Biological systems assemble living materials that are autonomously patterned, can self-repair and can sense and respond to their environment. The field of engineered living materials aims to create novel materials with properties similar to those of natural biomaterials using genetically engineered organisms. Here, we describe an approach to fabricating functional bacterial cellulose-based living materials using a stable co-culture of Saccharomyces cerevisiae yeast and bacterial cellulose-producing Komagataeibacter rhaeticus bacteria. Yeast strains can be engineered to secrete enzymes into bacterial cellulose, generating autonomously grown catalytic materials and enabling DNA-encoded modification of bacterial cellulose bulk properties. Alternatively, engineered yeast can be incorporated within the growing cellulose matrix, creating living materials that can sense and respond to chemical and optical stimuli. This symbiotic culture of bacteria and yeast is a flexible platform for the production of bacterial cellulose-based engineered living materials with potential applications in biosensing and biocatalysis.
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All produced data that support the main figures of this study are included in this published article. Data points for the mechanical and rheological tests are provided as Source data files. Additional data are available from the corresponding author upon request.
Chen, A. Y., Zhong, C. & Lu, T. K. Engineering living functional materials. ACS Synth. Biol. 4, 8–11 (2015).
Nguyen, P. Q. Synthetic biology engineering of biofilms as nanomaterials factories. Biochem. Soc. Trans. 45, 585–597 (2017).
Nguyen, P. Q., Courchesne, N. D., Duraj-Thatte, A., Praveschotinunt, P. & Joshi, N. S. Engineered living materials: prospects and challenges for using biological systems to direct the assembly of smart materials. Adv. Mater. 30, e1704847 (2018).
Gilbert, C. & Ellis, T. Biological engineered living materials: growing functional materials with genetically programmable properties. ACS Synth. Biol. 8, 1–15 (2019).
Blanco, L. P., Evans, M. L., Smith, D. R., Badtke, M. P. & Chapman, M. R. Diversity, biogenesis and function of microbial amyloids. Trends Microbiol. 20, 66–73 (2012).
Kalyoncu, E., Ahan, R. E., Olmez, T. T. & Safak Seker, U. O. Genetically encoded conductive protein nanofibers secreted by engineered cells. RSC Adv. 7, 32543–32551 (2017).
Seker, U. O. S., Chen, A. Y., Citorik, R. J. & Lu, T. K. Synthetic biogenesis of bacterial amyloid nanomaterials with tunable inorganic-organic interfaces and electrical conductivity. ACS Synth. Biol. 6, 266–275 (2017).
Dorval Courchesne, N.-M. et al. Biomimetic engineering of conductive curli protein film. Nanotechnology 29, 509501 (2018).
Chen, A. Y. et al. Synthesis and patterning of tunable multiscale materials with engineered cells. Nat. Mater. 13, 515–523 (2014).
Moser, F., Voigt, C. A., Tham, E., González, L. M. & Lu, T. K. Light-controlled, high-resolution patterning of living engineered bacteria onto textiles, ceramics, and plastic. Adv. Funct. Mater. 29, 1901788 (2019).
Nguyen, P. Q., Botyanszki, Z., Tay, P. K. R. & Joshi, N. S. Programmable biofilm-based materials from engineered curli nanofibres. Nat. Commun. 5, 4945 (2014).
Nussbaumer, M. G. et al. Bootstrapped biocatalysis: Biofilm-derived materials as reversibly functionalizable multienzyme surfaces. ChemCatChem 9, 4328–4333 (2017).
Duraj-Thatte, A. M. et al. Genetically programmable self‐regenerating bacterial hydrogels. Adv. Mater. 31, e1901826 (2019).
Dorval Courchesne, N.-M., Duraj-Thatte, A., Tay, P. K. R., Nguyen, P. Q. & Joshi, N. S. Scalable production of genetically engineered nanofibrous macroscopic materials via filtration. ACS Biomater. Sci. Eng. 3, 733–741 (2016).
Park, S. J. et al. Phototactic guidance of a tissue-engineered soft-robotic ray. Science 353, 158–162 (2016).
Van Tittelboom, K., De Belie, N., De Muynck, W. & Verstraete, W. Use of bacteria to repair cracks in concrete. Cem. Concr. Res. 40, 157–166 (2010).
Wang, J., Van Tittelboom, K., De Belie, N. & Verstraete, W. Use of silica gel or polyurethane immobilized bacteria for self-healing concrete. Constr. Build. Mater. 26, 532–540 (2012).
Gerber, L. C., Koehler, F. M., Grass, R. N. & Stark, W. J. Incorporating microorganisms into polymer layers provides bioinspired functional living materials. Proc. Natl Acad. Sci. USA 109, 90–94 (2012).
Gerber, L. C., Koehler, F. M., Grass, R. N. & Stark, W. J. Incorporation of penicillin-producing fungi into living materials to provide chemically active and antibiotic-releasing surfaces. Angew. Chem. Int. Ed. 124, 11455–11458 (2012).
Liu, X. et al. Stretchable living materials and devices with hydrogel–elastomer hybrids hosting programmed cells. Proc. Natl Acad. Sci. USA 114, 2200–2205 (2017).
Chawla, P. R., Bajaj, I. B., Survase, S. A. & Singhal, R. S. Microbial cellulose: fermentative production and applications. Food Technol. Biotechnol. 47, 107–124 (2009).
Huang, Y. et al. Recent advances in bacterial cellulose. Cellulose 21, 1–30 (2014).
Hsieh, Y. C., Yano, H., Nogi, M. & Eichhorn, S. J. An estimation of the Young’s modulus of bacterial cellulose filaments. Cellulose 15, 507–513 (2008).
Kondo, T., Rytczak, P. & Bielecki, S. in Bacterial Nanocellulose (eds. Gama, M. et al.) 59–71 (Elsevier, 2016).
Wang, J., Tavakoli, J. & Tang, Y. Bacterial cellulose production, properties and applications with different culture methods – a review. Carbohydr. Polym. 219, 63–76 (2019).
Ludwicka, K., Jedrzejczak-Krzepkowska, M., Kubiak, K., Kolodziejczyk, M. & Pankiewicz, T. in Bacterial Nanocellulose (eds. Gama, M. et al.) 145–165 (Elsevier, 2016).
Yadav, V. et al. Novel in vivo-degradable cellulose-chitin copolymer from metabolically engineered gluconacetobacter xylinus. Appl. Environ. Microbiol. 76, 6257–6265 (2010).
Fang, J., Kawano, S., Tajima, K. & Kondo, T. In vivo curdlan/cellulose bionanocomposite synthesis by genetically modified Gluconacetobacter xylinus. Biomacromolecules 16, 3154–3160 (2015).
Gwon, H. et al. A safe and sustainable bacterial cellulose nanofiber separator for lithium rechargeable batteries. Proc. Natl Acad. Sci. USA 116, 19288–19293 (2019).
Florea, M. et al. Engineering control of bacterial cellulose production using a genetic toolkit and a new cellulose-producing strain. Proc. Natl Acad. Sci. USA 113, E3431–E3440 (2016).
Teh, M. Y. et al. An expanded synthetic biology toolkit for gene expression control in Acetobacteraceae. ACS Synth. Biol. 8, 708–723 (2019).
Jacek, P., Ryngajłło, M. & Bielecki, S. Structural changes of bacterial nanocellulose pellicles induced by genetic modification of Komagataeibacter hansenii ATCC 23769. Appl. Microbiol. Biotechnol. 103, 5339–5353 (2019).
Walker, K. T., Goosens, V. J., Das, A., Graham, A. E. & Ellis, T. Engineered cell-to-cell signalling within growing bacterial cellulose pellicles. Microb. Biotechnol. 12, 611–619 (2018).
Jayabalan, R., Malini, K., Sathishkumar, M., Swaminathan, K. & Yun, S. E. Biochemical characteristics of tea fungus produced during kombucha fermentation. Food Sci. Biotechnol. 19, 843–847 (2010).
Lee, M. E., DeLoache, W. C., Cervantes, B. & Dueber, J. E. A highly-characterized yeast toolkit for modular, multi-part assembly. ACS Synth. Biol. 4, 975–986 (2015).
Ong, E., Gilkes, N. R., Miller, R. C., Warren, R. A. & Kilburn, D. G. The cellulose-binding domain (CBD(Cex)) of an exoglucanase from Cellulomonas fimi: production in Escherichia coli and characterization of the polypeptide. Biotechnol. Bioeng. 42, 401–409 (1993).
Antošová, Z., Herkommerová, K., Pichová, I. & Sychrová, H. Efficient secretion of three fungal laccases from Saccharomyces cerevisiae and their potential for decolorization of textile industry effluent – a comparative study. Biotechnol. Prog. 34, 69–80 (2018).
Villares, A. et al. Lytic polysaccharide monooxygenases disrupt the cellulose fibers structure. Sci. Rep. 7, 40262 (2017).
Lee, C.-R. et al. Co-fermentation using recombinant Saccharomyces cerevisiae yeast strains hyper-secreting different cellulases for the production of cellulosic bioethanol. Sci. Rep. 7, 4428 (2017).
Bhagia, S., Dhir, R., Kumar, R. & Wyman, C. E. Deactivation of cellulase at the air–liquid interface is the main cause of incomplete cellulose conversion at low enzyme loadings. Sci. Rep. 8, 1350 (2018).
Yamanaka, S. et al. The structure and mechanical properties of sheets prepared from bacterial cellulose. J. Mater. Sci. 24, 3141–3145 (1989).
Soykeabkaew, N., Sian, C., Gea, S., Nishino, T. & Peijs, T. All-cellulose nanocomposites by surface selective dissolution of bacterial cellulose. Cellulose 16, 435–444 (2009).
Shi, X., Zheng, F., Pan, R., Wang, J. & Ding, S. Engineering and comparative characteristics of double carbohydrate binding modules as a strength additive for papermaking applications. Bioresources 9, 3117–3131. (2014).
Butchosa, N., Leijon, F., Bulone, V. & Zhou, Q. Stronger cellulose microfibril network structure through the expression of cellulose-binding modules in plant primary cell walls. Cellulose 26, 3083–3094 (2019).
McIsaac, R. S., Gibney, P. A., Chandran, S. S., Benjamin, K. R. & Botstein, D. Synthetic biology tools for programming gene expression without nutritional perturbations in Saccharomyces cerevisiae. Nucleic Acids Res. 42, e48 (2014).
McIsaac, R. S. et al. Fast-acting and nearly gratuitous induction of gene expression and protein depletion in Saccharomyces cerevisiae. Mol. Biol. Cell 22, 4447–4459 (2011).
Pothoulakis, G. & Ellis, T. Synthetic gene regulation for independent external induction of the Saccharomyces cerevisiae pseudohyphal growth phenotype. Commun. Biol. 1, 7 (2018).
Ostrov, N. et al. A modular yeast biosensor for low-cost point-of-care pathogen detection. Sci. Adv. 3, e1603221 (2017).
Cardinal-Watkins, C. & Nicell, J. A. Enzyme-catalyzed oxidation of 17β-estradiol using immobilized laccase from trametes versicolor. Enzyme Res. 2011, 725172 (2011).
Adeel, M., Song, X., Wang, Y., Francis, D. & Yang, Y. Environmental impact of estrogens on human, animal and plant life: a critical review. Environ. Int. 99, 107–119 (2017).
Avar, P. et al. β-Estradiol and ethinyl-estradiol contamination in the rivers of the Carpathian Basin. Environ. Sci. Pollut. Res. 23, 11630–11638 (2016).
Pathak, G. P., Strickland, D., Vrana, J. D. & Tucker, C. L. Benchmarking of optical dimerizer systems. ACS Synth. Biol. 3, 832–838 (2014).
Jarque, S., Bittner, M., Blaha, L. & Hilscherova, K. Yeast biosensors for detection of environmental pollutants: current state and limitations. Trends Biotechnol. 34, 408–419 (2016).
Adeniran, A., Stainbrook, S., Bostick, J. & Tyo, K. Detection of a peptide biomarker by engineered yeast receptors. ACS Synth. Biol. 7, 696–705 (2018).
We thank G. Pothoulakis, C. Bricio-Garberi, B. E. Wolfe and E. Landis for advice and discussions, J. van der Hilst for contributions to co-culture methods and B. An for assisting with photo taking. Work at Imperial College London was funded by UK Engineering and Physical Sciences Research Council (EPSRC) awards EP/M002306/1 and EP/N026489/1 and an Imperial College London President’s Scholarship to C.G. W.O. was supported by a research fellowship (OT 577/1-1) from the German Research Foundation (DFG). Work at MIT was funded by Army Research Office award W911NF-11-1-0281 and Institute for Soldier Nanotechnologies award W911NF-13-D-0001, T.O. 4. T.C.T. was supported by the MIT J-WAFS Fellowship. Work across both institutions was funded by the MIT-MISTI MIT-Imperial College London Seed Fund.
C.G., T.-C.T., W.O., T.K.L. and T.E. are co-inventors on patent applications (International Patent Application no. PCT/US2020/047330) filed by MIT and Imperial College London relating to all the work covered in this article.
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Gilbert, C., Tang, TC., Ott, W. et al. Living materials with programmable functionalities grown from engineered microbial co-cultures. Nat. Mater. 20, 691–700 (2021). https://doi.org/10.1038/s41563-020-00857-5
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