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Enhancing translation efficiency and exploring constraints in high-level 4-hydroxyvaleric acid production from levulinic acid in Escherichia coli

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Abstract

4-Hydroxyvaleric acid (4-HV) holds promise as a sustainable monomer for biodegradable polyesters and liquid transportation fuels. This study achieved high-level 4-HV production from levulinic acid using an antibiotic-free, substrate-inducible system in Escherichia coli. Enzymes involved in the conversion of levulinic acid to 4-HV were expressed with a bicistronic design of ribosome binding sites. The engineered strain demonstrated a 28% higher productivity compared to its counterpart, reaching a significant concentration of 107 g/L 4-HV with a production rate of 4.5 g/L/h and a molar conversion of 95% from levulinic acid in fed-batch cultivation. Recombinant cells from the initial cultivation were reused for a second round of biotransformation, demonstrating 73% efficiency of fresh cells. The study identified specific factors contributing to decreased system efficiency, including medium conditions, increased ionic strength, and high product concentration. Overall, the reported system and our findings hold significant potential for cost-effective microbial production of 4-HV at scale from levulinic acid.

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The study’s original contributions and findings have been comprehensively documented within the article itself and the accompanying Supplementary Material. Should any further clarification or additional inquiries arise, readers are encouraged to direct them to the corresponding author, whose contact information is provided.

References

  1. Stoklosa RJ, García-Negrón V, Latona RJ, Toht M. Limiting acetoin generation during 2,3-butanediol fermentation with Paenibacillus polymyxa using lignocellulosic hydrolysates. Bioresour Technol. 2024;393:130053. https://doi.org/10.1016/j.biortech.2023.130053.

    Article  CAS  PubMed  Google Scholar 

  2. Zhang Y, Yu J, Wu Y, Li M, Zhao Y, Zhu H, et al. Efficient production of chemicals from microorganism by metabolic engineering and synthetic biology. Chin J Chem Eng. 2021;30:14–28. https://doi.org/10.1016/j.cjche.2020.12.014.

    Article  CAS  Google Scholar 

  3. Sathesh-Prabu C, Lee SK. Engineering the lva operon and optimization of culture conditions for enhanced production of 4-hydroxyvalerate from levulinic acid in Pseudomonas putida KT2440. J Agric Food Chem. 2019;67:2540–6. https://doi.org/10.1021/acs.jafc.8b06884.

    Article  CAS  PubMed  Google Scholar 

  4. Cho JS, Kim GB, Eun H, Moon CW, Lee SY. Designing microbial cell factories for the production of chemicals. JACS Au. 2022;2:1781–99. https://doi.org/10.1021/jacsau.2c00344.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Gorenflo V, Schmack G, Vogel R, Steinbüchel A. Development of a process for the biotechnological large-scale production of 4-hydroxyvalerate-containing polyesters and characterization of their physical and mechanical properties. Biomacromol. 2001;2:45–57. https://doi.org/10.1021/bm0000992.

    Article  CAS  Google Scholar 

  6. Yu J. Biosynthesis of polyhydroxyalkanoates from 4-ketovaleric acid in bacterial cells. In: Cheng HN, Gross RA, editors. Green polymer chemistry: biocatalysis and biomaterials. Washington: American Chemical Society; 2010. p. 161–73. https://doi.org/10.1021/bk-2010-1043.ch012.

    Chapter  Google Scholar 

  7. Bond JQ, Alonso DM, Wang D, West RM, Dumesic JA. Integrated catalytic conversion of gamma-valerolactone to liquid alkenes for transportation fuels. Science. 2010;327:1110–4. https://doi.org/10.1126/science.1184362.

    Article  CAS  PubMed  Google Scholar 

  8. Martin CH, Prather KLJ. High-titer production of monomeric hydroxyvalerates from levulinic acid in Pseudomonas putida. J Biotechnol. 2009;139:61–7. https://doi.org/10.1016/j.jbiotec.2008.09.002.

    Article  CAS  PubMed  Google Scholar 

  9. Yeon YJ, Park HY, Yoo YJ. Enzymatic reduction of levulinic acid by engineering the substrate specificity of 3-hydroxybutyrate dehydrogenase. Bioresour Technol. 2013;134:377–80. https://doi.org/10.1016/j.biortech.2013.01.078.

    Article  CAS  PubMed  Google Scholar 

  10. Moon M, Yeon YJ, Park HJ, Park J, Park GW, Kim GH, et al. Chemoenzymatic valorization of agricultural wastes into 4-hydroxyvaleric acid via levulinic acid. Bioresour Technol. 2021;337:125479. https://doi.org/10.1016/j.biortech.2021.125479.

    Article  CAS  PubMed  Google Scholar 

  11. Kim D, Sathesh-Prabu C, JooYeon Y, Lee SK. High-level production of 4-hydroxyvalerate from levulinic acid via whole-cell biotransformation decoupled from cell metabolism. J Agric Food Chem. 2019;67:10678–84. https://doi.org/10.1021/acs.jafc.9b04304.

    Article  CAS  PubMed  Google Scholar 

  12. Werpy T, Petersen G. Top value added chemicals from biomass: volume I—results of screening for potential candidates from sugars and synthesis gas. (No. DOE/GO-102004-1992). Golden: National Renewable Energy Lab (NREL); 2004.

    Google Scholar 

  13. Rand JM, Pisithkul T, Clark RL, Thiede JM, Mehrer CR, Agnew DE, et al. A metabolic pathway for catabolizing levulinic acid in bacteria. Nat Microbiol. 2017;2:1624–34. https://doi.org/10.1038/s41564-017-0028-z.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Sathesh-Prabu C, Tiwari R, Lee SK. Substrate-inducible and antibiotic-free high-level 4-hydroxyvaleric acid production in engineered Escherichia coli. Front Bioeng Biotechnol. 2022;10:960907. https://doi.org/10.3389/fbioe.2022.960907.

    Article  PubMed  PubMed Central  Google Scholar 

  15. Mutalik VK, Guimaraes JC, Cambray G, Lam C, Christoffersen MJ, Mai Q-A, et al. Precise and reliable gene expression via standard transcription and translation initiation elements. Nat Methods. 2013;10:354–60. https://doi.org/10.1038/nmeth.2404.

    Article  CAS  PubMed  Google Scholar 

  16. Seo SW, Yang J, Min BE, Jang S, Lim JH, Lim HG, et al. Synthetic biology: tools to design microbes for the production of chemicals and fuels. Biotechnol Adv. 2013;31:811–7. https://doi.org/10.1016/j.biotechadv.2013.03.012.

    Article  CAS  PubMed  Google Scholar 

  17. Seo SW, Yang J-S, Kim I, Yang J, Min BE, Kim S, et al. Predictive design of mRNA translation initiation region to control prokaryotic translation efficiency. Metab Eng. 2013;15:67–74. https://doi.org/10.1016/j.ymben.2012.10.006.

    Article  CAS  PubMed  Google Scholar 

  18. Zhou L, Zuo Z-R, Chen X-Z, Niu D-D, Tian K-M, Prior BA, et al. Evaluation of genetic manipulation strategies on d-lactate production by Escherichia coli. Curr Microbiol. 2011;62:981–9. https://doi.org/10.1007/s00284-010-9817-9.

    Article  CAS  PubMed  Google Scholar 

  19. Sathesh-Prabu C, Tiwari R, Kim D, Lee SK. Inducible and tunable gene expression systems for Pseudomonas putida KT2440. Sci Rep. 2021;11:18079. https://doi.org/10.1038/s41598-021-97550-7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Chen F, Cocaign-Bousquet M, Girbal L, Nouaille S. 5′UTR sequences influence protein levels in Escherichia coli by regulating translation initiation and mRNA stability. Front Microbiol. 2022;13:1088941. https://doi.org/10.3389/fmicb.2022.1088941.

    Article  PubMed  PubMed Central  Google Scholar 

  21. Dvir S, Velten L, Sharon E, Zeevi D, Carey LB, Weinberger A, et al. Deciphering the rules by which 5′-UTR sequences affect protein expression in yeast. Proc Natl Acad Sci USA. 2013;110:E2792–801. https://doi.org/10.1073/pnas.1222534110.

    Article  PubMed  PubMed Central  Google Scholar 

  22. Komarova AV, Tchufistova LS, Dreyfus M, Boni IV. AU-rich sequences within 5′ untranslated leaders enhance translation and stabilize mRNA in Escherichia coli. J Bacteriol. 2005;187:1344–9. https://doi.org/10.1128/jb.187.4.1344-1349.2005.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Habe H, Sato Y, Kirimura K. Microbial and enzymatic conversion of levulinic acid, an alternative building block to fermentable sugars from cellulosic biomass. Appl Microbiol Biotechnol. 2020;104:7767–75. https://doi.org/10.1007/s00253-020-10813-7.

    Article  CAS  PubMed  Google Scholar 

  24. Bozell J, Moens L, Elliott D, Wang Y, Neuenscwander G, Fitzpatrick S, et al. Production of levulinic acid and use as a platform chemical for derived products. Resour Conserv Recycl. 2000;28:227–39. https://doi.org/10.1016/S0921-3449(99)00047-6.

    Article  Google Scholar 

  25. Rackemann DW, Doherty WOS. The conversion of lignocellulosics to levulinic acid. Biofuels Bioprod Biorefin. 2011;5:198–214. https://doi.org/10.1002/bbb.267.

    Article  CAS  Google Scholar 

  26. Cha D, Ha HS, Lee SK. Metabolic engineering of Pseudomonas putida for the production of various types of short-chain-length polyhydroxyalkanoates from levulinic acid. Bioresour Technol. 2020;309:123332. https://doi.org/10.1016/j.biortech.2020.123332.

    Article  CAS  PubMed  Google Scholar 

  27. Hayes GC, Becer CR. Levulinic acid: a sustainable platform chemical for novel polymer architectures. Polym Chem. 2020;11:4068–77. https://doi.org/10.1039/D0PY00705F.

    Article  CAS  Google Scholar 

  28. Pileidis FD, Titirici M-M. Levulinic acid biorefineries: new challenges for efficient utilization of biomass. Chemsuschem. 2016;9:562–82. https://doi.org/10.1002/cssc.201501405.

    Article  CAS  PubMed  Google Scholar 

  29. Mital S, Christie G, Dikicioglu D. Recombinant expression of insoluble enzymes in Escherichia coli: a systematic review of experimental design and its manufacturing implications. Microb Cell Factories. 2021;20:1–20. https://doi.org/10.1186/s12934-021-01698-w.

    Article  CAS  Google Scholar 

  30. Xu P, Gu Q, Wang W, Wong L, Bower AGW, Collins CH, et al. Modular optimization of multi-gene pathways for fatty acids production in E. coli. Nat Commun. 2013;4:1409. https://doi.org/10.1038/ncomms2425.

    Article  CAS  PubMed  Google Scholar 

  31. Mohedano MT, Konzock O, Chen Y. Strategies to increase tolerance and robustness of industrial microorganisms. Synth Syst Biotechnol. 2022;7:533–40. https://doi.org/10.1016/j.synbio.2021.12.009.

    Article  CAS  PubMed  Google Scholar 

  32. Konzock O, Zaghen S, Norbeck J. Tolerance of Yarrowia lipolytica to inhibitors commonly found in lignocellulosic hydrolysates. BMC Microbiol. 2021;21:1–10. https://doi.org/10.1186/s12866-021-02126-0.

    Article  CAS  Google Scholar 

  33. Park C, Raines RT. Quantitative analysis of the effect of salt concentration on enzymatic catalysis. J Am Chem Soc. 2001;123:11472–9. https://doi.org/10.1021/ja0164834.

    Article  CAS  PubMed  Google Scholar 

  34. Weimberg R. Effect of sodium chloride on the activity of a soluble malate dehydrogenase from pea seeds. J Biol Chem. 1967;242:3000–6. https://doi.org/10.1016/S0021-9258(18)99604-3.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We would like to acknowledge the financial support from the Basic Science Research Program, funded by the Ministry of Science, ICT and Future Planning (MSIT) through the National Research Foundation of Korea (NRF) (Grant No. NRF RS-2023-00208026). We are also grateful to the Circle Foundation for their support through the 2020 Innovative Science Project.

Funding

1. National Research Foundation of Korea (NRF) (Grant No. NRF RS-2023-00208026). 2. Innovative science project in 2020 of the Circle Foundation.

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Authors and Affiliations

  1. School of Energy and Chemical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan, 44919, Republic of Korea

    Chandran Sathesh-Prabu, Rameshwar Tiwari & Sung Kuk Lee

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  1. Chandran Sathesh-Prabu
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Contributions

C.S-P. conceived and designed the experiments, conducted all procedures, acquired and processed data, and drafted the initial manuscript. R.T. contributed to data processing and provided review. S.K.L. conceptualized and designed the study, validated the data, and critically revised the manuscript. All authors thoroughly reviewed and approved the manuscript's content.

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Correspondence to Sung Kuk Lee.

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Sathesh-Prabu, C., Tiwari, R. & Lee, S.K. Enhancing translation efficiency and exploring constraints in high-level 4-hydroxyvaleric acid production from levulinic acid in Escherichia coli. Syst Microbiol and Biomanuf (2024). https://doi.org/10.1007/s43393-024-00258-8

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