Engineering control of bacterial cellulose production PNAS PLUS using a genetic toolkit and a new cellulose- producing strain Michael Floreaa,b,1, Henrik Hagemanna,c, Gabriella Santosaa,b, James Abbottd,e, Chris N. Micklema,b, Xenia Spencer-Milnesa,b, Laura de Arroyo Garciaa,b, Despoina Paschoua,c, Christopher Lazenbatta,b, Deze Konga,c, Haroon Chughtaia,c, Kirsten Jensena,f, Paul S. Freemonta,f, Richard Kitneya,c, Benjamin Reevea,c, and Tom Ellisa,c,2 aCentre for Synthetic Biology and Innovation, Imperial College London, London SW7 2AZ, United Kingdom; bDepartment of Life Sciences, Imperial College London, London SW7 2AZ, United Kingdom; cDepartment of Bioengineering, Imperial College London, London SW7 2AZ, United Kingdom; dBioinformatics Support Service, Department of Surgery and Cancer, Imperial College London, London SW7 2AZ, United Kingdom; eCentre for Integrative Systems Biology and Bioinformatics, Imperial College London, London SW7 2AZ, United Kingdom; and fDepartment of Medicine, Imperial College London, London SW7 2AZ, United Kingdom Edited by Jef D. Boeke, New York University School of Medicine, New York, NY, and approved April 29, 2016 (received for review November 20, 2015) Bacterial cellulose is a strong and ultrapure form of cellulose advantage in colonization over other microorganisms (10). In ma- produced naturally by several species of the Acetobacteraceae. Its terials science, genetic engineering has been used to create several high strength, purity, and biocompatibility make it of great interest novel biomaterials, such as strong underwater protein-based ad- to materials science; however, precise control of its biosynthesis has hesives (11), self-assembling, functionalized amyloid-based biofilms remained a challenge for biotechnology. Here we isolate a strain of (12), biodegradable bacterial cellulose-based tissue engineering Komagataeibacter rhaeticus (K. rhaeticus iGEM) that can produce scaffolds (13), and many others. Bacterial cellulose has long been a cellulose at high yields, grow in low-nitrogen conditions, and is focus of research because, unlike plant-based cellulose, it is pure of highly resistant to toxic chemicals. We achieved external control other chemical species (lignin and pectin) and is synthesized as a over its bacterial cellulose production through development of a continuous highly interconnected lattice (14). This makes the ma- modular genetic toolkit that enables rational reprogramming of terial mechanically strong [nanocellulose fibers possess tensile MICROBIOLOGY the cell. To further its use as an organism for biotechnology, we stiffness of between 100 and 160 GPa and tensile strength of at sequenced its genome and demonstrate genetic circuits that enable least 1 GPa (15, 16)] while still flexible, biocompatible, and highly functionalization and patterning of heterologous gene expression hydrophilic, capable of storing water over 90% of its total weight within the cellulose matrix. This work lays the foundations for using (17, 18). Due to these properties, bacterial cellulose is commercially genetic engineering to produce cellulose-based materials, with numerous applications in basic science, materials engineering, Significance and biotechnology. synthetic biology | bacterial cellulose | genetic engineering | biomaterials | Bacterial cellulose is a remarkable material that is malleable, genomics biocompatible, and over 10-times stronger than plant-based cel- lulose. It is currently used to create materials for tissue engi- neering, medicine, defense, electronics, acoustics, and fabrics. We he emergence of synthetic biology now enables model mi- describe here a bacterial strain that is readily amenable to genetic Escherichia coli Tcroorganisms such as to be easily reprog- engineering and produces high quantities of bacterial cellulose in rammed with modular DNA code to perform a variety of new low-cost media. To reprogram this organism for biotechnology tasks for useful purposes (1). However, for many application applications, we created a set of genetic tools that enables bio- areas, it is instead preferable to exploit the natural abilities of synthesis of patterned cellulose, functionalization of the cellulose nonmodel microbes as specialists at consuming or producing surface with proteins, and tunable control over cellulose pro- molecules or thriving within niche environments (2). Recent duction. This greatly expands our ability to control and engineer work has described adapting common E. coli synthetic biology new cellulose-based biomaterials, offering numerous applica- tools to work across different bacterial phyla (3, 4) and has tions for basic research, materials science, and biotechnology. produced genetic toolkits for new bacteria, where collections of DNA constructs and methods for precise control of heterologous Author contributions: M.F., H.H., and B.R., isolated and characterized the iGEM strain; M.F. gene expression have been developed for engineering strains and B.R. sequenced the genome; M.F. and J.A. scaffolded and analyzed the genome; M.F., naturally specialized for photosynthesis or survival within the gut H.H., G.S., C.N.M., X.S.-M., L.d.A.G., and D.P. created the genetic engineering toolkit; M.F. created the sRNA construct; H.H., G.S., C.N.M., and X.S.-M. created CBD fusion proteins; microbiome (5, 6). An important application area for biotech- M.F., H.H., G.S., C.N.M., and X.S.-M. created patterned and functionalized biomaterials; nology is the production of materials, and bacteria that naturally M.F., H.H., G.S., and T.E. conducted temporal patterning experiments; H.H., G.S., C.N.M., secrete high yields of cellulose have attracted significant attention X.S.-M., L.d.A.G., and B.R. created sfGFP-functionalized garments; M.F., C.N.M., D.P., C.L., D.K., and H.C. analyzed data; M.F. and T.E. wrote the manuscript; K.J., P.S.F., R.K., B.R., not just from people in industry and research (7) but also from and T.E. supervised the work; all authors contributed to planning the study. those in art, fashion, and citizen science (8). However, despite Conflict of interest statement: H.H. and G.S. are researching the industrial uses of cellulose their widespread use, no toolkit for genetic modification of these functionalized with cellulose binding domain fusion proteins as part of CustoMem Ltd. cellulose-producing bacteria has previously been described. This article is a PNAS Direct Submission. Komagataeibacter is a genus from the Acetobacteraceae family Freely available online through the PNAS open access option. of which multiple species produce bacterial cellulose. Cellulose Data deposition: The sequence reported in this paper has been deposited in the European nanofibers are synthesized from UDP-glucose by the acs (Acetobacter Nucleotide Archive, www.ebi.ac.uk/ena (accession no. PRJEB10933). cellulose synthase) operon proteins AcsA and AcsB (9) and secreted 1Present address: Department of Biosystems Science and Engineering, Eidgenössische by AcsC and AcsD, forming an interconnected cellulose “pellicle” Technische Hochschule Zürich, 4058 Basel, Switzerland. around cells (7). Although it is still unclear why Acetobacteraceae 2To whom correspondence should be addressed. Email: [email protected]. produce bacterial cellulose in nature (7), it has been shown to This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. confer the host with a high resistance to UV light and a competitive 1073/pnas.1522985113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1522985113 PNAS Early Edition | 1of10 Downloaded by guest on September 30, 2021 Fig. 1. Characterization of K. rhaeticus iGEM. (A) Morphology of a typical cellulose pellicle produced by K. rhaeticus iGEM. White patches are light reflected by the pellicle surface. (B) Cellulose productivity of K. rhaeticus iGEM and G. hansenii ATCC 53582 on different growth media, shown as pellicle dry weight after a 10-d incubation in 20 mL liquid Hestrin–Schramm (HS) media. Cellulose production of K. rhaeticus exceedsthatofG. hansenii in sucrose-containing media (HS sucrose and Kombucha tea, adjusted P = 0.0128 and P = 0.039, respectively), but is lower than that of G. hansenii in HS glucose (adjusted P = 0.027). (C and D) Growth and production of a cellulose pellicle (denoted by an arrow) by K. rhaeticus iGEM in nitrogen-free LGI medium. K. rhaeticus iGEM shows significant growth compared with negative controls G. hansenii and E. coli (adjusted P = 0.026 and P = 0.011, respectively), whereas G. hansenii and E. coli donotdiffer(adjustedP = 0.742). ns, not significant. (E) Comparison of K. rhaeticus and E. coli survival after incubation for 5, 30, or 90 min with toxic chemicals [0.1 M HCl, 0.1 M NaOH, 70% (vol/vol) EtOH, and 10% (vol/vol) bleach]. Survival is defined as the fraction of survived cells compared with PBS-treated cells. (F) Scanning electron micrographs of K. rhaeticus iGEM encased in bacterial cellulose, taken after 8 d of growth, at 6,000× magnification. *P < 0.05. n = 3 biological replicates for all experiments; error bars indicate SD. Statistical significance was determined for B with two-way ANOVA and Bonferroni’s multiple comparisons test and for D with one-way ANOVA with Tukey’s multiple comparisons tests. For A and C, images were taken 9 d postinoculation. Images were cropped, and contrast was adjusted to improve clarity, without affecting details. See Methods for experimental details. used in medical wound dressings, high-end acoustics, and many (24) that can grow in low-nitrogen