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Engineering Escherichia coli lifespan for enhancing chemical production

Abstract

Industrial chemical production from renewable feedstocks by microbial cell factories provides a promising avenue towards sustainability. However, the small size of bacterial cells and environmental stress significantly affect microbial cell factory performance. Here, we engineered the Escherichia coli lifespan to improve the chemical production of poly(lactate-co-3-hydroxybutyrate) and butyrate. The replicative lifespan was shortened by deleting a carbon storage regulator, and the chronological lifespan was extended by deleting a response regulator and overexpressing sigma-38 in Escherichia coli. The replicative lifespan was fine-tuned using a two-output recombinase-based state machine, and the cell size was enlarged 13.4-fold. The highest poly(lactate-co-3-hydroxybutyrate) content of 52 wt% was achieved in a 5-l fermenter. The chronological lifespan was modulated through a multi-output recombinase-based state machine, resulting in the highest butyrate titre of 29.8 g l−1, by programming cell differentiation according to different fermentation stages. These results highlight the applicability of engineering the bacterial lifespan to increase microbial cell factory performance.

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Fig. 1: Characterization of CLS regulation by genetic manipulation.
Fig. 2: Characterization of RLS regulation by genetic manipulation.
Fig. 3: Overview of the design and validation of the TRSM.
Fig. 4: Overview of engineering RLS for PLH production.
Fig. 5: Overview of the design and validation of the MRSM.
Fig. 6: Overview of engineering CLS for butyrate production.

Data availability

The data that support the figures within this paper and other findings of this study are available from the corresponding author upon reasonable request. Supplementary Table 9 provides a list of the GenBank accession numbers of the 14 key plasmids constructed in this study.

References

  1. 1.

    Nielsen, J. & Keasling, J. D. Engineering cellular metabolism. Cell 164, 1185–1197 (2016).

    CAS  PubMed  Google Scholar 

  2. 2.

    Wang, X., Zhang, H. & Quinn, P. J. Production of L-valine from metabolically engineered Corynebacterium glutamicum. Appl. Microbiol. Biotechnol. 102, 4319–4330 (2018).

    CAS  PubMed  Google Scholar 

  3. 3.

    Steensels, J. et al. Improving industrial yeast strains: exploiting natural and artificial diversity. FEMS Microbiol. Rev. 38, 947–995 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Park, J. H. & Lee, S. Y. Towards systems metabolic engineering of microorganisms for amino acid production. Curr. Opin. Biotechnol. 19, 454–460 (2008).

    CAS  PubMed  Google Scholar 

  5. 5.

    Chen, X. et al. DCEO biotechnology: tools to design, construct, evaluate, and optimize the metabolic pathway for biosynthesis of chemicals. Chem. Rev. 118, 4–72 (2017).

    PubMed  Google Scholar 

  6. 6.

    Kerfeld, C. A. Rewiring Escherichia coli for carbon-dioxide fixation. Nat. Biotechnol. 34, 1035–1036 (2016).

    CAS  PubMed  Google Scholar 

  7. 7.

    Luo, Y. Z., Enghiad, B. & Zhao, H. M. New tools for reconstruction and heterologous expression of natural product biosynthetic gene clusters. Nat. Prod. Rep. 33, 174–182 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Chen, X. et al. Metabolic engineering of Torulopsis glabrata for malate production. Metab. Eng. 19, 10–16 (2013).

    CAS  PubMed  Google Scholar 

  9. 9.

    Yu, T. et al. Reprogramming yeast metabolism from alcoholic fermentation to lipogenesis. Cell 174, 1–10 (2018).

    Google Scholar 

  10. 10.

    Ksiazek, K. Bacterial aging: from mechanistic basis to evolutionary perspective. Cell. Mol. Life Sci. 67, 3131–3137 (2010).

    CAS  PubMed  Google Scholar 

  11. 11.

    Boehm, A. et al. Genetic manipulation of glycogen allocation affects replicative lifespan in E. coli. PLoS Genet. 12, 1–17 (2016).

    Google Scholar 

  12. 12.

    Lindner, A. B., Madden, R., Demarez, A., Stewart, E. J. & Taddei, F. Asymmetric segregation of protein aggregates is associated with cellular aging and rejuvenation. Proc. Natl Acad. Sci. USA 105, 3076–3081 (2008).

    CAS  PubMed  Google Scholar 

  13. 13.

    Longo, V. D., Shadel, G. S., Kaeberlein, M. & Kennedy, B. Replicative and chronological aging in Saccharomyces cerevisiae. Cell Metab. 16, 18–31 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Kaeberlein, M. Lessons on longevity from budding yeast. Nature 464, 513–519 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Gonidakis, S., Finkel, S. E. & Longo, V. D. Genome-wide screen identifies Escherichia coli TCA-cycle-related mutants with extended chronological lifespan dependent on acetate metabolism and the hypoxia-inducible transcription factor ArcA. Aging Cell 9, 868–881 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Hill, S. M., Hao, X., Liu, B. & Nyström, T. Life-span extension by a metacaspase in the yeast Saccharomyces cerevisiae. Science 344, 1389–1392 (2014).

    CAS  PubMed  Google Scholar 

  17. 17.

    Fabrizio, P., Pozza, F., Pletcher, S. D., Gendron, C. M. & Longo, V. D. Regulation of longevity and stress resistance by Sch9 in yeast. Science 292, 288–290 (2001).

    CAS  PubMed  Google Scholar 

  18. 18.

    Orozco, H., Matallana, E. & Aranda, A. Two-carbon metabolites, polyphenols and vitamins influence yeast chronological life span in winemaking conditions. Microb. Cell Fact. 11, 1–10 (2012).

    Google Scholar 

  19. 19.

    Finkel, S. E. Long-term survival during stationary phase: evolution and the GASP phenotype. Nat. Rev. Microbiol. 4, 113–120 (2006).

    CAS  PubMed  Google Scholar 

  20. 20.

    Piper, P. W., Harris, N. L. & MacLean, M. Preadaptation to efficient respiratory maintenance is essential both for maximal longevity and the retention of replicative potential in chronologically ageing yeast. Mech. Ageing Dev. 127, 733–740 (2006).

    PubMed  Google Scholar 

  21. 21.

    Arlia-Ciommo, A., Piano, A., Leonov, A., Svistkova, V. & Titorenko, V. I. Quasi-programmed aging of budding yeast: a trade-off between programmed processes of cell proliferation, differentiation, stress response, survival and death defines yeast lifespan. Cell Cycle 13, 3336–3349 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Wahl, A., Schuth, N., Pfeiffer, D., Nussberger, S. & Jendrossek, D. PHB granules are attached to the nucleoid via PhaM in Ralstonia eutropha. BMC Microbiol. 12, 1–11 (2012).

    Google Scholar 

  23. 23.

    Farzadfard, F. & Lu, T. K. Emerging applications for DNA writers and molecular recorders. Science 361, 870–875 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Roquet, N., Soleimany, A. P., Ferris, A. C., Aaronson, S. & Lu, T. K. Synthetic recombinase-based state machines in living cells. Science 353, 1–13 (2016).

    Google Scholar 

  25. 25.

    Nagy, A. Cre recombinase: the universal reagent for genome tailoring. Genesis 26, 99–109 (2000).

    CAS  PubMed  Google Scholar 

  26. 26.

    Grindley, N. D., Whiteson, K. L. & Rice, P. A. Mechanisms of site-specific recombination. Annu. Rev. Biochem. 75, 567–605 (2006).

    CAS  PubMed  Google Scholar 

  27. 27.

    Karzai, A. W., Roche, E. D. & Sauer, R. T. The SsrA–SmpB system for protein tagging, directed degradation and ribosome rescue. Nat. Struct. Biol. 7, 449–455 (2000).

    CAS  PubMed  Google Scholar 

  28. 28.

    Weinberg, B. H. et al. Large-scale design of robust genetic circuits with multiple inputs and outputs for mammalian cells. Nat. Biotechnol. 35, 453–462 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Gonidakis, S., Finkel, S. E. & Longo, V. D. E. coli hypoxia-inducible factor ArcA mediates lifespan extension in a lipoic acid synthase mutant by suppressing acetyl-CoA synthetase. Biol. Chem. 391, 1139–1147 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Fontaine, F., Stewart, E. J., Lindner, A. B. & Taddei, F. Mutations in two global regulators lower individual mortality in Escherichia coli. Mol. Microbiol. 67, 2–14 (2008).

    CAS  PubMed  Google Scholar 

  31. 31.

    Fredriksson, A. & Nystrom, T. Conditional and replicative senescence in Escherichia coli. Curr. Opin. Microbiol. 9, 612–618 (2006).

    CAS  PubMed  Google Scholar 

  32. 32.

    Gao, Y. et al. Charged residues in the H-NS linker drive DNA binding and gene silencing in single cells. Proc. Natl Acad. Sci. USA 114, 12560–12565 (2017).

    CAS  PubMed  Google Scholar 

  33. 33.

    Choi, S. Y. et al. One-step fermentative production of poly(lactate-co-glycolate) from carbohydrates in Escherichia coli. Nat. Biotechnol. 34, 1–6 (2016).

    Google Scholar 

  34. 34.

    Maisonneuve, E., Ezraty, B. & Dukan, S. Protein aggregates: an aging factor involved in cell death. J. Bacteriol. 190, 6070–6075 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Pant, K. et al. Butyrate induces ROS-mediated apoptosis by modulating miR-22/SIRT-1 pathway in hepatic cancer cells. Redox Biol. 12, 340–349 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Yukihiro, F. et al. Commensal microbe-derived butyrate induces the differentiation of colonic regulatory T cells. Nature 504, 446–450 (2013).

    Google Scholar 

  37. 37.

    Alvarez, H. & Steinbüchel, A. Triacylglycerols in prokaryotic microorganisms. Appl. Microbiol. Biotechnol. 60, 367–376 (2002).

    CAS  PubMed  Google Scholar 

  38. 38.

    Elena, G. F. et al. Bacterial inclusion bodies: making gold from waste. Trends Biotechnol. 30, 65–70 (2012).

    Google Scholar 

  39. 39.

    Zhang, X. C. et al. Engineering cell wall synthesis mechanism for enhanced PHB accumulation in E. coli. Metab. Eng. 45, 32–42 (2017).

    PubMed  Google Scholar 

  40. 40.

    Jiang, X. R. & Chen, G. Q. Morphology engineering of bacteria for bio-production. Biotechnol. Adv. 34, 435–440 (2016).

    CAS  PubMed  Google Scholar 

  41. 41.

    Elhadi, D., Lv, L., Jiang, X. R., Wu, H. & Chen, G. Q. CRISPRi engineering E. coli for morphology diversification. Metab. Eng. 38, 358–369 (2016).

    CAS  PubMed  Google Scholar 

  42. 42.

    Galan, B. et al. Nucleoid-associated PhaF phasin drives intracellular location and segregation of polyhydroxyalkanoate granules in Pseudomonas putida KT2442. Mol. Microbiol. 79, 402–418 (2011).

    CAS  PubMed  Google Scholar 

  43. 43.

    Dorman, C. J. Horizontally acquired homologues of the nucleoid-associated protein H-NS: implications for gene regulation. Mol. Microbiol. 75, 264–267 (2010).

    CAS  PubMed  Google Scholar 

  44. 44.

    Jawed, K. et al. Engineered production of short chain fatty acid in Escherichia coli using fatty acid synthesis pathway. PLoS One 11, 1–20 (2016).

    Google Scholar 

  45. 45.

    Saini, M., Wang, Z. W., Chiang, C. J. & Chao, Y. P. Metabolic engineering of Escherichia coli for production of butyric acid. J. Agric. Food Chem. 62, 4342–4348 (2014).

    CAS  PubMed  Google Scholar 

  46. 46.

    Luo, H. et al. Recent advances and strategies in process and strain engineering for the production of butyric acid by microbial fermentation. Bioresour. Technol. 253, 343–354 (2018).

    CAS  PubMed  Google Scholar 

  47. 47.

    Qi, Y., Liu, H., Chen, X. & Liu, L. Engineering microbial membranes to increase stress tolerance of industrial strains. Metab. Eng. 53, 24–34 (2019).

    CAS  PubMed  Google Scholar 

  48. 48.

    Dai, Z. J. & Nielsen, J. Advancing metabolic engineering through systems biology of industrial microorganisms. Curr. Opin. Biotechnol. 36, 8–15 (2015).

    PubMed  Google Scholar 

  49. 49.

    Wu, C., Huang, J. & Zhou, R. Progress in engineering acid stress resistance of lactic acid bacteria. Appl. Microbiol. Biotechnol. 98, 1055–1063 (2014).

    CAS  PubMed  Google Scholar 

  50. 50.

    Wu, H., Tuli, L., Bennett, G. N. & San, K. Y. Metabolic transistor strategy for controlling electron transfer chain activity in Escherichia coli. Metab. Eng. 28, 159–168 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. 51.

    Jung, Y. K., Kim, T. Y., Park, S. J. & Lee, S. Y. Metabolic engineering of Escherichia coli for the production of polylactic acid and its copolymers. Biotechnol. Bioeng. 105, 161–171 (2010).

    CAS  PubMed  Google Scholar 

  52. 52.

    Datsenko, K. A. & Wanner, B. L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl Acad. Sci. USA 97, 6640–6645 (2000).

    CAS  PubMed  Google Scholar 

  53. 53.

    Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Meth 6, 343–347 (2009).

    CAS  Google Scholar 

  54. 54.

    Guo, L. et al. Enhancement of malate production through engineering of the periplasmic rTCA pathway in Escherichia coli. Biotechnol. Bioeng. 115, 1571–1580 (2018).

    CAS  PubMed  Google Scholar 

  55. 55.

    Li, Z. J. et al. Production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) from unrelated carbon sources by metabolically engineered Escherichia coli. Metab. Eng. 12, 352–359 (2010).

    CAS  PubMed  Google Scholar 

  56. 56.

    Young, J. W. et al. Measuring single-cell gene expression dynamics in bacteria using fluorescence time-lapse microscopy. Nat. Protoc. 7, 80–88 (2011).

    PubMed  PubMed Central  Google Scholar 

  57. 57.

    Durante-Rodriguez, G., de Lorenzo, V. & Nikel, P. I. A post-translational metabolic switch enables complete decoupling of bacterial growth from biopolymer production in engineered Escherichia coli. ACS Synth. Biol. 7, 2686–2697 (2018).

    CAS  PubMed  Google Scholar 

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Acknowledgements

This work was supported by the National Key R&D Program of China (2018YFA0901401), the National Natural Science Foundation of China (21808083, 21878126), the Key Field R & D Program of Guangdong Province (2019B020218001) and the National First-Class Discipline Program of Light Industry Technology and Engineering (LITE2018-08).

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L.G. and L.L. designed the project. L.G. and W.D. conducted and analysed the experiments. C.G., G.H., Q.D. and X.C. provided technical assistance. L.G. analysed the data and wrote the manuscript with input from C.Y., J.L. and L.L. All authors reviewed and approved the manuscript.

Corresponding author

Correspondence to Liming Liu.

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Supplementary Figures 1–54, Tables 1–9, Notes 1–9 and references

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Guo, L., Diao, W., Gao, C. et al. Engineering Escherichia coli lifespan for enhancing chemical production. Nat Catal 3, 307–318 (2020). https://doi.org/10.1038/s41929-019-0411-7

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