands, the critical residues were identified for creating the NDT-based saturation or site-directed mutagenesis library. In brief, the primers containing NDT degenerate codons were introduced during PCR and assembled into the corresponding plasmid backbone. All primer sequences can be found in Supplementary Data 4. The plasmids carrying the mixture of mutants were then assembled, and the assembly mixture was transformed into E. coli DH5α, which were plated on LB agar with appropriate antibiotics and incubated overnight at 37 °C. Next, the colonies formed the next day were washed out 5-10 times using 5 mL LB medium until all the colonies were collected, followed by plasmid extraction to obtain the mixed mutant plasmid pool. The plasmids carrying the mixture of mutants were transformed into E. coli MG1655(DE3) ∆endA ∆recA or other derivative strains and cultured overnight at 37 °C. To assess library quality, six colonies were sequenced to confirm the presence of the desired mutations. Then, 30 colonies, approximately converging at three times of library size, were cultured to evaluate the production of MEs (the alcohol concentration is maintained at 1 g/L of final concentration, with acid employed as a co-substrate at a fixed molar ratio. The acid-to-alcohol molar ratio is set at 1:4 for screening butyl butyrate, benzyl butyrate, and geranyl propionate, and at 2:1 for all other combinations) (Details can be found in “Bioconversion of paired MT with acid into ME”). Site-directed mutagenesis was conducted by designing the primers for the other 19 mutations (Supplementary Data 4). After the plasmid assembly, the plasmid with the mutant gene was transformed into E. coli DH5α. A single colony was selected and cultured overnight in 10 mL LB medium with appropriate antibiotics at 37 °C and 250 rpm. The plasmid was then extracted for sequencing to confirm the mutation, followed by transformation into the corresponding E. coli MG1655(DE3) ∆endA ∆recA or other derivative strains. The resulting strains were then used for evaluating the production of MEs. The strains with top-performing mutants were sequenced to determine the substitution of amino acids (Details can be found in “Bioconversion of paired MT with acid into ME”).

Profiling the formations of MTEs at varied dual-substrate molar ratios

Following initial screening, the strains harboring the mixture of the NDT-based saturation AAT mutants or site-directed mutants were investigated to determine the effect of dual-substrate molar ratio on the formation of acetate MTE and target MTE. A single colony was inoculated into 1 mL of LB medium containing 50 μg/mL of ampicillin for overnight incubation at 37 °C and 250 rpm to generate the seed culture. Subsequently, 2% (v/v) of the seed culture was transferred into 2 mL of M2 medium containing 50 μg/mL ampicillin in test tubes and incubated at 37 °C with shaking at 250 rpm. Once the OD600 of the culture reached 2.0, isopropyl-β-d-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM to induce enzyme expression. The alcohol substrate was added at a final concentration of 1 g/L with 20% (v/v) of organic solvent added to each culture (5 g/L alcohol stock in dodecane or hexadecane; for the synthesis of benzyl benzoate, 1.5 g/L benzyl alcohol in dodecane). Acid substrates were prepared as 200 g/L aqueous stock and added into the cultures with varying volumes to adjust molar ratios of acid-to-alcohol at 0 (no additional acid), 1:4, 1:2, 1:1, 2:1, and 4:1 (for the combination of geraniol and propionate, the molar ratio of acid-to-alcohol was adjusted from 2:1 to 10:1). The cultures were then incubated at 30 °C with shaking at 250 rpm for 24 h. After fermentation, cells were separated by centrifugation at 16,000 g for 5 min, and 20 μL from the upper organic phase was mixed with 80 μL of ethyl acetate and subjected to GC analysis. To evaluate the selectivity of AAT or its mutants to GB formation, the percentage of GB titer to the sum of GB and GA titers was calculated.

Curve fitting of MTE formation in bioconversion

To evaluate the formation kinetics of MTEs by various AATs and their mutants, a nonlinear regression model Equation (1) was applied to experimental data obtained under varying dual-substrate molar ratios. The course of dose-response data was fitted to a three-parameter exponential model of the form:

y = a × exp(b × x) + c (1)

where y represents the product titer (g/L), and x is the independent variable of the acid-to-alcohol molar ratio. The fitting parameters a, b, and c in the exponential model represent the initial amplitude, rate of exponential change, and final asymptotic value, respectively. The fitting procedure was implemented using the curve_fit function from the scipy.optimize module in Python 3.1271. Initial parameter guesses were set as (1, -1, 1). Model parameters (a, b, c) and the coefficient of determination (R²) were calculated for each product using least-squares regression. The fitted curves were plotted alongside the corresponding experimental data points and error bars. Fitting results, including equations and R² values, were detailed in Supplementary Data 3.

Enzyme purification

E. coli BL21(DE3) cells harboring the plasmid pHisTag-AAT with T7 promoter were cultured in a 500 mL shake flask containing 200 mL of LB medium at 37 °C for the overexpression of N-terminally His6-tagged AAT. When the OD600 of the culture reached 2.0, IPTG was added to a final concentration of 0.05 mM, and incubation continued for an additional 24 h at 30 °C. After that, the cells were harvested by centrifugation at 16,000 g for 10 min and washed once with 10 mL phosphate-buffered saline (PBS, pH 7.2-7.6, B548117, Sangon Biotech Shanghai Co., Ltd., China). The cell pellet was then resuspended in 10 mL of the same buffer. Cell lysis was performed using an ultrasonic homogenizer (WM-650E, Shanghai Weimi Technology Co., Ltd., China), and the lysate was clarified by centrifugation at 16,000 g for 10 min at 4 °C. The resulting supernatant was subjected to Ni2+-affinity chromatography (PrePacked Gravity Column, C600801, Sangon Biotech Shanghai Co., Ltd., China). A total of 10 mL of the supernatant was loaded onto the pre-equilibrated Ni2+ column. The elute solution was reloaded three times to ensure maximal binding of His6-tagged AAT protein. The column was washed with an equilibration buffer containing 20 mM imidazole to remove nonspecifically bound proteins. Elution was performed stepwise with the buffers containing 40 mM, 100 mM, 200 mM, and 500 mM imidazole. Eluted fractions were collected and analyzed to determine the elution efficiency and purity of the target protein. The concentration of purified AAT was quantified using the Bradford assay in 96-well plates. Briefly, 20 μL of protein sample was mixed with 180 μL of Bradford reagent (Modified Bradford Protein Assay Kit, C503041, Sangon Biotech Shanghai Co., Ltd., China) and incubated at room temperature. The sample was measured at absorbance at 595 nm using a microplate reader (Varioskan LUX, Thermo Fisher Scientific Inc., USA).

in vitro enzymatic activity assay and kinetic analysis

Following protein purification, the enzymatic reactions were conducted with a volume of 500 μL. The enzyme concentration was fixed at 20 mg/L in all assays. Two experimental groups were designed to determine kinetic parameters and reaction mechanism. Group A was conducted under a fixed geraniol concentration while varying acyl-CoA (acetyl-CoA or butyryl-CoA). Geraniol was dissolved in DMSO at 1.5 g/L, and 10.2 μL was added to each reaction to a final concentration of 20 mg/L. Acyl-CoA substrates were freshly prepared at 1 g/L in 1× PBS buffer (pH 7.2–7.6), and the corresponding volume was adjusted to achieve molar ratios of geraniol-to-acyl-CoA from 1:4 to 4:1. Group B was designed with fixed acyl-CoA concentration while varying geraniol. Specifically, 10 μL of 1 g/L acetyl-CoA or butyryl-CoA was added to 20 mg/L final concentration, and geraniol was added in varying volumes from 1 g/L stock to establish molar ratios ranging from 1:2 to 8:1. In both groups, the final reaction volume was adjusted to 500 μL using PBS buffer. Reactions were incubated at 30 °C and 250 rpm for 6 h. Upon completion, 500 μL of ethyl acetate was added to each reaction for extraction, followed by shaking at 800 rpm and 30 °C for 10 min. The mixture was then centrifuged at 16,000 g for 5 min. A 200 μL aliquot of the upper organic layer was collected and analyzed via GC-MS. Steady-state kinetic data were analyzed using Python 3.12. Reaction rates from triplicate assays under varying substrate concentrations were fitted to the Michaelis–Menten equation using scipy.optimize.curve_fit to obtain Vmax and Km. Double-reciprocal (Lineweaver–Burk) plots were generated using matplotlib, with reciprocal values (1/[S], 1/V) calculated via numpy and fitted by linear regression. Standard deviations were calculated and used as error bars in all plots.

Protein and ligand structure optimization under dynamic simulation

All MD simulations were conducted using Amber1372 with the amber14SB force field for proteins. The simulation system was solvated in a TIP3P water model within a cubic water box, and neutralized by the addition of sodium counterions. Energy minimization was performed using the steepest descent algorithm for 50,000 steps. The system was equilibrated through 30 ps of NVT followed by 30 ps of NPT simulations at 300 K. A 100-ns production run was subsequently carried out. Temperature coupling was maintained via velocity rescaling73, and pressure control was achieved using the Berendsen barostat74. The SHAKE algorithm was employed to constrain all covalent bonds involving hydrogen atoms, and long-range electrostatic interactions were treated using the particle mesh Ewald method75. Short-range van der Waals interactions were truncated at 12 Å. All equations of motion were integrated using the leapfrog algorithm. Ligand structures of acetyl-CoA, butyryl-CoA, and geraniol were retrieved from PubChem76, and 3D conformers were generated using RDKit's Chem.AllChem.EmbedMolecule function77 with the ETKDG algorithm78. The generated conformations were optimized with the MMFF94 force field79, and the lowest-energy conformation was selected as the input for docking.

MD simulation of docking-derived enzyme-ligand complex

Initial coordinates for MD simulations were generated from molecular docking to provide reasonable starting configurations of enzyme-ligand complexes. To avoid bias from any single docking pose, all simulations were subsequently allowed to freely relax and sample conformational space without geometric restraints, and each MD simulation was carried out for 100 ns. The General AMBER Force Field (GAFF2)80 was used to parameterize ligand molecules, and all other simulation settings were identical to those described above. MD trajectories were analyzed to characterize the dynamic behavior of active-site regions and substrate conformations. Root mean square deviation (RMSD) analysis was used solely to assess overall trajectory stability rather than as a proxy for catalytic competence. Where applicable, binding free energy estimates were obtained using the Gmx_MMPBSA tool81. Representative trajectories and raw simulation data are available via Zenodo67.

Quantum mechanical calculations

Quantum mechanical (QM) calculations were performed using Gaussian 1682. Near-attack conformations were extracted from pre-reaction state (PRS) MD ensembles and converted into Gaussian input files83. The QM cluster models consisted of truncated CoA thioester and geraniol substrates together with key active-site residues, resulting in systems of approximately 80 atoms. Each residue was truncated at the Cα atom and capped with hydrogen atoms to satisfy valence requirements. Reaction pathways were investigated using the B3LYP-D3(BJ)/6-31+G(d,p) level of theory with implicit solvation modeled by SMD (methanol). Optimized geometries and corresponding XYZ coordinates are provided, and all raw QM data are available via Zenodo67.

Modular construction of MT biosynthetic pathway in E. coli.

A two-plasmid system comprising upstream and downstream modules was constructed to enable MT overproduction in E. coli. The upstream plasmid pMVA1 was built on a pAC-derived backbone with spectinomycin resistance and harbors seven genes involved in the entire mevalonate pathway. The genes of EcatoB (E. coli acetoacetyl-CoA thiolase), Sahmgs, and Sahmgr (Staphylococcus aureus HMG-CoA synthase and reductase) were placed under the control of the pLac promoter (inducible by IPTG). Scmk (Saccharomyces cerevisiae BY4741 mevalonate kinase), Scpmk (S. cerevisiae phosphomevalonate kinase), Scpmd (S. cerevisiae phosphomevalonate decarboxylase), and EcIDI (E. coli isopentenyl diphosphate isomerase) were driven by the pTrc promoter (inducible by IPTG), enabling tunable flux from acetyl-CoA to IPP/DMAPP through IPTG induction with varied concentration. For the downstream biosynthetic pathway, the plasmid of pLina1+EUP, pGer1+EUP, pLav5+EUP, pNer1+EUP, pBor3+EUP, or pTerp1+EUP was constructed based on a pMB1-based backbone carrying an ampicillin resistance marker, respectively. This plasmid encodes one of three prenyl diphosphate synthases—trAgGPPS (truncated Abies grandis geranyl pyrophosphate synthase), trLiLPPS (truncated Lavandula x intermedia lavandulyl pyrophosphate synthase), or trSlNPPS (truncated Solanum lycopersicum neryl pyrophosphate synthase) under the control of a pT7 promoter (inducible by IPTG), in combination with various MTSs (Supplementary Data 5) under control of the pLux promoter that was induced by 3-oxohexanoyl-homoserine lactone (3OC6-HSL). Additionally, DpADA (Dickeya parazeae Ech586 acetaldehyde dehydrogenase) and ScADSH2 (S. cerevisiae alcohol dehydrogenase) in EUP were also included to boost the supply of acetyl-CoA using ethanol as substrate. The EUP module was driven by a constitutive promoter GryA44. Co-transformation of pMVA1 and pLina1+EUP, pGer1+EUP, pLav5+EUP_mut2, pNer1+EUP, pBor3+EUP, or pTerp1+EUP in to E. coli MG1655(DE3) ∆endA ∆recA or other derivative strains enabled the engineered strains to overproduce MTs using glycerol and/or ethanol as carbon source. The detailed plasmid and strain information is provided in Supplementary Data 7-9.

A single colony of the MT-producing strain was inoculated into 2 mL LB medium supplemented with 50 μg/mL spectinomycin and 50 μg/mL ampicillin. The culture was incubated overnight at 37 °C with shaking at 250 rpm. Subsequently, 2% (v/v) of the seed cultures were transferred into 2 mL M2 medium containing the same antibiotics. The culture was incubated at 37 °C with shaking at 250 rpm until the OD600 reached ~1.0 or ~2.0, IPTG and 3OC6-HSL were added with appropriate concentrations, overlaying with 400 μL of hexadecane for in situ product extraction. Culture fermentation was carried out at 30 °C with shaking at 250 rpm for 48 h. After the fermentation was completed, the mixture was centrifuged at 16,000 g for 5 min, and 10 μL of the obtained upper organic phase was mixed with 190 μL of ethyl acetate for GC and GC-MS analysis.

Monoculture and coculture for de novo biosynthesis of MTEs

To biosynthesize LA using a single-strain mode, LiAAT4 was employed for strain construction based on substrate pairing evaluation. The plasmid pLiAAT4K (pCDF backbone with kanamycin resistance) was constructed under the control of the pCym promoter (inducible by cumic acid) to express LiAAT4, capable of converting linalool to LA. Transformation of pLiAAT4K into the strain Lina1 yielded strain LinaA1 for LA de novo biosynthesis from glycerol and ethanol. Further optimization of IPTG, 3OC6-HSL, and cumic acid concentration was conducted to balance the pathway modules. A single colony of LinaA1 from plate was inoculated into 2 mL LB medium supplemented with 50 μg/mL spectinomycin and 50 μg/mL ampicillin. The culture was incubated overnight at 37 °C with shaking at 250 rpm. Subsequently, 2% (v/v) of the seed cultures were transferred into 2 mL M2 medium containing the same antibiotics. The culture was incubated at 37 °C with shaking at 250 rpm until the OD600 reached ~2.0, IPTG, 3OC6-HSL, and cumic acid were added with appropriate concentrations, overlaying with 400 μL of hexadecane for in situ product extraction. Culture fermentation was carried out at 30 °C with shaking at 250 rpm for 48 h. After the fermentation, the mixture was centrifuged at 16,000 g for 5 min, and 20 μL of the obtained upper organic phase was mixed with 80 μL of ethyl acetate for GC and GC-MS analysis.

To biosynthesize LA using a coculture mode, co-transformation of pLiAAT4 and pEmpty, pCaBACT+LiAAT4 and pEmpty1, or pCaBAPCT+LiAAT4 and pEup1 into E. coli MG1655(DE3) ΔendA ΔrecA ΔtnaA Δpta were conducted to obtain the strains AATE1, AATE2, and AATE3, capable of converting linalool to LA with and without CaBACT and EUP, allowing us to investigate whether maintaining CaBACT and enhancing the supply of acetyl-CoA would affect the production titer of LA. Further coculturing of linalool-producing strain Lina3 and ester-producing strain AATE3 was conducted with varying the inoculation ratio (20:1 to 1:10). For the coculture fermentation setup, single colonies of each strain were inoculated into 2 mL LB medium supplemented with 50 μg/mL spectinomycin and 50 μg/mL ampicillin. The culture was incubated overnight at 37 °C with shaking at 250 rpm. Subsequently, 2% (v/v) of the primary seed cultures were transferred into 30 mL of fresh M2 medium containing the same antibiotics in 50 mL shake flasks to prepare secondary seed cultures. Once both secondary cultures reached an OD600 of ~2.0, equal volumes (1 mL + 1 mL; inoculation ratio set as 1:1) of each culture were mixed to initiate coculture fermentation. To vary the inoculation ratio, one of the secondary seed cultures was diluted with fresh M2 medium containing the same antibiotics, after which equal volumes (1 mL + 1 mL) were combined to maintain a constant total inoculum volume. Induction was performed by adding appropriate concentrations of IPTG and 3OC6-HSL, followed by adding 10 g/L ethanol depending on the strain used and overlaying with 400 μL of hexadecane for in situ product extraction. Coculture fermentation was carried out at 30 °C and 250 rpm for 48 h. After the fermentation, the obtained upper organic phase was used for GC and GC-MS analysis.

To biosynthesize GB using a single-strain mode, FaAAT was employed for strain construction based on substrate pairing evaluation. A plasmid pFaAATK (pCDF backbone with kanamycin resistance) was constructed to coexpress FaAAT along with a butyryl-CoA biosynthetic module comprising EcatoB, Cahbd, Cacrt, and Tdter. The genes EcatoB and Cahbd were placed under the control of the pLac promoter, while Cacrt and Tdter were placed under the control of the pTrc promoter, and FaAAT was placed under the control of the pCym promoter. Transformation of pFaAATK into the strain Ger1 yielded strain GerB1 for GB de novo biosynthesis from glycerol using the single-strain mode. However, a preliminary optimization of inducer concentration did not bring a detectable titer of GB (Data not shown). To biosynthesize GB using a coculture mode, the ester-producing strain with a two-plasmid system was constructed, which contained (1) plasmid pButy1 (pAC backbone with spectinomycin resistance) carrying the butyryl-CoA biosynthetic module under the control of the T7 and trc promoter and (2) plasmid pFaAAT or pCaBACT+FaAAT (pMB1 backbone with ampicillin resistance) carrying FaAAT, with or without CaBACT under the control of the pLac promoter. Co-transformation of these two plasmids into E. coli MG1655(DE3) ΔendA ΔrecA Δpta ΔldhA ΔadhE yielded strain But1 and But2 capable of converting geraniol into GB. Subsequently, the FaAAT in pCaBACT+FaAAT was replaced with a mutant, yielding pCaBACT+F303GFaAAT and pCaBACT+I303GFaAAT2, with pButy1 co-transformed producing strains But3 and But4. Coculturing of the geraniol-producing strain Ger3 and the ester-producing strain But4 was conducted with varying the inoculation ratio (20:1 to 1:10) upon induction. After the coculture fermentation setup, the induction was performed by adding appropriate concentrations of IPTG and 3OC6-HSL, followed by overlaying with 400 μL of dodecane/hexadecane. The fermentation was conducted at 30 °C and 250 rpm for 48 h. The sample of the upper organic phase was collected for GC and GC-MS analysis.

To biosynthesize lavandulyl acetate, neryl acetate, bornyl acetate, lavandulyl butyrate, and neryl butyrate, coculture fermentation was performed using optimized MT-producing strains (Lav15, Ner3, Bor5) and inducer concentrations. Based on preliminary screening, LiAAT4 (for acetate esters) and I303GFaAAT2 (for butyrate esters) were expressed in AAT-expressing strains AATE3 and But4, respectively. Single colonies of each strain were inoculated into 2 mL LB medium supplemented with 50 μg/mL spectinomycin and 50 μg/mL ampicillin, and the culture was cultured overnight at 37 °C with shaking at 250 rpm. Then, 2% (v/v) of the seed cultures were transferred into 30 mL M2 medium with proper antibiotics in 50 mL shake flasks for secondary growth. Once the OD600 reached ~2.0, coculture was initiated by mixing the secondary cultures at an inoculation ratio of either 15:1 for lavandulyl acetate, neryl acetate, bornyl acetate biosynthesis or 1:1 for lavandulyl butyrate, neryl butyrate biosynthesis. Induction was performed by adding IPTG and 3OC6-HSL at the optimized concentrations, followed by adding 10 g/L ethanol with the strain containing the plasmid with EUP, and 400 μL hexadecane overlay for in situ product extraction. Fermentation was carried out at 30 °C and 250 rpm for 48 h. After fermentation, the hexadecane phase was collected for GC-MS analysis.

Scale-up of MTE production in a bioreactor

Fed-batch fermentations for LA and GB production were conducted in a 1-L bioreactor (BLBIO-1L, BaiLun Biotechnology Co., Ltd, Shanghai, China) containing 400 mL of M2 medium supplemented with 50 μg/mL spectinomycin and 50 μg/mL ampicillin. Single colonies of each strain used in the coculture system were inoculated into 2 mL LB medium supplemented with spectinomycin 50 μg/mL and ampicillin 50 μg/mL, and cultured overnight at 37 °C with shaking at 250 rpm. Next, two seed cultures were transferred into two independent 50 mL flasks containing 30 mL of LB medium supplemented with spectinomycin 50 μg/mL and ampicillin 50 μg/mL, and the culture was cultured for 5-7 h at 37 °C in a shaking incubator at 250 rpm. Subsequently, two seed cultures with OD600 reaching ~2 from two independent shake flasks were inoculated into the fermenter at the defined ratio (Lina3:AATE3=15:1, or Ger3:But4=1:1, v/v), and the culture temperature was set to 37 °C. The pH of fermentation broth in the bioreactor was maintained at 7.0 using 30% (v/v) ammonia solution and 30% (v/v) phosphoric acid. Air was continuously supplied at a rate of 0.4 L/min. The dissolved oxygen level was maintained at 20% by automatically adjusting the agitation speed to 300-800 rpm. Induction was initiated when the OD600 reached ~2.0 by adding 0.05 mM IPTG and 0.2 μM 3OC6-HSL for LA production, or 0.1 mM IPTG and 1 μM 3OC6-HSL for GB production. Approximately 220 mL of hexadecane for LA production or dodecane for GB production was added to capture the produced esters. After induction, the culture temperature was changed to 30 °C, and a feeding strategy was implemented every ~12 h to maintain glycerol concentration to be ~10 g/L using 600 g/L glycerol feeding medium during the entire fermentation process.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript reports the development of a dual-substrate microbial platform for the programmable biosynthesis of monoterpenoate esters (MTEs). The topic is timely and relevant to synthetic biology and enzyme engineering. Although the study contains a large amount of experimental data and presents potentially valuable ideas, the current version suffers from substantial issues in experimental logic, mechanistic interpretation, and data presentation. The central claim of establishing a “programmable and generalizable” MTE biosynthetic platform is not convincingly demonstrated, as key mechanistic and structural evidence remains incomplete. Therefore, in its current form, I do not consider the manuscript suitable for publication in Nature Communications.\n\nMajor Comments\n1. Overall, the manuscript lacks conceptual coherence—for instance, the discussion of substrate tunnel determinants is not logically connected to the subsequent single-site (Phe302) mutagenesis, and several mechanistic claims rely solely on qualitative molecular dynamics observations rather than quantitative or energetic analyses. Figures and supplementary data are poorly organized, with inconsistent labeling, missing statistical information, and unclear residue numbering. In addition, undefined abbreviations, enzyme names, and inconsistent figure references further undermine readability.\n2. Line 137–186: The section aims to explore the diversity of MTE biosynthesis. In Lines 144–160, the authors examined AAT-catalyzed reactions using various short-chain acyl-CoAs, whereas in Lines 162–186, only benzoic acid was used as the representative aromatic acid substrate. Please clarify the rationale for choosing benzoic acid instead of a broader set of aromatic acids. Discuss whether the platform can be generalized for MTEs derived from other aromatic acids.\n3. In the first section, “Biosynthesis of monoterpenoate ester,” the catalytic capacity of AATs is demonstrated. However, the following discussion (Lines 188–213) shifts to AAT tunnel architecture and substrate specificity, whereas the subsequent section focuses again on product biosynthesis. This abrupt structural transition disrupts the logical flow. Please reassess the organization to ensure that sections follow a consistent conceptual progression.\n4. In lines 200–213, The authors emphasize tunnel determinants (length, bottleneck radius, steric-gate residues) as key to substrate selectivity but subsequently target only Phe302 near the active site. Please clarify why tunnel-level mutations were not prioritized. Specifically: (1) Clarify why tunnel engineering was not prioritized; (2) If tunnel-level modification was not attempted, discuss the advantages and limitations of tunnel versus single-residue engineering, and how these approaches could be integrated; (3) Propose one or two feasible tunnel engineering schemes (e.g., multi-site substitutions near the bottleneck, tunnel expansion, or curvature modification) to demonstrate conceptual completeness and forward-looking design.\n5. The current explanation for altered substrate selectivity relies mainly on geometric parameters (e.g., D2 distance from MD simulations). Interatomic distances alone cannot justify energetic or catalytic effects. To strengthen the mechanistic argument, quantitative analyses are recommended: (1) Binding free energy calculations (MMGBSA or MMPBSA): Compare the binding affinities (ΔG_bind) of acetyl-CoA and butyryl-CoA before and after mutation, and decompose the energetic components (van der Waals, electrostatics, solvation, and nonpolar terms); (2) Free energy profiling (umbrella sampling, WHAM, or metadynamics) to map the binding and reaction energy landscapes and assess whether the mutation raises the acetyl-CoA barrier.\n6. The discussion on Phe302 mutations (e.g., F302G) attributes substrate selectivity changes to geometric alterations near the catalytic pocket. Docking and MD simulations, though informative, are insufficient to describe transition-state stabilization or reaction barriers. QM/MM calculations would provide quantitative insight into how these mutations reshape the catalytic energy surface and selectivity, and are strongly encouraged.\n7. Line 380: The statement “For GPP-derived MTs, the titers obtained were varied when MTPS was changed” lacks definition and mechanistic explanation. Please define MTPS, describe how its variation affects MTE titers, and provide the corresponding quantitative data, as this relationship is not evident in Supplementary Fig. 46b–e.\n8. Lines 364–386: This section describes de novo biosynthesis of six distinct MTs involving four modules: the mevalonate (MVA) module, downstream MVA module, PDPS module, and MTS module. However, these modules are not clearly delineated in the metabolic schematic (Supplementary Fig. 46a). Please redraw or reorganize the figure into clearly labeled modules to enhance interpretability.\n\nMinor Comments\n1.In the first section “Biosynthesis of monoterpenoate ester,” Fig. 2d, which demonstrates AAT catalytic capability, is difficult for readers to interpret. Please consider presenting this data using a clearer schematic or a tabular format to improve readability.\n2.Line 199: The text refers to residue Phe299, but Supplementary Fig. 26d shows only Phe309. Please verify and correct the numbering.\n3.Line 276: Residues Gly162 and Ser378 mentioned in the text do not correspond to those in Supplementary Fig. 32a (which shows G160 and S377). Please ensure residue numbering consistency throughout the manuscript.\n4.Line 280: The term “NDT mutagenesis” appears for the first time without being spelled out. Please provide the full name upon first mention.\n5.Line 284: The abbreviations GA titer and GB titer appear without full definitions. Please define them at first occurrence.\n6.Line 310: The SDS-PAGE image in Supplementary Fig. 36a does not clearly show the claimed “poor soluble expression of both wtFaAAT and F302GFaAAT.” Please provide a higher-resolution image or a clearer version of the gel.\n7.In both the main text and Supplementary Information, the x-axis labels of Supplementary Figs. 32b–e are unclear. Please clarify the axis descriptions or revise the figures.\n8.When describing the effect of the F302G mutation, several statements are overly qualitative (e.g., “significantly reduced acetyl-CoA catalytic efficiency”). Quantitative data such as the exact fold-change or kinetic parameters (kcat, Km, kcat/Km) should be provided.\n9.Figures related to enzyme engineering (e.g., Fig. 4 and Supplementary Figs. 32–35) should explicitly label the mutated residues, substrate orientations, and key molecular interactions to enhance interpretability.\n10.In the Discussion section, the authors are encouraged to summarize general design principles derived from AAT engineering and discuss the broader applicability of this strategy to other ester-forming enzymes.\n11.Please verify the consistency among figure legends and supplementary data numbering, as cross-references in Supplementary Figs. 32–35 appear slightly misordered.\n12.The protein–ligand interaction diagrams in Supplementary Figs. 26/27/32 have overly complex backgrounds and inconsistent color schemes, making them difficult to interpret. Additionally, some labels are inconsistent with the textual description. Please simplify and standardize these figures.\n13.The layout of Supplementary Figs. 5n, 7g, 17e, and 23b is inconsistent with other figures. Please unify the figure formatting.\n14.Lines 384–386: The text states, “In lavandulol biosynthesis, a variant pyrophosphatase (F118D-H182C EcRdgB) engineered through structure-guided mutagenesis enhanced the titer of lavandulol by 7.64-fold.” However, it does not clarify that this engineered enzyme is an original design in this study, nor does it explain why this enzyme was targeted for modification. Please provide this rationale.\n15.Lines 409–416: During de novo GB synthesis, the authors first used wtFaAAT instead of the previously optimized I302GFaAAT2 variant, which had demonstrated superior selectivity. Please justify this choice and assess whether the use of wtFaAAT is logically consistent.\n16.In Fig. 5a/c/d/f, several schematic elements are unclear and cannot be cross-referenced with data in panels b/e. For example, CaBACT is not shown in the figure. Please label all relevant enzymes and components clearly to avoid reader confusion.\n17.The reference list contains numerous and significant formatting errors that must be corrected. Specifically, journal names are inconsistently presented (some appear as full names while others are abbreviated; e.g. refs. 43, 44, 45, 49), several references are missing volume and page numbers (e.g. refs. 1, 7, 50–52), and title capitalization is inconsistent (some titles use sentence case whereas others capitalize every word; e.g. ref. 8). The entire list of references needs a careful and comprehensive revision to ensure consistency with the journal’s formatting requirements.", "reviewer_2": "Review of the manuscript titled “A dual-substrate microbial platform for selective biosynthesis of monoterpene esters”\nYang et al. have developed a dual-substrate-wheeled microbial system. Using this system, they successfully produced three types of monoterpene esters: monoterpenyl esters, monoterpenoate esters, and monoterpenyl monoterpenoate esters, which include several new-to-nature molecules. Enzyme engineering was applied to shift the preference of alcohol acyltransferases (AAT) towards various acyl-CoA or alcohol substrates. Finally, the co-culture strategy enabled the production of linalyl acetate to reach 11.5 g/L, which is an impressive titre. Overall, this is a solid study with originality, comprehensive data and clear demonstration of biosynthetic versatility achieved by using enzymes with broader acceptability of various substrates, such as AATs. I recommend that the manuscript be accepted after the following revisions.\n\nMajor Comments:\n1. Figure 3, the authentic standards should be plotted together with reactions to show the identity between biosynthesized monoterpene esters and standards. The retention times of 5’a’, 5’b’ and 5’c’ in Figure 3d differ from those of chemically synthesized compounds in Supplementary Fig 28. In addition, the TIC chromatograms of newly produced monoterpene esters should be provided in addition to EIC chromatograms, which can assess matrix complexity and method robustness.\n2. The authors produced many novel monoterpene esters, however, the authors selected linalyl acetate and geranyl butyrate to fed-batch fermentation. Why were these two molecules chosen instead of others? What is their commercial value?\n3. The manuscript reports that the F302G mutation enhances selectivity toward butyryl-CoA (GB), resulting in a 98.95% selectivity at a 4:1 butyrate-to-geraniol ratio. However, in Figure 4 and supplementary figures, the data range markers are extremely small, making it difficult to visually distinguish the different mutants by color. More importantly, the improvement attributed to the mutation appears modest compared to the effect of adjusting the substrate molar ratio. For instance, the wild-type FaAAT also reaches >95% selectivity under the 4:1 ratio, which is comparable to that of the mutant. As currently presented, the figure and discussion may overstate the impact of mutagenesis relative to substrate ratio optimization. The authors are encouraged to clarify and separate the respective contributions of enzyme mutation and substrate ratio adjustment in the text.\n4. The manuscript concludes that the F302G mutation reduces steric hindrance and the contact distance between geraniol and acyl-CoAs based solely on molecular docking results (Fig. 4d–e). However, docking typically generates multiple possible binding poses and represents only static predictions. Therefore, it is difficult to draw definitive conclusions about steric effects or substrate interactions from docking alone. Moreover, the subsequent MD simulations reveal that this mutation actually destabilizes the acetyl-CoA complex. These findings suggest that the docking-derived conformations may not accurately represent the catalytically relevant states. The authors should revise the manuscript accordingly to reflect the MD simulation results.\n5. Supplementary Fig. 39 shows that after the I302G mutation of FaAAT2, the catalytic distances increase when binding butyryl-CoA, which may imply less stable binding. However, this seems inconsistent with the results in Fig. 4 j, k, where the mutant shows enhanced catalytic activity and selectivity for butyryl-based esters. It is recommended to further discuss and clarify the relationship between the change in catalytic distances and the catalytic performance in the revised manuscript.\n6. “Reconstructing the precise biosynthesis of structurally complex natural esters, such as monoterpene esters, in engineered microbes remains a major challenge, owing to the limited repertoire of highly selective alcohol acyltransferases and the lack of compatible pathway modularity.” This statement is partially true, as there are several excellent examples of the biosynthesis of monoterpene esters, such as geranyl acetate, geranyl butyrate.\n7. “Coculture engineering further redistributed metabolic fluxes between acyl-CoA and alcohol precursors, achieving >10 g/L production of monoterpene ester.” This sentence is not wrong, but gives an impression that it seems all the monoterpene esters have reached such a high titre in this study. The fact is only one linalyl acetate has reached this titre, and >90% of the monoterpene esters were produced only in detectable amounts and without any quantitative data. I suggest to rewrite the sentence to achieving >10 g/L production of “linalyl acetate” specifically, not “monoterpene esters” in general.\n\nMinor Comments:\n1. A few minor issues remain, and the authors are advised to carefully check the manuscript for similar errors.\n2. In the Results section (line 405), the phrase “the third the plasmid” appears to be a typographical error. It should likely be “the third plasmid”. Please revise this for grammatical accuracy and clarity.\n3. In line 502, the phrase “a type pf dimeric MEs” contains a typographical error. The word “pf” should be corrected to “of”. Please revise this for accuracy.\n4. The “100 nm” in the legend of Fig. 4m-n (line 592) appears incorrect, as catalytic distances in enzyme active sites are typically on the Å scale; this likely refers to the 100 ns MD simulation duration mentioned subsequently.", "reviewer_3": "Yang et al describe a \"dual-substrate-wheeled microbial platform\" to engineer alcohol acyltransferase to synthesize monoterpenyl, monoterpenoate and monoterpenyl monoterpenoate esters, including some apparently new-to-nature compounds. I was asked to specifically comment on the enzymology and protein engineering. More generally, the study looks interesting and these may be important results as the authors correctly note that they make a new class of sesquiterpenes and other intersting esters.\nUnfortunately, I found much of the paper very difficult to follow, as the writing style was lacking in what I consider to be a lot of important detail. Phrasing is also unusual. for example, \"dual-substrate-wheeled\" is not a term I am familiar with, although with the help of Figure 1, I think I understand what the authors mean. Other terms such as \"dual-substrate molar ratio tuning\" can be guessed at, but in my opinion the paper needs to be rewritten to improve clarity and to add missing detail.\n\nSpecific comments:\n\"The kinetic data support a model in which AAT and its mutants operate via a random sequential bi–bi mechanism within the cellular environment, allowing both acetyl-CoA and butyryl-CoA to independently access the active site in the presence of geraniol (Supplementary Note 1).\"\nThe proposed mechanism is shown in Fig 4. Presumably acetyl-CoA or butyryl-CoA can bind to the enzyme-alcohol complex, not at the same time (\"independently\" is confusing here). The kinetics then should establish whether the binding order is random (e.g. alcohol always binds first) or random, and Michaelis-Menten kinetics analysis will give Km values for both substates, and potentially a Kd for binding of the first substrate. It's not clear which Km values are reported in the paper and what the fixed substrate concentration (which I think was geraniol) was. These should be given in the caption and Km's should be measured with multiple fixed substrate concentrations to ensure they are not convoluted.\nThe molecular modelling appears to be well done, but in SI Fig 26 the residue/ligand coloring doesn't appear to match the caption. Does this provide insight into substrate binding order?\n\"both wt FaAAT and F302G FaAAT exhibited high exponential curve fitting confidence for GA and GB formation (R 2 >0.99, Fig. 4f,300 Supplementary Table 7).\"\nThis could be phrased better and the equation used for fitting (in the methods) numbered and referred to here. Why is the dependence of the titre exponentially dependent on the ratio of substrates? Can this be rationalised based on the kinetics?\np.15 \"...representing 61.2% of the theoretical chemical structure space of esters.\" The authors have not made 61% of all possible esters.", "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": "Response to Reviewers’ Comments\nWe sincerely appreciate the reviewers’ valuable comments and suggestions. Please\nfind the point-to-point response to reviewers’ comments below. Residue numbering\nhas been unified throughout the manuscript and responses; residue labels in the\nresponses may therefore differ from those used in the original reviewer comments.\n[Responses are highlighted in blue]\nReviewer #1\nComments:\nThis manuscript reports the development of a dual-substrate microbial platform for the\nprogrammable biosynthesis of monoterpenoate esters (MTEs). The topic is timely and\nrelevant to synthetic biology and enzyme engineering. Although the study contains a\nlarge amount of experimental data and presents potentially valuable ideas, the current\nversion suffers from substantial issues in experimental logic, mechanistic\ninterpretation, and data presentation. The central claim of establishing a\n“programmable and generalizable” MTE biosynthetic platform is not convincingly\ndemonstrated, as key mechanistic and structural evidence remains incomplete.\nTherefore, in its current form, I do not consider the manuscript suitable for publication\nin Nature Communications.\nResponse:\nWe sincerely appreciate Reviewer #1 for the constructive comments, which have\nsignificantly improved the manuscript’s clarity, organization, and scientific rigor. In\nresponse, we have substantially revised the manuscript to address concerns regarding\nconceptual coherence, mechanistic support, and data presentation to more clearly\narticulate the scope and generalizability of the proposed ME biosynthetic platform.\nMajor Comments\n1. Overall, the manuscript lacks conceptual coherence—for instance, the discussion\nof substrate tunnel determinants is not logically connected to the subsequent single-\nsite (Phe302) mutagenesis, and several mechanistic claims rely solely on qualitative\nmolecular dynamics observations rather than quantitative or energetic analyses.\nFigures and supplementary data are poorly organized, with inconsistent labelling,\nmissing statistical information, and unclear residue numbering. In addition, undefined\n1\nabbreviations, enzyme names, and inconsistent figure references further undermine\nreadability.\nResponse:\nAs suggested, we have revised the manuscript to improve conceptual consistency and\nmechanistic clarity. Specifically, we have (1) reorganized the Results section to\nestablish a clear progression from AAT catalytic capacity, docking analysis with tunnel\narchitecture, rational mutagenesis, pathway-level MTE biosynthesis (Line 136-476);\n(2) strengthened the mechanistic interpretation by incorporating quantitative\nquantitative MD and QM analyses rather than relying solely on qualitative observations\n(Line 241-391); (3) corrected all residue numbering based on the final curated\nstructural model and unified numbering across the revised manuscript and\nSupplementary Material; and (4) reorganized Supplementary Figures and improved\nlabeling, statistical information, and definition of abbreviations and enzyme names.\n2. Line 137–186: The section aims to explore the diversity of MTE biosynthesis. In\nLines 144–160, the authors examined AAT-catalyzed reactions using various short-\nchain acyl-CoAs, whereas in Lines 162–186, only benzoic acid was used as the\nrepresentative aromatic acid substrate. Please clarify the rationale for choosing\nbenzoic acid instead of a broader set of aromatic acids. Discuss whether the platform\ncan be generalized for MTEs derived from other aromatic acids.\nResponse:\nIn our initial experimental design, we selected benzoic acid as the representative\naromatic acyl donor. Benzoate-derived monoterpenoate esters constitute an important\nclass of aroma compounds, widely used in the flavor, fragrance, and cosmetic\nindustries, where they contribute sweet, floral, and fruity notes. Besides, benzoic acid\nrepresents a structurally minimal yet rigid aromatic acyl scaffold, making it an ideal\nprobe to evaluate whether AAT variants can accommodate planar aromatic acyl\ndonors that differ substantially from short-chain aliphatic acyl-CoAs. As suggested, we\nhave revised the Results section to clearly present the outcomes for all three aromatic\nacids (including phenylacetyl-CoA- and phenylpropionyl-CoA-derived MTEs). These\ndata collectively demonstrate that multiple AAT variants can accept structurally diverse\naromatic acyl donors, supporting the applicability of our dual-substrate platform for\ngenerating aromatic-derived monoterpenoate esters. A corresponding discussion\n2\nemphasizing the generalizability of the platform toward broader aromatic MTEs has\nnow been added to the revised manuscript (Lines 177-186).\n3. In the first section, “Biosynthesis of monoterpenoate ester,” the catalytic capacity of\nAATs is demonstrated. However, the following discussion (Lines 188–213) shifts to\nAAT tunnel architecture and substrate specificity, whereas the subsequent section\nfocuses again on product biosynthesis. This abrupt structural transition disrupts the\nlogical flow. Please reassess the organization to ensure that sections follow a\nconsistent conceptual progression.\nResponse:\nWe thank the reviewer for this comment and agree that the original manuscript did not\nsufficiently clarify the logical connection between AAT catalytic characterization,\nstructural analysis, and subsequent enzyme engineering. In the revised manuscript,\nwe have reorganized this part to follow a clearer conceptual progression. We first\ndescribe the biosynthetic scope and catalytic diversity of AATs in monoterpenoate\nester (MTE), monoterpenoate ester (MTaE), and monoterpenyl monoterpenoate ester\n(MtMtaE) formation. We then introduce the structural analysis as a hypothesis-\ngenerating step, aimed at rationalizing these functional differences. Comparative\ndocking and tunnel analyses show that while global tunnel architecture explains\ndifferences in substrate accessibility across AAT families, it does not sufficiently\naccount for the fine discrimination between acetyl- and butyryl-CoA observed for\nFaAAT. This observation naturally motivates a more focused investigation into\nlocalized structural determinants governing acyl-CoA selectivity, which is further\nexplored in the subsequent section. These revisions ensure a coherent narrative from\nbiosynthetic capability, through structure-informed reasoning, to targeted enzyme\nengineering. The corresponding changes have been incorporated into the revised\nmanuscript (Lines 136-391).\n4. In lines 200–213, The authors emphasize tunnel determinants (length, bottleneck\nradius, steric-gate residues) as key to substrate selectivity but subsequently target\nonly Phe302 near the active site. Please clarify why tunnel-level mutations were not\nprioritized. Specifically: (1) Clarify why tunnel engineering was not prioritized; (2) If\ntunnel-level modification was not attempted, discuss the advantages and limitations of\ntunnel versus single-residue engineering, and how these approaches could be\n3\nintegrated; (3) Propose one or two feasible tunnel engineering schemes (e.g., multi-\nsite substitutions near the bottleneck, tunnel expansion, or curvature modification) to\ndemonstrate conceptual completeness and forward-looking design.\nResponse:\nWe thank the reviewer for raising the point. (1) The identification of the residue for\nsingle-site mutagenesis engineering of FaAAT indeed began with AAT-ligand docking\nand tunnel-geometry analysis, in which tunnel length and bottleneck radius across\nrepresentative AATs were analysed. This analysis identified a critical residue of F303\nin FaAAT at the junction of the dual-substrate channels (Supplementary Fig. 32),\nwhich was predicted to exert a particularly strong influence on dual-substrate\ndiscrimination, prompting the further experimental validation.\n(2) To experimentally validate the key residues influencing acyl-CoA selectivity, we\nfirst performed single-site saturation mutagenesis on five structurally relevant\npositions identified from the acyl-CoA with FaAAT docking analysis—F303, G162,\nH250, S378, and W380 (Fig. R1, Supplementary Fig. 34). The F303 mutations\ndisplayed the most substantial shifts in acyl-CoA specificity, whereas the mutations at\nG162, H250, S378, and W380 in FaAAT resulted in minimal, no improvements, or\neven abolished product formation (Fig. R1). We then generated the variants of\nF303GFaAA with the residues of G162, H250, S378, and W380 to test whether double-\nsite substitution in tunnel reshaping could enhance selectivity, but these double-site\nmutations did not lead to additional improvement in specificity or product titer\n(Supplementary Fig. 35), likely due to the disruption of the desired interaction of acyl-\nCoA. These results again support the conclusion that a localized single residue\nengineering could be used efficiently to discriminate between C2/C4 acyl-CoA in\nFaAAT.\nenhance geranyl butyrate biosynthesis. a, Molecular docking model of wtFaAAT\nin complex with butyryl-CoA and geraniol, highlighting residues surrounding the\nactive site that are potentially involved in substrate accommodation and catalysis\n(H157, H250, G162, S378, W380, and F303). b-f, Single-site saturation\nmutagenesis of five active-site-proximal residues (F303, G162, H250, S378, and\nW380) using NDT-based libraries and their effects on geranyl butyrate (GB) and\ngeranyl acetate (GA) production. g-j, Second-round mutational scanning of four\nresidues (G162, H250, S378, and W380) based on the F303GFaAAT using NDT-\nbased mutagenesis libraries, revealing combinatorial effects on geranyl acetate and\nbutyrate formation.\n(3) As supported above, these double-site mutations were attempted to modify the\nacyl-CoA accommodation space; however, the resulting double-site mutations did not\nimprove product titer and selectivity, and even in several cases, compromised catalytic\nperformance. This outcome indicates that simple tunnel expansion is not effective for\nshort-chain acyl-CoA discrimination, likely because excessive widening disrupts\nproductive acyl-CoA positioning/stabilization rather than facilitating catalysis. Based\non these findings, future tunnel engineering will focus on structure-guided fine-tuning\nrather than global expansion. In future work, we would develop in silico\ncomputationally guided multiple-site fine-tuning mutagenesis strategies in which the\nmolecule, can be mutated simultaneously to subtly modulate conformational features.\nCorresponding revisions have been made to the revised manuscript (Lines 278-307).\n5. The current explanation for altered substrate selectivity relies mainly on geometric\nparameters (e.g., D2 distance from MD simulations). Interatomic distances alone\ncannot justify energetic or catalytic effects. To strengthen the mechanistic argument,\nquantitative analyses are recommended: (1) Binding free energy calculations\n(MMGBSA or MMPBSA): Compare the binding affinities (ΔG_bind) of acetyl-CoA and\nbutyryl-CoA before and after mutation, and decompose the energetic components\n(van der Waals, electrostatics, solvation, and nonpolar terms); (2) Free energy profiling\n(umbrella sampling, WHAM, or metadynamics) to map the binding and reaction energy\nlandscapes and assess whether the mutation raises the acetyl-CoA barrier.\nResponse:\nWe thank the reviewer for raising the critical point for quantitative energetic analyses\nto strengthen the mechanistic model. We have performed comprehensive binding free\nenergy calculations using the MMGBSA approach to compare the affinities of acetyl-\nCoA and butyryl-CoA for both the wild-type of wtFaAAT and the mutant of F303GFaAAT.\nOur results reveal a pronounced divergent effect of the mutation. Both acetyl-CoA and\nbutyryl-CoA binding are weakened F303GFaAAT, with more disfavoured for acetyl-CoA\nby ~7.9 kcal/mol (a net selective advantage for butyryl-CoA). To elucidate the origin of\nthis selectivity shift, we decomposed the energetic components in Fig. R2. This\nanalysis identified the 284Asn of FaAAT as the pivotal residue responsible for the\ndifferential response. In F303GFaAAT, the interaction between the 284Asn of FaAAT and\nacetyl-CoA is severely compromised (energy penalty ~3.6 kcal/mol), whereas its\ninteraction with butyryl-CoA is markedly strengthened (energy gain ~3.6 kcal/mol).\nThis reversal accounts for ~90% of the net selective advantage. Importantly, the 378Ser\nof FaAAT further modulates selectivity: the F303G mutation in FaAAT enlarged the\nsubstrate cavity, amplifying pre-existing differences in electrostatic interaction\nbetween the acetyl-CoA and butyryl-CoA. Consequently, altered hydrogen-bonding\nnetworks with the 378Ser of FaAAT selectively disfavor acetyl-CoA binding.\nCorresponding revisions have been made in the revised manuscript (Lines 294-300).\nbutyryl-CoA binding to FaAAT. Scatter plot showing the calculated changes in\nbinding free energy (ΔΔG, kcal/mol) for acetyl-CoA (x-axis) and butyryl-CoA (y-axis)\nupon single-site mutations in FaAAT, relative to the wild-type enzyme, as estimated\nby the MMGBSA method. Each point represents one mutation, with F303G highlighted.\nDashed lines indicate ΔΔG=0. Quadrants classify mutations according to their relative\neffects on C2- and C4-acyl donor binding: both favored, both disfavored, C2\nfavored/C4 disfavored, or C2 disfavored/C4 favored.\nThe F303G mutation in FaAAT directly destabilizes binding for both acetyl-CoA and\nbutyryl-CoA (ΔΔG 1.8-1.9 kcal/mol). Its role in substrate selectivity is thus indirect - it\nmagnifies inherent energy differences between the two substrates rather than creating\nnew favorable interactions. This mechanistic interpretation replaces our initial reliance\non geometric parameters (e.g., D2 distances). Accordingly, distance-based analyses\nare no longer primarily used as the mechanism. Importantly, the selectivity effect of\ncavity-enlarging mutations is highly context-dependent, as supported by contrasting\nresults from the homologous FaAAT2.\nbutyryl-CoA binding to FaAAT2. Scatter plot showing the calculated changes in\nbinding free energy (ΔΔG, kcal/mol) for acetyl-CoA (x-axis) and butyryl-CoA (y-axis)\nupon single-site mutations in FaAAT2, relative to the wild-type enzyme, as estimated\nby the MMGBSA method. Each point represents one mutation, with I303G highlighted.\nDashed lines indicate ΔΔG=0. Quadrants classify mutations according to their relative\neffects on C2- and C4-acyl donor binding: both favored, both disfavored, C2\nfavored/C4 disfavored, or C2 disfavored/C4 favored.\nIn FaAAT2, an equivalent I303G mutation (homologous to F303GFaAAT) also weakened\nbinding for both substrates (0.9-1.3 kcal/mol penalty). However, decomposition\nrevealed opposing roles of key residues: (i) 284Asn (homologous to 284Asn)\nunexpectedly favored acetyl-CoA (ΔΔG = -2.9 kcal/mol) while disfavoring butyryl-CoA\n(ΔΔG = +1.0 kcal/mol); (ii) 288Arg exhibited strong electrostatic destabilization\nspecifically for acetyl-CoA (ΔΔG = +4.3 kcal/mol). Consequently, I303G in FaAAT2\nreduces butyryl-CoA selectivity, directly opposing the effect of F303G in FaAAT.\nThis stark divergence suggests that substrate selectivity in FaAAT2 is more strongly\ngoverned by global electrostatic and steric networks than by isolated residue changes.\nCorresponding revisions have been made in the revised manuscript (Lines 334-337).\nthermodynamic integration), we acknowledge their potential value. In this study, we\nfocused on MMGBSA analysis because (i) the F303G mutation of FaAAT lies outside\nthe core CoA-binding channel and does not alter substrate loading in MD trajectories;\n(ii) MMGBSA and residue-level energy decomposition already explain >90% of the\nobserved selectivity shift; and (iii) enhanced-sampling free-energy calculations will be\npursued in future work to further refine the energetic landscape of acyl-CoA\ndiscrimination.\n6. The discussion on Phe302 mutations (e.g., F302G) attributes substrate selectivity\nchanges to geometric alterations near the catalytic pocket. Docking and MD\nsimulations, though informative, are insufficient to describe transition-state\nstabilization or reaction barriers. QM/MM calculations would provide quantitative\ninsight into how these mutations reshape the catalytic energy surface and selectivity,\nand are strongly encouraged.\nResponse:\nWe sincerely appreciate the reviewer's insightful suggestion regarding transition-state\nstabilization analysis. To quantitatively address the catalytic mechanism, we have\nperformed ab initio QM calculations on a theozyme model1, 2 that encompasses the\ncore reaction center (157His in FaAAT, geraniol, and CoA thioester). This model\nconfirms the proposed mechanism: 157His facilitates geraniol deprotonation followed\nby nucleophilic attack on the thioester carbonyl, with a rate-determining barrier of 17.6\nkcal/mol (ΔHǂ). Notably, the subsequent proton transfer from the imidazolium to\nthiolate is essentially barrierless, and the overall reaction is slightly exergonic (ΔG = -\n3.1 kcal/mol). The computed energy profile as shown in Fig. R4 aligns well with the\nmechanistic scheme in Fig. 4I, suggesting the critical role of pre-organized active-site\narchitecture in transition-state stabilization.\nRegarding the recommendation for full-enzyme QM/MM calculations, we fully agree\nthat such approaches represent a powerful framework for quantitatively dissecting\nmutational effects on catalytic energy landscapes. In the present system, however,\nextensive MD simulations reveal pronounced conformational heterogeneity in\ncatalytically competent states of FaAAT, particularly in regions distal to the active site.\nsnapshots would be highly sensitive to conformational selection and may complicate\nstatistically robust interpretation associated with mutations such as F303G in FaAAT.\nWe therefore propose that the observed selectivity changes are at least partly reflected\nin thermodynamic shifts of ground-state substrate interactions, which shape the\npopulation of catalytically competent conformations consistent with our mechanistic\nmodel. While QM/MM studies could provide finer mechanistic detail, in highly flexible\nsystems with distal mutations, such calculations can be sensitive to conformational\nsampling, potentially complicating statistically robust interpretation.\nFigure R4. DFT-calculated reaction potential energy surface for the FaAAT\nmodel. The reaction profile for FaAAT-catalyzed geranyl ester formation was\ncalculated at the B3LYP(D3)/6-31+G*/SMD(methanol) level of theory. Relative\nenthalpy (ΔH) and Gibbs free energy (ΔG, in parentheses) values are shown in\nkcal/mol. The pathway proceeds from the pre-reaction complex, in which geraniol and\nacyl-CoA are co-positioned near the catalytic residue 157His, through the first transition\nstate (TS1) to form a tetrahedral intermediate (IM), followed by a second transition\nstate (TS2) leading to geranyl ester formation and HS-CoA release. Key interatomic\ndistances (Å) along the reaction coordinate are indicated to illustrate bond formation\nand cleavage events.\n7. Line 380: The statement “For GPP-derived MTs, the titers obtained were varied\nwhen MTPS was changed” lacks definition and mechanistic explanation. Please define\nMTPS, describe how its variation affects MTE titers, and provide the corresponding\nquantitative data, as this relationship is not evident in Supplementary Fig. 46b–e.\nIn the revised manuscript, we have corrected the definition of MTPS to MTS with a\nclear definition of monoterpene synthase (Line 396, 409). We now explicitly state that\nvarying MTPS alters both the catalytic efficiency toward GPP and the effective flux of\nMT We have added the absolute titers achieved by various strains and the fold\nchanges in the most variable titers obtained between two MTPs, along with the\ncorresponding statistical information (p-values). The changes have been listed in the\nrevised manuscript (Lines 413-414).\n8. Lines 364–386: This section describes de novo biosynthesis of six distinct MTs\ninvolving four modules: the mevalonate (MVA) module, downstream MVA module,\nPDPS module, and MTS module. However, these modules are not clearly delineated\nin the metabolic schematic (Supplementary Fig. 46a). Please redraw or reorganize the\nfigure into clearly labelled modules to enhance interpretability.\nResponse:\nAs suggested, we have redrawn the metabolic schematic to delineate the four modules\n(Fig. R5): Module 1-Upstream MVA pathway (coded by the genes of EcatoB, Sahmgs,\nSahmgr, and Scmk), Module 2-Downstream MVA pathway (coded by the genes of\nScpmk, Scmvd, and Ecidi), Module 3-PDPS (coded by the genes of trAgGPPS,\ntrSlNPPS, or trLiLPPS), and Module 4-MTS (coded by the genes of varied\nmonoterpene synthases or phosphatases). Each module is now color-coded, labelled,\nand separated by dashed boundaries to guide interpretation. We also added arrows\nand annotations to highlight how flux passes through each module and leads to the\nformation of varied monoterpenes. The changes have been listed in the revised\nSupplementary Fig. 55a.\nnovo biosynthetic pathway for monoterpene overproduction from glycerol in E.\ncoli. EcatoB: acetyl-CoA acetyltransferase from E. coli; Sahmgs:\nhydroxymethylglutaryl-CoA synthase from Staphylococcus aureus; Sahmgr:\nhydroxymethylglutaryl-CoA reductase from Staphylococcus aureus; Scmk:\nmevalonate kinase from Saccharomyces cerevisiae; Scpmk: phosphomevalonate\nkinase from Saccharomyces cerevisiae; Scpmd: diphosphomevalonate\ndecarboxylase from Saccharomyces cerevisiae; Ecidi: isopentenyl diphosphate Delta-\nisomerase from E. coli; DpADA: aldehyde dehydrogenase from Dickeya parazeae\nEch586; ScADH2: alcohol dehydrogenase ADH2 from Saccharomyces cerevisiae;\ntrAgGPPS: truncated geranyl pyrophosphate synthase from Abies grandis; trSlNPPS:\ntruncated neryl pyrophosphate synthase from Solanum lycopersicum; trLiLPPS:\nlavandulyl pyrophosphate synthase from Lavandula x intermedia; ApLinS: linalool\nsynthase from Agrocybe pediades; trObGES: truncated geraniol synthase from\nOcimum basilicum; DiBPPS: bornyl pyrophosphate synthase from Dipterocarpus\nintricatus; trVvTS: truncated alpha-terpineol synthase from Vitis vinifera; EcnudJ:\nphosphatase NudJ from E. coli; EcRdgB: dITP/XTP pyrophosphatase from E. coli.\nMinor Comments\n1. In the first section “Biosynthesis of monoterpenoate ester,” Fig. 2d, which\ndemonstrates AAT catalytic capability, is difficult for readers to interpret. Please\nconsider presenting this data using a clearer schematic or a tabular format to improve\nreadability.\nResponse:\nWe have revised Fig. 2d and used a tabular format with higher resolution to improve\nreadability in the revised manuscript, and also include a high-resolution figure in PDF\nformat in the resubmitted supplementary materials for your reference.\n12\n2. Line 199: The text refers to residue Phe299, but Supplementary Fig. 26d shows\nonly Phe309. Please verify and correct the numbering.\nResponse:\nThe residue numbering has been corrected; “Phe299” is now consistent with the\nstructural model and Supplementary Fig. 26d and revised to “310Phe”.\n3. Line 276: Residues Gly162 and Ser378 mentioned in the text do not correspond to\nthose in Supplementary Fig. 32a (which shows G160 and S377). Please ensure\nresidue numbering consistency throughout the manuscript.\nResponse:\nWe have unified all residue numbering across the revised manuscript and\nsupplementary figures in the revised manuscript.\n4. Line 280: The term “NDT mutagenesis” appears for the first time without being\nspelled out. Please provide the full name upon first mention.\nResponse:\nWe have spelled out the term at its first occurrence as “NDT codon-based saturation\nmutagenesis (N = A/C/G/T, D = A/G/T, T = thymine)” and added the detailed\nexplanation of the NDT codon design to the Methods section in the revised manuscript\n(Lines 273-276).\n5. Line 284: The abbreviations GA titer and GB titer appear without full definitions.\nPlease define them at first occurrence.\nResponse:\nWe have added the full names of GA and GB in the revised manuscript (Lines 108,\n278).\n6. Line 310: The SDS-PAGE image in Supplementary Fig. 36a does not clearly show\nthe claimed “poor soluble expression of both wtFaAAT and F302GFaAAT.” Please\nprovide a higher-resolution image or a clearer version of the gel.\nResponse:\nWe have provided a higher-resolution SDS-PAGE image with figure caption specifying\nwhich elution gradient fractions were used for the subsequent in vitro enzyme activity\n13\nassay in Supplementary Fig. 40a and the source data of SDS-PAGE image has also\nbeen included in Source data file.\n7. In both the main text and Supplementary Information, the x-axis labels of\nSupplementary Figs. 32b–e are unclear. Please clarify the axis descriptions or revise\nthe figures.\nResponse:\nWe have clarified the x-axis labels and revised Supplementary Figs. 34b–f in the\nrevised manuscript.\n8. When describing the effect of the F302G mutation, several statements are overly\nqualitative (e.g., “significantly reduced acetyl-CoA catalytic efficiency”). Quantitative\ndata such as the exact fold-change or kinetic parameters (k , K , k /K ) should be\ncat m cat m\nprovided.\nResponse:\nFigure R6. Steady-state kinetic parameters of wtFaAAT and F303GFaAAT using\nacetyl-CoA (C2) or butyryl-CoA (C4) as acyl donors at a fixed geraniol\nconcentration. a, K ; b, k /K . #p=0.66; ***p< 0.001; n.a.: not available.\nm cat m\nAs suggested, we have revised the manuscript to include quantitative data on the fold\nchange in kinetic parameters of FaAAT before and after mutation (Lines 330-334). For\nthe acetyl-CoA/geraniol reaction, no detectable product formation was observed for\nF303GFaAAT under the experimental conditions used. Consequently, reliable kinetic\nparameters (K , k /K ) could not be determined for acetyl-CoA, and this reaction is\nm cat m\ntherefore reported as not applicable (n.a., Fig. R6). This result indicates a near-\ncomplete loss of catalytic activity toward acetyl-CoA upon introduction of the F303G\nmutation in FaAAT under the tested conditions in vitro, which is consistent with the\nmarkedly reduced formation of GA observed in vivo (7.24-fold reduced titer without\nsupplementing butyrate, p < 0.0001, Fig. 4f). In contrast, for the butyryl-CoA/geraniol\nreaction, quantitative kinetic analysis was feasible. The K of F303GFaAAT to wild-type\nm\nshowed no significant change upon mutation (1.04-fold, p=0.66, Fig. R6), indicating\nthat substrate binding affinity for butyryl-CoA was largely preserved. However, the\ncatalytic efficiency (k /K ) of F303GFaAAT was reduced to 0.35-fold of the wild-type\ncat m\n(p<0.001, Fig. R6). These quantitative data have supplemented in the revised\nmanuscript (Lines 330-334).\n9. Figures related to enzyme engineering (e.g., Fig. 4 and Supplementary Figs. 32–\n35) should explicitly label the mutated residues, substrate orientations, and key\nmolecular interactions to enhance interpretability.\nResponse:\nWe have updated Fig. 4 and Supplementary Figs. 34–41 to explicitly label mutated\nresidues, substrate orientations, and key molecular interactions in the revised\nmanuscript.\n10. In the Discussion section, the authors are encouraged to summarize general\ndesign principles derived from AAT engineering and discuss the broader applicability\nof this strategy to other ester-forming enzymes.\nResponse:\nWe have supplemented the Discussion with generalized design principles for AAT\nengineering and its applicability to other ester-forming enzymes in the revised\nmanuscript (Lines 501-527).\n11. Please verify the consistency among figure legends and supplementary data\nnumbering, as cross-references in Supplementary Figs. 32–35 appear slightly\nmisordered.\nResponse:\nSupplementary Figs. 34–41 in the revised manuscript.\n12. The protein–ligand interaction diagrams in Supplementary Figs. 26/27/32 have\noverly complex backgrounds and inconsistent color schemes, making them difficult to\ninterpret. Additionally, some labels are inconsistent with the textual description. Please\nsimplify and standardize these figures.\nResponse:\nWe have simplified the protein–ligand interaction diagrams, standardized color\nschemes, and corrected inconsistent labels in Supplementary Figs. 32, 33, and 34\nin the revised manuscript.\n13. The layout of Supplementary Figs. 5n, 7g, 17e, and 23b is inconsistent with other\nfigures. Please unify the figure formatting.\nResponse:\nWe have unified the layout and formatting of Supplementary Figs. 5n, 7g, 17e, and\n23b in the revised manuscript.\n14. Lines 384–386: The text states, “In lavandulol biosynthesis, a variant\npyrophosphatase (F118D-H182C EcRdgB) engineered through structure-guided\nmutagenesis enhanced the titer of lavandulol by 7.64-fold.” However, it does not clarify\nthat this engineered enzyme is an original design in this study, nor does it explain why\nthis enzyme was targeted for modification. Please provide this rationale.\nResponse:\nWe have clarified that F118D-H182CEcRdgB is a structure-guided engineered\npyrophosphate originally designed in this study and added the rationale for targeting\nthis enzyme in the revised manuscript (Lines 417-423).\n15. Lines 409–416: During de novo GB synthesis, the authors first used wtFaAAT\ninstead of the previously optimized I302GFaAAT2 variant, which had demonstrated\nsuperior selectivity. Please justify this choice and assess whether the use of\nwtFaAAT is logically consistent.\nResponse:\nand clarified the consistency with previous optimization results in the revised\nmanuscript (Lines 454-457). Specifically, wtFaAAT was initially used to optimize co-\nculture conditions; we then replaced it with F303GFaAAT and I303GFaAAT2 for direct\nperformance comparison.\n16. In Fig. 5a/c/d/f, several schematic elements are unclear and cannot be cross-\nreferenced with data in panels b/e. For example, CaBACT is not shown in the figure.\nPlease label all relevant enzymes and components clearly to avoid reader confusion.\nResponse:\nWe have revised Fig. 5a/c/d/f to label all relevant enzymes (including CaBACT) and\nensured clear cross-referencing to panels b/e in the revised manuscript.\n17. The reference list contains numerous and significant formatting errors that must\nbe corrected. Specifically, journal names are inconsistently presented (some appear as\nfull names while others are abbreviated; e.g., refs. 43, 44, 45, 49), several\nreferences are missing volume and page numbers (e.g., refs. 1, 7, 50–52), and title\ncapitalization is inconsistent (some titles use sentence case whereas others capitalize\nevery word; e.g., ref. 8). The entire list of references needs a careful and\ncomprehensive revision to ensure consistency with the journal’s formatting\nrequirements.\nResponse:\nWe thank the reviewer for raising the format issue in reference. We have thoroughly\nrevised the entire reference list to ensure formatting consistency, correct journal styles,\nand complete volume/page information in the revised manuscript.\nComments:\nYang et al. have developed a dual-substrate-wheeled microbial system. Using this\nsystem, they successfully produced three types of monoterpene esters: monoterpenyl\nesters, monoterpenoate esters, and monoterpenyl monoterpenoate esters, which\ninclude several new-to-nature molecules. Enzyme engineering was applied to shift the\npreference of alcohol acyltransferases (AAT) towards various acyl-CoA or alcohol\nsubstrates. Finally, the co-culture strategy enabled the production of linalyl acetate to\nreach 11.5 g/L, which is an impressive titre. Overall, this is a solid study with originality,\ncomprehensive data and clear demonstration of biosynthetic versatility achieved by\nusing enzymes with broader acceptability of various substrates, such as AATs. I\nrecommend that the manuscript be accepted after the following revisions.\nResponse:\nWe sincerely appreciate Reviewer #2 for the valuable comments and suggestions,\nwhich have greatly helped us refine the manuscript and strengthen its scientific\npresentation.\nMajor Comments:\n1. Figure 3, the authentic standards should be plotted together with reactions to show\nthe identity between biosynthesized monoterpene esters and standards. The retention\ntimes of 5’a’, 5’b’ and 5’c’ in Figure 3d differ from those of chemically synthesized\ncompounds in Supplementary Fig 28. In addition, the TIC chromatograms of newly\nproduced monoterpene esters should be provided in addition to EIC chromatograms,\nwhich can assess matrix complexity and method robustness.\nResponse:\nWe have redrawn Fig. 3 to plot the chemically synthesized authentic standards\ntogether with biosynthesized monoterpene esters (MEs) in the same EIC panels,\nenabling direct comparison of retention time and peak shape. We have also added the\nTIC chromatograms of newly produced MEs with the chemically synthesized authentic\nstandards to Supplementary Fig. 27 and 30 to illustrate matrix complexity and\nmethod robustness in the revised manuscript.\n18\n2. The authors produced many novel monoterpene esters, however, the authors\nselected linalyl acetate and geranyl butyrate to fed-batch fermentation. Why were\nthese two molecules chosen instead of others? What is their commercial value?\nResponse:\nWe thank the reviewer for the insightful comment. We have supplemented the\nrationale for selecting linalyl acetate and geranyl butyrate to the revised manuscript\n(Lines 431-434). First, both esters have clear industrial relevance: linalyl acetate is the\ndominant high-value fragrance component in lavender essential oil and is extensively\nused in perfumery, while geranyl butyrate is a commercially important fruity/floral flavor\ncompound widely applied in food, beverage, and fragrance formulations. Their\nestablished market demand makes them ideal candidates for scale-up validation.\nSecond, their biosynthetic precursors—linalool and geraniol—are among the\nmonoterpene alcohols with the higher achievable microbial titers in this study and\nother extensive studies, enabling a representative demonstration of monoterpenoate\nester biosynthesis. Together, these attributes made linalyl acetate and geranyl\nbutyrate suitable and industrially meaningful examples for fed-batch fermentation.\n3. The manuscript reports that the F302G mutation enhances selectivity toward\nbutyryl-CoA (GB), resulting in a 98.95% selectivity at a 4:1 butyrate-to-geraniol ratio.\nHowever, in Figure 4 and supplementary figures, the data range markers are\nextremely small, making it difficult to visually distinguish the different mutants by color.\nMore importantly, the improvement attributed to the mutation appears modest\ncompared to the effect of adjusting the substrate molar ratio. For instance, the wild-\ntype FaAAT also reaches >95% selectivity under the 4:1 ratio, which is comparable to\nthat of the mutant. As currently presented, the figure and discussion may overstate the\nimpact of mutagenesis relative to substrate ratio optimization. The authors are\nencouraged to clarify and separate the respective contributions of enzyme mutation\nand substrate ratio adjustment in the text.\nResponse:\nWe thank the reviewer for the comment. We have further clarified in the text that,\nalthough the WT enzyme also achieves >95% GB selectivity at a 4:1 butyrate-to-\ngeraniol molar ratio, the F303G variant exhibits substantially enhanced selectivity at\nother molar ratios, including a 2.21-fold improvement (p < 0.001) at the 1:4 butyrate-\nto-geraniol molar ratio (Fig. 4g). This data indicate that F303GFaAAT maintains higher\n19\nselectivity across a broader range of physiological dual-substrate ratios intracellularly,\nparticularly under low butyryl-CoA and high geranyl-CoA conditions that better reflect\nintracellular metabolism, where acetyl-CoA as the precursor is typically more abundant\nthan butyryl-CoA. We have revised the figures to include fold-changes in selectivity\nacross all tested dual-substrate molar ratios for FaAAT and its variant as shown in Fig.\nR7, improving readability in the revised manuscript (Lines 314-319).\nFigure R7. Selectivity of geranyl butyrate produced by strains expressing\nwtFaAAT or F303GFaAAT with varying butyrate-to-geraniol molar ratios. ***p< 0.001.\n4. The manuscript concludes that the F302G mutation reduces steric hindrance and\nthe contact distance between geraniol and acyl-CoAs based solely on molecular\ndocking results (Fig. 4d–e). However, docking typically generates multiple possible\nbinding poses and represents only static predictions. Therefore, it is difficult to draw\ndefinitive conclusions about steric effects or substrate interactions from docking alone.\nMoreover, the subsequent MD simulations reveal that this mutation actually\ndestabilizes the acetyl-CoA complex. These findings suggest that the docking-derived\nconformations may not accurately represent the catalytically relevant states. The\nauthors should revise the manuscript accordingly to reflect the MD simulation results.\nResponse:\nWe thank the reviewer for this important comment and fully agree that molecular\ndocking alone provides only static, non-unique binding hypotheses and should not be\n20\nused to draw definitive conclusions regarding steric effects or catalytically relevant\nsubstrate interactions. In the revised manuscript, we have therefore toned down static\ndocking-based interpretations and revised the relevant text to emphasize its\nexploratory nature rather than as mechanistic evidence. To more appropriately reflect\nthe dynamic behavior of substrates within the active site, we focused the analysis on\nthe pre-reaction states (PRS) of molecular dynamics (MD) simulations, defined by\ncatalytically competent arrangements consistent with established residue functions\n(e.g., 157His as the general base). These simulations do not assume a single\nproductive pose, instead, examine how mutations reshape the ensemble of near-\nattack conformations3.\nAs shown in Fig. R8, PRS-MD simulations reveal that the F303G mutation in FaAAT\ninduces an allosteric reorganization of the CoA thioester environment rather than a\nuniform reduction in steric hindrance. Specifically, F303G in FaAAT substantially\nweakens interactions between 284Asn and acetyl-CoA, consistent with destabilization\nobserved in MD trajectories, while simultaneously enhancing interactions between\n284Asn and butyryl-CoA. 378Ser likewise exhibits mutation-dependent, substrate-\nspecific changes in interaction. These differential dynamic effects correlate with\nsubstrate-specific binding free energy trends and align with the experimentally\nobserved shift in acyl-donor preference. Accordingly, we have revised the manuscript\nto avoid overemphasizing steric effects and to clarify that the impact of F303G on\nsubstrate selectivity arises from mutation-induced redistribution of dynamic substrate\ninteractions and PRS populations, rather than from simple steric distance shortening\ninferred from docking poses. Besides, the identified residues (e.g., 284Asn and 378Ser)\nin PRS-MD exhibiting strong substrate-dependent dynamic responses provide\npromising targets for future multiple mutational validation. Corresponding revisions\nhave been made in the revised manuscript (Lines 291-300).\nmolecular dynamics simulations. (a) Acetyl-CoA-geraniol complex in wtFaAAT;\n(b) Acetyl-CoA-geraniol complex in F303GFaAAT; (c) Butyryl-CoA-geraniol complex in\nwtFaAAT; (d) Butyryl-CoA-geraniol complex in F303GFaAAT. Key interacting residues\n(284Asn and 378Ser) are shown as sticks, and dashed lines indicate representative\ninteratomic distances observed in PRS conformations.\n5. Supplementary Fig. 39 shows that after the I302G mutation of FaAAT2, the catalytic\ndistances increase when binding butyryl-CoA, which may imply less stable binding.\nHowever, this seems inconsistent with the results in Fig. 4 j, k, where the mutant shows\nenhanced catalytic activity and selectivity for butyryl-based esters. It is recommended\nto further discuss and clarify the relationship between the change in catalytic distances\nand the catalytic performance in the revised manuscript.\nResponse:\nWe thank the reviewer for this thoughtful comment highlighting the apparent\ninconsistency between the increased catalytic distances observed for butyryl-CoA in\nI303GFaAAT2 (Supplementary Fig. 39 in the original manuscript) and the enhanced\nselectivity and catalytic efficiency toward butyryl-derived esters as shown in Fig. 4j\nand 4k. This observation suggests the importance of interpreting static geometric\nmetrics within the context of the full catalytic reaction pathway. The distance analysis\ncharacterizes the Michaelis complex, where an increased nucleophile-carbonyl\nseparation for butyryl-CoA indeed reflects a modest weakening of ground-state\nbinding upon mutation. However, catalytic efficiency is not determined by ground-state\n22\nbinding alone but by how effectively the enzyme guides the substrate ensemble toward\ncatalytically competent, pre-reaction conformations.\nFigure R9. Distinct modulation of Bürgi–Dunitz attack geometry by homologous\nmutations in FaAAT and FaAAT2. Two-dimensional probability density maps of\nreactive geometries obtained from pre-reaction state of molecular dynamics\nsimulations for FaAAT and FaAAT2 in complex with acetyl-CoA (C2) or butyryl-CoA\n(C4). Panel (a) shows wtFaAAT and F303GFaAAT, and panel (b) shows wtFaAAT2 and\nI303GFaAAT2. In FaAAT, the F303G mutation predominantly alters the S–C(=O)–O–C\ndihedral angle (SOCO), whereas in FaAAT2, the homologous I303G mutation mainly\naffects the O–C(=O)–O attack angle (OCO).\nTo reconcile this apparent discrepancy, we performed additional PRS-MD simulations\nthat explicitly probe near-attack conformations preceding nucleophilic transfer. These\nanalyses reveal that the mechanistic role of the corresponding mutation differs\nfundamentally between FaAAT and FaAAT2 (Fig. R9). In F303GFaAAT, the mutation\nprimarily reshapes the reactive ensemble by modulating the S–C(=O)–O–C (SOCO)\ndihedral angle, thereby adjusting the torsional orientation of the thioester and favoring\nconformations compatible with acyl transfer. In contrast, the I303G mutation in\nFaAAT2 predominantly alters the O–C(=O)–O (OCO) angle, corresponding to the\nBürgi-Dunitz attack angle, indicating a distinct reaction coordination mode. Importantly,\nthis divergence implies that a single geometric distance metric cannot be universally\ninterpreted as a proxy for catalytic competence across different AAT variants. In\nI303GFaAAT2, although the average Michaelis-state distance increases, the mutation\nenhances sampling of pre-reactive conformations along the dominant reaction\ncoordinate, thereby promoting butyryl-CoA turnover and selectivity. We have revised\nthe manuscript accordingly to de-emphasize distance-based metrics and clarify that\nchanges in catalytic performance arise from mutation-specific reorganization of\n23\ndynamic reaction coordinates rather than from static, atomic-distance shortening\nalone (Line 300-307).\n6. “Reconstructing the precise biosynthesis of structurally complex natural esters, such\nas monoterpene esters, in engineered microbes remains a major challenge, owing to\nthe limited repertoire of highly selective alcohol acyltransferases and the lack of\ncompatible pathway modularity.” This statement is partially true, as there are several\nexcellent examples of the biosynthesis of monoterpene esters, such as geranyl\nacetate, geranyl butyrate.\nResponse:\nWe appreciate the reviewer’s comment and agree that several MEs, most notably\nacetyl-CoA–derived products such as geranyl acetate and related acetate esters, have\nbeen successfully biosynthesized in engineered microbes. However, we would like to\nclarify that microbial biosynthesis of butyryl-CoA-derived MEs (e.g., geranyl butyrate)\nwith high selectivity and titer has not been previously established, largely due to the\ndominance of intracellular acetyl-CoA and the lack of AATs with sufficient acyl-donor\nselectivity toward C>2 acyl-CoAs. As discussed in the Introduction (Lines 106-108),\ncurrent engineered systems predominantly access a narrow subset of MEs derived\nfrom acetyl-CoA, whereas precise and modular biosynthesis of structurally diverse\nMEs remains constrained by AAT promiscuity and incompatibility between alcohol and\nacyl-CoA precursor pathways. Besides, to date there has been no report of de novo\nmicrobial biosynthesis of geranyl butyrate via intracellular AAT-mediated pathways;\nexisting studies achieve geranyl butyrate only through integrated processes combining\nmicrobial geraniol production with subsequent lipase-catalyzed esterification4, rather\nthan de novo biosynthesis from endogenous acyl-CoA pools. Our statement in the\nAbstract therefore does not deny prior successes for individual acetate esters, but\nrather emphasizes the broader challenge of reconstructing generalizable, selective,\nand modular biosynthetic routes for diverse MEs. Accordingly, we believe the current\nAbstract accurately reflects the state of the field and the scope of our contribution.\n7. “Coculture engineering further redistributed metabolic fluxes between acyl-CoA and\nalcohol precursors, achieving >10 g/L production of monoterpene ester.” This\nsentence is not wrong, but gives an impression that it seems all the monoterpene\nesters have reached such a high titre in this study. The fact is only one linalyl acetate\n24\nhas reached this titre, and >90% of the monoterpene esters were produced only in\ndetectable amounts and without any quantitative data. I suggest to rewrite the\nsentence to achieving >10 g/L production of “linalyl acetate” specifically, not\n“monoterpene esters” in general.\nResponse:\nWe have rewritten the sentence to specifically state that >10 g/L was achieved for\nlinalyl acetate, rather than MEs in general, in the revised manuscript (Lines 46-48).\nMinor Comments:\n1. A few minor issues remain, and the authors are advised to carefully check the\nmanuscript for similar errors.\nResponse:\nWe have carefully rechecked the entire manuscript for remaining minor errors and\ncorrected them accordingly in the revised manuscript.\n2. In the Results section (line 405), the phrase “the third the plasmid” appears to be a\ntypographical error. It should likely be “the third plasmid”. Please revise this for\ngrammatical accuracy and clarity.\nResponse:\nWe have corrected the typographical error and revised the wording to “the third\nplasmid” in the revised manuscript (Lines 448).\n3. In line 502, the phrase “a type pf dimeric MEs” contains a typographical error. The\nword “pf” should be corrected to “of”. Please revise this for accuracy.\nResponse:\nWe have corrected “pf” to “of” to improve accuracy and clarity in the revised manuscript\n(Lines 1339).\n4. The “100 nm” in the legend of Fig. 4m-n (line 592) appears incorrect, as catalytic\ndistances in enzyme active sites are typically on the Å scale; this likely refers to the\n100 ns MD simulation duration mentioned subsequently.\nResponse:\nWe have corrected this labelling error. The “100 nm” in the original figure legend was\na typographical mistake and has been corrected to “100 ns,” referring to the duration\n25\nof the MD simulations. In the revised manuscript, we have de-emphasized distance-\nbased geometric metrics as mechanistic evidence. MD simulations are now primarily\nused to characterize conformational ensembles and dynamic reorganization of the\nactive site, rather than to infer catalytic competence from absolute interatomic\ndistances.\nComments:\nYang et al describe a \"dual-substrate-wheeled microbial platform\" to engineer alcohol\nacyltransferase to synthesize monoterpenyl, monoterpenoate and monoterpenyl\nmonoterpenoate esters, including some apparently new-to-nature compounds. I was\nasked to specifically comment on the enzymology and protein engineering. More\ngenerally, the study looks interesting and these may be important results as the\nauthors correctly note that they make a new class of sesquiterpenes and other\ninteresting esters. Unfortunately, I found much of the paper very difficult to follow, as\nthe writing style was lacking in what I consider to be a lot of important detail. Phrasing\nis also unusual. for example, \"dual-substrate-wheeled\" is not a term I am familiar with,\nalthough with the help of Figure 1, I think I understand what the authors mean. Other\nterms such as \"dual-substrate molar ratio tuning\" can be guessed at, but in my opinion\nthe paper needs to be rewritten to improve clarity and to add missing detail.\nResponse:\nWe sincerely appreciate Reviewer #3 for the detailed comments, and we have\nthoroughly rewritten the manuscript to improve clarity, structure, and completeness.\nWe have also removed uncommon terms (e.g., dual-substrate-wheeled), added\nexplicit explanations upon first use, and revised the description of catalytic mechanism\nin enzyme kinetics to ensure readers can easily understand the concepts and enzyme\ncatalytic data presented in the revised manuscript.\n1. \"The kinetic data support a model in which AAT and its mutants operate via a\nrandom sequential bi–bi mechanism within the cellular environment, allowing both\nacetyl-CoA and butyryl-CoA to independently access the active site in the presence of\ngeraniol (Supplementary Note 1).\" The proposed mechanism is shown in Fig 4.\nPresumably acetyl-CoA or butyryl-CoA can bind to the enzyme-alcohol complex, not\nat the same time (\"independently\" is confusing here). The kinetics then should\nestablish whether the binding order is random (e.g. alcohol always binds first) or\nrandom, and Michaelis-Menten kinetics analysis will give Km values for both substates,\nand potentially a Kd for binding of the first substrate. It's not clear which Km values\nare reported in the paper and what the fixed substrate concentration (which I think was\ngeraniol) was. These should be given in the caption and Km's should be measured\nwith multiple fixed substrate concentrations to ensure they are not convoluted.\nTo avoid misunderstanding, we have removed the term “independently” from the\ndescription of the proposed mechanism in both the main text and supplementary file.\nAs suggested, we have clarified in the Figure caption (Line 1396-1399) and Methods\n(Line 836-859) how and which kinetic parameters were measured, the substrate that\nwas fixed during each assay, and the experimental conditions used to determine k\ncat\nand K . Specifically, we describe the kinetic setup as follows: enzymatic reactions\nm\n(500 μL total volume) were performed with purified enzyme at 20 mg/L. Two assay\ngroups were conducted, in each of which the concentration of one substrate was fixed\nwhile the other was varied. In Group A, geraniol concentration was fixed (20 mg/L),\nwhile acetyl-CoA or butyryl-CoA concentrations were varied across geraniol-to-acyl-\nCoA molar ratios of 1:4 to 4:1. In Group B, acyl-CoA concentration was fixed (20 mg/L),\nwhile geraniol was varied to achieve molar ratios from 1:2 to 8:1. All substrates were\nfreshly prepared, reactions were incubated at 30 °C and 250 rpm for 6 h, extracted\nwith ethyl acetate, and analyzed by GC-MS. Steady-state kinetic parameters (k and\ncat\nK ) were obtained by fitting triplicate reaction rates to the Michaelis–Menten equation\nm\nand Lineweaver–Burk reciprocal plots were generated for mechanistic evaluation.\nThe kinetic parameters reported here represent conditional/apparent K values\nm\ndetermined under steady-state conditions with the second substrate held at a fixed\nconcentration. These measurements do not directly provide equilibrium dissociation\nconstants (K ) for individual substrates, nor do they resolve the precise substrate\nd\nbinding order. Accordingly, we have revised the manuscript to avoid over-\ninterpretation and now describe the kinetic behavior as consistent with a sequential\nbi–bi mechanism, without assigning a specific binding sequence.\n2. The molecular modelling appears to be well done, but in SI Fig 26 the residue/ligand\ncoloring doesn't appear to match the caption. Does this provide insight into substrate\nbinding order?\nResponse:\nWe thank the reviewer for pointing this out. We have corrected the residue and ligand\ncolor schemes in Supplementary Fig. 32 to ensure full consistency with the caption.\ninstead of to the visual coloring of residues in the ligand-enzyme complex.\nRegarding mechanistic interpretation, the molecular docking in this figure was\nintended to identify key interacting residues between acyl-CoAs and different AATs,\nrather than to infer a substrate-binding order. The revised text now clarifies that the\nmodelling supports multiple feasible binding orientations but does not provide\ndefinitive evidence for a strict substrate-binding sequence. Accordingly, we have\navoided overinterpretation and explicitly state in the revised manuscript that the\nbinding order cannot be conclusively determined from the current modelling alone\n(Lines 363–367).\n3. \"both wt FaAAT and F302G FaAAT exhibited high exponential curve fitting\nconfidence for GA and GB formation (R2 >0.99, Fig. 4f, Supplementary Table 7).\" This\ncould be phrased better and the equation used for fitting (in the methods) numbered\nand referred to here. Why is the dependence of the titre exponentially dependent on\nthe ratio of substrates? Can this be rationalised based on the kinetics?\nResponse:\nWe have revised the phrasing for clarity and now explicitly refer to the numbered fitting\nequation provided in Supplementary Data 3. Product formation in Fig. 4f by both\nwtFaAAT and F303GFaAAT is well described by an empirical fitting function, with high\ngoodness-of-fit (R² > 0.99, number 31 to 34), without implying an intrinsic exponential\nkinetic behavior.\nWe emphasize that the exponential-like dependence observed in Fig. 4f reflects an\nempirical saturation-type response of GA and GB accumulation to changes in the acyl-\ndonor/geraniol ratio at the cellular level, rather than a direct representation of the\nfundamental enzymatic rate law. Under the experimental conditions, the concentration\nof geraniol is fixed, and increasing butyrate supplementation enhances the intracellular\navailability of acyl-donor equivalents through uptake and activation to acyl-CoA. As a\nresult, product accumulation increases rapidly at low supplementation levels and\nprogressively approaches a plateau as the system becomes constrained by enzyme\ncapacity, fixed alcohol concentration, or cellular and metabolic limitations. From a\nkinetic perspective, this behavior is qualitatively consistent with a Michaelis–Menten–\n29\ntype dependence of reaction rate on acyl-CoA concentration, integrated over a fixed\nreaction time to yield final product titers. This interpretation is supported by the in vitro\nenzyme assays shown in Supplementary Fig. 41a and 41c, where FaAAT activity\nwas measured under well-defined conditions by varying butyryl-CoA concentration at\nfixed geraniol. These assays directly report the intrinsic catalytic properties of the\nenzyme (rate versus [acyl-CoA]) and were used to extract kinetic parameters.\nAccordingly, while the whole-cell biotransformation dataset (Fig. 4f) and the in vitro\nenzyme assays (Supplementary Fig. 41a and 41c) are not expected to show a direct\none-to-one correspondence, they are mechanistically linked in that increasing acyl-\ndonor availability enhances product formation through the AAT step in a saturable\nmanner under fixed geraniol conditions. Under such circumstances, the exponential\napproaches to a maximum can adequately capture the observed curvature. Therefore,\nthe exponential fitting equation is used here as a quantitative descriptor of trends in\nproduct formation in the whole-cell biotransformation system, rather than as a\nmechanistic kinetic model defining reaction order or transition states.\n4. p.15 \"...representing 61.2% of the theoretical chemical structure space of esters.\"\nThe authors have not made 61% of all possible esters.\nResponse:\nWe have revised the statement to avoid implying coverage of the global ester chemical\nspace. In this study, 71 unique MEs were biosynthesized, including 64 MTEs (out of\n88 [11 MTs*8 acyl-CoAs]) (Fig. 2d), 3 MTaEs (out of 6 [3 SCFAlcs*2 acyl-CoAs]) (Fig.\n3b), and 4 MtMtaEs (out of 22 [11 MTs*2 acyl-CoAs]) (Fig. 3f), achieving a coverage\nrate of 61.2% (71 out of 116 theoretical number of MEs). The description now clarifies\nthat the 61.2% refers only to the percentage of the biosynthesized MEs in this study\nagainst the theoretical ones, which is the number of the subset of monoterpene–acyl\nester combinations tested in this study (Fig. 2d, Fig. 3b, and Fig. 3f), based on the\ndefined ester design matrix. This correction has been incorporated in the revised\nmanuscript (Lines 482-487).\nComments:\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nResponse:\nWe sincerely thank Reviewer #4 for the thorough evaluation and constructive\ncomments.\nReference:\n1. Houk, K.N., Leach, A.G., Kim, S.P. & Zhang, X. Binding Affinities of Host–Guest,\nProtein–Ligand, and Protein–Transition-State Complexes. Angewandte\nChemie International Edition 42, 4872-4897 (2003).\n2. Ahern, W. et al. Atom-level enzyme active site scaffolding using RFdiffusion2.\nNat Methods 23, 96-105 (2026).\n3. Bruice, T.C. & Lightstone, F.C. Ground State and Transition State Contributions\nto the Rates of Intramolecular and Enzymatic Reactions. Accounts of Chemical\nResearch 32, 127-136 (1999).\n4. Yang, D., Li, X., Zhang, C., Liang, H. & Ma, X. Bioproduction of geranyl esters\nby integrating microbial biosynthesis and enzymatic conversion. Journal of\nAgricultural and Food Chemistry 72, 24677-24686 (2024).\nIn Title:\n1. The title of “A dual-substrate-wheeled microbial platform for selective\nbiosynthesis of monoterpene esters” has been changed “A microbial platform\nfor selective biosynthesis of monoterpene esters” to avoid uncommon terms\nused on Page 1 Line 1-2.\nIn Author's Name and Information:\n2. The additional authors and their order have been added and updated based\non the contributions on Page 1 Line 4-6, 16-25.\nIn Abstract:\n1. The word of “dual-substrate-wheeled” has been changed to “dual-substrate”\nthroughout the revised manuscript.\n2. The word of “achieved” has been changed to “achieve” on Page 2 Line 44.\n3. The sentence of “Coculture engineering further redistributed metabolic fluxes\nbetween acyl-CoA and alcohol precursors, achieving >10 g/L production of\nmonoterpene ester” has been changed to “Coculture engineering further\nredistributed metabolic fluxes between acyl-CoA and alcohol precursors,\nyielding 11.50 g/L linalyl acetate and 3.16 g/L geranyl butyrate in 1-L bioreactor”\non Page 2 Line 46-48.\n4. The word of “reprogram” has been changed to “control” on Page 2 Line 49.\nIn Main:\n1. The word of “(GA)” has been added on Page 4 Line 108.\n2. The sentence of “titers of MEs” has been changed to “titer of ME” on Page 4\nLine 114.\n3. The sentence of “significantly enhancing product diversity, substrate specificity,\nand pathway modularity” has been changed to “significantly enhancing the\ndiversity, substrate specificity, and pathway modularity” on Page 5 Line 133-\n134.\nIn Biosynthesis of monoterpenoate ester:\n32\n1. The word of “enzymes” has been changed to “AATs” on Page 5 Line 155.\n2. The sentence of “Among all the validated AATs, LiAAT4 and ScAAT1 showed\nthe activities of catalyzing primary to tertiary MTs” has been changed to\n“Among all the validated AATs, LiAAT4 and ScAAT1 showed catalytic activity\ntoward primary-to-tertiary MTs” on Page 6 Line 159-160.\n3. The word of “the” has been deleted on Page 6 Line 169.\n4. The sentence of “FvAAT and FaAAT exhibited a wide range of substrate\nspecificity for various MTs for benzoyl-CoA-derived MTEs. PmBABT showed\nnarrow catalytic activity for the accessed MTs and exclusively generated three\nbenzoate MTEs, namely geranyl benzoate, neryl benzoate, and (S)-(-)-perillyl\nbenzoate (Fig. 2d, Supplementary Figs. 9, 11, and 13). Building on these\nfindings, we profiled the capacity of FvAAT and FaAAT to form phenylacetyl-\nCoA- and phenylpropionyl-CoA-derived MTEs. The identification shows that\nFaAAT possesses broad catalytic activities for esterifying aromatic acid-derived\nacyl-CoA with multiple MTs (Fig. 2d, Supplementary Figs. 4-25). These\nresults suggest that FaAAT holds the potential as a versatile catalytic module\nfor aromatic acid-derived MTE biosynthesis” has been changed to “FvAAT\nand FaAAT exhibited a wide range of substrate specificity for various MTs for\nbenzoyl-CoA-derived MTEs, which are widely used in the flavor, fragrance, and\ncosmetic industries, where they contribute sweet, floral, and fruity notes39.\nPmBABT showed better catalytic activity for the accessed MTs and could\nexclusively generate benzoate MTEs (e.g., lavandulyl benzoate, geranyl\nbenzoate, neryl benzoate, and perillyl benzoate, Fig. 2d, Supplementary Figs.\n7, 9, 11, and 13). Building on these findings, we profiled the capacity of FvAAT\nand FaAAT to form phenylacetyl-CoA- and phenylpropionyl-CoA-derived MTEs.\nThe identification shows that FaAAT possesses broad catalytic activities for\nesterifying aromatic acid-derived acyl-CoA with multiple MTs (Fig. 2d,\nSupplementary Figs. 4-25)” on Page 6 Line 177-186.\nIn Biosynthesis of monoterpenoate and monoterpenyl monoterpenoate ester:\n1. The word of “thereby” has been changed to “for” on Page 7 Line 208.\n2. The sentence of “Importantly, the conversion of volatile and membrane-\npermeable monoterpenes into bulkier MtMtaEs alters their physicochemical\nproperties, reducing volatility and cytotoxicity while favoring sequestration into\n33\nsubcellular storage compartments such as lipid droplets or peroxisomes. Such\ncompartmentalization not only alleviates product toxicity but also establishes a\nstable reservoir that supports more efficient and sustainable monoterpene\nbiosynthesis” has been changed to “Importantly, the conversion of volatile and\nmembrane-permeable monoterpenes into bulkier MtMtaEs alters their\nphysicochemical properties, reducing volatility while favoring sequestration in\nsubcellular storage compartments such as lipid droplets or peroxisomes. Such\ncompartmentalization not only possibly alleviates product toxicity but also\nestablishes a stable reservoir that supports more efficient and sustainable\nmonoterpene biosynthesis” on Page 8 Line 233-239.\nIn Reprogramming AAT selectivity via single-residue mutagenesis and dual-\nsubstrate ratio:\n1. The sentence of “We conducted the molecular docking of the AlphaFold2-\npredicted FaAAT, LiAAT4, FvAAT, and PmBABT with acyl-CoAs, which\nrevealed marked differences in active site configuration that are the basis of\ntheir distinct substrate specificities (Supplementary Fig. 26). All AATs share a\nconserved acyl-CoA binding architecture coordinated by key catalytic residues\n(e.g., 155His/148His/165His, 159Asp/152Asp/169Asp), yet the acyl pocket\ncomposition diverges significantly. FaAAT and FvAAT, both of which\naccommodate acetyl-CoA, feature narrow and hydrophobic acyl-binding sites\nshaped by residues such as 280Ala/280Val and 302Phe/300Leu (Supplementary\nFig. 26a-b), which form a tight cavity around the acetyl group. In LiAAT4\n(Supplementary Fig. 26c), the substitution of bulky hydrophobic residues by\n291Tyr may narrow the cavity, and PmBABT shows a dramatic adaptation for\naromatic substrates, showing that benzoyl-CoA is stabilized by expanded\nhydrophobic contacts from 299Phe and 261Phe, as well as polar interactions with\n370Asp and 373Arg (Supplementary Fig. 26d). These differences are mirrored\nin the tunnel topology accordingly. Substrate tunnel architectures of FaAAT,\nLiAAT4, FvAAT, and PmBABT exhibit distinct configurations in length,\nbottleneck radius, and steric gating residues (Supplementary Fig. 27a-d),\nunderlying their substrate specificities. FaAAT and FvAAT display extended\nright-side tunnels (~28 Å), potentially favouring elongated substrates (Fig. 2d).\nIn contrast, PmBABT presents relatively shorter and narrower paths on both\n34\nsides (Supplementary Fig. 27e). Steric gate residues at tunnel define distinct\nspatial constraints, with LiAAT4 showing a much narrower bottleneck radius\n(<1.0 Å) than FaAAT and FvAAT (Supplementary Fig. 27f), consistent with its\nlimited acceptance of bulky acid-derived acyl-CoA (Fig. 2d). These analyses\nindicate that the varying shape of substrate tunnels modulates substrate\ningress and product egress, providing a structure-to-function basis for\nelucidating catalytic differences or engineering substrate specificity” has been\nremoved and changed to “We performed molecular docking of the\nAlphaFold2-predicted FaAAT, LiAAT4, FvAAT, and PmBABT in complex with\nacyl-CoAs, revealing distinct differences in active-site organization that underlie\ntheir distinct substrate preferences (Supplementary Fig. 32). Although these\nAATs share a conserved acyl-CoA binding framework coordinated by key\ncatalytic residues (e.g., His and Asp), the composition and geometry of the acyl-\nbinding pocket vary substantially. FaAAT and FvAAT, which preferentially\naccept short-chain acyl donors, possess relatively narrow and hydrophobic acyl\npockets shaped by residues such as Ala/Val and Phe/Leu, forming a compact\nenvironment around acetyl-CoA (Supplementary Fig. 32a-b). In contrast,\nLiAAT4 exhibits a more restricted cavity due to substitution by bulkier residues\n(e.g., Tyr), whereas PmBABT displays a distinct adaptation for aromatic\nsubstrates, with benzoyl-CoA stabilized by expanded hydrophobic contacts and\nadditional polar interactions (Supplementary Fig. 32c-d). Consistent with\nthese pocket-level differences, substrate tunnel architectures across these\nAATs differ in length, bottleneck radius, and residue composition\n(Supplementary Fig. 33). FaAAT and FvAAT feature extended tunnels that\ncan accommodate elongated aliphatic substrates, whereas PmBABT exhibits\nshorter and more constrained pathways. These global tunnel features\nrationalize broad differences in substrate scope among AATs; however, they\nprimarily govern substrate accessibility rather than fine discrimination between\nstructurally similar acyl donors. In particular, the precise selectivity of FaAAT\ntoward acetyl- versus butyryl-CoA cannot be explained by tunnel geometry\nalone, implicating localized interactions and dynamic effects near the catalytic\ncenter” on Page 8 Line 243-264.\n2. The sentence of “The specificity of AAT towards the selected acyl-CoA is a\nkey determinant in tailoring microbial ester biosynthesis, particularly in\n35\nintracellular environments rich in acetyl-CoA. To precisely direct ester formation,\nwe focused on FaAAT using geraniol as a fixed alcohol donor, with butyryl-CoA\nand acetyl-CoA as competing acyl donors. Molecular docking simulations using\nthe AlphaFold2-predicted FaAAT structure revealed that butyryl-CoA binding\nwithin a shared pocket adjacent to the catalytic residue 155His, with 302Phe,\n162Gly, 250His, and 378Ser as the critical interaction residues to acyl-CoA\n(Supplementary Fig. 32a). Among them, 302Phe of FaAAT was proposed as a\ncrucial steric gate residue proximal to the catalytic residue of 162His,\ndominantly affecting the acyl-CoA to geraniol binding geometry (Fig. 4a-b, Supplementary\nFig. 32a). To confirm the hypothesis, structure-guided NDT mutagenesis\nconducted at the 302Phe steric gate residue uncovered mutants with selectively\nenhanced butyryl-CoA specificity over acetyl-CoA (Supplementary Fig. 32b-\ne). The replacement of 302Phe by the residues with small-sized side chains (e.g.,\nGly, Ser, Asn) significantly reduced GA titer while not causing a substantial\ndecrease of GB titer. Phylogenetic analysis revealed diversity among plant-\nderived AATs, with a conserved aromatic or bulky aliphatic residue at position\n302 (Tyr, Phe, Ile), which likely imposes steric hindrance for C>2 acyl-CoA\nbinding (Fig. 4c, Supplementary Fig. 33). The molecular docking of butyryl-\nCoA-geraniol and acetyl-CoA-geraniol complexes with the AlphaFold2-\npredicted F302GFaAAT confirmed the reduced steric hindrance and contact\ndistance between geraniol and acyl-CoAs (Fig. 4d-e). Moreover, the mutants\nof FaAAT at 302Phe also modulated the formation of MTaEs and MtMtaEs\n(Supplementary Fig. 34), revealing a structurally conserved gate residue that\nacts as a steric control point for acyl-CoA discrimination. This finding challenges\nthe long-standing assumption that the intrinsic, low plasticity of AATs limits their\nuse in precision biosynthesis” has been changed to “Accordingly, we focused\non FaAAT to further investigate how ligand-binding residues govern acetyl-CoA\nversus butyryl-CoA selectivity using geraniol as a fixed alcohol donor. Molecular\ndocking using the AlphaFold2-predicted FaAAT suggested that acyl-CoA binds\nwithin a shared pocket adjacent to the catalytic residue His157, with 303Phe,\n162Gly, 250His, 378Ser, and 380Trp located in close proximity to the acyl-CoA\nmoiety (Supplementary Fig. 34). Among them, 303Phe was proposed as a key\nresidue proximal to 157His that influences the relative binding geometry of C2\nand C4 acyl-CoAs to geraniol (Fig. 4a-b, Supplementary Fig. 34a). To test\n36\nthis hypothesis, structure-guided NDT codon-based saturation mutagenesis (N\n= A/C/G/T, D = A/G/T, T = thymine) was performed. Several variants exhibited\nselectively enhanced on butyryl-CoA over acetyl-CoA (Supplementary Fig.\n34b-f). Replacement of 303Phe with small side-chain residues (e.g., Gly, Ser,\nand Asn) significantly reduced GA formation without causing a substantial\ndecrease in geranyl butyrate (GB) titer. Additional substitutions at G162, H250,\nS378, and W380 on F303GFaAAT did not yield further improvements in selectivity\nor product titer (Supplementary Fig. 35), indicating that, for short-chain acyl-\nCoA discrimination in FaAAT, double-site tunnel reshaping is less effective than\nlocalized residue engineering near the catalytic center, likely as excessive\ntunnel perturbation compromises productive acyl-CoA positioning.\nPhylogenetic analysis revealed substantial diversity among plant-derived AATs;\nhowever, a conserved aromatic or bulky aliphatic residue (Tyr, Phe, or Ile) is\nfrequently observed at position 303 (Fig. 4c, Supplementary Fig. 36).\nFurthermore, mutagenesis at position 303 in FaAAT resulted in substrate-\ndependent changes in the production profiles of MTaEs and MtMtaEs\n(Supplementary Fig. 37), indicating that this residue influences acyl-donor\npreference beyond the geraniol-based system” on Page 9 Line 266-289.\n3. The sentence of “Representative poses of acetyl-CoA-geraniol and butyryl-\nCoA-geraniol complexes generated from the AlphaFold2-predicted F303GFaAAT\nrevealed altered substrate orientations and contact patterns in the vicinity of the\nactive site after mutation (Fig. 4d-e). However, because molecular docking\nprovides only static and non-unique binding hypotheses, we then examined\nmutation-induced effects using pre-reaction state (PRS) molecular dynamics\n(MD) simulations. These analyses revealed that the F303G mutation does not\nuniformly shorten substrate contact distances but instead redistributes dynamic\ninteraction patterns within the acyl-CoA thioester environment, particularly\ninvolving 284Asn and 378Ser, in a substrate-dependent manner (Supplementary\nFig. 38). Moreover, comparative PRS-MD analyses revealed that homologous\nmutations in closely related FaAAT2 can modulate distinct reaction coordinates.\nIn FaAAT, the F303G mutation primarily reshapes thioester torsional\nconformations, whereas in FaAAT2 with an isoleucine at the corresponding\nposition, the analogous I303G mutation in FaAAT2 alters the Bürgi-Dunitz\nattack geometry. This divergence indicates that mutation-induced selectivity\n37\narises from enzyme-specific reorganization of reactive conformational\nensembles rather than static distance changes alone (Supplementary Fig. 39)”\nhas been added on Page 10 Line 291-307.\n4. The sentence of “The intracellular availability of acyl-CoA and MT affects the\napparent specificity of dual-substrate esterification conducted by AATs, which\ncould be regulated by varying the molar ratio of the dual substrates. To confirm\nthis, we profiled the formations of GB and GA at varying dual-substrate molar\nratios, both wtFaAAT and F302GFaAAT exhibited high exponential curve fitting\nconfidence for GA and GB formation (R2>0.99, Fig. 4f, Supplementary Table\n7). The selectivity for GB formation was improved at all tested dual-substrate\nmolar ratios (Fig. 4g). The selectivity obtained using the strain harboring\nF302GFaAAT reached 98.95% under a 4:1 molar ratio of butyrate-to-geraniol,\nwhich is a 3.12-fold increase compared with the one obtained using the strain\nharboring wtFaAAT under a 1:4 molar ratio of butyrate-to-geraniol (Fig. 4g).\nF302G mutation drastically reduced catalytic efficiency toward acetyl-CoA,\nprecluding reliable kinetic parameter estimation (Fig. 4h, Supplementary Fig.\n35). Profiling the other two mutants at position 302 of FaAAT with enhanced\nGB production titer exhibited improved selectivity when a higher molar ratio of\nbutyrate-to-geraniol was adopted (Supplementary Fig. 32f-h). We observed\npoor soluble expression of both wtFaAAT and F302GFaAAT (Supplementary Fig.\n36a), which likely caused the reduced catalytic activity for GB formation (Fig.\n4f and 4i). Phylogenetic analysis and pairwise sequence alignment revealed\nthat FaAAT2, blasted from NCBI protein database (Supplementary Fig. 36b-\nc), contains an Ile at position 302 and differs from FaAAT by five amino acid\nresidues (Supplementary Fig. 33), showing improved solubility compared with\nFaAAT (Supplementary Fig. 36d). Profiling GB and GA production by the\nstrains expressing wtFaAAT2 and I302GFaAAT2 varying molar ratios of butyrate-\nto-geraniol generated high-confidence exponential curve fitting\n(Supplementary Fig. 37, R²>0.99, Supplementary Table 7) and a GB titer\n(Fig. 4i) approaching the theoretical yield (1.45 g/L, Supplementary Fig. 38).\nThe GB selectivity achieved by the strain harboring I302GFaAAT2 (97.62%) was\nslightly lower than that of F302GFaAAT under a 4:1 molar ratio of butyrate-to-\ngeraniol, but still represented a 4.22-fold increase compared to wtFaAAT2 under\n1:4 molar ratio of butyrate-to-geraniol (Fig. 4j). The enhanced GB selectivity\n38\nobserved with I302GFaAAT2 was consistent with improved catalytic efficiency,\nas confirmed by steady-state kinetic assays (Fig. 4k, Supplementary Fig. 36e-\nf). These results suggest that the dual-substrate ratio control enhanced the\nspecificity of butyrate ME biosynthesis. The kinetic data support a model in\nwhich AAT and its mutants operate via a random sequential bi–bi mechanism\nwithin the cellular environment, allowing both acetyl-CoA and butyryl-CoA to\nindependently access the active site in the presence of geraniol\n(Supplementary Note 1). To dissect the mechanism underlying altered\nsubstrate specificity of mutants, we conducted molecular dynamics (MD)\nsimulations of AAT-dual-substrate complex and analyzed the catalytic\nmechanism-guided substrate atom distance profiles across the catalytic\nprocess (Fig. 4l, Supplementary Note 2). Substitution of 302Phe with Gly\ndestabilized acetyl-CoA, enlarging the contacting distance between geraniol's\noxygen and the acetyl-CoA carbonyl carbon during the nucleophilic attack step\n(D2: Distance 2, Supplementary Video 1 and 2), subsequently reducing the\ncatalytic efficiency as confirmed by steady-state kinetic assays (Fig. 4m). In\ncontrast, F302GFaAAT exhibits a similar trend of distance varying between\ngeraniol's oxygen and the carbonyl carbon of butyrate-CoA (D2) compared with\nwtFaAAT, possibly suggesting the almost unchanged substrate binding affinity\n(Fig. 4n), as confirmed by steady-state kinetic assays (Fig. 4h). The similar\ntrends observed in wtFaAAT2 and I302GFaAAT2 through steady-state kinetic\nassays and MD simulations confirmed that the reduced affinity for acetyl-CoA\nrelative to butyryl-CoA likely leads to the shift in acyl-CoA specificity\n(Supplementary Fig. 39). To validate the combined strategy of protein\nengineering and dual-substrate ratio control for precision ester biosynthesis, we\ncharacterized more substrate pairings: butyryl-CoA with nerol, butanol, and\nbenzyl alcohol; geraniol with propionyl-CoA and benzoyl-CoA; and benzoyl-\nCoA with benzoyl-CoA. Exponential fittings of the product titers on the selected\ndual-substrate molar ratio capitulated high confidence (average R²>0.99)\nacross all dual-substrate pairings (Supplementary Table 7). Modulating molar\nratios of acyl-CoA-to-alcohol from 4:1 to 1:4 with wtAAT and its mutants\nsignificantly enhanced selectivity by 3.71-fold (neryl butyrate), 2.08-fold (butyl\nbutyrate), 3.19-fold (benzyl butyrate), 2.10-fold (geranyl propionate), 60.13-fold\n(geranyl benzoate), and 1.37-fold (benzyl benzoate) (Supplementary Figs. 40-\n39\n45), outperforming the reported AATs under comparable conditions\n(Supplementary Fig. 38). These results demonstrate that dual-substrate molar\nratio modulation adjusts the apparent AAT selectivity, while steric gate residue\nengineering alters the enzyme’s intrinsic specificity, both of which aided\nprecision ester biosynthesis” has been changed to “The intracellular\navailability of acyl-CoA and MT affects the apparent specificity of dual-substrate\nesterification catalyzed by AATs, which can be modulated by varying the molar\nratio of the substrates. To confirm this, we profiled the formations of GB and\nGA at varying dual-substrate molar ratios, both wtFaAAT and F303GFaAAT\nexhibited high exponential curve fitting confidence for GA and GB formation\n(R2>0.99, Fig. 4f, Supplementary Data 3, No. 31-34). The selectivity for GB\nformation was improved at all tested dual-substrate molar ratios (Fig. 4g). The\nstrain harboring F303GFaAAT achieved a GB selectivity of 98.95% under a 4:1\nbutyrate-to-geraniol molar ratio, representing a 3.12-fold (p < 0.001) increase\nrelative to wtFaAAT under a 1:4 ratio; even under 1:4 butyrate-to-geraniol molar\nratio, F303GFaAAT still exhibited a 2.21-fold improvement in GB selectivity\ncompared with wtFaAAT (p < 0.001; Fig. 4g). Consistent with this trend, the\nother two beneficial substitutions at position 303 of FaAAT also showed\nimproved GB selectivity under higher butyrate-to-geraniol ratios\n(Supplementary Fig. 34g-i). To elucidate the enzymatic basis underlying\nthese mutation-enhanced selectivity effects, we next characterized the intrinsic\ncatalytic properties of wtFaAAT and F303GFaAAT in vitro. The F303G mutation in\nFaAAT resulted in no detectable product in the acetyl-CoA/geraniol reaction,\nindicating a loss of enzyme catalytic activity toward acetyl-CoA upon\nintroduction of the F303G mutation under the tested conditions in vitro (Fig. 4h,\nSupplementary Figs. 40-41). In contrast, for the butyryl-CoA/geraniol reaction,\nquantitative kinetic analysis was feasible. The K value showed no significant\nm\nchange upon mutation (1.04-fold difference relative to the wild type, p = 0.66),\nindicating that substrate binding affinity for butyryl-CoA was largely preserved.\nHowever, the catalytic efficiency (k /K ) was significantly reduced to 0.35-fold\ncat m\nof wtFaAAT (p < 0.001) (Fig. 4h, Supplementary Fig. 40). These kinetics are\nconsistent with mutation-induced destabilization of catalytically productive\nacetyl-CoA conformations, arising from altered local hydrogen-bonding\nnetworks involving 284Asn and 378Ser, while largely preserving butyryl-CoA\n40\nbinding (Supplementary Fig. 42). Despite the favorable shift in acyl-donor\npreference conferred by the F303G mutation in FaAAT, the overall catalytic\nperformance of FaAAT remained constrained under cellular conditions,\nprompting us to examine whether enzyme solubility represents an additional\nlimiting factor. We observed poor soluble expression of both wtFaAAT and\nF303GFaAAT (Supplementary Fig. 40a), which likely caused the reduced\ncatalytic activity for GB formation (Fig. 4f and 4i). Phylogenetic analysis and\npairwise sequence alignment revealed that FaAAT2, identified from the NCBI\nprotein database (Supplementary Fig. 40b-c), contains an Ile at position 303\nand differs from FaAAT by five amino acid residues (Supplementary Fig. 36),\nshowing improved solubility compared with FaAAT (Supplementary Fig. 40d).\nProfiling GB and GA production by the strains expressing wtFaAAT2 and\nI303GFaAAT2 varying molar ratios of butyrate-to-geraniol generated high-\nconfidence exponential curve fitting (Supplementary Fig. 43, R²>0.98,\nSupplementary Data 3, No. 39-42) and a GB titer (Fig. 4i) approaching the\ntheoretical yield (1.45 g/L, Supplementary Fig. 44). The GB selectivity\nachieved by the strain harboring I303GFaAAT2 (97.62%) was slightly lower than\nthat of F303GFaAAT under a 4:1 molar ratio of butyrate-to-geraniol, but still\nrepresented a 4.22-fold increase compared to wtFaAAT2 under 1:4 molar ratio\nof butyrate-to-geraniol (p < 0.001) (Fig. 4j). The enhanced GB selectivity\nobserved with I303GFaAAT2 was consistent with altered catalytic behavior, as\nconfirmed by steady-state kinetic assays (Fig. 4k, Supplementary Fig. 40e-f),\nwhich suggests that, in FaAAT2, the apparent acyl-CoA selectivity is governed\nby a combined contribution of catalytic turnover, enzyme solubility, and global\nactive-site interaction networks (Supplementary Fig. 45), rather than being\nsolely dictated by local acyl-CoA binding energetics. To place these enzyme\ndeterminants within a unified catalytic framework, we next examined the\nintrinsic reaction mechanism shared by AAT and its variants. The kinetic data\nare consistent with a sequential bi–bi mechanism for AAT and its variants under\nthe tested conditions, in which geraniol and acyl-CoA form a catalytically\ncompetent ternary complex (Fig. 4I, Supplementary Fig. 46-47). To\nquantitatively address the catalytic mechanism, we have performed ab initio\nQM calculations on an enzyme model that encompasses the core reaction\ncenter (157His in FaAAT, geraniol, and CoA thioester, Supplementary Fig. 48).\ndeprotonation followed by nucleophilic attack on the thioester carbonyl, with a\nrate-determining barrier of 17.6 kcal/mol (ΔHǂ). Notably, the subsequent proton\ntransfer from the imidazolium to thiolate is essentially barrierless, and the\noverall reaction is slightly exergonic (ΔG = -3.1 kcal/mol). The computed energy\nprofile aligns well with the mechanistic scheme in Fig. 4I, suggesting the critical\nrole of pre-organized active-site architecture in transition-state stabilization.\nBuilding on this mechanistic understanding, to validate the combined strategy\nof single-residue engineering and dual-substrate ratio control for precision ester\nbiosynthesis, we characterized more substrate pairings: butyryl-CoA with nerol,\nbutanol, and benzyl alcohol; geraniol with propionyl-CoA and benzoyl-CoA; and\nbenzoyl-CoA with benzoyl-CoA. Exponential fittings of the product titers on the\nselected dual-substrate molar ratio showed high confidence (average R²>0.98\nacross all dual-substrate pairings (Supplementary Data 3, No. 1-30, 47-72).\nModulating molar ratios of acyl-CoA-to-alcohol from 4:1 to 1:4 with varied wtAAT\nand its mutants significantly enhanced selectivity by 3.71-fold (neryl butyrate, p\n< 0.001), 1.94-fold (butyl butyrate, p < 0.001), 3.19-fold (benzyl butyrate, p <\n0.001), 2.10-fold (geranyl propionate, p < 0.001), 60.13-fold (geranyl benzoate,\np < 0.001), and 1.37-fold (benzyl benzoate, p < 0.001) (Supplementary Figs.\n49-54), outperforming the reported AATs under comparable conditions\n(Supplementary Fig. 44). These results support that dual-substrate molar ratio\nmodulation adjusts the apparent AAT selectivity, while critical residue\nengineering alters the AAT’s intrinsic specificity, both of which aided precision\nester biosynthesis” on Page 11 Line 309-391.\nIn Coculture to de novo monoterpenoate ester biosynthesis:\n1. The word of “pyrophosphate” has been changed to “diphosphate” on Page 13\nLine 395.\n2. The sentence of “Additionally, the flux of the downstream module would be\nprimarily determined by the catalytic efficiency of PDPS and MTS. For GPP-\nderived MTs, the titers obtained were varied when MTPS was changed\n(Supplementary Fig. 46b-e). The titer of linalool was 21.04-fold higher relative\nto α-terpineol using optimized strain and induction conditions. Protein\n42\nengineering of MTS to enhance their activities was therefore critical for\nimproving MT biosynthesis. In lavandulol biosynthesis, a variant\npyrophosphatase (F118D-H182CEcRdgB) engineered through structure-guided\nmutagenesis enhanced the titer of lavandulol by 7.64-fold (Supplementary Fig.\n52)” has been changed to “For GPP-derived MTs, changing MTSs in the\ndownstream biosynthetic pathway resulted in evident differences in product\ntiters (Supplementary Fig. 55b-e). Under optimized strain and induction\nconditions, the titers of linalool, geraniol, α-terpineol, borneol, and nerol\nreached 3.33 g/L, 1.18 g/L, 198.98 mg/L, 3.84 mg/L, and 224.42 mg/L,\nrespectively. The titer of linalool achieved was 16.74-fold higher (p < 0.001)\nthan that of α-terpineol. This disparity is attributable to differences among MTSs\nin both catalytic efficiency toward GPP and effective expression levels24,\nresulting in varied production titers of MTs. Besides, protein engineering of MTS\nto enhance their activities was therefore critical for improving MT biosynthesis.\nIn lavandulol biosynthesis, we screened a panel of E. coli endogenous\nphosphatases and identified EcRdgB as the most active enzyme toward\nlavandulyl pyrophosphate. We further performed structure-guided multiple-\nround single-site mutagenesis to enhance its catalytic efficiency, obtaining the\nengineered variant F118D-H182CEcRdgB, which significantly improved the\nhydrolysis of lavandulyl pyrophosphate and consequently increased the\nlavandulol titer by 7.64-fold (p < 0.001) (Supplementary Fig. 61)” on Page 14\nLine 408-423.\n3. The sentence of “With the above-achieved MT-producing strains, we coupled\nthe MT-producing strains with an esterification module containing AATs with\ndefined substrate specificity, producing linalyl acetate (LA) and GB” has been\nchanged to “By combining the above MT-producing strains with an\nesterification module containing AATs with defined substrate specificity, we\nproduced natural linalyl acetate (LA) and GB via microbial biosynthesis. LA and\nGB have high industrial relevance: LA is the dominant high-value fragrance\ncomponent in lavender essential oil and is widely used in perfumery, while GB\nis a commercially important fruity/floral flavor compound used in food, beverage,\nand fragrance formulations27, 46. Besides, their biosynthetic precursors—linalool\nand geraniol—are among the monoterpene alcohols with the higher achievable\n43\nmicrobial titers in this study and other extensive studies, enabling a\nrepresentative demonstration of MTEs biosynthesis” on Page 14 Line 429-437.\n4. The sentence of “(p < 0.001)” has been added on Page 15 Line 447.\n5. The sentence of “Subsequent optimizing the inoculation ratio of both strains\nand using I302GFaAAT2 in the ester-producing strain led to a titer of 0.32 g/L GB,\nwith a GB selectivity of 97.81% (Fig. 5c, Supplementary Fig. 55)” has been\nchanged to “Because wtFaAAT provided a consistent baseline for optimizing\ncoculture conditions, we subsequently replaced it with F303GFaAAT and\nI303GFaAAT2 in the ester-producing strain, which increased the GB titer to 0.32\ng/L with a selectivity of 97.81% (Fig. 5e, Supplementary Fig. 64)” on Page 15\nLine 454-457.\n6. The sentence of “To scale up and validate the stability of coculture engineering\nfor ME biosynthesis” has been changed to “To scale up the coculture process\nfor ME biosynthesis” on Page 16 Line 463.\n7. The word of “our” has been changed to “the” on Page 16 Line 476.\nIn Discussion:\n1. The sentence of “enabling the generation of 71 MEs, representing 61.2% of the\ntheoretical chemical structure space of esters (Supplementary Fig. 1)” has\nbeen changed to “enabling the generation of 71 MEs, representing 61.2% of\nthe theoretical chemical structure space of MEs, which was the number of the\nsubset of monoterpene–acyl ester combinations tested in this study\n(Supplementary Fig. 1)” on Page 16 Lines 485-487.\n2. The sentence of “The strategy of structure-guided steric gate residue\nengineering improved selectivity of FaAAT toward longer-chain acyl-CoAs such\nas butyryl-CoA, achieving >99% selectivity for GB biosynthesis (Fig. 4i). By\ncombining rational protein design and dual-substrate molar ratio control, this\napproach proved broadly generalizable across a wide range of alcohol-to-acyl-\nCoA pairing substrates, highlighting the necessity of fine-tuning AAT selectivity\nto enhance ester biosynthesis in vivo” has been changed to “By combining\nrational protein engineering and dual-substrate molar ratio control, this\napproach proved broadly generalizable across a wide range of alcohol-to-acyl-\nCoA pairing substrates, highlighting the necessity of fine-tuning AAT selectivity\n44\nto enhance the specificity of ester biosynthesis in vivo (Supplementary Fig.\n49-54). Interestingly, comparative analysis of FaAAT and its close homolog\nFaAAT2 shows that although single-residue engineering at position 303\nconsistently modulates acyl-donor selectivity, the underlying enzymatic kinetic\nand mechanistic basis is not conserved across diverse AAT scaffolds. In FaAAT,\nthe F303G mutation biases a dominant reaction coordinate, leading to\nconcordant changes in energetics, dynamics, and kinetics, whereas in FaAAT2\nthe homologous I303G mutation acts within a distributed energetic network,\npartially decoupling binding and selectivity. Thus, single-residue engineering is\ntransferable at the strategy level, particularly for tuning discrimination between\nstructurally close C2-C4 acyl donors, but the mechanistic differences in\nselectivity cannot be unified by a single reaction-coordinate model for varied\nAATs” on Page 17 Line 501-515.\n3. The word of “Critically” has been changed to “Besides” on Page 17 Line 517.\nIn Methods:\n1. The relevant data website link has been updated.\n2. The sentence of “When exporting the extracted ion chromatograms (EIC) based\non the information provided in Supplementary Table 3-4 and Supplementary\nData 2, fixed offsets were applied to the Y-axis to distinguish the\nchromatographic traces of different samples for clearer visualization, and the\nX-axis range was constrained to focus on the regions of interest. It should be\nnoted that these adjustments were made solely to optimize the graphical\npresentation, and all original raw data remain complete and unaltered, which\nare fully provided in Supplementary Data 9. GC-MS spectra exported from the\ninstrument software (MassHunter Workstation Software, Version B.07.00). For\neach spectrum, the exported table (CSV format, Supplementary Data 10) was\nparsed by skipping the first two metadata/header lines and extracting the m/z\nand ion-count columns. Peak intensities were normalized to relative intensity\n(%) by setting the base peak to 100 (intensity/max intensity × 100). The\nnormalized spectra were then rendered as mass spectra with uniform peak-line\n45\nwidth, and the 15 most intense ions were annotated by their m/z values.” has\nbeen added on Page 20 Lines 626-640.\n3. The sentence of “(the alcohol concentration is maintained at 1 g/L of final\nconcentration, with acid employed as a co-substrate at a fixed molar ratio. The\nacid-to-alcohol molar ratio is set at 1:4 for screening butyl butyrate, benzyl\nbutyrate, and geranyl propionate, and at 2:1 for all other combinations)” has\nbeen added on Page 24 Lines 782-785.\n4. The sentence of “The docking pose, which is consistent with the hypothesized\ncatalytic mechanism, was used as the initial structure for MD simulations. The\nGeneral AMBER Force Field (GAFF2) was applied to assign topology and\nparameters for ligand molecules. All other simulation settings were consistent\nwith those described above. Root mean square deviation (RMSD) analysis was\nused to monitor conformational dynamics of active site residues during the\ntrajectory. Where applicable, binding free energies were calculated using the\nGmx_MMPBSA tool. As shown in Fig. 4l-n, the three-step catalytic distances\nof the atoms in 156His of AAT or its mutants, acyl-CoA, and geraniol were\nmeasured using Pymol, and the pse format video files of each 100-ns run for\neight enzyme-ligand complexes generated in PyMOL were provided, and the\nraw data are available via Zenodo at https://doi.org/10.5281/zenodo.17279732”\nhas been changed to “Initial coordinates for MD simulations were generated\nfrom molecular docking to provide reasonable starting configurations of\nenzyme-ligand complexes. To avoid bias from any single docking pose, all\nsimulations were subsequently allowed to freely relax and sample\nconformational space without geometric restraints, and each MD simulation\nwas carried out for 100 ns. To avoid bias from any single docking pose, all\nsimulations were subsequently allowed to freely relax and sample\nconformational space without geometric restraints. The General AMBER Force\nField (GAFF2)79 was used to parameterize ligand molecules, and all other\nsimulation settings were identical to those described above. MD trajectories\nwere analyzed to characterize the dynamic behavior of active-site regions and\nsubstrate conformations. Root mean square deviation (RMSD) analysis was\nused solely to assess overall trajectory stability rather than as a proxy for\ncatalytic competence. Where applicable, binding free energy estimates were\n46\nobtained using the Gmx_MMPBSA tool80. Representative trajectories and raw\nsimulation data are available via Zenodo\n(https://doi.org/10.5281/zenodo.18308115)” on Page 27 Line 906-920.\n5. The sentence of “Quantum mechanical (QM) calculations were performed using\nGaussian 1681. Near-attack conformations were extracted from pre-reaction\nstate (PRS) MD ensembles and converted into Gaussian input files82. The QM\ncluster models consisted of truncated CoA thioester and geraniol substrates\ntogether with key active-site residues, resulting in systems of approximately 80\natoms. Each residue was truncated at the Cα atom and capped with hydrogen\natoms to satisfy valence requirements. Reaction pathways were investigated\nusing the B3LYP-D3(BJ)/6-31+G(d,p) level of theory with implicit solvation\nmodeled by SMD (methanol). Optimized geometries and corresponding XYZ\ncoordinates are provided, and all raw QM data are available via Zenodo\n(https://doi.org/10.5281/zenodo.18308115)” has been added on Page 27 Line\n923-932.\nIn Reference:\n1. The relevant literature has been updated, and the format has been uniformly\nrevised.\nIn Author contributions:\nIn response to the revised experimental design and additional analyses required\nduring manuscript revision, we have updated the author list and corresponding\ncontributions accordingly. All authors have reviewed and approved these changes.\n1. The sentence of “Z.C. conducted molecular dynamics simulations. C.Z.\nassisted with data collection and experimental execution. B.W. provided\nresources, performed formal analysis, and manuscript revision. M.Y. supported\ndata collection and experimental execution. K.Z. contributed to data analysis\nand manuscript editing” has been changed to “B.W. conducted the AAT-\ncontaining strain isolation, performed formal analysis, and manuscript revision.\nC.Z., and M.Y. supported data collection and experimental execution. K.Z.\n47\ncontributed to data analysis and manuscript editing. Z.C., P.N., and Y.Z.\nconducted molecular docking, dynamics simulation, and quantum mechanical\ncalculations” on Page 36 Line 1315-1319.\nIn Competing interests:\n1. The sentence of “X.M., D.Y., and H.L. are inventors on patent application\n(application number CN116926130A and CN119320806A)” has been\nchanged to “X.M., D.Y., and H.L. are inventors on patent applications\n(CN202511509374.0, CN202511621358.0, and CN202511621364.6)” on Page\nIn Figure:\n1. The sentence of “Microbial wheeled biosynthesis of monoterpene esters (MEs)”\nhas been changed to “Dual-substrate microbial biosynthesis of monoterpene\nesters (MEs)” on Page 38 Line 1332.\n2. The word of “pf” has been changed to “of” on Page 38 Line 1339.\n3. The sentence of “AATs: alcohol acyltransferases; CaBACT: butyrate-\nacetoacetate CoA-transferase from Clostridium acetobutylicum; TtACSL: ATP-\ndependent fatty/aromatic acid-CoA ligase from Thauera terpenica; MT:\nmonoterpenol; FvAAT: AAT from Fragaria vesca; FaAAT: AAT from Fragaria x\nananassa; LiAAT4: AAT4 from Lavandula x intermedia; ScAAT1: AAT1 from S.\ncerevisiae BY4741; ScEEB1: medium-chain fatty acid ethyl ester\nsynthase/esterase 1 from S. cerevisiae BY4741; ScEHT1-1: medium-chain\nfatty acid ethyl ester synthase/esterase 2 from S. cerevisiae BY4741; ScEHT1-\n2: medium-chain fatty acid ethyl ester synthase/esterase 2 from S. cerevisiae\nLBGJP-510-1; LaAAT1: AAT1 from Lavandula angustifolia; MdAAT: AAT from\nMalus domestica; CsCAT: chloramphenicol O-acetyltransferase from\nClostridium scindens ATCC 35704; ScAAT2: AAT2 from S. cerevisiae BY4741;\nMsBABT: benzyl alcohol acyltransferase from Magnolia sinica; PhBABT:\nbenzyl alcohol acyltransferase from Petunia × hybrida; PmBABT: benzyl\nalcohol acyltransferase from Prunus mume” has been added on Page 39 Line\n1362-1373.\n48\n4. The sentence of “Source data are provided as a Source Data file” has been\nadded on Page 39 Lines 1373, 1387-1388, 1412, 1436.\n5. The sentence of “STD refers to chemically synthesized standards” has been\nadded on Page 40 Line 1387.\n6. The sentence of “C2-C5” has been changed to “C2-C4” on Page 40 Line 1377,\n1381.\n7. The sentence of “C1-C3” has been changed to “C2-C4” on Page 40 Line 1379.\n8. The sentence of “Molecular docking” has been changed to “Representative\nposes” on Page 41 Line 1391, 1393.\n9. The sentence of “In box plots, center lines indicate medians, box bounds\nindicate the 25th-75th percentiles, whiskers indicate minima and maxima (0th-\n100th percentiles), and circles indicate individual five data points” has been\nadded on Page 41 Lines 1397-1399, 1405-1407.\n10. The sentence of “#p=0.66; ***p< 0.001” has been added on Page 41 Line 1406.\n11. The sentence of “l, Proposed catalytic mechanism of FaAAT indicating three\ncritical distances: alcohol deprotonation (D1), nucleophilic attack (D2), and\nintermediate collapse (D3). m-n, Molecular dynamics simulation quantifying\n100 nm catalytic distances between His156 and substrate atoms in acetyl-CoA\n(m) and butyryl-CoA (n) complexes of wtFaAAT and F302GFaAAT. MD\nsimulations were run for 100 ns, and individual snapshots were analyzed to\nquantify distance fluctuations” has been changed to “l, Proposed catalytic\nmechanism of FaAAT with numbered key steps, including alcohol\ndeprotonation, nucleophilic attack on the acyl-CoA thioester, and collapse of\nthe tetrahedral intermediate to form the ester product” on Page 41 Line 1409-\n1412.\n12. The sentence of “Data are mean ± s.e.m.; n=5 biological replicates” has been\nadded on Page 42 Line 1417, 1422-1423.\nIn Supplementary information and files:\n1. The needed Supplementary Figs have been updated and reordered.\n2. The Supplementary Figs of “5, 13, 17, 19, 23, 34, 36, 40, 50, 55” has been\nrevised.\n49\n3. The Supplementary Figs of “27, 28, 30, 31, 35, 38, 39, 42, 45, 46, 47, 48” has\nbeen added.\n4. The Supplementary Table 1, 5, 7, 8, 9, 10, 11, 12 have been revised to\nSupplementary Data 1, 2, 3, 4, 5, 6, 7, 8.\n5. The Figures in Supplementary Note. 1 have been revised to Supplementary\nFigs 45, 46.\n6. The Supplementary Video 1 has been deleted to avoid using the distance-\nbased illustrations to elucidate catalytic mechanism.\n7. The Original images of Uncropped scans of Supplementary Fig. 40a-b have\nbeen added.\n8. The sentence of “Source data are provided as a Source Data file” has been\nadded in the appropriate location.\n9. The sentence of “Supplementary References” has been added in the end of\nSupplementary Data 5.\n10.\nOther revisions:\n1. The residue numbering has been corrected throughout the manuscript.\n2. The numbering of all the Figures, Supplementary Figs and Tables has been\nuniformly revised throughout the manuscript.\n50"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors gave a point-to-point response to the comments and addredded my concerns. The manuscript is now recommneded for publication in Nature Communications.", "reviewer_2": "In the revised manuscript, the authors have adequately addressed my concerns regarding the investigation of the enzyme mechanism. The manuscript is now clearer than the previous version. I am agreeable to the acceptance of the current version.", "reviewer_3": "The authors have made quite extensive changes to the manuscript, which have adequately addressed my previous comments. I now support publication."}, "author_response": ""}]

Decision letter