Expanding the Application of Threonine: Industrial Biomanufacturing of Threonine and Its Derivatives

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Please see the attached.

Comments for author File: Comments.pdf

Author Response

Reviewer #1: The review manuscript titled‘Expanding the application of threonine: industrial biomanufacturing of threonine and its derivatives” provides an overview of the current state of L-threonine production, the routes of production including the comparison of the production economics, and applications of L-threonine derivatives. The manuscript is relevant, and the language is clear and easy to follow. However, the manuscript requires revision to meet the standards for publication in Fermentation journal.

Comment 1: The authors need to organize the manuscript to show the use of systematic combination of genetic engineering and optimization strategies to improve L-Thr production in specific organisms. As is now, it looks disjointed, and readers cannot appreciate what genetic engineering strategies achieved and what optimization strategies achieved. For example, readers would like to know that the use of genetic engineering strategies improved L-Thr production from, say, X g/L to Y g/L. The reporting currently does not show why specific genes improved L-Thr production. Readers ought to be able to deduce which genes affect the production of L-Thr in specific organisms. Also, the authors should also give their perspectives on what they think should be improved, in particular the target genes, in order to further improve L-Thr fermentation.

Response: Thank you for your comments. We have made revisions based on your recommendation.

In lines 183-189 on page 5, “Key strategies include: strengthening the expression of key enzymes in the synthesis pathway to increase carbon flux; weakening competitive branch metabolism; alleviating feedback inhibition of enzymes by product accumulation; and reducing intracellular degradation of L-Thr. The integrated application of these methods is crucial for improving L-Thr synthesis efficiency.” was revised as “Based on established metabolic engineering principles, this section systematically elaborates four key strategies: flux amplification to strengthen precursor supply and key enzyme expression, elimination of competing pathways to reduce by-product formation, cofactor engineering to optimize reducing power balance, and transport engineering to enhance product efflux and alleviate feedback inhibition. The integrated application of these methods is crucial for improving L-Thr synthesis efficiency.”

In lines 213-218 on page 7,“Beyond precursor supply, direct enhancement of the L-Thr biosynthesis pathway enzymes critical, particularly given the feedback inhibition exerted by terminal amino acids on key enzymes such as AK and HDH. To overcome these constraints, introduction of feedback-resistant mutants has proven effective. Site-directed mutagenesis can generate mutants completely insensitive to high concentrations of L-Thr.” was added.

In lines 238-240 on page 7,“Elimination of competing pathways directs metabolic flux toward threonine by knocking out genes involved in branch pathways and degradation routes, thereby minimizing carbon loss and reducing by-product accumulation.” was added.

In lines 285-288 on page 8,“Cofactor engineering focuses on optimizing the supply of reducing equivalents, particularly NADPH, which is essential for several steps in L-Thr biosynthesis. HD utilizes NADPH as a reducing equivalent to catalyze the decarboxylation of aspartate semialdehyde to generate L-homoserine.” was added.

In lines 330-335 on page 9,“Transport engineering addresses the critical bottleneck of product accumulation within the cell, which leads to feedback inhibition of key enzymes and limits further synthesis. Reducing excessive intracellular product accumulation is achieved by promoting extracellular secretion to decrease intracellular threonine concentration, thereby alleviating feedback inhibition on key enzymes in the synthesis pathway and enabling sustained high-rate synthesis.” was added.

Comment 2: Line 32: What is the global market value of L-Threonine production?

Response: Thank you for your comments. We have made revisions based on your recommendation.

In lines 50-52 on page 2, “The important functions and extensive applications of L-Thr have driven market demand, with the global annual production reported to reach 700,000 tons.” has already appeared.

Comment 3: Line 42: How does the use of L-Threonine amino acid infusions help reduce nutrient losses in food during production, transportation or storage? The authors should provide evidence and mechanism of the process with impact of L-Thr in nutrient loss reduction.

Response: Thank you for your comments. Sorry, we made such a mistake. We have made revisions based on your recommendation.

In lines 40-45 on page 1, “In the food sector, L-Thr is commonly co-heated with glucose to generate burnt and chocolate aromas for flavor enhancement. It is also employed in the formulation of amino acid infusions and comprehensive amino acid preparations to balance nutrition or maintain protein nutritional value, thereby reducing nutrient losses in food during production, transportation, and storage to a certain exten.” was revised as “In the food sector, L-Thr is commonly co-heated with glucose to generate burnt and chocolate aromas for flavor enhancement. It is also employed in the formulation of amino acid infusions and comprehensive amino acid preparations, where its polarity and aqueous solubility enable hydrophobic, hydrophilic, and dipole–dipole interactions with solvent and solute molecules. These interactions stabilize macromolecular conformations, prevent protein denaturation and aggregation, and thereby preserve nutritional integrity.”

Comment 4: Lines 76-78: This is repetition and should be removed.

Response: Thank you for your comments. We have made revisions based on your recommendation.We have done the deletion processing.

Comment 5: Lines 97-98: The two metabolic pathways shown in Figure 2 can be converted into one pathway as oxaloacetate is not fed to L-Thr producing microorganisms.

Response: Thank you for your comments. We have made revisions based on your recommendation.We have made the modifications as required, as shown in Figure 2.

Comment 6: Line 117: In the earlier comparison of L-Thr fermentation to L-glutamate, the authors mentioned that L-Thr is far lower than the 160 g/L and above of L-glutamate production. Here, the authors also mentioned that L-Thr production has reached 160.3 g/L. The two statements are contradictory.

Response: Thank you for your comments. We have made revisions based on your recommendation.

In lines 68-71 on page 2, “In recent years, metabolic engineering modification of microorganisms has significantly improved the yield of L-Thr, but its fermentation level is still far lower than that of amino acids such as L-lysine and L-glutamic acid, the latter can reach above 160 g/L.” was revised as “In recent years, metabolic engineering modification of microorganisms has significantly improved the yield of L-Thr, but its fermentation level is still far lower than that of amino acids such as L-lysine and L-glutamic acid, the latter can reach above 181.5 g/L [18].”

[18]Xu, J.-Z.; Wu, Z.-H.; Gao, S.-J.; Zhang, W.J.M.c.f. Rational modification of tricarboxylic acid cycle for improving L-lysine production in Corynebacterium glutamicum. 2018, 17, 105.

Comment 7: Line 124: What ratio are the authors referring to here? Do they mean strain ratios? If yes, they should provide a brief description of the strains’ co-culture and the rationale for the use of the co culture in the study.

Response: Thank you for your comments. We have made revisions based on your recommendation.

In lines 226-231 on page 7, “Hao et al. systematically analyzed the effects of the ratio of thrAB to thrC on the growth and threonine production of E. coli strains. The study found that when the ratio of thrAB to thrC was 3:5, the L-Thr yield was the highest, reaching 40.06 g/L after 60 h of fermentation, which was 96.85% higher than that of the initial control strain TH.” was revised as “Hao et al. systematically investigated the effect of varying the thrAB-to-thrC expression ratio on E. coli growth and L-Thr production, rather than adjusting strain ratios. This approach exemplifies single-strain metabolic engineering that circumvents the complexities of co-culture systems. The study found that when the ratio of thrAB to thrC was 3:5, the L-Thr yield was the highest, reaching 40.06 g/L after 60 h of fermentation, which was 96.85% higher than that of the initial control strain TH.”

Comment 8: Line 128: “…4-fold increase in yield to 1.36 g/L”, from what initial concentration?

Response: Thank you for your comments. We have made the following revisions in the revised manuscript.

In lines 231-236 on page 7, “Su et al. constructed a dual-responsive biosensor based on the L-Thr inducible effect and riboswitch for ribosome binding site (RBS) library metabolic pathway optimization, resulting in a 4-fold increase in yield to 1.36 g/L; and drove site-directed saturation mutagenesis of the ACT domain of thrA enzyme, obtaining mutant strain WA1 with a 7-fold increase in yield.” was revised as “Su et al. constructed a dual-responsive biosensor based on the L-Thr inducible effect and riboswitch for ribosome binding site library metabolic pathway optimization, which increased the L-Thr yield from 0.34 g/L to 1.36 g/L; and drove site-directed saturation mutagenesis of the ACT domain of thrA enzyme, obtaining mutant strain WA1 with a 7-fold increase in yield. ”

Comment 9: Line 133: What are the functions of rhtC and sstT/tdh?

Response: Thank you for your comments. We have made the following revisions in the revised manuscript.

In lines 231-236 on page 7, “Du et al. systematically engineered Halomonas bluephagenesis using Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9 (CRISPR/Cas9) genome editing and modular transcriptional regulation. By introducing the exogenous exporter rhtC, knocking out endogenous uptake and degradation genes sstT/tdh, and integrating and optimizing the feedback inhibition-resistant synthetic module thrABC and lysC, the resulting engineered strain TDHR3-42-p226 produced 7.5 g/L L-Thr in shake flasks under open non-sterile conditions, and achieved a yield of 33 g/L in 7 L fed-batch fermentation after 24 h.” was revised as “Du et al. engineered Halomonas bluephagenesis using CRISPR/Cas9 and modular regulation. They introduced the exporter rhtC to secrete threonine, deleted sstT (importer) and tdh (degradation enzyme) to prevent intracellular loss, and integrated feedback-resistant thrABC and lysC to enhance synthesis. The final strain produced 7.5 g/L in shake flasks and 33 g/L in a 7-L fermentor within 24 h, demonstrating the potential of this host for low-cost amino acid production.”

Comment 10: Lines 121-137: Authors should provide more context for the studies conducted by the researchers, their findings and rationale for their studies.

Response: Thank you for your comments. We have made the following revisions in the revised manuscript.

In lines 213-218 on page 7, “Beyond precursor supply, direct enhancement of the L-Thr biosynthesis pathway enzymes critical, particularly given the feedback inhibition exerted by terminal amino acids on key enzymes such as AK and HDH. To overcome these constraints, introduction of feedback-resistant mutants has proven effective. Site-directed mutagenesis can generate mutants completely insensitive to high concentrations of L-Thr.”was added.

Comment 11: Lines 148-149: What are the functions of dapA, dhaM, metL, aspC and ppc genes?

Response: Thank you for your comments. We have made the following revisions in the revised manuscript.

In lines 311-328 on page 8-9, “Hanke et al. employed combinatorial cloning and a hybrid deep learning model to guide multi-gene optimization of the threonine synthesis pathway in E. coli, with specific deletion of tdh, dapA, dhaM, and metL genes and overexpression of pntAB, aspC, and ppc genes, achieving a yield increase from 2.7 g/L to 8.4 g/L through three rounds of iteration.” was revised as “ A compelling example of integrating cofactor engineering with complementary metabolic strategies is the work by Hanke et al. Employing combinatorial cloning coupled with a hybrid deep learning model, the authors iteratively engineered the L-Thr biosynthetic pathway in E. coli across three rounds of optimization. To augment NADPH availability, they overexpressed pntAB (encoding membrane-bound transhydrogenase). Concurrently, they eliminated competing metabolic branches to redirect carbon flux toward L-Thr by deleting tdh (encoding threonine dehydrogenase, which catalyzes threonine degradation), dapA (encoding dihydrodipicolinate synthase, committing carbon to the lysine branch), dhaM (encoding a phosphotransferase system component influencing carbon partitioning), and metL (encoding a bifunctional aspartate kinase/homoserine dehydrogenase directing flux toward methionine). Furthermore, they enhanced precursor supply by overexpressing aspC (encoding aspartate aminotransferase, increasing aspartate availability) and ppc (encoding PEPC, replenishing the oxaloacetate pool). This multi-pronged, machine learning-guided optimization progressively elevated L-Thr titers from 2.7 g/L to 8.4 g/L. Although this study integrated multiple engineering strategies, the inclusion of pntAB overexpression specifically underscores the pivotal contribution of cofactor engineering to enhanced L-Thr production and exemplifies how synergistic implementation of diverse approaches can achieve substantial yield improvements.”

Comment 12: Figure 2: ‘Pyruvata’ should be changed to pyruvate. ‘Asparty’ should be changed to aspartyl.

Response: Thank you for your comments. We have made the following revisions in the revised manuscript.As shown in Figure 2, we have made the revisions.

Comment 13: Line 192: What are the key enzymes of L-Thr biosynthesis?

Response: Thank you for your comments. We have made the following revisions in the revised manuscript.

In lines 116-132 on page 4, “ In E. coli and C. glutamicum, the key enzyme systems involved in threonine biosynthesis exhibit significant differences in organizational form. Specifically, E. coli AK exists as three isoenzymes (AKI, AKII, and AKIII) [20], whereas HD has two isoenzymes (HDI and HDII). Among these, AKI and HDI form a bifunctional enzyme encoded by thrA; similarly, AKII and HDII also constitute a bifunctional enzyme encoded by metL [21]. In contrast, AKIII is a monofunctional enzyme encoded by lysC [22]. Among the three AK isoenzymes, AKI exhibits the highest protein expression level, while AKII shows the lowest expression level. As for ASD, HK, and TS, they all exist as single enzymes in E. coli, encoded by asd, thrB, and thrC, respectively [23].In contrast, in C. glutamicum, the genetic organization of the above five enzymes is more streamlined, with no isoenzymes identified: AK is encoded by lysC, ASD by asd, HD by hom, HK by thrB, and TS by thrC [24,25]. In microbial production of L-Thr, the transport of product from intracellular to extracellular space is generally regarded as a critical rate-limiting step. For this step, three functional proteins responsible for L-Thr efflux have been identified in E. coli, namely RhtA, RhtB, and RhtC, encoded by rhtA, rhtB, and rhtC, respectively [26]. In C. glutamicum, however, only one L-Thr efflux protein, ThrE, has been discovered, encoded by thrE [27].” was added.

[20]Simic, P.; Sahm, H.; Eggeling, L.J.J.o.b. L-threonine export: use of peptides to identify a new translocator from Corynebacterium glutamicum. 2001, 183, 5317-5324.

[21]Katinka, M.; Cossart, P.; Sibilli, L.; Saint-Girons, I.; Chalvignac, M.; Le Bras, G.; Cohen, G.; Yaniv, M.J.P.o.t.N.A.o.S. Nucleotide sequence of the thrA gene of Escherichia coli. 1980, 77, 5730-5733.

[22]Schrumpf, B.; Schwarzer, A.; Kalinowski, J.; Pühler, A.; Eggeling, L.; Sahm, H.J.J.o.b. A functionally split pathway for lysine synthesis in Corynebacterium glutamicium. 1991, 173, 4510-4516.

[23]Lee, H.-S.; Hwang, B.-J.J.A.m.; biotechnology. Methionine biosynthesis and its regulation in Corynebacterium glutamicum: parallel pathways of transsulfuration and direct sulfhydrylation. 2003, 62, 459-467.

[24]Follettie, M.; Shin, H.; Sinskey, A.J.M.m. Organization and regulation of the Corynebacterium glutamicum hom‐thrB and thrC loci. 1988, 2, 53-62.

[25]Cremer, J.; Treptow, C.; Eggeling, L.; Sahm, H.J.M. Regulation of enzymes of lysine biosynthesis in Corynebacterium glutamicum. 1988, 134, 3221-3229.

[26]Livshits, V.A.; Zakataeva, N.P.; Aleshin, V.V.; Vitushkina, M.V.J.R.i.m. Identification and characterization of the new gene rhtA involved in threonine and homoserine efflux in Escherichia coli. 2003, 154, 123-135.

[27]Kotaka, M.; Ren, J.; Lockyer, M.; Hawkins, A.R.; Stammers, D.K.J.J.o.B.C. Structures of R-and T-state Escherichia coli aspartokinase III: Mechanisms of the allosteric transition and inhibition by lysine. 2006, 281, 31544-31552.

Comment 14: Line 198: What enzymes do crr, iclR and gltA genes code for? What are their role in L-Thr biosynthesis? Please provide names and possible functions of genes throughout the manuscript.

Response: Thank you for your comments. We have made the following revisions in the revised manuscript.

In lines 202-212 on page 6, “Zhu et al. investigated the effects of glucose uptake, glyoxylate bypass, and threonine synthesis pathway on L-Thr accumulation in E. coli using chromosomal gene editing and plasmid introduction techniques. By deleting the crr and iclR genes and overexpressing gltA, the engineered strain WMZ016 was successfully constructed, with its L-Thr yield increased to 17.98 g/L.” was revised as “Zhu et al. [38] investigated the effects of glucose uptake, the glyoxylate shunt, and threonine biosynthesis on L-Thr accumulation in E. coli through chromosomal editing and plasmid-based expression. Key modifications included overexpression of gltA (enhancing TCA cycle flux) to increase OAA supply, enabling the engineered strain to produce 17.98 g/L L-Thr in shake-flask fermentation, compared to 0.68 g/L from the control.”

Comment 15:Line 203: “glucose titer of 0.57 g/g” is wrong. I assume the authors are using glucose substrates; this should be the yield of the product and not the substrate.

Response: Thank you for your comments. We have made the following revisions in the revised manuscript.

In lines 346-350 on page 9 “Song et al. designed an artificial quorum sensing system that, through the combination of a switch module and a production module, enhances the L-Thr titer to 118.2 g/L by auto-inducing the expression of pyruvate carboxylase (PYC) and threonine extracellular transporter after cells reach a certain growth level, with a glucose titer of 0.57 g/g and a productivity of 2.46 g/(L·h).” was revised as “Song et al. designed an artificial quorum sensing system that, through the combination of a switch module and a production module, enhances the L-Thr titer to 118.2 g/L by auto-inducing the expression of PYC and threonine extracellular transporter after cells reach a certain growth level, with a yield of 0.57 g/g glucose and a productivity of 2.46 g/(L·h).”

Comment 16:Line 302: Change ‘chapter’ to section.

Response: Thank you for your comments. We have made the following revisions in the revised manuscript.

In lines 373-375 on page 10 “This chaper reviews the following compounds (Table 2): pyridoxine (PN), 2,3,5-trimethylpyrazine (TMP) and 2,5-dimethylpyrazine (2,5-DMP), L-ABA, PA, 2-OBA, etc.”was revised as “This sectionr reviews the following compounds (Table 2): pyridoxine (PN), 2,3,5-trimethylpyrazine (TMP) and 2,5-dimethylpyrazine (2,5-DMP), L-ABA, PA, 2-OBA, etc.”

Comment 17:Table 2: The authors used ±value for TMP titer. The authors should also include this for the titer of all other compounds or remove it for TMP to be consistent across board.  

Response: Thank you for your comments. We have made the following revisions in the revised manuscript. We have uniformly removed the ± values of TMP to maintain consistency, as shown in Table 2.

Products	Strategies	Substrate	titer (g/L)	Productivity (%)	Reference
Pyridoxine	Pathway engineering, RBS optimization, medium optimization, fed-batch fermentation	Glucose	174.6	NR	[65]
2,3,5-Trimethylpyrazine	Overexpression of BITDH (N157A), fermentation condition optimization (substrate ratio 1:2, IPTG 1.0 mM, fermentation for 4 d)	Glucose, L-Thr	44.52	NR	[66]
2,5-Dimethylpyrazine	Knockout of the kbl gene	L-Thr	2.82	17	[67]
L-2-Aminobutyric acid	Whole-cell catalysis using BL21/pET28a-R3ilvA-Esldh72Δ-fdh	L-Thr	121	95	[68]
Propionic Acid	Sequential fermentation using MG1655 - Pseudomonas putida	L-Thr	62	＞98	[69]
2-Oxobutyric Acid	Whole-cell catalysis using Pseudomonas stutzeri SDM	L-Thr	25.6	99.6	[70]

 

Reviewer 2 Report

Comments and Suggestions for Authors

In the manuscript by Lu et al., entitled “Expanding the Application of Threonine: Industrial Biomanufacturing of Threonine and Its Derivatives”, the authors aim to summarize the metabolic pathways involved in L-threonine biosynthesis and describe metabolic engineering strategies for enhancing its production and that of its derivatives. While the topic is relevant and timely for the field of industrial biotechnology, the manuscript currently lacks sufficient depth, cohesion, and critical analysis. Although the authors outline the threonine biosynthetic pathway and discuss several engineering strategies, only a limited number of representative examples are provided. Moreover, the discussion of current bottlenecks, technical challenges, and future perspectives in biological threonine production is either superficial or insufficiently developed. A more comprehensive and critical evaluation of recent advances, limitations, and emerging strategies would significantly strengthen the manuscript. Therefore, a thorough revision is required before the work can be considered for publication in Fermentation.

Comment 1: The description of the threonine biosynthetic pathway is brief and does not sufficiently address the genetic and enzymatic basis of pathway regulation. Rather than broadly summarizing engineering approaches in this section, the authors are suggested to provide a more detailed account of the genes involved, their genetic organization, and the biochemical properties and regulation of the corresponding enzymes (e.g., feedback inhibition, transcriptional control). The discussion of engineering strategies would be more appropriately placed in Section 3.

Comment 2: Section 3 is currently divided into four subsections; however, the content appears repetitive and lacks a clear conceptual structure. The section would benefit from reorganization based on established metabolic engineering principles. The authors are encouraged to restructure this section as. 3.1 Flux amplification, 3.2 Removal of competing pathways, 3.3 Cofactor engineering, 3.4, Transport engineering.

Comment 3: In Figure 2, the authors are encouraged to include key regulatory nodes and feedback inhibition points within the threonine biosynthetic pathway. Additionally, metabolic branches leading to threonine-derived products should be clearly illustrated to highlight the metabolic connectivity and engineering targets for derivative production.

Comments on the Quality of English Language

Need to rectify some gramatical mistake

Author Response

Reviewer #2:In the manuscript by Lu et al., entitled “Expanding the Application of Threonine: Industrial Biomanufacturing of Threonine and Its Derivatives”, the authors aim to summarize the metabolic pathways involved in L-threonine biosynthesis and describe metabolic engineering strategies for enhancing its production and that of its derivatives. While the topic is relevant and timely for the field of industrial biotechnology, the manuscript currently lacks sufficient depth, cohesion, and critical analysis. Although the authors outline the threonine biosynthetic pathway and discuss several engineering strategies, only a limited number of representative examples are provided. Moreover, the discussion of current bottlenecks, technical challenges, and future perspectives in biological threonine production is either superficial or insufficiently developed. A more comprehensive and critical evaluation of recent advances, limitations, and emerging strategies would significantly strengthen the manuscript. Therefore, a thorough revision is required before the work can be considered for publication in Fermentation.

Comment 1: The description of the threonine biosynthetic pathway is brief and does not sufficiently address the genetic and enzymatic basis of pathway regulation. Rather than broadly summarizing engineering approaches in this section, the authors are suggested to provide a more detailed account of the genes involved, their genetic organization, and the biochemical properties and regulation of the corresponding enzymes (e.g., feedback inhibition, transcriptional control). The discussion of engineering strategies would be more appropriately placed in Section 3.

Response: Thank you for your comments. We have made revisions based on your recommendation.

In lines 116-132 on page 4,“In E. coli and C. glutamicum, the key enzyme systems involved in threonine biosynthesis exhibit significant differences in organizational form. Specifically, E. coli AK exists as three isoenzymes (AKI, AKII, and AKIII) [20], whereas HD has two isoenzymes (HDI and HDII). Among these, AKI and HDI form a bifunctional enzyme encoded by thrA; similarly, AKII and HDII also constitute a bifunctional enzyme encoded by metL [21]. In contrast, AKIII is a monofunctional enzyme encoded by lysC [22]. Among the three AK isoenzymes, AKI exhibits the highest protein expression level, while AKII shows the lowest expression level. As for ASD, HK, and TS, they all exist as single enzymes in E. coli, encoded by asd, thrB, and thrC, respectively [23].In contrast, in C. glutamicum, the genetic organization of the above five enzymes is more streamlined, with no isoenzymes identified: AK is encoded by lysC, ASD by asd, HD by hom, HK by thrB, and TS by thrC [24,25]. In microbial production of L-Thr, the transport of product from intracellular to extracellular space is generally regarded as a critical rate-limiting step. For this step, three functional proteins responsible for L-Thr efflux have been identified in E. coli, namely RhtA, RhtB, and RhtC, encoded by rhtA, rhtB, and rhtC, respectively [26]. In C. glutamicum, however, only one L-Thr efflux protein, ThrE, has been discovered, encoded by thrE [27].”was added.

[20]Simic, P.; Sahm, H.; Eggeling, L.J.J.o.b. L-threonine export: use of peptides to identify a new translocator from Corynebacterium glutamicum. 2001, 183, 5317-5324.

[21]Katinka, M.; Cossart, P.; Sibilli, L.; Saint-Girons, I.; Chalvignac, M.; Le Bras, G.; Cohen, G.; Yaniv, M.J.P.o.t.N.A.o.S. Nucleotide sequence of the thrA gene of Escherichia coli. 1980, 77, 5730-5733.

[22]Schrumpf, B.; Schwarzer, A.; Kalinowski, J.; Pühler, A.; Eggeling, L.; Sahm, H.J.J.o.b. A functionally split pathway for lysine synthesis in Corynebacterium glutamicium. 1991, 173, 4510-4516.

[23]Lee, H.-S.; Hwang, B.-J.J.A.m.; biotechnology. Methionine biosynthesis and its regulation in Corynebacterium glutamicum: parallel pathways of transsulfuration and direct sulfhydrylation. 2003, 62, 459-467.

[24]Follettie, M.; Shin, H.; Sinskey, A.J.M.m. Organization and regulation of the Corynebacterium glutamicum hom‐thrB and thrC loci. 1988, 2, 53-62.

[25]Cremer, J.; Treptow, C.; Eggeling, L.; Sahm, H.J.M. Regulation of enzymes of lysine biosynthesis in Corynebacterium glutamicum. 1988, 134, 3221-3229.

[26]Livshits, V.A.; Zakataeva, N.P.; Aleshin, V.V.; Vitushkina, M.V.J.R.i.m. Identification and characterization of the new gene rhtA involved in threonine and homoserine efflux in Escherichia coli. 2003, 154, 123-135.

[27]Kotaka, M.; Ren, J.; Lockyer, M.; Hawkins, A.R.; Stammers, D.K.J.J.o.B.C. Structures of R-and T-state Escherichia coli aspartokinase III: Mechanisms of the allosteric transition and inhibition by lysine. 2006, 281, 31544-31552.

In lines 134-166 on page 4-6,“In E. coli, the transcription of genes involved in the L-Thr biosynthetic pathway and their corresponding enzymatic activities are subject to exquisite regulation by terminal amino acids. The thrA, thrB, and thrC genes, located within the thrABC operon [28], are transcriptionally repressed by L-Thr and L-isoleucine through attenuation mediated by the upstream thrL leader sequence; introduction of mutations at specific positions within this sequence can alleviate this regulatory control. Meanwhile, transcription of the metL gene is repressed by L-methionine, whereas the lysC gene is regulated by L-lysine through a translational attenuation mechanism [29]. Additionally, transcription of the asd gene is subject to multivalent repression by L-lysine, L-Thr, and L-methionine [30]. At the enzymatic level, the bifunctional enzyme AKI-HDI is subject to incomplete feedback inhibition by L-Thr, wherein AKI activity is modulated by allosteric regulation and substrate competition, while HDI activity exhibits non-competitive inhibition [31]. AKIII activity can be completely inhibited by L-lysine through conformational change, and specific point mutations can partially relieve this inhibition [27,31]. The feedback inhibition mechanism of HK is rather complex, being subject to competitive inhibition by L-Thr and non-competitive inhibition by L-lysine, as well as modulation by high concentrations of the substrate L-homoserine and ATP [31].

In C. glutamicum, transcriptional regulation of the L-Thr biosynthetic pathway is relatively limited, primarily targeting the operon composed of hom and thrB. This operon is arranged in the 5'-hom-thrB-3' orientation, with a long inverted repeat sequence upstream, suggesting that feedback repression of these two genes by L-methionine may be mediated through an attenuator mechanism [32]. At the enzymatic activity level, three key enzymes are subject to feedback inhibition by terminal amino acids. Among them, CgAK (encoded by lysC) is an allosteric enzyme, whose activity is synergistically inhibited by feedback from L-Thr and L-lysine; this inhibition can be completely abolished by introducing specific mutations [33]. CgHD (encoded by hom) activity is feedback-inhibited by L-Thr, an effect mediated by its C-terminal domain [34]; site-directed mutagenesis (e.g., G378E) can generate mutants completely insensitive to high concentrations of L-Thr [35]. CgHK (encoded by thrB) is subject to competitive feedback inhibition by L-Thr, with the degree of inhibition decreasing as substrate L-homoserine concentration increases; consequently, this inhibition can only be alleviated by elevating substrate concentration rather than by altering the enzyme protein structure [36].” was added.

[27]Kotaka, M.; Ren, J.; Lockyer, M.; Hawkins, A.R.; Stammers, D.K.J.J.o.B.C. Structures of R-and T-state Escherichia coli aspartokinase III: Mechanisms of the allosteric transition and inhibition by lysine. 2006, 281, 31544-31552.

[28]Theze, J.; Saint-Girons, I.J.J.o.B. Threonine locus of Escherichia coli K-12: genetic structure and evidence for an operon. 1974, 118, 990-998.

[29]Grundy, F.J.; Lehman, S.C.; Henkin, T.M.J.P.o.t.N.A.o.S. The L box regulon: lysine sensing by leader RNAs of bacterial lysine biosynthesis genes. 2003, 100, 12057-12062.

[30]Boy, E.; Patte, J.-C.J.J.o.B. Multivalent repression of aspartic semialdehyde dehydrogenase in Escherichia coli K-12. 1972, 112, 84-92.

[31]Chassagnole, C.; RAÏS, B.; Quentin, E.; FELL, D.A.; MAZAT, J.-P.J.B.J. An integrated study of threonine-pathway enzyme kinetics in Escherichia coli. 2001, 356, 415-423.

[32]Mateos, L.M.; Pisabarro, A.; Pátek, M.; Malumbres, M.; Guerrero, C.; Eikmanns, B.J.; Sahm, H.; Martin, J.F.J.J.o.b. Transcriptional analysis and regulatory signals of the hom-thrB cluster of Brevibacterium lactofermentum. 1994, 176, 7362-7371.

[33]Yoshida, A.; Tomita, T.; Kurihara, T.; Fushinobu, S.; Kuzuyama, T.; Nishiyama, M.J.J.o.m.b. Structural insight into concerted inhibition of α2β2-type aspartate kinase from Corynebacterium glutamicum. 2007, 368, 521-536.

[34]Archer, J.; Solow-Cordero, D.; Sinskey, A.J.G. A C-terminal deletion in Corynebacterium glutamicum homoserine dehydrogenase abolishes allosteric inhibition by L-threonine. 1991, 107, 53-59.

[35]Reinscheid, D.J.; Eikmanns, B.J.; Sahm, H.J.J.o.b. Analysis of a Corynebacterium glutamicum hom gene coding for a feedback-resistant homoserine dehydrogenase. 1991, 173, 3228-3230.

[36]Colón, G.E.; Jetten, M.; Nguyen, T.T.; Gubler, M.E.; Follettie, M.T.; Sinskey, A.J.; Stephanopoulos, G.J.A.; microbiology, e. Effect of inducible thrB expression on amino acid production in Corynebacterium lactofermentum ATCC 21799. 1995, 61, 74-78.

Comment 2:Section 3 is currently divided into four subsections; however, the content appears repetitive and lacks a clear conceptual structure. The section would benefit from reorganization based on established metabolic engineering principles. The authors are encouraged to restructure this section as. 3.1 Flux amplification, 3.2 Removal of competing pathways, 3.3 Cofactor engineering, 3.4, Transport engineering.

Response: Thank you for your comments. Sorry, we made such a mistake. We have made revisions based on your recommendation.

In lines 191-235 on page 6-7,“3.1 Efficient expression of key enzymes in the L-Thr synthesis pathway: Efficient expression of key enzymes in the synthesis pathway is achieved through genetic engineering techniques to strengthen the key enzyme encoding genes of the L-Thr synthesis pathway, increase the supply of precursor substances, and effectively improve the yield of L-Thr. Zhu et al. [27] investigated the effects of glucose uptake, glyoxylate bypass, and threonine synthesis pathway on L-Thr accumulation in E. coli using chromosomal gene editing and plasmid introduction techniques. By deleting the crr and iclR genes and overexpressing gltA, the engineered strain WMZ016 was successfully constructed, with its L-Thr yield increased to 17.98 g/L. Song et al. [33] designed an artificial quorum sensing system that, through the combination of a switch module and a production module, enhances the L-Thr titer to 118.2 g/L by auto-inducing the expression of pyruvate carboxylase (PYC) and threonine extracellular transporter after cells reach a certain growth level, with a glucose titer of 0.57 g/g and a productivity of 2.46 g/(L·h). Zhao et al. [34] constructed a genetic circuit based on thrL and cysteine promoters (P-cys) to dynamically regulate the expression of seven key genes (iclR, aspC, arcA, cpxR, gadE, pykF, and fadR), increasing the L-Thr yield of the engineered strain TWF083 to 116.62 g/L. Jin et al. [35] modified the E. coli threonine synthesis system using modular pathway optimization, dynamic transport regulation, and global transcription factor engineering strategies. After obtaining the Fur mutant through directed evolution, the engineered strain THR-48 was constructed, achieving a threonine yield of 154.2 g/L using glucose as the substrate. Based on THR-48, the THR-50 strain was constructed by integrating the sucrose utilization operon, which could directly produce 92.46 g/L threonine using untreated cane sugar as the carbon source, reducing production costs by 48%. Jin et al. [36] developed a dual-responsive biosensor for NADPH and L-Thr, and combined it with fluorescence-activated cell sorting (FACS) to obtain a high-yield (0.65 g/g) L-Thr-producing strain. Zhao et al. [37] employed site-directed saturation mutagenesis and rational design to modify the critical Arg212 site of HK in C. glutamicum, obtaining the optimal mutant R212Q. By overexpressing the efflux transporter ThrE in the engineered strain R212Q/pXTuf-thrE, they achieved an L-Thr titer of 86.4 g/L and a glucose yield of 0.28 g/g.” was revised as “3.1 Flux amplification: Flux amplification aims to increase carbon flux toward L-Thr biosynthesis by enhancing the expression of rate-limiting enzymes and bolstering precursor metabolite supply. The L-Thr synthesis module utilizes OAA as a direct precursor, which is replenished through anaplerotic reactions of the TCA cycle. Notably, the mechanism of anaplerotic OAA formation differs between E. coli and C. glutamicum: in E. coli, phosphoenolpyruvate carboxylase (PEPC; encoded by ppc) catalyzes the carboxylation of phosphoenolpyruvate to OAA, whereas in C. glutamicum, pyruvate carboxylase (PYC; encoded by pyc) mediates the ATP-dependent carboxylation of pyruvate to OAA [45]. Consequently, targeted overexpression of these species-specific anaplerotic genes constitutes a fundamental strategy for enhancing precursor availability. Zhu et al. [38] investigated the effects of glucose uptake, the glyoxylate shunt, and threonine biosynthesis on L-Thr accumulation in E. coli through chromosomal editing and plasmid-based expression. Key modifications included overexpression of gltA (enhancing TCA cycle flux) to increase OAA supply, enabling the engineered strain to produce 17.98 g/L L-Thr in shake-flask fermentation, compared to 0.68 g/L from the control. Zhao et al. [46] constructed a genetic circuit based on thrL and cysteine promoters (P-cys) to dynamically regulate the expression of seven key genes (iclR, aspC, arcA, cpxR, gadE, pykF, and fadR), increasing the L-Thr yield of the engineered strain TWF083 to 116.62 g/L, demonstrating the importance of optimizing central metabolism for precursor supply.

Beyond precursor supply, direct enhancement of the L-Thr biosynthesis pathway enzymes critical, particularly given the feedback inhibition exerted by terminal amino acids on key enzymes such as AK and HDH. To overcome these constraints, introduction of feedback-resistant mutants has proven effective. Site-directed mutagenesis can generate mutants completely insensitive to high concentrations of L-Thr. Jin et al. [47] modified the E. coli threonine synthesis system using modular pathway optimization, dynamic transport regulation, and global transcription factor engineering strategies. After obtaining the Fur mutant through directed evolution, the engineered strain THR-48 was constructed, achieving a threonine yield of 154.2 g/L using glucose as the substrate. Based on THR-48, the THR-50 strain was constructed by integrating the sucrose utilization operon, which could directly produce 92.46 g/L threonine using untreated cane sugar as the carbon source, reducing production costs by 48%. Optimization of gene expression ratios also plays a key role in flux amplification. Hao et al. [48] systematically investigated the effect of varying the thrAB-to-thrC expression ratio on E. coli growth and L-Thr production, rather than adjusting strain ratios. This approach exemplifies single-strain metabolic engineering that circumvents the complexities of co-culture systems. The study found that when the ratio of thrAB to thrC was 3:5, the L-Thr yield was the highest, reaching 40.06 g/L after 60 h of fermentation, which was 96.85% higher than that of the initial control strain TH. Su et al. [49]constructed a dual-responsive biosensor based on the L-Thr inducible effect and riboswitch for ribosome binding site library metabolic pathway optimization, which increased the L-Thr yield from 0.34 g/L to 1.36 g/L; and drove site-directed saturation mutagenesis of the ACT domain of thrA enzyme, obtaining mutant strain WA1 with a 7-fold increase in yield. ”

In lines 236-282 on page 7-8 “3.2. Interruption or attenuation of carbon flux in branch pathways : High yields are achieved by efficiently expressing feedback-resistant TS genes while knocking out competing branch and degradation pathway genes, thereby directing metabolic flux toward threonine and minimizing carbon loss. For L-Thr biosynthesis, carbon flux wastage primarily occurs through four pathways: 1) the Lys branch pathway in its synthetic route; 2) the L-methionine branch pathway; 3) intracellular degradation to glycine after synthesis; 4) utilization for L-isoleucine synthesis. The Lys branch pathway is initiated by dihydrodipicolinate synthase (DDPS) encoded by the dapA gene in both C. glutamicum and E. coli [38]. Diesveld et al. [39] performed site-directed mutagenesis of the ilvA gene on the chromosome in C. glutamicum DM1800-T to inactivate the encoded threonine dehydratase (TD), increasing L-Thr production from 2.5 g/L to 4 g/L. Zhao et al. [40] performed a series of genetic engineering modifications on C. glutamicum WM001. By regulating the activities of phosphoenolpyruvate carboxylase, PYC, pyruvate kinase (PK), phosphoenolpyruvate carboxykinase, and oxaloacetate decarboxylase to finely adjust carbon flux, and inhibiting DDPS to reduce by-products, they achieved efficient synthesis of L-Thr. The final engineered strain TWZ024/pXTuf-thrE produced 78.3 g/L L-Thr in a 7 L bioreactor. Wang et al. [41] achieved L-Thr production of 96.4 g/L in a 3 L fermenter and 133.5 g/L in a 10 L fermenter with strain TWF001/pFW01-phaCAB through upregulation of key genes in the L-Thr biosynthesis pathway, downregulation of genes related to acetate formation, and upregulation of the acs gene encoding the enzyme that converts acetate to acetyl-coenzyme A.” was revised as “3.2. Elimination of competing: Elimination of competing pathways directs metabolic flux toward threonine by knocking out genes involved in branch pathways and degradation routes, thereby minimizing carbon loss and reducing by-product accumulation. For L-Thr biosynthesis, carbon flux wastage primarily occurs through four pathways: (1) the lysine branch pathway initiated by dihydrodipicolinate synthase (DDPS) encoded by dapA; (2) the L-methionine branch pathway; (3) intracellular degradation to glycine after synthesis; and (4) utilization for L-isoleucine synthesis [50]. The lysine branch represents the most significant competitive sink. In both C. glutamicum and E. coli, DDPS encoded by dapA catalyzes the first committed step toward lysine synthesis. Inhibition of DDPS activity has been shown to effectively redirect carbon flux toward threonine. Zhao et al. [51] performed a series of genetic engineering modifications on C. glutamicum WM001. By regulating the activities of PEPC, PYC, pyruvate kinase (PK), phosphoenolpyruvate carboxykinase, and oxaloacetate decarboxylase to finely adjust carbon flux, and inhibiting DDPS to reduce by-products, they achieved efficient synthesis of L-Thr. The final engineered strain TWZ024/pXTuf-thrE produced 78.3 g/L L-Thr in a 7 L bioreactor. The isoleucine pathway also competes for threonine as a substrate. Threonine dehydratase (TD), encoded by ilvA, catalyzes the conversion of L-Thr to 2-oxobutyrate, the first step in isoleucine biosynthesis. Diesveld et al. [52] performed site-directed mutagenesis of the ilvA gene on the chromosome in C. glutamicum DM1800-T to inactivate the encoded TD, increasing L-Thr production from 2.5 g/L to 4 g/L.Yan et al. [53] increased L-Thr titer from 5.55 g/L to 8.65 g/L after knocking out the ilvA gene, and further improved L-Thr titer to 13.6 g/L by additionally knocking out the metA gene. Wu et al. [54] utilized the molecular dynamics-based allosteric prediction method and molecular mechanics-generalized born surface area energy decomposition to investigate the isoleucine allosteric inhibition mechanism of TD. They found that isoleucine concentrations above 0.1 mM caused wild-type TD activity loss exceeding 95%, confirming that intracellular accumulation severely inhibits enzyme function. Through computer-aided screening, the mutant P441L was obtained, which maintained 80% activity at 2.5 mM isoleucine and increased leucine yield by over 400% in E. coli fermentation.Intracellular degradation of L-Thr represents another significant carbon loss. The main degradation products are glycine and acetyl-CoA, catalyzed by threonine dehydrogenase (TDH, encoded by tdh) and serine hydroxymethyltransferase (SHMT, encoded by glyA). Lee et al. [55] employed systematic metabolic engineering strategies to construct an engineered E. coli with a clear genetic background by deleting tdh in the L-Thr intracellular degradation pathway and mutating ilvA to decrease L-Thr intracellular consumption, achieving an L-Thr yield of 82.4 g/L and a productivity of 0.393 g/g glucose in fed-batch culture. Acetate formation represents an additional carbon loss in E. coli. Wang et al. [56] achieved L-Thr production of 96.4 g/L in a 3 L fermenter and 133.5 g/L in a 10 L fermenter with strain TWF001/pFW01-phaCAB through upregulation of key genes in the L-Thr biosynthesis pathway, downregulation of genes related to acetate formation, and upregulation of the acs gene encoding the enzyme that converts acetate to acetyl-coenzyme A.Xie et al. [57] systematically knocked out phosphofructokinase genes pfkA and pfkB and PK genes pykF and pykA in the E. coli L-Thr producer THRD to weaken glycolytic flux and redirect it toward the PPP, thereby significantly reducing acetate accumulation and improving L-Thr yield. The double knockout strain THRDΔpfkBΔpykF achieved an L-Thr yield of 111.37 g/L.”

In lines 283-327 on page 8-9 “3.3. Reduction of excessive intracellular product accumulation inhibiting enzyme activity: Reducing excessive intracellular product accumulation that inhibits enzyme activity is achieved by promoting extracellular secretion or process control to decrease intracellular threonine concentration, thereby alleviating feedback inhibition on key enzymes in the synthesis pathway (particularly AK) and enabling sustained high-speed synthesis reactions. Xie et al. [42] systematically knocked out phosphofructokinase genes pfkA and pfkB and PK genes pykF and pykA in the E. coli L-Thr producer THRD to weaken glycolytic flux and redirect it toward the PPP, thereby significantly reducing acetate accumulation and improving L-Thr yield. The double knockout strain THRDΔpfkBΔpykF achieved an L-Thr yield of 111.37 g/L. Kruse et al. [43] overexpressed endogenous rhtB and rhtC genes and the thrE gene from C. glutamicum in E. coli, confirming that RhtB and RhtC are stereoselective threonine exporters. The study found that intracellular threonine concentration in the high-producing strain BKIIM B-3996 pVIC40 remained consistently higher than extracellular concentration, reaching up to tenfold, indicating that efflux capacity constitutes a production bottleneck. Overexpression of rhtB, rhtC, and heterologous thrE increased the yield of strain MG442 by 290%, 200%, and 250% to 290%, respectively. Wu et al. [44] utilized the molecular dynamics-based allosteric prediction method and molecular mechanics-generalized born surface area energy decomposition to investigate the isoleucine allosteric inhibition mechanism of TD. They found that isoleucine concentrations above 0.1 mM caused wild-type TD activity loss exceeding 95%, confirming that intracellular accumulation severely inhibits enzyme function. Through computer-aided screening, the mutant P441L was obtained, which maintained 80% activity at 2.5 mM isoleucine and increased leucine yield by over 400% in E. coli fermentation.” was revised as “3.3. Cofactor engineering: Cofactor engineering focuses on optimizing the supply of reducing equivalents, particularly NADPH, which is essential for several steps in L-Thr biosynthesis. HD utilizes NADPH as a reducing equivalent to catalyze the decarboxylation of aspartate semialdehyde to generate L-homoserine. Therefore, adequate NADPH supply is critical for maintaining high flux through the L-Thr synthesis pathway.Several strategies have been employed to enhance NADPH availability. Jin et al. [58] developed a dual-responsive biosensor for NADPH and L-Thr, and combined it with fluorescence-activated cell sorting (FACS) to obtain a high-yield (0.65 g/g) L-Thr-producing strain,demonstrating the importance of NADPH optimization. Overexpression of membrane-bound pyridine nucleotide transhydrogenase (PNT, encoded by pntAB) represents a direct approach to increase NADPH supply by converting NADH to NADPH. The engineered strain THPE5, with additional expression of pntAB, achieved an L-Thr yield of 70.8 g/L [44]. Toan et al. [59] adopted a modular metabolic engineering strategy to systematically optimize the acetate assimilation, TCA cycle, and coenzyme A metabolic pathways of E. coli W3110, constructing the high-efficiency threonine-producing strain E. coli W-H31/pM2/pR1P, which achieved maximum titers of 44.1 g/L L-Hse and 45.8 g/L L-Thr in fed-batch fermentation, demonstrating the importance of cofactor balance. In the context of L-Thr derivative production, NADH oxidase has been employed to modulate redox balance. Liu et al. [60] engineered the L-Thr metabolic pathway in E. coli by overexpressing L-Thr dehydrogenase and NADH oxidase, constructing the engineered strain BL21(DE3)/pACYCDuet-1-Ectdh-Efnox, which successfully converted L-Thr to 2,5-dimethylpyrazine with a yield of 2009.31 mg/L. This approach demonstrates how cofactor engineering can be applied not only to enhance L-Thr production but also to improve the efficiency of downstream conversion processes. A compelling example of integrating cofactor engineering with complementary metabolic strategies is the work by Hanke et al. [61]. Employing combinatorial cloning coupled with a hybrid deep learning model, the authors iteratively engineered the L-Thr biosynthetic pathway in E. coli across three rounds of optimization. To augment NADPH availability, they overexpressed pntAB (encoding membrane-bound transhydrogenase). Concurrently, they eliminated competing metabolic branches to redirect carbon flux toward L-Thr by deleting tdh (encoding threonine dehydrogenase, which catalyzes threonine degradation), dapA (encoding dihydrodipicolinate synthase, committing carbon to the lysine branch), dhaM (encoding a phosphotransferase system component influencing carbon partitioning), and metL (encoding a bifunctional aspartate kinase/homoserine dehydrogenase directing flux toward methionine). Furthermore, they enhanced precursor supply by overexpressing aspC (encoding aspartate aminotransferase, increasing aspartate availability) and ppc (encoding PEPC, replenishing the oxaloacetate pool). This multi-pronged, machine learning-guided optimization progressively elevated L-Thr titers from 2.7 g/L to 8.4 g/L. Although this study integrated multiple engineering strategies, the inclusion of pntAB overexpression specifically underscores the pivotal contribution of cofactor engineering to enhanced L-Thr production and exemplifies how synergistic implementation of diverse approaches can achieve substantial yield improvements.”

In lines 328-357 on page 9 “3.4 Attenuation of intracellular L-Thr degradation: The main by-products generated during L-Thr fermentation production are L-glycine, L-isoleucine, and Lys. Among these, the first two are intracellular degradation metabolites of L-Thr. Since transporters and degradation metabolic pathways compete for the common substrate L-Thr, reducing intracellular degradation can improve L-Thr yield and decrease by-product formation, thereby lowering downstream extraction costs. Simic et al. [45] cloned and purified serine hydroxymethyltransferase (SHMT) and confirmed its threonine aldol cleavage activity. They then constructed an isopropyl-β-D-thiogalactopyranoside (IPTG)-dependent glyA-regulated strain to reduce SHMT activity to 8%, resulting in a 41% decrease in glycine production and a 49% increase in L-Thr accumulation. Overexpression of the threonine exporter gene thrE further increased threonine production in the high-yielding strain DR-17 from 49 mM to 67 mM, demonstrating that inhibiting intracellular degradation and enhancing efflux can synergistically improve L-Thr synthesis efficiency. Lee et al. [46] employed systematic metabolic engineering strategies to construct an engineered E. coli with a clear genetic background by deleting tdh in the L-Thr intracellular degradation pathway and mutating ilvA to decrease L-Thr intracellular consumption, achieving an L-Thr yield of 82.4 g/L and a productivity of 0.393 g/g glucose in fed-batch culture. Yan et al. [47] increased L-Thr titer from 5.55 g/L to 8.65 g/L after knocking out the ilvA gene, and further improved L-Thr titer to 13.6 g/L by additionally knocking out the metA gene. Therefore, blocking the degradation pathway of L-Thr can effectively reduce L-Thr consumption and promote L-Thr accumulation.”was revised as “3.4 Transport engineering: Transport engineering addresses the critical bottleneck of product accumulation within the cell, which leads to feedback inhibition of key enzymes and limits further synthesis. Reducing excessive intracellular product accumulation is achieved by promoting extracellular secretion to decrease intracellular threonine concentration, thereby alleviating feedback inhibition on key enzymes in the synthesis pathway and enabling sustained high-rate synthesis. In microbial production of L-Thr, the transport of product from intracellular to extracellular space is generally regarded as a critical rate-limiting step. Kruse et al. [62] overexpressed endogenous rhtB and rhtC genes and the thrE gene from C. glutamicum in E. coli, confirming that RhtB and RhtC are stereoselective threonine exporters. The study found that intracellular threonine concentration in the high-producing strain BKIIM B-3996 pVIC40 remained consistently higher than extracellular concentration, reaching up to tenfold, indicating that efflux capacity constitutes a production bottleneck. Overexpression of rhtB, rhtC, and heterologous thrE increased the yield of strain MG442 by 290%, 200%, and 250% to 290%, respectively. Zhao et al. [63] similarly overexpressed ThrE in the HK mutant R212Q strain, obtaining 86.4 g/L L-Thr. Dynamic control of transporter expression has emerged as an advanced strategy to balance growth and production.Song et al. [64] designed an artificial quorum sensing system that, through the combination of a switch module and a production module, enhances the L-Thr titer to 118.2 g/L by auto-inducing the expression of PYC and threonine extracellular transporter after cells reach a certain growth level, with a yield of 0.57 g/g glucose and a productivity of 2.46 g/(L·h). Fang et al. [41] developed a thermal switch vector pFT24rp that overexpresses rhtC under dynamic thermal control in strain TWF113/pFT24rpa1, achieving 25.85 g/L. Du et al. [42] engineered Halomonas bluephagenesis using CRISPR/Cas9 and modular regulation. They introduced the exporter rhtC to secrete threonine, deleted sstT (importer) and tdh (degradation enzyme) to prevent intracellular loss, and integrated feedback-resistant thrABC and lysC to enhance synthesis. The final strain produced 7.5 g/L in shake flasks and 33 g/L in a 7-L fermentor within 24 h, demonstrating the potential of this host for low-cost amino acid production.”

Comment 3: In Figure 2, the authors are encouraged to include key regulatory nodes and feedback inhibition points within the threonine biosynthetic pathway. Additionally, metabolic branches leading to threonine-derived products should be clearly illustrated to highlight the metabolic connectivity and engineering targets for derivative production.

Response: Thank you very much for your insightful suggestion regarding Figure 2. We agree that including these details significantly enhances the clarity and comprehensiveness of the pathway illustration. In response to your comment, we have revised Figure 2 to include the key regulatory nodes and feedback inhibition points within the threonine biosynthetic pathway. Additionally, the metabolic branches leading to threonine-derived products are now clearly illustrated to highlight the metabolic connectivity and engineering targets for derivative production. We believe these modifications have substantially improved the figure, making it more informative for the readers. The changes are clearly visible in the revised Figure 2 in the manuscript. Thank you again for your valuable feedback.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Authors responded to reviewer query properly and modified the draft accordingly. The paper is acceptable for publication.

Comments on the Quality of English Language

Need to rectify some gramatical mistake