De novo engineering of programmable and multi-functional biomolecular condensates for controlled biosynthesis.
Journal
Nature communications
ISSN: 2041-1723
Titre abrégé: Nat Commun
Pays: England
ID NLM: 101528555
Informations de publication
Date de publication:
12 Sep 2024
12 Sep 2024
Historique:
received:
15
12
2023
accepted:
05
09
2024
medline:
17
9
2024
pubmed:
17
9
2024
entrez:
16
9
2024
Statut:
epublish
Résumé
There is a growing interest in the creation of engineered condensates formed via liquid-liquid phase separation (LLPS) to exert precise cellular control in prokaryotes. However, de novo design of cellular condensates to control metabolic flux or protein translation remains a challenge. Here, we present a synthetic condensate platform, generated through the incorporation of artificial, disordered proteins to realize specific functions in Bacillus subtilis. To achieve this, the "stacking blocks" strategy is developed to rationally design a series of LLPS-promoting proteins for programming condensates. Through the targeted recruitment of biomolecules, our investigation demonstrates that cellular condensates effectively sequester biosynthetic pathways. We successfully harness this capability to enhance the biosynthesis of 2'-fucosyllactose by 123.3%. Furthermore, we find that condensates can enhance the translation specificity of tailored enzyme fourfold, and can increase N-acetylmannosamine titer by 75.0%. Collectively, these results lay the foundation for the design of engineered condensates endowed with multifunctional capacities.
Identifiants
pubmed: 39284811
doi: 10.1038/s41467-024-52411-5
pii: 10.1038/s41467-024-52411-5
doi:
Substances chimiques
Hexosamines
0
Bacterial Proteins
0
N-acetylmannosamine
X80PR7P73R
Trisaccharides
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
7989Informations de copyright
© 2024. The Author(s).
Références
Huang, J. et al. Complete integration of carbene-transfer chemistry into biosynthesis. Nature 617, 403–408 (2023).
pubmed: 37138074
pmcid: 11334723
doi: 10.1038/s41586-023-06027-2
Baumschabl, M. et al. Conversion of CO
doi: 10.1073/pnas.2211827119
Jiang, Y. Q. et al. Manipulation of sterol homeostasis for the production of 24-epi-ergosterol in industrial yeast. Nat. Commun. 14, 437 (2023).
pubmed: 36707526
pmcid: 9883489
doi: 10.1038/s41467-023-36007-z
Scown, C. D. & Keasling, J. D. Sustainable manufacturing with synthetic biology. Nat. Biotechnol. 40, 304–306 (2022).
pubmed: 35190687
doi: 10.1038/s41587-022-01248-8
Jin, K. et al. Compartmentalization and transporter engineering strategies for terpenoid synthesis. Micro. Cell Fact. 21, 92 (2022).
doi: 10.1186/s12934-022-01819-z
Dusséaux, S., Wajn, W. T., Liu, Y. X., Ignea, C. & Kampranis, S. C. Transforming yeast peroxisomes into microfactories for the efficient production of high-value isoprenoids. Proc. Natl Acad. Sci. USA 117, 31789–31799 (2020).
pubmed: 33268495
pmcid: 7749339
doi: 10.1073/pnas.2013968117
Omidvar, M., Zdarta, J., Sigurdardóttir, S. B. & Pinelo, M. Mimicking natural strategies to create multi-environment enzymatic reactors: From natural cell compartments to artificial polyelectrolyte reactors. Biotechnol. Adv. 54, 107798 (2022).
pubmed: 34265377
doi: 10.1016/j.biotechadv.2021.107798
Moon, S. Y., Son, S. H., Oh, S. S. & Lee, J. Y. Harnessing cellular organelles to bring new functionalities into yeast. Biotechnol. Bioproc. E 28, 936–948 (2023).
doi: 10.1007/s12257-022-0195-5
Ma, Y. S., Li, J. B., Huang, S. W. & Stephanopoulos, G. Targeting pathway expression to subcellular organelles improves astaxanthin synthesis in Yarrowia lipolytica. Metab. Eng. 68, 152–161 (2021).
pubmed: 34634493
doi: 10.1016/j.ymben.2021.10.004
Grewal, P. S., Samson, J. A., Baker, J. J., Choi, B. & Dueber, J. E. Peroxisome compartmentalization of a toxic enzyme improves alkaloid production. Nat. Chem. Biol. 17, 96–103 (2021).
pubmed: 33046851
doi: 10.1038/s41589-020-00668-4
Jiang, S., da Silva, L. C., Ivanov, T., Mottola, M. & Landfester, K. Synthetic silica nano-organelles for regulation of cascade reactions in multi-compartmentalized systems. Angew. Chem. Int. Ed. 61, e202113784 (2022).
doi: 10.1002/anie.202113784
Kang, W. et al. Organizing enzymes on self-assembled protein cages for cascade reactions. Angew. Chem. Int. Ed. 61, e202214001 (2022).
doi: 10.1002/anie.202214001
Bobik, T. A. & Stewart, A. M. Selective molecular transport across the protein shells of bacterial microcompartments. Curr. Opin. Microbiol. 62, 76–83 (2021).
pubmed: 34087617
pmcid: 8286307
doi: 10.1016/j.mib.2021.05.006
Kerfeld, C. A., Aussignargues, C., Zarzycki, J., Cai, F. & Sutter, M. Bacterial microcompartments. Nat. Rev. Microbiol. 16, 277–290 (2018).
pubmed: 29503457
pmcid: 6022854
doi: 10.1038/nrmicro.2018.10
Qian, Z. G., Huang, S. C. & Xia, X. X. Synthetic protein condensates for cellular and metabolic engineering. Nat. Chem. Biol. 18, 1330–1340 (2022).
pubmed: 36400990
doi: 10.1038/s41589-022-01203-3
Banani, S. F., Lee, H. O., Hyman, A. A. & Rosen, M. K. Biomolecular condensates: organizers of cellular biochemistry. Nat. Rev. Mol. Cell Biol. 18, 285–298 (2017).
pubmed: 28225081
pmcid: 7434221
doi: 10.1038/nrm.2017.7
Wan, L., Zhu, Y. Y., Zhang, W. L. & Mu, W. M. Phase-separated synthetic organelles based on intrinsically disordered protein domain for metabolic pathway assembly in Escherichia coli. ACS Nano 17, 10806–10816 (2023).
pubmed: 37191277
doi: 10.1021/acsnano.3c02333
Alberti, S., Gladfelter, A. & Mittag, T. Considerations and challenges in studying liquid-liquid phase separation and biomolecular condensates. Cell 176, 419–434 (2019).
pubmed: 30682370
pmcid: 6445271
doi: 10.1016/j.cell.2018.12.035
Zhang, H. et al. Liquid-liquid phase separation in biology: mechanisms, physiological functions and human diseases. Sci. China Life Sci. 63, 953–985 (2020).
pubmed: 32548680
doi: 10.1007/s11427-020-1702-x
Dai, Y., You, L. & Chilkoti, A. Engineering synthetic biomolecular condensates. Nat. Rev. Bioeng. 1, 466–480 (2023).
doi: 10.1038/s44222-023-00052-6
Wang, Y. et al. Phase-separated multienzyme compartmentalization for terpene biosynthesis in a prokaryote. Angew. Chem. Int. Ed. 61, e202203909 (2022).
doi: 10.1002/anie.202203909
Wei, S. P. et al. Formation and functionalization of membraneless compartments in Escherichia coli. Nat. Chem. Biol. 16, 1143–1148 (2020).
pubmed: 32601486
doi: 10.1038/s41589-020-0579-9
Guo, H. T. et al. Spatial engineering of E. coli with addressable phase-separated RNAs. Cell 185, 3823–3837 (2022).
pubmed: 36179672
doi: 10.1016/j.cell.2022.09.016
Dai, Y. F. et al. Programmable synthetic biomolecular condensates for cellular control. Nat. Chem. Biol. 19, 518–528 (2023).
pubmed: 36747054
pmcid: 10786170
doi: 10.1038/s41589-022-01252-8
Hilditch, A. T. et al. Assembling membraneless organelles from de novo designed proteins. Nat. Chem. 16, 89–97 (2024).
pubmed: 37710047
doi: 10.1038/s41557-023-01321-y
Hirose, T., Ninomiya, K., Nakagawa, S. & Yamazaki, T. A guide to membraneless organelles and their various roles in gene regulation. Nat. Rev. Mol. Cell Biol. 24, 288–304 (2023).
pubmed: 36424481
doi: 10.1038/s41580-022-00558-8
Liu, Y. F., Liu, L., Li, J. H., Du, G. C. & Chen, J. Synthetic biology toolbox and chassis development in Bacillus subtilis. Trends Biotechnol. 37, 548–562 (2019).
pubmed: 30446263
doi: 10.1016/j.tibtech.2018.10.005
Qi, X. M., Sun, Y. F. & Xiong, S. D. A single freeze-thawing cycle for highly efficient solubilization of inclusion body proteins and its refolding into bioactive form. Micro. Cell Fact. 14, 24 (2015).
doi: 10.1186/s12934-015-0208-6
Kang, W. et al. Modular enzyme assembly for enhanced cascade biocatalysis and metabolic flux. Nat. Commun. 10, 4248 (2019).
pubmed: 31534134
pmcid: 6751169
doi: 10.1038/s41467-019-12247-w
Dzuricky, M., Rogers, B. A., Shahid, A., Cremer, P. S. & Chilkoti, A. De novo engineering of intracellular condensates using artificial disordered proteins. Nat. Chem. 12, 814–825 (2020).
pubmed: 32747754
pmcid: 8281385
doi: 10.1038/s41557-020-0511-7
Amiram, M. et al. Evolution of translation machinery in recoded bacteria enables multi-site incorporation of nonstandard amino acids. Nat. Biotechnol. 33, 1272–1279 (2015).
pubmed: 26571098
pmcid: 4784704
doi: 10.1038/nbt.3372
Zhao, E. M. et al. Light-based control of metabolic flux through assembly of synthetic organelles. Nat. Chem. Biol. 15, 589–597 (2019).
pubmed: 31086330
pmcid: 6755918
doi: 10.1038/s41589-019-0284-8
Adhikari, A. et al. Reprogramming natural proteins using unnatural amino acids. RSC Adv. 11, 38126–38145 (2021).
pubmed: 35498070
pmcid: 9044140
doi: 10.1039/D1RA07028B
Reinkemeier, C. D., Girona, G. E. & Lemke, E. A. Designer membraneless organelles enable codon reassignment of selected mRNAs in eukaryotes. Science 363, eaaw2644 (2019).
pubmed: 30923194
pmcid: 7611745
doi: 10.1126/science.aaw2644
Reinkemeier, C. D. & Lemke, E. A. Dual film-like organelles enable spatial separation of orthogonal eukaryotic translation. Cell 184, 4886–4903 (2021).
pubmed: 34433013
pmcid: 8480389
doi: 10.1016/j.cell.2021.08.001
Deng, J. Y. et al. Creating an in vivo bifunctional gene expression circuit through an aptamer-based regulatory mechanism for dynamic metabolic engineering in Bacillus subtilis. Metab. Eng. 55, 179–190 (2019).
pubmed: 31336181
doi: 10.1016/j.ymben.2019.07.008
Tian, R. Z. et al. Titrating bacterial growth and chemical biosynthesis for efficient N-acetylglucosamine and N-acetylneuraminic acid bioproduction. Nat. Commun. 11, 5078 (2020).
pubmed: 33033266
pmcid: 7544899
doi: 10.1038/s41467-020-18960-1
Zhu, Y. Y., Cao, H. Z., Wang, H. & Mu, W. M. Biosynthesis of human milk oligosaccharides via metabolic engineering approaches: current advances and challenges. Curr. Opin. Biotechnol. 78, 102841 (2022).
pubmed: 36371892
doi: 10.1016/j.copbio.2022.102841
Yu, W. W. et al. A pathway independent multi-modular ordered control system based on thermosensors and CRISPRi improves bioproduction in Bacillus subtilis. Nucleic Acids Res. 50, 6587–6600 (2022).
pubmed: 35670665
pmcid: 9226513
doi: 10.1093/nar/gkac476
Shin, Y. et al. Spatiotemporal control of intracellular phase transitions using light-activated optodroplets. Cell 168, 159–171 (2017).
pubmed: 28041848
doi: 10.1016/j.cell.2016.11.054
Schuster, B. S. et al. Controllable protein phase separation and modular recruitment to form responsive membraneless organelles. Nat. Commun. 9, 2985 (2018).
pubmed: 30061688
pmcid: 6065366
doi: 10.1038/s41467-018-05403-1
Carrillo, N. et al. Safety and efficacy of N-acetylmannosamine (ManNAc) in patients with GNE myopathy: an open-label phase 2 study. Genet. Med. 23, 2067–2075 (2021).
pubmed: 34257421
pmcid: 8553608
doi: 10.1038/s41436-021-01259-x
Bi, X. Y. et al. etiBsu1209: a comprehensive multiscale metabolic model for Bacillus subtilis. Biotechnol. Bioeng. 120, 1623–1639 (2023).
pubmed: 36788025
doi: 10.1002/bit.28355
Mayr, C. et al. Frontiers in biomolecular condensate research. Nat. Cell Biol. 25, 512–514 (2023).
pubmed: 36973422
doi: 10.1038/s41556-023-01102-2
Garabedian, M. V. et al. Designer membraneless organelles sequester native factors for control of cell behavior. Nat. Chem. Biol. 17, 998–1007 (2021).
pubmed: 34341589
pmcid: 8387445
doi: 10.1038/s41589-021-00840-4
Galvanetto, N. et al. Extreme dynamics in a biomolecular condensate. Nature 619, 876–883 (2023).
pubmed: 37468629
doi: 10.1038/s41586-023-06329-5
Lee, D. S. W. et al. Size distributions of intracellular condensates reflect competition between coalescence and nucleation. Nat. Phys. 19, 586–596 (2023).
pubmed: 37073403
pmcid: 10104779
doi: 10.1038/s41567-022-01917-0
Wang, K. H. et al. Defining synonymous codon compression schemes by genome recoding. Nature 539, 59–64 (2016).
pubmed: 27776354
pmcid: 5321499
doi: 10.1038/nature20124
Carlson, E. D. et al. Engineered ribosomes with tethered subunits for expanding biological function. Nat. Commun. 10, 3920 (2019).
pubmed: 31477696
pmcid: 6718428
doi: 10.1038/s41467-019-11427-y
Schmied, W. H. et al. Controlling orthogonal ribosome subunit interactions enables evolution of new function. Nature 564, 444–448 (2018).
pubmed: 30518861
pmcid: 6525102
doi: 10.1038/s41586-018-0773-z
Dunkelmann, D. L., Oehm, S. B., Beattie, A. T. & Chin, J. W. A 68-codon genetic code to incorporate four distinct non-canonical amino acids enabled by automated orthogonal mRNA design. Nat. Chem. 13, 1110–1117 (2021).
pubmed: 34426682
pmcid: 7612796
doi: 10.1038/s41557-021-00764-5
Johnson, G. E., Lalanne, J. B., Peters, M. L. & Li, G. W. Functionally uncoupled transcription-translation in Bacillus subtilis. Nature 585, 124–128 (2020).
pubmed: 32848247
pmcid: 7483943
doi: 10.1038/s41586-020-2638-5
Gray, W. T. et al. Nucleoid size scaling and intracellular organization of translation across bacteria. Cell 177, 1632–1648 (2019).
pubmed: 31150626
pmcid: 6629263
doi: 10.1016/j.cell.2019.05.017
Li, F. R. et al. Deep learning-based k
doi: 10.1038/s41929-022-00798-z