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. 2026 Jan 4;25:34. doi: 10.1186/s12934-025-02915-6

Enhanced L-tyrosine production in Bacillus amyloliquefaciens through alleviation of feedback Inhibition and coordinated pathway expression

Ziyue Zhao 1,3, Anying Ji 2,3,✉, Can Song 1, Zhengyuan Zhai 1, Xuetuan Wei 3,✉
PMCID: PMC12866237  PMID: 41486272

Abstract

Background

L-tyrosine is a commercially significant compound with broad applications in the food and pharmaceutical industries. The development of efficient microbial cell factories for its production is of great interest. This study describes the engineering of the food-safe Bacillus amyloliquefaciens as a host chassis for enhanced L-tyrosine biosynthesis.

Results

To overcome the key regulatory constraint of L-tyrosine feedback inhibition, a mutant prephenate dehydrogenase (Eco-TyrAM53I/A354V) from Escherichia coli was introduced. This intervention increased the L-tyrosine titer to 446.32 mg/L, representing a 53% improvement over the control strain. The underlying mechanism was also investigated. Subsequent optimization of expression elements (promoter, 5′-UTR, and terminator) generated strain A8, which produced 560.57 mg/L of L-tyrosine. Additionally, the co-expression of key genes Eco-tyrAM53I/A354V and Bao-aroA significantly enhanced metabolic flux, boosting the titer to 1104.02 mg/L, a 257% increase relative to the control.

Conclusions

This work significantly improves L-tyrosine production in a food-grade B. amyloliquefaciens chassis and provides a suite of efficient genetic tools for strain development. The findings and engineered systems established here offer a robust platform for advancing high-level microbial production of L-tyrosine, with strong potential for industrial application.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12934-025-02915-6.

Keywords: L-tyrosine, Prephenate dehydrogenase, Protein engineering, Expression elements, Bacillus amyloliquefaciens

Introduction

L-tyrosine, an essential aromatic amino acid, plays key roles in neurodevelopment, memory enhancement, and antioxidant activity [1–3]. It is widely used in the food, cosmetic, and pharmaceutical industries. Additionally, L-tyrosine can be converted into melanin via a single enzymatic reaction, making it a versatile functional biomaterial for applications in drug delivery, UV absorption, cation exchange, and amorphous semiconductors [4]. Furthermore, L-tyrosine serves as a biosynthetic precursor for various high-value natural products, including L-DOPA, as well as salvianic acid A, resveratrol, hydroxytyrosol, and benzylisoquinoline alkaloids [5–8]. Therefore, the development of efficient production methods for L-tyrosine is of significant importance.

Compared to chemical synthesis and enzymatic conversion, microbial fermentation offers significant advantages including environmental friendliness, sustainability, low cost, and ease of operation [9–11]. As a result, it has become the primary method for L-tyrosine production. The biosynthetic pathway of L-tyrosine has been systematically characterized in various host strains, such as E. coli, Corynebacterium glutamicum, Saccharomyces cerevisiae, Bacillus licheniformis, and B. amyloliquefaciens [12–14]. The synthesis of L-tyrosine begins with phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P) produced by the central carbon metabolism. These precursors are converted to the key intermediate chorismate, then to prephenate, and finally to L-tyrosine via oxidative decarboxylation and transamination reactions.

While metabolic engineering has markedly improved L-tyrosine production in microbial hosts, further yield enhancement remains crucial for commercial viability [15, 16]. Studies indicate that the oxidative decarboxylation of prephenate to 4-hydroxyphenylpyruvate is a key rate-limiting step in L-tyrosine biosynthesis, constraining the increase in L-tyrosine yield [17]. This reaction is catalyzed by dehydrogenases from the TyrA protein family, including prephenate dehydrogenase (PDH) from enteric bacteria, arogenate dehydrogenase from plants, and the dual-substrate-utilizing cyclohexadienyl dehydrogenase [18, 19]. Notably, among these enzymes, PDH is the most widely used in L-tyrosine biosynthesis, yet its activity is subject to strict feedback inhibition by the pathway end-product, L-tyrosine. Previous studies have shown that the addition of 1 mM L-tyrosine significantly reduced the activity of wild-type TyrA from 72.4 U/g to 37.6 U/g [20]. This result directly demonstrates strong feedback inhibition of TyrA by L-tyrosine. In contrast, the engineered mutant TyrAM53I/A354V, obtained through directed evolution, showed no significant change in enzymatic activity in the presence of 0 µM or 100 µM L-tyrosine [20]. The significant potential of this research direction is demonstrated by the dramatically enhanced yields achieved through expressing feedback-insensitive enzymes, such as cyclohexadienyl dehydrogenase TyrC in E. coli (6.6-fold) and the mutant Eco-TyrAM53I/A354V in B. licheniformis (4.5-fold) [17, 21]. Therefore, identifying or rationally designing dehydrogenases with high catalytic activity and resistance to feedback inhibition has become a central metabolic engineering strategy for enhancing L-tyrosine production.

In this study, B. amyloliquefaciens was selected as a host for L-tyrosine production. As a generally recognized as safe (GRAS) organism, it presents several advantages, including low cultivation costs, rapid growth, strong stress tolerance, and efficient protein secretion, which collectively support its potential as a robust microbial platform for industrial biotechnology [22]. Notably, B. amyloliquefaciens has already been successfully engineered to produce a range of high-value compounds, including SAM, iturin A, and spermidine [23–25]. However, the endogenous L-tyrosine pathway in this bacterium is inefficient, primarily limited by the suboptimal activity and regulation of prephenate dehydrogenase. To overcome this limitation, feedback inhibition was relieved through point mutagenesis of the TyrA from E. coli. Static regulatory strategies were then applied to optimize the expression of key genes, which were co-expressed with DAHP synthase to enhance the supply of precursors. The resulting high-activity, feedback-resistant TyrA variants and tailored expression elements provide essential genetic tools for constructing advanced B. amyloliquefaciens cell factories.

Materials and methods

Strains, plasmids, and cultivation conditions

The principal strains and plasmids used in this study are listed in Table 1 and Table S1, respectively. B. amyloliquefaciens A1 was used as the parental strain, and E. coli DH5α was used for cloning. The pHY300PLK was used for gene over-expression. The main primers are provided in Table S2.

Table 1.

Strains used in this study

Number Strains Description Source
A1 Bacillus amyloliquefaciens Wild type Stored in lab
A2 A1/pHY300PLK A1 with pHY300PLK This study
A3 A1/P43-Eco-tyrA A1 with pHY-P43-Eco-tyrA This study
A4 A1/P43-Eco-tyrAM53I A1 with pHY-P43-Eco-tyrAM53I This study
A5 A1/P43-Eco-tyrAA354V A1 with pHY-P43-Eco-tyrAA354V This study
A6 A1/P43-Eco-tyrAM53I/A354V A1 with pHY-P43-Eco-tyrAM53I/A354V This study
A7 A1/PgisB-Eco-tyrAM53I/A354V A1 with pHY-PgisB-Eco-tyrAM53I/A354V This study
A8 A1/PytzE- Eco-tyrAM53I/A354V A1 with pHY-PytzE-Eco-tyrAM53I/A354V This study
A9 A1/PbacA-Eco-tyrAM53I/A354V A1 with pHY-PbacA-Eco-tyrAM53I/A354V This study
A10 A1/Pbay-Eco-tyrAM53I/A354V A1 with pHY-Pbay-Eco-tyrAM53I/A354V This study
A11 A1/PHpaII-Eco-tyrAM53I/A354V A1 with pHY-PHpaII-Eco-tyrAM53I/A354V This study
A12 A1/PsrfA-Eco-tyrAM53I/A354V A1 with pHY-PsrfA-Eco-tyrAM53I/A354V This study
A13 A1/PluxS-Eco-tyrAM53I/A354V A1 with pHY-PluxS-Eco-tyrAM53I/A354V This study
A14 A1/Pylb-Eco-tyrAM53I/A354V A1 with pHY-Pylb-Eco-tyrAM53I/A354V This study
A15 A1/PU12-Eco-tyrAM53I/A354V A1 with pHY-PU12-Eco-tyrAM53I/A354V This study
A16 A1/PRBS6-Eco-tyrAM53I/A354V A1 with pHY-PRBS6-Eco-tyrAM53I/A354V This study
A17 A1/P382-Eco-tyrAM53I/A354V A1 with pHY-P382-Eco-tyrAM53I/A354V This study
A18 A1/PykzA-Eco-tyrAM53I/A354V A1 with pHY-PykzA-Eco-tyrAM53I/A354V This study
A19 A1/P43-PylB-Eco-tyrAM53I/A354V A1 with pHY-P43-PylB-Eco-tyrAM53I/A354V This study
A20 A1/P03468-Eco-tyrAM53I/A354V A1 with pHY-P03468-Eco-tyrAM53I/A354V This study
A21 A1/P43-Eco-tyrAM53I/A354V-BSUT1 A1 with pHY-P43-Eco-tyrAM53I/A354V-BSUT1 This study
A22 A1/P43-Eco-tyrAM53I/A354V-BSUT2 A1 with pHY-P43-Eco-tyrAM53I/A354V-BSUT2 This study
A23 A1/P43-Eco-tyrAM53I/A354V-BSUT3 A1 with pHY-P43-Eco-tyrAM53I/A354V-BSUT3 This study
A24 A1/P43-Eco-tyrAM53I/A354V-ECOT4 A1 with pHY-P43-Eco-tyrAM53I/A354V-ECOT4 This study
A25 A1/P43-Eco-tyrAM53I/A354V-ECOT5 A1 with pHY-P43-Eco-tyrAM53I/A354V-ECOT5 This study
A26 A1/P43-Eco-tyrAM53I/A354V-ECOT6 A1 with pHY-P43-Eco-tyrAM53I/A354V-ECOT6 This study
A27 A1/P43-UTR1-Eco-tyrAM53I/A354V A1 with pHY-P43-UTR1-Eco-tyrAM53I/A354V This study
A28 A1/P43-UTR2-Eco-tyrAM53I/A354V A1 with pHY-P43-UTR2-Eco-tyrAM53I/A354V This study
A29 A1/P43-UTR3-Eco-tyrAM53I/A354V A1 with pHY-P43-UTR3-Eco-tyrAM53I/A354V This study
A30 A1/P43-UTR4-Eco-tyrAM53I/A354V A1 with pHY-P43-UTR4-Eco-tyrAM53I/A354V This study
A31 A1/P43-UTR5-Eco-tyrAM53I/A354V A1 with pHY-P43-UTR5-Eco-tyrAM53I/A354V This study
A32 A1/P43-UTR6-Eco-tyrAM53I/A354V A1 with pHY-P43-UTR6-Eco-tyrAM53I/A354V This study
A33 A1/PgisB-Eco-tyrAM53I/A354V-RBS-Bao-aroA A1 with pHY-PgisB-Eco-tyrAM53I/A354V-RBS-Bao-aroA This study
A34 A1/PgisB-Eco-tyrAM53I/A354V-Linker-Bao-aroA A1 with pHY-PgisB-Eco-tyrAM53I/A354V-Linker-Bao-aroA This study
A35 A1/PgisB-aroA A1 with pHY-PgisB-aroA This study
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Construction of recombinant plasmids and engineered strains

The overexpression vectors and strains were constructed according to the method described by Jiang et al. [23]. Using the Eco-tyrA overexpression strain as an example, the construction procedure was as follows. The tyrA gene from E. coli MG1655, the P43 promoter from Bacillus subtilis 168, and the TamyL terminator from B. licheniformis WX-02 were fused by overlap extension PCR. The resulting fragment was cloned into the pHY300PLK vector using restriction digestion and ligation. The constructed plasmid was subsequently introduced into E. coli via chemical transformation for propagation and verified by DNA sequencing. Finally, the confirmed recombinant plasmid was electroporated into the wild-type B. amyloliquefaciens to generate the overexpression strain A3. All other strains were constructed following the same procedure.

Detection of L-tyrosine

L-tyrosine was quantified according to the method described by Ji et al. [12]. Briefly, 1 mL of fermentation broth was acidified with 0.6 mL of 1 M HCl and agitated for 1 h. After centrifugation, the supernatant was filtered and analyzed by HPLC (Agilent 1100) using a ZORBAX Eclipse XDB-C18 column (4.6 mm × 250 mm, 5 μm) with UV detection at 280 nm. The mobile phase consisted of 10% methanol and 90% sodium acetate (100 mM, pH 4.0) at a flow rate of 0.6 mL/min. Quantification was based on peak area comparison with an L-tyrosine standard.

Homologous modeling

The amino acid sequence of TyrA from E. coli was submitted to SWISS-MODEL for homology-based tertiary structure prediction [26]. The optimal model was selected based on the highest sequence identity, Global Model Quality Estimation (GMQE), and Qualitative Model Energy Analysis (QMEAN) scores. The three-dimensional structures of the substrate molecules (chorismate, prephenate, and L-tyrosine) were retrieved from the PubChem database.

Molecular docking

Molecular docking was performed using AutoDock Vina 1.2.3 [27]. The TyrA protein model was prepared by removing water molecules, ions, and small ligands, followed by adding hydrogen atoms and gasteiger charges. The processed model was saved in PDBQT format. A docking grid was defined to enclose the active site of the TyrA. It measures 20 × 20 × 20 Å and is used to define the search space for the ligand. The conformation with the highest score and lowest predicted binding energy was selected as the most reliable complex structure. For each complex structure, at least three independent docking runs were conducted. Specifically, the binding energy were − 5.374 kcal/mol for the TyrAWT/L-tyrosine complex, -5.374 kcal/mol for the TyrAWT/Prephenate complex, -4.864 kcal/mol for the TyrAM53I/A354V/Prephenate complex, and − 5.181 kcal/mol for the TyrAM53I/A354V/L-tyrosine complex. Protein-ligand interactions were analyzed using the PLIP web server (https://plip-tool.biotec.tu-dresden.de/plip-web/plip/index), and key residues were visualized using PyMOL.

Molecular dynamics simulation

Molecular dynamics (MD) simulations were performed on the solvated and charge-neutralized protein-ligand complex [28]. The system was simulated for 100 ns at a constant temperature of 313.15 K. Trajectory analysis was conducted to quantitatively evaluate the dynamic conformational properties of the complex. Two key metrics were examined: root mean square deviation (RMSD) for overall conformational stability, and root mean square fluctuation (RMSF) for residue-level flexibility. Furthermore, MM/GBSA analysis was performed on the docked complexes to further determine their binding affinities.

Statistical analysis

All experiments were conducted with three biological replicates, each comprising three technical replicates. Data are presented as mean ± standard deviation. Statistical significance was assessed using SPSS 20.0, and graphs were generated with GraphPad Prism 8.

Results and discussion

Relief of feedback inhibition enhances L-tyrosine production

In B. amyloliquefaciens, prephenate is converted to L-tyrosine in a two-step pathway. It is first oxidized to 4-hydroxyphenylpyruvate by prephenate dehydrogenase (TyrA), which is then aminated to form L-tyrosine. TyrA from E. coli is a bifunctional enzyme with both chorismate mutase and prephenate dehydrogenase activities [15]. This enables the sequential conversion of chorismate to prephenate and then to 4-hydroxyphenylpyruvate, which is subsequently aminated to form L-tyrosine (Fig. 1A). However, the wild-type TyrA is subject to potent feedback inhibition by L-tyrosine. A previously reported feedback-resistant mutant (TyrAM53I/A354V) was identified in E. coli through error-prone PCR [20]. It remains unknown whether the E. coli-derived TyrAM53I/A354V mutant is effective in B. amyloliquefaciens. To test this gene in B. amyloliquefaciens, the wild-type tyrA gene from E. coli, and corresponding mutations (M53I, A354V and M53I/A354V) were overexpressed in B. amyloliquefaciens, generating strains A3, A4, A5, and A6, respectively. Therein, the strain A3 produced 380.67 mg/L L-tyrosine, a 30% increase over the control strain A2, and the double mutant strain A6 exhibited the highest L-tyrosine titer of 446.32 mg/L, representing a 53% increase over A2 (Fig. 1B). These results indicate that the TyrAM53I/A354V variant effectively alleviates feedback inhibition and enhances L-tyrosine production in B. amyloliquefaciens without impairing cell growth (Fig. 1C).

Fig. 1.

Fig. 1

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Effect of releasing feedback inhibition on L-tyrosine titer and cell growth. A Schematic diagram of the L-tyrosine biosynthetic pathway and target genes. PEP, Phosphoenolpyruvate; E4P, Erythrose 4-phosphate, DAHP, 3-deoxyd-arabino-heptulosonic acid 7-phosphate, SHK, shikimate; CHA, chorismate; PHE, prephenate; 4HPP, 4-hydroxyphenylpyruvate; TYR, L-tyrosine. B Effect of releasing feedback inhibition on L-tyrosine production. C Effect of releasing feedback inhibition on cell growth. * (p < 0.05) and ** (p < 0.01) indicate the significance levels compared with the control

Binding mode analysis of TyrAM53I/A354V in enhancing L-tyrosine production through molecular docking

Studies have shown that changes in protein-ligand interactions can significantly affect enzyme activity [29, 30].To elucidate the molecular mechanism by which the TyrAM53I/A354V mutant enhances L-tyrosine production, a comparative analysis of ligand-binding interactions between the wild-type and mutant proteins was conducted using molecular docking. The tertiary structure of TyrA was generated by homology modeling with SWISS-MODEL, and the model quality was evaluated (Fig. S1). Molecular docking of TyrAWT and TyrAM53I/A354V with substrates (chorismate and prephenate) and the inhibitor (L-tyrosine) was performed using AutoDock Vina 1.2.3. As shown in Fig. 2A–B, the binding mode with chorismate was significantly altered in the mutant, where newly formed hydrogen bonds with Y (Tyrosine) 83, R (Arginine) 9, and E (Glutamic acid) 86 were observed. Combining MM/GBSA analysis, it was found that the binding free energy was decreased from − 13.41 to −30.59 kcal/mol (Table S3), indicating enhanced binding affinity for chorismate. Similarly, in the interaction with prephenate, additional hydrogen bonds at residues Q109 (Glutamine), Q253, and V (Valine) 150 were identified in the mutant (Fig. 2C–D). Combining MM/GBSA analysis, it was found that the binding free energy was further reduced from − 4.39 to − 4.64 kcal/mol (Table S3), suggesting a strengthened binding capability for prephenate. In contrast, the hydrogen bonding network with the inhibitor L-tyrosine was notably weakened in the mutant (Fig. 2E–F). Combining MM/GBSA analysis, it was found that the binding free energy was increased from − 24.33 to − 16.44 kcal/mol (Table S3), reflecting a reduced binding affinity for L-tyrosine and consequently a decreased sensitivity to feedback inhibition. In summary, the TyrAM53I/A354V mutant is proposed to optimize substrate binding preference by simultaneously strengthening interactions with the substrates (chorismate and prephenate) and weakening the binding to the inhibitor (L-tyrosine). This shift alleviates feedback inhibition and improves catalytic efficiency, ultimately leading to enhanced L-tyrosine accumulation.

Fig. 2.

Fig. 2

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Structural mechanism underpinning enhanced L-tyrosine production by the TyrAM53I/A354V mutant. A 3D interaction diagram of TyrAWT/Chorismate complex. B 3D interaction diagram of TyrAM53I/A354V/Chorismate complex. C 3D interaction diagram of TyrAWT/Prephenate complex. D 3D interaction diagram of TyrAM53I/A354V/Prephenate complex. E 3D interaction diagram of TyrAWT/L-tyrosine complex. F 3D interaction diagram of TyrAM53I/A354V/L-tyrosine complex. Magenta, chorismate; blue, NAD+; green, prephenate; orange, L-tyrosine

Conformational dynamics of TyrAM53I/A354V in enhancing L-tyrosine production through molecular dynamics simulations

It has been established that molecular dynamics simulations can visualize the dynamics of protein-ligand binding, offering key mechanistic insights into changes in enzyme activity [31, 32]. To further investigate the dynamic binding properties, 100 ns molecular dynamics (MD) simulations were performed on the enzyme-ligand complexes. Meanwhile, multiple analytical metrics consistently demonstrated that the mutations significantly stabilized the enzyme-substrate complex and enhanced its binding affinity. Root-mean-square deviation (RMSD) analysis revealed that the TyrAM53I/A354V/Prephenate complex exhibited lower conformational fluctuation and achieved a stable equilibrium faster than the wild-type, indicating enhanced structural stability (Fig. 3A). According to the molecular docking results, the key amino acid residues of the substrate prephenate binding pocket are mainly distributed in regions 197–203, 294–303, 302–306, and 350–362, among which ARG299, ARG294, and TYR285 play crucial roles in substrate binding (Fig. 2B). Molecular dynamics simulations revealed that the root mean square fluctuation (RMSF) values of the mutant in these regions were lower than those of the wild type, indicating that the mutation enhanced local rigidity, which is beneficial for stabilizing the substrate binding conformation (Fig. 3C). Furthermore, the overall RMSF analysis demonstrated that the key pocket residues themselves exhibited relatively small fluctuations, and the overall structure of this region remained stable, with no significant backbone rearrangement induced by the mutation.

Fig. 3.

Fig. 3

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Molecular dynamics insights into the enhanced L-tyrosine production by the TyrAM53I/A354V mutant. A RMSD profiles of the TyrA/Prephenate complex. B RMSF analysis of the TyrA/Prephenate complex. C The binding energy of the TyrA/Prephenate complex

Based on the MM/GBSA analysis, the binding free energy of the mutant complex was consistently more negative and stable throughout the simulation compared to the wild-type, demonstrating superior and sustained binding affinity (Fig. 3C). In summary, the TyrAM53I/A354V mutant provides a more favorable dynamic conformational landscape for catalysis by stabilizing enzyme-substrate complex and reducing flexibility. This molecular-level optimization explains the enhanced L-tyrosine production observed in the engineered strain.

Effect of expression system optimization for Eco-TyrAM53I/A354V on L-tyrosine biosynthesis

The expression level of a target gene is influenced by various genetic elements, including the promoter, terminator, and 5′-UTR [12, 33, 34]. To further enhance L-tyrosine biosynthesis, the promoter, terminator, and 5′-UTR of the Eco-tyrAM53I/A354V gene was systematically optimized (Fig. 4A). A panel of well-characterized Bacillus promoters with a wide range of strengths was firstly evaluated, including PbacA, PsrfA, PytzE, PU12, PylB, P43-PylB, Pbay, PykzA, PRBS6, PHpaII, PluxS, PgisB, P382, P03468, and PabrB [35–37]. Among them, recombinant strains equipped with the PgisB (A8) and PytzE (A9) promoters showed significantly higher L-tyrosine production than the P43 (A6) control strain. The titers reached 560.57 mg/L and 502.06 mg/L, representing increases of 26% and 13%, respectively (Fig. 4B). Subsequently, terminators of varying efficiencies, classified as high (BSUT1, BSUT2), medium (BSUT3, ECOT4), or low (ECOT5, ECOT6), were tested [38–40]. Results indicated that the BSUT1 terminator also increased L-tyrosine production by 5%. Additionally, its compact size (54 bp) facilitates subsequent genetic manipulations (Fig. 4C). In parallel, a series of redesigned 5′-UTR sequences were assessed. However, the native 5′-UTR was identified as the most favorable for L-tyrosine synthesis (Fig. 4D). The growth of all engineered strains at the fermentation endpoint was unaffected by the optimization of genetic elements (Fig. S2). Based on these findings, the PgisB promoter and BSUT1 terminator were selected for subsequent engineering efforts, establishing an optimized transcriptional framework for further enhancement of L-tyrosine production.

Fig. 4.

Fig. 4

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Effect of expression system optimization on L-tyrosine production. A Schematic diagram of the component optimization strategy. B Effect of promoter optimization on L-tyrosine biosynthesis. C Effect of terminator optimization on L-tyrosine biosynthesis. D Effect of 5′-UTR redesign on L-tyrosine biosynthesis. * (p < 0.05) and ** (p < 0.01) represent significance levels compared to the control; “ns” indicates no significant difference

Effect of co-expressing Eco-tyrAM53I/A354V and Bao-aroA on L-tyrosine biosynthesis

Tandem gene expression facilitates the coordinated regulation of multiple enzymes, thereby optimizing metabolic flux to prevent bottlenecks and the accumulation of intermediates [41]. Given that DAHP synthase (AroA) overexpression is known to enhance L-tyrosine yield, Bao-aroA was identified as another key target al.ongside Eco-tyrAM53I/A354V [16]. Therefore, leveraging the previously optimized expression elements, this study employed RBS and linker strategies to co-express these genes and investigate their synergistic effect on L-tyrosine production. As shown in Fig. 5, linker-mediated fusion expression resulted in an L-tyrosine titer of 779.32 mg/L. In contrast, RBS-mediated tandem expression was more effective, yielding a titer of 1104.02 mg/L (a 257% increase over the control), which corresponds to a volumetric productivity of 30.67 mg/L/h. This represents an increase of 97% and 170% compared to the individual overexpression of Eco-tyrAM53I/A354V or Bao-aroA alone, respectively (Fig. 5). Meanwhile, tandem expression had no significant effect on bacterial growth (Fig. S3). These results demonstrate that key gene co-expression effectively strengthens the L-tyrosine biosynthetic pathway by redirecting carbon flux toward downstream conversion, leading to product overaccumulation. This strategy is corroborated by parallel findings in other biosynthetic systems, as exemplified by a 6.7-fold increase in sclareol yield using fused diterpene synthases (Tps/Lpps) [42].

Fig. 5.

Fig. 5

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Effect of Co-expressing Eco-tyrAM53I/A354V and Bao-aroA on L-tyrosine biosynthesis. *** (p < 0.001) indicates the significance levels compared with the control

Conclusion

In summary, this study developed a comprehensive multi-level engineering strategy for significantly enhancing L-tyrosine production in B. amyloliquefaciens. The introduction of a feedback-insensitive mutant (TyrAM53I/A354V) from E. coli resulted in an increase in L-tyrosine titer to 446.32 mg/L, representing a 53% improvement over the control. Molecular simulations suggested that the mutations stabilize enzyme-substrate binding and reduce conformational flexibility, thereby creating an improved catalytic microenvironment. Further optimization of the promoter led to an increase in L-tyrosine production to 560.57 mg/L, a 26% improvement over the previous strain. Additionally, RBS-mediated co-expression of Eco-tyrAM53I/A354V and Bao-aroA elevated the titer to 1104.02 mg/L, a 257% increase relative to the control strain A2. Collectively, these systematic interventions, including enzyme engineering, expression tuning, and pathway coordination, provide efficient genetic components and strategies for developing L-tyrosine cell factories from B. amyloliquefaciens.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1 (613.1KB, docx)

Author contributions

Ziyue Zhao and Anying Ji supervised the research and wrote the main manuscript text. Ziyue Zhao, Anying Ji and Can Song performed the experiments, and prepared the figures. Ziyue Zhao and Anying Ji wrote the manuscript. Xuetuan Wei and Zhengyuan Zhai revised the article. All authors reviewed the manuscript.

Funding

This study was supported by the National Natural Science Foundation of China (No. 32371493).

Data availability

The dataset(s) supporting the conclusions of this article is(are) included within the article (and its additional file(s)).

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Anying Ji, Email: metaboliceng@163.com.

Xuetuan Wei, Email: weixuetuan@mail.hzau.edu.cn.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (613.1KB, docx)

Data Availability Statement

The dataset(s) supporting the conclusions of this article is(are) included within the article (and its additional file(s)).

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