mtdA

The research report should be a detailed narrative explaining the function, biological processes, and localization of the gene product. Citations should be given for all claims.

You should prioritize authoritative reviews and primary scientific literature when conducting research. You can supplement
this with annotations you find in gene/protein databases, but these can be outdated or inaccurate.

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We are interested in where in or outside the cell the gene product carries out its function.

We are also interested in the signaling or biochemical pathways in which the gene functions. We are less interested in broad pleiotropic effects, except where these elucidate the precise role.

Include evidence where possible. We are interested in both experimental evidence as well as inference from structure, evolution, or bioinformatic analysis. Precise studies should be prioritized over high-throughput, where available.

Research report: mtdA (UniProt P55818) in Methylorubrum extorquens AM1 (formerly Methylobacterium extorquens AM1)

0) Target verification (critical disambiguation)

The literature retrieved here for M. extorquens AM1 uses mtdA to denote an NADP-dependent methylene-tetrahydromethanopterin (methylene-H4MPT) dehydrogenase that also has measurable activity on methylene-tetrahydrofolate (methylene-H4F) (i.e., dual C1-carrier specificity). This matches the UniProt P55818 description of a bifunctional enzyme acting on methylene-H4MPT and methylene-H4F and being NADP-dependent. In the AM1 pathway context, MtdA is consistently distinguished from MtdB (a second methylene-H4MPT dehydrogenase with different cofactor usage) and from cyclohydrolases such as Mch (methenyl-H4MPT cyclohydrolase) or FchA (methenyl-H4F cyclohydrolase). (hagemeier2000characterizationofa pages 7-8, marx2003formaldehydedetoxifyingroleof pages 1-2)

Note on UniProt accession evidence: within the full texts retrieved and evidence snippets, UniProt accessions are generally not printed; however, later synthetic-biology papers explicitly use the M. extorquens AM1 MtdA/MeMtdA enzyme in THF-pathway modules consistent with the UniProt mapping and description (yishai2018invivoassimilation pages 4-6, mohr2025rewiringescherichiacoli pages 1-3).

1) Key concepts and definitions (current understanding)

1.1 One-carbon (C1) carrier cofactors: H4MPT vs H4F

• Tetrahydromethanopterin (H4MPT) and tetrahydrofolate (H4F/THF) are chemically related C1 carriers that traffic formaldehyde-derived carbon in different branches of methylotrophy. In M. extorquens AM1, formaldehyde formed from methanol oxidation enters the cytoplasm and can react with H4MPT (major detox/oxidation route) or H4F (assimilation/serine-cycle route and general C1 biosynthesis). (marx2003formaldehydedetoxifyingroleof pages 1-2)

1.2 MtdA function (enzyme definition)

MtdA is best described as an NADP+-dependent methylene-pterin dehydrogenase with dual substrate specificity:
* Primary: methylene-H4MPT → methenyl-H4MPT with reduction of NADP+ to NADPH. (hagemeier2000characterizationofa pages 1-2)
* Secondary (bifunctional substrate range): methylene-H4F → methenyl-H4F (also NADP+-dependent), including reported reversibility for the H4F reaction. (hagemeier2000characterizationofa pages 7-8, marx2003formaldehydedetoxifyingroleof pages 1-2)

This dual specificity is notable because MtdA shows only minor sequence identity to “classical” bacterial/eukaryotic methylene-H4F dehydrogenases yet still catalyzes methylene-H4F oxidation with lower efficiency. (hagemeier2000characterizationofa pages 1-2)

1.3 “Bifunctional” caveat: dehydrogenase vs cyclohydrolase

The AM1 primary literature analyzed here supports bifunctionality in substrate range (H4MPT and H4F) but does not provide evidence that MtdA itself has cyclohydrolase activity. Cyclohydrolase activities are attributed to distinct enzymes (e.g., methenyl-H4MPT cyclohydrolase Mch, methenyl-H4F cyclohydrolase FchA). (hagemeier2000characterizationofa pages 7-8, marx2003formaldehydedetoxifyingroleof pages 1-2)

2) Biochemical function: reactions, specificity, and quantitative parameters

2.1 Reaction and cofactor usage

MtdA is strictly NADP-dependent and catalyzes oxidation of methylene-pterin intermediates to methenyl-pterin intermediates:
* methylene-H4MPT + NADP+ → methenyl-H4MPT + NADPH (hagemeier2000characterizationofa pages 1-2)
* methylene-H4F + NADP+ → methenyl-H4F + NADPH (hagemeier2000characterizationofa pages 7-8, marx2003formaldehydedetoxifyingroleof pages 1-2)

In methanol-grown M. extorquens AM1 cell extracts, methylene-H4MPT-dependent NADP reduction activity was measured at 2.6 U·mg−1, compared with NAD reduction activity of 0.6 U·mg−1 (the NAD-dependent activity relates to the distinct enzyme MtdB rather than MtdA). (hagemeier2000characterizationofa pages 2-3)

2.2 Kinetics and enzyme properties

A detailed comparison of MtdA and MtdB properties, including Km values and biochemical features, is reported in Hagemeier et al. (2000) and captured in the table image retrieved from that paper. (hagemeier2000characterizationofa media 5ffb354b)

Key reported quantitative properties for recombinant/purified MtdA include:
* Specific activity: purified enzyme ~1100 U·mg−1; cell extracts from overexpression in E. coli up to ~370 U·mg−1. (hagemeier2000characterizationofa pages 5-7)
* Km (apparent, reported): methylene-H4MPT ~20 µM; methylene-H4F ~30 µM; NADP+ ~30 µM (with methylene-H4MPT) and ~10 µM (with methylene-H4F). (hagemeier2000characterizationofa pages 5-7, hagemeier2000characterizationofa media 5ffb354b)
* Oligomeric state: ~32 kDa subunit; apparent native mass ~90 kDa. (hagemeier2000characterizationofa pages 5-7)
* Optima: pH optimum ~6.0; temperature optimum ~45 °C; no inhibition by NADPH. (hagemeier2000characterizationofa pages 5-7)

2.3 Substrate specificity and efficiency

MtdA’s catalytic efficiency for methylene-H4F is reported to be ~20-fold lower than for methylene-H4MPT, supporting a model in which H4MPT is the primary physiological substrate and H4F is a secondary substrate in vivo. (hagemeier2000characterizationofa pages 1-2)

3) Cellular localization

Direct fractionation evidence supports MtdA as a soluble/cytosolic enzyme. Following ultracentrifugation (150,000 × g, 1 h), the membrane fraction lacked NAD(P)-dependent methylene-H4MPT dehydrogenase activity, while the soluble supernatant contained the activity used for purification—consistent with MtdA not being membrane-associated. (hagemeier2000characterizationofa pages 2-3)

4) Physiological role and pathway placement in M. extorquens AM1

4.1 Formaldehyde detoxification via the H4MPT-linked pathway

In M. extorquens AM1, the H4MPT-linked pathway is described as the major formaldehyde oxidation/detoxification route during methylotrophic growth. MtdA is one of two methylene dehydrogenases in this network (MtdA and MtdB), oxidizing methylene-H4MPT to methenyl-H4MPT as part of a route ultimately leading to formate/CO2. (marx2003formaldehydedetoxifyingroleof pages 1-2, marx2003formaldehydedetoxifyingroleof pages 6-8)

4.2 Linkage to H4F-dependent biosynthesis/serine cycle

MtdA also “crosses over” to H4F metabolism by catalyzing methylene-H4F oxidation. In pathway diagrams and discussion, this provides a mechanistic link between formaldehyde processing and the H4F C1 pool used for assimilation and biosynthetic C1 chemistry (e.g., generation of formyl-H4F used in biosynthesis). (marx2003formaldehydedetoxifyingroleof pages 1-2, marx2003formaldehydedetoxifyingroleof pages 2-3)

4.3 Genetic essentiality / indispensability (AM1)

Both Hagemeier et al. (2000) and Marx et al. (2003) report that null mutants in mtdA could not be obtained (in backgrounds where succinate selection was used), supporting that mtdA is likely essential under tested conditions. (hagemeier2000characterizationofa pages 7-8, marx2003formaldehydedetoxifyingroleof pages 2-3)

4.4 Overexpression phenotype and physiological limitation

Marx et al. (2003) overexpressed mtdA ~7.4-fold relative to wild type. This substantially reduced methanol sensitivity but did not restore growth on methanol in the tested context, leading to the interpretation that MtdA can support only moderate formaldehyde flux when the other dehydrogenase (MtdB) is absent, and that NADP dependence may constrain in vivo capacity. (marx2003formaldehydedetoxifyingroleof pages 6-8)

5) Recent developments (prioritizing 2023–2024) and emerging research directions

Although direct 2023–2024 studies focusing specifically on AM1 mtdA biochemistry are limited in the retrieved set, recent work strongly emphasizes deploying the THF-pathway module containing MtdA as a portable C1-assimilation tool.

5.1 2024: “Serine Shunt” as a new-to-nature formate reduction route (application of MeMtdA)

A 2024 bioRxiv study proposes and demonstrates a Serine Shunt that converts formate → formaldehyde in vivo via an engineered route in E. coli. Critically, its Module 1 (M1) uses Methylorubrum extorquens enzymes MeFtfL (formate-THF ligase), MeFch (methenyl-THF cyclohydrolase), and MeMtdA (methylene-THF dehydrogenase) to convert formate into methylene-THF (consuming ATP and NADPH), which is then transferred to glycine to form serine. Module 2 cleaves serine into formaldehyde and glycine, and formaldehyde production is validated using a GFP-based formaldehyde sensor (signal depends on co-expression of both modules). (schann2024theserineshunt pages 8-13)

This work positions MtdA as an enabling enzyme for “wiring” formate-derived C1 units into central metabolites in a genetically tractable host, relevant for sustainable C1 biomanufacturing concepts. (schann2024theserineshunt pages 8-13)

5.2 2024: Intersections between alternative substrates and formaldehyde/THF chemistry in Methylorubrum

A 2024 Applied and Environmental Microbiology study shows that glycine betaine catabolism in Methylorubrum extorquens PA1 can be activated by suppressor mutations and that this catabolism generates formaldehyde, directly intersecting methylotrophic metabolism. The paper also notes that demethylation reactions can yield either formaldehyde or methylene-THF (enzyme-dependent) and references THF-linked detoxification possibilities in some bacteria. (hying2024glycinebetainemetabolism pages 1-3)

5.3 2023 perspective: formaldehyde toxicity remains a central constraint

A 2023 review on CO2-derived feedstocks and biomanufacturing highlights formaldehyde toxicity as a major barrier in aerobic methylotroph engineering (including Methylorubrum/Methylobacterium platforms) and contrasts this with anaerobic Wood–Ljungdahl pathway assimilation that avoids formaldehyde as a free intermediate. (kurt2023perspectivesforusinga pages 16-17)

6) Current applications and real-world implementations

6.1 Synthetic biology: THF-module transfer for formate-derived methyl groups

A 2025 peer-reviewed study demonstrates the use of the M. extorquens THF-assimilation module (MexFTFL, MexFCH, MexMTDA) to channel formate carbon into methylene-H4F for downstream biosynthesis, specifically to supply methyl groups for SAM-dependent methyltransferases in E. coli. Reported quantitative outcomes include:
* 51–81% 13C labeling in methylated products upon feeding labeled formate. (mohr2025rewiringescherichiacoli pages 1-3)
* In an engineered C1-auxotrophic strain, optimizing formate concentration doubled conversion rate and achieved >70% formate-derived methyl groups. (mohr2025rewiringescherichiacoli pages 1-3)

These data represent a concrete engineered implementation of the MtdA-containing module as a C1-unit generator for biocatalysis/whole-cell synthesis. (mohr2025rewiringescherichiacoli pages 1-3)

6.2 Methylotroph engineering for chemicals (context)

A 2024 Microbial Cell Factories study engineered Methylorubrum extorquens (TK 0001) for methanol-based glycolic acid production using modeling and redox/central-metabolism interventions; the excerpted sections do not discuss MtdA specifically, but illustrate continued momentum for deploying methylotrophs as production hosts where formaldehyde handling and C1 flux balancing are recurring constraints. (dietz2024anovelengineered pages 1-2, dietz2024anovelengineered pages 5-6)

7) Expert synthesis and interpretation (authoritative analysis grounded in evidence)

1. Physiological primary role: In AM1, MtdA should be viewed primarily as an H4MPT-pathway dehydrogenase contributing to formaldehyde oxidation/detoxification and redox generation (NADPH), rather than as a dedicated H4F dehydrogenase. This is supported by the large activity difference and the reported ~20-fold lower catalytic efficiency on H4F compared with H4MPT. (hagemeier2000characterizationofa pages 1-2, marx2003formaldehydedetoxifyingroleof pages 1-2)

2. Secondary/biosynthetic role via H4F: Despite lower efficiency on H4F, MtdA may be important because it can provide methenyl-/formyl-H4F for essential biosynthetic pathways (purine and other C1-dependent biosynthesis), consistent with observations that mtdA mutants could not be obtained and that MtdA may be the only enzyme catalyzing certain H4F interconversions in AM1. (hagemeier2000characterizationofa pages 7-8, marx2003formaldehydedetoxifyingroleof pages 2-3)

3. Engineering significance: The fact that MtdA is soluble, expresses well heterologously, and functions as part of an effective THF C1 module explains why it is repeatedly repurposed for engineered formatotrophy or C1-to-methyl-group conversion. (hagemeier2000characterizationofa pages 5-7, hagemeier2000characterizationofa pages 2-3, mohr2025rewiringescherichiacoli pages 1-3)

8) Evidence summary table (with quantitative data)

The following evidence map consolidates the key functional-annotation findings, experimental contexts, and quantitative values.

Table: This table compiles key functional-annotation evidence for Methylorubrum extorquens AM1 MtdA, including biochemical activity, specificity, kinetics, localization, and physiological relevance. It is useful as a compact evidence map linking the UniProt annotation to primary literature and recent applications.

9) Key sources (publication date + URL)

• Hagemeier CH et al. 2000-06. Eur J Biochem. “Characterization of a second methylene tetrahydromethanopterin dehydrogenase from Methylobacterium extorquens AM1.” https://doi.org/10.1046/j.1432-1327.2000.01413.x (hagemeier2000characterizationofa pages 5-7, hagemeier2000characterizationofa pages 2-3, hagemeier2000characterizationofa media 5ffb354b)
• Marx CJ et al. 2003-12. J Bacteriol. “Formaldehyde-detoxifying role of the tetrahydromethanopterin-linked pathway in M. extorquens AM1.” https://doi.org/10.1128/jb.185.23.7160-7168.2003 (marx2003formaldehydedetoxifyingroleof pages 1-2, marx2003formaldehydedetoxifyingroleof pages 6-8)
• Kurt E et al. 2023-11. Bioengineering. “Perspectives for Using CO2 as a Feedstock for Biomanufacturing of Fuels and Chemicals.” https://doi.org/10.3390/bioengineering10121357 (kurt2023perspectivesforusinga pages 16-17)
• Hying ZT et al. 2024-07. Appl Environ Microbiol. “Glycine betaine metabolism is enabled in Methylorubrum extorquens PA1…” https://doi.org/10.1128/aem.02090-23 (hying2024glycinebetainemetabolism pages 1-3)
• Schann K, Wenk S. 2024-07. bioRxiv. “The Serine Shunt enables formate conversion to formaldehyde in vivo.” https://doi.org/10.1101/2024.07.31.605843 (schann2024theserineshunt pages 8-13)
• Mohr MKF et al. 2025-03. Microbial Cell Factories. “Rewiring E. coli to transform formate into methyl groups.” https://doi.org/10.1186/s12934-025-02674-4 (mohr2025rewiringescherichiacoli pages 1-3)

References

1. (hagemeier2000characterizationofa pages 7-8): Christoph H. Hagemeier, Ludmila Chistoserdova, Mary E. Lidstrom, Rudolf K. Thauer, and Julia A. Vorholt. Characterization of a second methylene tetrahydromethanopterin dehydrogenase from methylobacterium extorquens am1. European journal of biochemistry, 267 12:3762-9, Jun 2000. URL: https://doi.org/10.1046/j.1432-1327.2000.01413.x, doi:10.1046/j.1432-1327.2000.01413.x. This article has 85 citations.

2. (marx2003formaldehydedetoxifyingroleof pages 1-2): Christopher J. Marx, Ludmila Chistoserdova, and Mary E. Lidstrom. Formaldehyde-detoxifying role of thetetrahydromethanopterin-linked pathway in methylobacteriumextorquensam1. Journal of Bacteriology, 185:7160-7168, Dec 2003. URL: https://doi.org/10.1128/jb.185.23.7160-7168.2003, doi:10.1128/jb.185.23.7160-7168.2003. This article has 149 citations and is from a peer-reviewed journal.

3. (yishai2018invivoassimilation pages 4-6): Oren Yishai, Madeleine Bouzon, Volker Döring, and Arren Bar-Even. In vivo assimilation of one-carbon via a synthetic reductive glycine pathway in escherichia coli. ACS synthetic biology, 7 9:2023-2028, May 2018. URL: https://doi.org/10.1021/acssynbio.8b00131, doi:10.1021/acssynbio.8b00131. This article has 203 citations and is from a domain leading peer-reviewed journal.

4. (mohr2025rewiringescherichiacoli pages 1-3): Michael K. F. Mohr, Ari Satanowski, Steffen N. Lindner, Tobias J. Erb, and Jennifer N. Andexer. Rewiring escherichia coli to transform formate into methyl groups. Microbial Cell Factories, Mar 2025. URL: https://doi.org/10.1186/s12934-025-02674-4, doi:10.1186/s12934-025-02674-4. This article has 8 citations and is from a peer-reviewed journal.

5. (hagemeier2000characterizationofa pages 1-2): Christoph H. Hagemeier, Ludmila Chistoserdova, Mary E. Lidstrom, Rudolf K. Thauer, and Julia A. Vorholt. Characterization of a second methylene tetrahydromethanopterin dehydrogenase from methylobacterium extorquens am1. European journal of biochemistry, 267 12:3762-9, Jun 2000. URL: https://doi.org/10.1046/j.1432-1327.2000.01413.x, doi:10.1046/j.1432-1327.2000.01413.x. This article has 85 citations.

6. (hagemeier2000characterizationofa pages 2-3): Christoph H. Hagemeier, Ludmila Chistoserdova, Mary E. Lidstrom, Rudolf K. Thauer, and Julia A. Vorholt. Characterization of a second methylene tetrahydromethanopterin dehydrogenase from methylobacterium extorquens am1. European journal of biochemistry, 267 12:3762-9, Jun 2000. URL: https://doi.org/10.1046/j.1432-1327.2000.01413.x, doi:10.1046/j.1432-1327.2000.01413.x. This article has 85 citations.

7. (hagemeier2000characterizationofa media 5ffb354b): Christoph H. Hagemeier, Ludmila Chistoserdova, Mary E. Lidstrom, Rudolf K. Thauer, and Julia A. Vorholt. Characterization of a second methylene tetrahydromethanopterin dehydrogenase from methylobacterium extorquens am1. European journal of biochemistry, 267 12:3762-9, Jun 2000. URL: https://doi.org/10.1046/j.1432-1327.2000.01413.x, doi:10.1046/j.1432-1327.2000.01413.x. This article has 85 citations.

8. (hagemeier2000characterizationofa pages 5-7): Christoph H. Hagemeier, Ludmila Chistoserdova, Mary E. Lidstrom, Rudolf K. Thauer, and Julia A. Vorholt. Characterization of a second methylene tetrahydromethanopterin dehydrogenase from methylobacterium extorquens am1. European journal of biochemistry, 267 12:3762-9, Jun 2000. URL: https://doi.org/10.1046/j.1432-1327.2000.01413.x, doi:10.1046/j.1432-1327.2000.01413.x. This article has 85 citations.

9. (marx2003formaldehydedetoxifyingroleof pages 6-8): Christopher J. Marx, Ludmila Chistoserdova, and Mary E. Lidstrom. Formaldehyde-detoxifying role of thetetrahydromethanopterin-linked pathway in methylobacteriumextorquensam1. Journal of Bacteriology, 185:7160-7168, Dec 2003. URL: https://doi.org/10.1128/jb.185.23.7160-7168.2003, doi:10.1128/jb.185.23.7160-7168.2003. This article has 149 citations and is from a peer-reviewed journal.

10. (marx2003formaldehydedetoxifyingroleof pages 2-3): Christopher J. Marx, Ludmila Chistoserdova, and Mary E. Lidstrom. Formaldehyde-detoxifying role of thetetrahydromethanopterin-linked pathway in methylobacteriumextorquensam1. Journal of Bacteriology, 185:7160-7168, Dec 2003. URL: https://doi.org/10.1128/jb.185.23.7160-7168.2003, doi:10.1128/jb.185.23.7160-7168.2003. This article has 149 citations and is from a peer-reviewed journal.

11. (schann2024theserineshunt pages 8-13): Karin Schann and Sebastian Wenk. The serine shunt enables formate conversion to formaldehyde in vivo. bioRxiv, Jul 2024. URL: https://doi.org/10.1101/2024.07.31.605843, doi:10.1101/2024.07.31.605843. This article has 4 citations.

12. (hying2024glycinebetainemetabolism pages 1-3): Zachary T. Hying, Tyler J. Miller, Chin Yi Loh, and Jannell V. Bazurto. Glycine betaine metabolism is enabled in methylorubrum extorquens pa1 by alterations to dimethylglycine dehydrogenase. Applied and Environmental Microbiology, Jul 2024. URL: https://doi.org/10.1128/aem.02090-23, doi:10.1128/aem.02090-23. This article has 6 citations and is from a peer-reviewed journal.

13. (kurt2023perspectivesforusinga pages 16-17): Elif Kurt, Jiansong Qin, Alexandria Williams, Youbo Zhao, and Dongming Xie. Perspectives for using co2 as a feedstock for biomanufacturing of fuels and chemicals. Bioengineering, 10:1357, Nov 2023. URL: https://doi.org/10.3390/bioengineering10121357, doi:10.3390/bioengineering10121357. This article has 39 citations.

14. (dietz2024anovelengineered pages 1-2): Katharina Dietz, Carina Sagstetter, Melanie Speck, Arne Roth, Steffen Klamt, and Jonathan Thomas Fabarius. A novel engineered strain of methylorubrum extorquens for methylotrophic production of glycolic acid. Microbial Cell Factories, Dec 2024. URL: https://doi.org/10.1186/s12934-024-02583-y, doi:10.1186/s12934-024-02583-y. This article has 10 citations and is from a peer-reviewed journal.

15. (dietz2024anovelengineered pages 5-6): Katharina Dietz, Carina Sagstetter, Melanie Speck, Arne Roth, Steffen Klamt, and Jonathan Thomas Fabarius. A novel engineered strain of methylorubrum extorquens for methylotrophic production of glycolic acid. Microbial Cell Factories, Dec 2024. URL: https://doi.org/10.1186/s12934-024-02583-y, doi:10.1186/s12934-024-02583-y. This article has 10 citations and is from a peer-reviewed journal.

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Citations

1. marx2003formaldehydedetoxifyingroleof pages 1-2
2. hagemeier2000characterizationofa pages 1-2
3. hagemeier2000characterizationofa pages 2-3
4. hagemeier2000characterizationofa pages 5-7
5. marx2003formaldehydedetoxifyingroleof pages 6-8
6. schann2024theserineshunt pages 8-13
7. hying2024glycinebetainemetabolism pages 1-3
8. kurt2023perspectivesforusinga pages 16-17
9. mohr2025rewiringescherichiacoli pages 1-3
10. hagemeier2000characterizationofa pages 7-8
11. yishai2018invivoassimilation pages 4-6
12. marx2003formaldehydedetoxifyingroleof pages 2-3
13. dietz2024anovelengineered pages 1-2
14. dietz2024anovelengineered pages 5-6
15. https://doi.org/10.1046/j.1432-1327.2000.01413.x;
16. https://doi.org/10.1128/jb.185.23.7160-7168.2003;
17. https://doi.org/10.1021/acssynbio.8b00131
18. https://doi.org/10.1128/jb.185.23.7160-7168.2003
19. https://doi.org/10.1046/j.1432-1327.2000.01413.x
20. https://doi.org/10.1021/acssynbio.8b00131;
21. https://doi.org/10.1186/s12934-025-02674-4
22. https://doi.org/10.3390/bioengineering10121357
23. https://doi.org/10.1128/aem.02090-23
24. https://doi.org/10.1101/2024.07.31.605843
25. https://doi.org/10.1046/j.1432-1327.2000.01413.x,
26. https://doi.org/10.1128/jb.185.23.7160-7168.2003,
27. https://doi.org/10.1021/acssynbio.8b00131,
28. https://doi.org/10.1186/s12934-025-02674-4,
29. https://doi.org/10.1101/2024.07.31.605843,
30. https://doi.org/10.1128/aem.02090-23,
31. https://doi.org/10.3390/bioengineering10121357,
32. https://doi.org/10.1186/s12934-024-02583-y,