Engineering control of bacterial cellulose productionusing a genetic toolkit and a new cellulose-producing strainMichael 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 CollegeLondon, London SW7 2AZ, United Kingdom; cDepartment of Bioengineering, Imperial College London, London SW7 2AZ, United Kingdom; dBioinformaticsSupport Service, Department of Surgery and Cancer, Imperial College London, London SW7 2AZ, United Kingdom; eCentre for Integrative Systems Biologyand Bioinformatics, Imperial College London, London SW7 2AZ, United Kingdom; and fDepartment of Medicine, Imperial College London, London SW72AZ, 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 celluloseproduced naturally by several species of the Acetobacteraceae. Itshigh strength, purity, and biocompatibility make it of great interestto materials science; however, precise control of its biosynthesis hasremained a challenge for biotechnology. Here we isolate a strain ofKomagataeibacter rhaeticus (K. rhaeticus iGEM) that can producecellulose at high yields, grow in low-nitrogen conditions, and ishighly resistant to toxic chemicals. We achieved external controlover its bacterial cellulose production through development of amodular genetic toolkit that enables rational reprogramming ofthe cell. To further its use as an organism for biotechnology, wesequenced its genome and demonstrate genetic circuits that enablefunctionalization and patterning of heterologous gene expressionwithin the cellulose matrix. This work lays the foundations for usinggenetic engineering to produce cellulose-based materials, withnumerous applications in basic science, materials engineering,and biotechnology.

synthetic biology | bacterial cellulose | genetic engineering | biomaterials |genomics

The emergence of synthetic biology now enables model mi-croorganisms such as Escherichia coli to be easily reprog-

rammed with modular DNA code to perform a variety of newtasks for useful purposes (1). However, for many applicationareas, it is instead preferable to exploit the natural abilities ofnonmodel microbes as specialists at consuming or producingmolecules or thriving within niche environments (2). Recentwork has described adapting common E. coli synthetic biologytools to work across different bacterial phyla (3, 4) and hasproduced genetic toolkits for new bacteria, where collections ofDNA constructs and methods for precise control of heterologousgene expression have been developed for engineering strainsnaturally specialized for photosynthesis or survival within the gutmicrobiome (5, 6). An important application area for biotech-nology is the production of materials, and bacteria that naturallysecrete high yields of cellulose have attracted significant attentionnot just from people in industry and research (7) but also fromthose in art, fashion, and citizen science (8). However, despitetheir widespread use, no toolkit for genetic modification of thesecellulose-producing bacteria has previously been described.Komagataeibacter is a genus from the Acetobacteraceae family

of which multiple species produce bacterial cellulose. Cellulosenanofibers are synthesized from UDP-glucose by the acs (Acetobactercellulose synthase) operon proteins AcsA and AcsB (9) and secretedby AcsC and AcsD, forming an interconnected cellulose “pellicle”around cells (7). Although it is still unclear why Acetobacteraceaeproduce bacterial cellulose in nature (7), it has been shown toconfer the host with a high resistance to UV light and a competitive

advantage in colonization over other microorganisms (10). In ma-terials science, genetic engineering has been used to create severalnovel biomaterials, such as strong underwater protein-based ad-hesives (11), self-assembling, functionalized amyloid-based biofilms(12), biodegradable bacterial cellulose-based tissue engineeringscaffolds (13), and many others. Bacterial cellulose has long been afocus of research because, unlike plant-based cellulose, it is pure ofother chemical species (lignin and pectin) and is synthesized as acontinuous highly interconnected lattice (14). This makes the ma-terial mechanically strong [nanocellulose fibers possess tensilestiffness of between 100 and 160 GPa and tensile strength of atleast 1 GPa (15, 16)] while still flexible, biocompatible, and highlyhydrophilic, capable of storing water over 90% of its total weight(17, 18). Due to these properties, bacterial cellulose is commercially

Significance

Bacterial cellulose is a remarkable material that is malleable,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. Wedescribe here a bacterial strain that is readily amenable to geneticengineering and produces high quantities of bacterial cellulose inlow-cost media. To reprogram this organism for biotechnologyapplications, we created a set of genetic tools that enables bio-synthesis of patterned cellulose, functionalization of the cellulosesurface with proteins, and tunable control over cellulose pro-duction. This greatly expands our ability to control and engineernew cellulose-based biomaterials, offering numerous applica-tions for basic research, materials science, and biotechnology.

Author contributions: M.F., H.H., and B.R., isolated and characterized the iGEM strain; M.F.and B.R. sequenced the genome; M.F. and J.A. scaffolded and analyzed the genome; M.F.,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;M.F., H.H., G.S., C.N.M., and X.S.-M. created patterned and functionalized biomaterials;M.F., H.H., G.S., and T.E. conducted temporal patterning experiments; H.H., G.S., C.N.M.,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.,and T.E. supervised the work; all authors contributed to planning the study.

Conflict of interest statement: H.H. and G.S. are researching the industrial uses of cellulosefunctionalized with cellulose binding domain fusion proteins as part of CustoMem Ltd.

This article is a PNAS Direct Submission.

Freely available online through the PNAS open access option.

Data deposition: The sequence reported in this paper has been deposited in the EuropeanNucleotide Archive, www.ebi.ac.uk/ena (accession no. PRJEB10933).1Present address: Department of Biosystems Science and Engineering, EidgenössischeTechnische Hochschule Zürich, 4058 Basel, Switzerland.

2To whom correspondence should be addressed. Email: t.ellis@imperial.ac.uk.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1522985113/-/DCSupplemental.

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used in medical wound dressings, high-end acoustics, and manyother products (7), and in the laboratory has been used to createbiodegradable tissue scaffolds (13), nanoreinforcements (19), andartificial blood vessels (18), as well as sensors (20), flexible elec-trodes (21), organic light-emitting diode (OLED) displays, andother materials (22).Functionalization or modification of bacterial cellulose has

mainly been achieved by chemical or mechanical modificationsof the cellulose matrix or via changing culturing conditions (7,22), whereas only a few attempts at genetic engineering havebeen made (13, 23). However, genetic engineering may allow agreater range of materials to be produced, by enabling finecontrol over cellulose synthesis genes and production of protein-cellulose composite biomaterials. Here we isolate a strain ofKomagataeibacter rhaeticus (previouslyGluconacetobacter rhaeticus)

(24) that can grow in low-nitrogen conditions and produce cel-lulose at high yields, sequence its genome, and develop a syn-thetic biology toolkit for its genetic engineering. This toolkitprovides the characterization data necessary for the engineeringof K. rhaeticus iGEM, and enables transformation, controlledexpression of constitutive and inducible transgenes, and controlover endogenous gene expression of this strain. We use thesetools to engineer a system that allows tunable control over nativecellulose production, and produce novel patterned and func-tionalized cellulose-based biomaterials.

ResultsIsolation, Characterization, and Genome Sequencing of K. rhaeticusiGEM. As part of the International Genetically Engineered Ma-chine Competition (iGEM) (25), we evaluated Gluconacetobacter

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 thepellicle surface. (B) Cellulose productivity ofK. rhaeticus iGEM andG. hanseniiATCC 53582 on different growthmedia, shown as pellicle dryweight after a 10-d incubationin 20 mL liquid Hestrin–Schramm (HS) media. Cellulose production of K. rhaeticus exceeds that of G. 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 cellulosepellicle (denoted by an arrow) by K. rhaeticus iGEM in nitrogen-free LGImedium.K. rhaeticus iGEM shows significant growth comparedwith negative controlsG. hanseniiand E. coli (adjusted P = 0.026 and P = 0.011, respectively), whereas G. hansenii and E. coli do not differ (adjusted P = 0.742). ns, not significant. (E) Comparison ofK. 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 isdefined as the fraction of survived cells compared with PBS-treated cells. (F) Scanning electronmicrographs of K. rhaeticus iGEM encased in bacterial cellulose, taken after8 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 Bwithtwo-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 taken9 d postinoculation. Images were cropped, and contrast was adjusted to improve clarity, without affecting details. See Methods for experimental details.

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hansenii ATCC 53582 [one of the highest-reported cellulose-producing strains (26), recently reclassified as Komagataeibacterhansenii ATCC 53582 (27)] and a strain isolated from Kombuchatea as potential new synthetic biology hosts (Fig. 1A). We chosethe latter strain (hereafter called “iGEM”) for further work, aspreliminary experiments showed that it can be transformed morereadily with plasmid DNA than G. hansenii ATCC 53582. Fur-thermore, the iGEM strain produced more cellulose thanG. hansenii ATCC 53582 on sucrose in small-scale tests (Fig. 1B)and also produced cellulose at high yields on cheap, low-nitrogenKombucha tea medium (Fig. 1B). Surprisingly, it could alsogrow on the defined nitrogen-free LGI medium (Fig. 1 C and Dand SI Appendix, Figs. S1 and S2). Because several species ofAcetobacteraceae, notably Gluconacetobacter diazotrophicus,have been confirmed to fix atmospheric nitrogen (28, 29), thissuggested possible nitrogen fixation (see below). Finally, becausecellulose has been reported to increase resistance toward UVlight and environmental stresses in closely related species (10), wetested whether cellulose could also confer resistance towardchemical stresses, which may occur in unrefined feedstocks orduring industrial production. We tested the susceptibility of theiGEM strain to 70% (vol/vol) ethanol, 10% (vol/vol) bleach, 0.1 MNaOH, and 0.1 M HCl (Fig. 1E), and found that when encased incellulose it is highly tolerant to chemical stressors, being over1,000-fold more resistant than E. coli to all treatments (Fig.1E). Finally, scanning electron microscopy of a cellulose pelli-cle confirmed that, as with other closely related species, cells ofK. rhaeticus iGEM are rod-like, ∼2 μm in length, and heavilyencased in cellulose during normal growth (Fig. 1F and SI Ap-pendix, Fig. S3).To determine the genetic basis of high cellulose productivity

and to provide background information for genetic engineeringof the iGEM strain, we sequenced its genome to 400× coverage andassembled the genome using the genome of Gluconacetobacterxylinus NBRC 3288 (30) as the reference genome for scaffolding(European Nucleotide Archive accession no. PRJEB10933).Sequencing showed that the genome totals 3.87 Mbp with aGC% of 62.7 and contains a predicted 3,573 genes, with an N50(shortest sequence length at 50% of the genome) of 3.16 Mbp.The genome is divided between a chromosome of 3.16 Mbp, atleast two plasmids: pKRi01 (238 kbp) and pKRi02 (3 kbp), and 37unplaced contigs (in total 460 kbp), which may be part of thechromosome or additional plasmids, and could not be confidentlyassigned due to being flanked by repetitive sequences (Fig. 2A).The genome sequence revealed several interesting aspects aboutthe biology of K. rhaeticus iGEM. First, a 16S rRNA phylogenysuggests the iGEM strain to be a previously unidentified strain ofK. rhaeticus (SI Appendix, Fig. S4), rather than G. xylinus, which isnormally thought to be associated with Kombucha tea. Further-more, the sequence shows the presence of four acs operons onthe chromosome, sharing 40–65% amino acid identity (Fig. 2).Up to three acs operons have been reported in other bacterialcellulose-producing species [G. xylinusATCC 23769 andG. hanseniiATCC 53582 (31, 32)], indicating that the high cellulose synthasecopy number may be a possible contributor to the high celluloseproductivity observed here (Fig. 1B). These operons also differ instructure. The acs1 operon contains separate acsA and acsB,whereas they are fused in the other operons, and the only genomiccopy of acsD is found in acs1. Operon acs4 uniquely contains onlyacsAB, and phylogenetic analysis indicates that acs4 is most closelyrelated to the acs2 operon (Fig. 2B), and possibly arose via du-plication and subsequent translocation. From the genes flankingacs operons, cmcAX, ccpAX, bglxA, bcsX, and bcsY have beenpreviously shown to contribute to cellulose production in closelyrelated species (26, 33, 34). We found two other, stand-alonecopies of bglxA from the genome (genomic positions 517401–519440 and 3029825–3032221), and also identified genes close toacs2 that are associated with extracellular matrix formation (kpsC,

kpsS, and rfaB) and may play a role in cellulose productivity (35, 36).Finally, to determine whether the iGEM strain can fix atmosphericnitrogen similar to G. diazotrophicus, we searched its genome forgenes associated with nitrogen fixation. We located five genes (ntrB,-C, -X, and -Y and nifU) (SI Appendix, Table S1) associated withnitrogen fixation in Acetobacteraceae (37); however, interestingly, wedid not find the genes homologous to nifHDK, which form the mainnitrogenase subunits in G. diazotrophicus.

Genetic Engineering Toolkit for Komagataeibacter.Very few genetictools are available for the engineering of Acetobacteraceae. Wetherefore developed a complete set of tools for its engineering,consisting of protocols, modular plasmids, promoters, reporterproteins, and inducible constructs that enable external control ofgene expression (Fig. 3A).Protocols and plasmid backbones. We first used the plasmid pBla-Vhb-122 [previously described to replicate in Acetobacteraceae (38)]to develop protocols for the preparation of electrocompetent cells,transformation, plasmid purification, and genomic DNA extractionof K. rhaeticus iGEM (SI Appendix, Supplementary Protocols).Using these protocols, we then assessed eight plasmids forpropagation in K. rhaeticus iGEM: pSEVA311, pSEVA321,pSEVA331, pSEVA341, pSEVA351, pBAV1K-T5-sfGFP, pSB1C3,and pBca1020 (see SI Appendix, Table S2 for details). From these,pSEVA321, 331, 351, pBAV1K-T5-sfGFP, and pBla-Vhb-122showed replication in iGEM (SI Appendix, Fig. S5), giving a totalof five different plasmids to act as vectors. We further engineeredpSEVA321 and pSEVA331 into pSEVA321Bb and pSEVA331Bb,making them compatible with the widely used BioBrick standard(25), to enable rapid cloning of publicly available DNA parts. Wethen used pSEVA331Bb for all subsequent studies, due to its likelyhigher copy number.Reporter proteins and constitutive and inducible promoters. We nexttested expression of seven reporter proteins (mRFP1, GFPmut3,sfGFP, and chromoproteins tsPurple, aeBlue, gfasPurple, andspisPink) (see SI Appendix, Tables S3 and S4 for details), of whichmRFP1, GFPmut3, and sfGFP showed visually detectable ex-pression. We then chose 10 promoters from an open-access col-lection of synthetic minimal E. coli promoters and, using mRFP1as the reporter, characterized these in K. rhaeticus iGEM (Fig. 3Band SI Appendix, Table S4; also see SI Appendix, Fig. S6 for acomparison with promoter strengths in E. coli). For induciblepromoters, we engineered four constructs allowing gene expres-sion to be induced externally by anhydrotetracycline (ATc) orN-acylhomoserine lactone (AHL) (see SI Appendix, Fig. S7 for an overviewof constructs). From these, the AHL-inducible constructs (pLux01and pLux02) showed higher induction and lower leakiness thanthe ATc-induced constructs (pTet01 and pTet02) (Fig. 3C) and,contrary to our initial expectations, they also gave robust inductionof mRFP1 expression when cells were encased in the pellicle,showing visible fluorescence (Fig. 3 D and E and SI Appendix,Fig. S8). This is notable, as it shows that cells in the pellicle caneffectively receive signals from their environment despite theircellulose encasing. Because K. rhaeticus is highly resistant tovarious environmental hazards within cellulose (Fig. 1E), theability to receive signals while being protected by cellulose makesit a potentially suitable host for applications requiring tolerance totoxic chemicals and long-term survival.

Engineering Control over Cellulose Production. Because wild-typespecies produce cellulose constitutively, a major goal of geneticengineering of Acetobacteraceae has been to achieve control overcellulose production. Constitutive cellulose production complicatesgenetic engineering techniques and is not always desirable forindustrial applications as it imparts a high metabolic cost, which inwell-aerated conditions typically leads to the emergence of cellu-lose-nonproducing mutants (39). It is therefore desirable to inhibitcellulose production during periods when it is not required, to

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prevent the proliferation of these mutants. Furthermore, finecontrol over cellulose production levels may allow control over thedensity of cellulose fibrils, and thus the macroscale properties ofthe cellulose. To achieve controlled cellulose production, we engi-neered a system in which an E. coli Hfq and an sRNA targetingUGPase mRNA (UDP-glucose pyrophosphorylase) are coexpressedfrom a plasmid in response to AHL (plasmid J-sRNA-331Bb) (Fig.4A; also see SI Appendix, Fig. S9 for a detailed overview). ThesRNA contains a 24-base region complementary to UGPasemRNA and an E. coli Hfq-binding region. When expressed, itbinds to the target UGPase mRNA and recruits E. coli Hfq,inhibiting UGPase translation. We targeted the UGPase gene, asit catalyzes the production of UDP-glucose critical for cellulosesynthesis (40) and is present in single copy in the genome,allowing knockdown by a single sRNA. We found this system tobe highly efficient, as cellulose production was suppressed com-pletely upon full induction and could be fine-tuned using differentconcentrations of AHL (Fig. 4B). The observed reduction in cellulose

production was not related to toxicity, as growth rate did not decreasecompared with wild-type levels (Fig. 4C and SI Appendix, Fig. S10).This system was engineered to be a general platform for targetedknockdowns in Komagataeibacter and other bacterial species, as ex-pression of E. coli Hfq makes it independent from the host Hfq, andthe broad host range pSEVA331Bb backbone enables replication ina wide range of species. Furthermore, new sRNAs can be added tothe plasmid, and the 24-base sRNA region can be recoded rapidly bysite-directed mutagenesis, making the construct easily modifiable forother targets.

Genetic Engineering of Patterned and Functionalized Biomaterials.Owing to the discovery that K. rhaeticus gene expression canbe induced even when inside a cellulose pellicle, we hypothesizedthat it may be possible to generate spatially and temporallypatterned biomaterials that are controlled by the diffusion of theinducer AHL and timing of exposure to induction during pelliclegrowth. To test this, we induced growing cellulose pellicles with

Fig. 2. K. rhaeticus iGEM genome. (A) Overview of the K. rhaeticus iGEM genome. The iGEM genome totals 3.87 Mbp with a GC% of 62.7, and contains apredicted 3,505 protein-coding genes, 3 rRNAs, 52 tRNAs, and 13 other noncoding RNAs. The genome consists of a chromosome of ∼3.16 Mbp and at leasttwo plasmids, pKRi01 (238 kbp) and pKRi02 (3 kbp). The chromosome contains 2,899 predicted genes with 63% GC content and four copies of acs operons. Forthe chromosome, consecutive rings show (from the outside in): (i) read coverage, (ii and iii), genes on (ii) forward and (iii) reverse strands, (iv) acs operonsinvolved in cellulose synthesis, (v) GC%, and (vi) GC skew. Additionally, the genome contains 37 scaffolds (totaling 460 kbp) that could not be confidentlyplaced due to repetitive sequences. These scaffolds may be part of the chromosome or plasmids, or may belong to additional plasmids (the closely relatedspecies G. xylinus NBRC 3288 and K. xylinus E25 contain five and seven plasmids, respectively). (B and C) Phylogenetic relationships (B) and amino acid se-quence identity (C) of acs operons. Phylogeny and sequence identity indicate that acs2 and acs4 operons are most closely related. Amino acid sequences werealigned and percent identity was calculated using MUSCLE (54) and the tree was generated using the neighbor-joining method (55). The tree is drawn toscale, with bootstrap values (56) from 1,000 replicates shown next to the branches. All positions containing gaps were eliminated from analysis. See Methodsfor further details on sequencing and analysis.

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cells containing the AHL-inducible construct pLux01 with dif-ferent concentrations of AHL, at different locations and timepoints (Fig. 5 A and B). We found that both spatial and temporalcontrol were possible. When a limited amount of inducer wasadded to one side of the pellicle, cells produced mRFP1 followingthe diffusion gradient of AHL (Fig. 5A). Furthermore, becausecells are active only in the top layer of the cellulose pellicle (7),when inducer was added at different times midway through pel-licle growth, only cells at the growing top layer produced mRFP1,capturing the temporal difference between uninduced cells in thebottom layers and induced cells at the top (Fig. 5B).To produce functionalized cellulose materials where the nano-

cellulose matrix is coated by proteins of interest, we consideredtwo strategies: genetic engineering of K. rhaeticus to produce theseproteins in situ, or separate expression of proteins in E. coli, whichare then purified and applied directly to bacterial cellulose (Fig.5C). Although the latter requires a three-step process (proteinand cellulose production separately, followed by combining thetwo), it may be preferred for medical and other applications wherevery high purity of material is required, as it would allow defined

and purified components to be used for functionalization. To testfor the possibility of post hoc functionalization, we producedmRFP1 in E. coli, extracted and added it to bacterial cellulose,and compared it by fluorescence microscopy with cellulose pro-duced by K. rhaeticus with in vivo constitutive mRFP1 expression.We found that extracted proteins can diffuse well throughout thepellicle and functionalize the cellulose evenly (Fig. 5D), whereasthe granular fluorescence exhibited by expression from pellicle-based cells (Fig. 3E; also see SI Appendix, Fig. S11 for a full-sizecomparison) indicates that mRFP1 remains largely in theK. rhaeticus cells and would likely require active secretion orlysis of cell membranes to access the extracellular cellulose.To further increase the efficiency of functionalization, we

engineered expression vectors that allow easy fusion of proteinsto one of four different cellulose-binding domains (CBDs):CBDclos (41), CBDCex (42), dCBD (43), and CBDcipA (44).CBDs are short peptides that bind tightly to cellulose fibrils, thusincreasing protein adhesion to cellulose (45). In these constructs,proteins can be modularly fused to CBDs via restriction enzymecloning. We assessed the cellulose binding strengths of these CBDs

GFPmut3 sfGFP mRFP1

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Fig. 3. K. rhaeticus synthetic biology tookit. (A) Overview of the toolkit contents. (B and C) Constitutive promoter strengths (B) and AHL (pLux01, pLux02)and ATc (pTet01, pTet02) inducible construct expression strengths (C) in K. rhaeticus iGEM, as measured by total mRFP1 fluorescence per cell (fluorescence at630 nm divided by OD600). Promoter strengths in B and C were assayed in liquid HS with cellulase to remove formation of cellulose fibrils interfering withmeasurements. (D) Total mRFP1 fluorescence expressed from the pLux01 construct when K. rhaeticus cells were induced with AHL inside the cellulose pellicle(induced in pellicle) compared with uninduced or wild-type (WT) cells. Induction caused a significant increase in fluorescence compared with uninduced orwild-type cells (P < 0.001 for both induced vs. uninduced, and induced vs. wild-type, determined with one-way ANOVA and Tukey’s post hoc tests). Cells weregrown in HS (without cellulase), and fluorescence was quantified by fluorescence microscopy image analysis. (E and F) Induction in the pellicle results in visiblemRFP1 production compared with uninduced cells (E) (indicated by the arrow; also see SI Appendix, Fig. S8), and results in granular fluorescence due tolocalization within cells (F). n = 3 for all experiments; error bars indicate SD. For E and F, images were cropped and contrast was adjusted to improve clarity.See SI Appendix, Fig. S7 for a detailed overview of constructs and Methods for details of characterization assays.

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by washing four different E. coli-extracted CBD-sfGFP fusionproteins with different solvents (dH2O, EtOH, BSA, and PBS) andmeasuring the fluorescence that remained bound. Addition ofCBDs to sfGFP gave up to a fivefold increase in binding to cellu-lose compared with GFP alone (SI Appendix, Fig. S12). Finally,because bacterial cellulose is a candidate for new textile materialsand of high interest to the fashion industry, we used our approachto demonstrate production of functionalized garments. Using theCBDcipA-sfGFP fusion protein extract and dried pellicle materialfrom K. rhaeticus cultures, we created bacterial cellulose fashionaccessories by functionalization of cellulose fibrils with greenfluorescent protein (SI Appendix, Fig. S13), indicating that thisapproach is scalable to produce macroscale objects.

DiscussionHere we isolated a strain of bacterial cellulose-producingK. rhaeticus (K. rhaeticus iGEM), which can grow on acidic andlow-nitrogen media, is highly resistant to damaging chemicalagents when encased in cellulose, and is notable for its highcellulose production. Although it is clear that K. rhaeticus iGEMcan effectively produce cellulose in low-nitrogen media, whetherit fixes nitrogen is still an open question. Nitrogen fixation seemsto be prevalent in Acetobacteraceae, as multiple species (mostnotably G. diazotrophicus) have been recorded to be nitrogen-fixing (46). However, in the case of K. rhaeticus iGEM, althoughit can grow in nitrogen-free LGI medium (Fig. 1 C and D and SIAppendix, Figs. S1 and S2) and its genome contains a set of genesassociated with nitrogen fixation (SI Appendix, Table S1), wewere unable to find genes homologous to G. diazotrophicusnifHDK, which form the structural subunits of the nitrogenasecomplex (47). This either suggests that despite careful handling,very low level nitrogen contamination may have been present,allowing K. rhaeticus to produce cellulose in LGI medium or,alternatively, that K. rhaeticus uses a different set of nitrogenase

genes for nitrogen fixation, as alternative nitrogenases have beenisolated in other species (48). However, in either case, from theperspective of manufacturing, low nitrogen tolerance allows theuse of cheap, low-nitrogen media, potentially reducing cellulose-manufacturing costs.To use this strain for biotechnology applications, it was first

necessary to develop methods and tools for its genetic manipu-lation. For a genetic toolkit to be useful, it should minimally allowintroduction of foreign DNA, plasmid propagation, and a degreeof control over heterologous gene expression levels. Although therequired tools can differ between species and the applications inwhich they are used, they should ideally also allow tunable regu-lation of transgene expression through inducible or repressiblesystems and regulation over native gene expression. A toolkitshould further provide relevant characterization data about thehost, such as a genome sequence and growth and productivityrates in different conditions. Indeed, many of these elements havebeen part of toolkits created for other species (5, 6, 49). Thetoolkit described here for K. rhaeticus iGEM contains all of thesefeatures, providing protocols and DNA constructs to allow trans-formation, precisely controlled constitutive and inducible expressionof heterologous genes, control over endogenous gene expression,and a genome sequence with characterization data.Using this toolkit, we created two approaches for engineering

new cellulose-based materials, first through genetic engineeringof K. rhaeticus itself, and second through application of extractedproteins to the bacterial cellulose. Genetic engineering offerssimple, one-step in situ production of materials, and would beideal for applications where the cellulose matrix is modified as itis being made. The physical and biochemical properties of cel-lulose are largely dependent on the microscale morphology andstructure of cellulose fibrils, so modification of these duringproduction by expressed heterologous enzymes could result innew material properties. For example, by engineering cells to

Fig. 4. sRNA construct (J-sRNA-331Bb) for control of cellulose production. (A) Overview of the sRNA silencing construct. Constitutively produced LuxR bindsto pLux in the presence of AHL and up-regulates production of E. coli Hfq and an sRNA targeting UGPase mRNA. The 5′ end of the sRNA contains a 24-basesequence complementary to UGPase mRNA, and binds to it in the presence of E. coli Hfq (53), leading to silencing of the UGPase gene. (B) Cellulose pro-duction of induced and uninduced cultures shown as cellulose dry weight measured 40 h postinoculation. “No cellulose” indicates empty weighing boats;“pSEVA331Bb 100 nM AHL” and “pSEVA331Bb” indicate iGEM strain with empty pSEVA331Bb vector with and without 100 nM AHL, respectively. Full in-duction with 100 and 500 nM AHL results in complete suppression of cellulose synthesis (adjusted P < 0.0001 for uninduced vs. 100 nM AHL and 500 nM AHL).Addition of AHL itself does not decrease cellulose productivity (adjusted P > 0.999 for pSEVA331Bb vs. pSEVA331Bb with 100 nM AHL), and uninduced cellsare not different from negative controls (adjusted P = 0.12 for uninduced vs. pSEVA331Bb 100 nM AHL). (C) OD600 of cultures 3 h postinoculation. Differencesbetween OD600 are not significant for any comparisons (adjusted P > 0.05), showing that induction of the sRNA silencing construct does not reduce growthrate. n = 5 for all samples (except n = 3 for pSEVA331Bb and pSEVA331Bb with 100 nM AHL in B); error bars indicate SD. Statistical significance was de-termined with one-way ANOVA and Tukey’s multiple comparisons tests for B and C. See Methods for further details of all assays.

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incorporate N-acetylglucosamine residues into cellulose, Yadavet al. (13) created biodegradable, low-immunogenicity cellulosetissue scaffolds. Our second approach (addition of purified pro-teins to already-produced cellulose) is likely to be preferable forapplications where bulk material properties are not changed butthe material is functionalized with new properties. In our experi-ments, post hoc functionalization resulted in an even, thoroughdistribution of functionalizing proteins (Fig. 5D), and may bepreferable in medical and tissue engineering applications wherehighly pure materials are required, as both the cellulose andfunctionalizing proteins can be purified before formation of thecomposite material. Together, these two approaches complementeach other, as the ability to modify cellulose via genetic engi-neering as well as functionalize it with purified proteins allows fora wider range of materials to be engineered.Here, genetic engineering allowed us to control cellulose pro-

duction by sRNA-mediated knockdown and create spatial andtemporal patterning through induction with N-acyl homoserinelactone. We used an AHL-inducible system here; however, forindustrial applications, it is likely straightforward to exchange itfor a control system using cheaper chemical or physical inducersif required. Although we did not test for this specifically, it isimportant to note that as knockdown of cellulose production did

not significantly change growth rate, control over cellulose pro-duction levels may also allow control of the specific cell-vs.-cel-lulose and thus protein-vs.-cellulose concentrations in thematerial. In the case of functionalization of cellulose with CBDfusion proteins, in principle any protein stable enough to func-tion in the intended extracellular environment could be usedfor functionalization.By providing genetic engineering tools that allow control of

production of bacterial cellulose and modification of it as amaterial, this work enables a variety of potential applications.The physical properties and pore size of the cellulose might betunable by altering gene expression of the K. rhaeticus acs op-erons or by complexing cellulose in situ with secreted structuralproteins such as curli fimbriae (12). Cellulose can be function-alized with specific proteins (Fig. 5C and SI Appendix, Figs. S12and S13), which may be used to create advanced materials suchas bacterial cellulose wound dressings coated with antimicrobialpeptides or novel high-specificity water filters coated with pro-teins binding specific contaminants. In another area, the toolkitallows creating patterned cellulose structures in three dimensions(Fig. 5 A and B). In combination with functionalization, this maybe used in tissue engineering for one-step production of cellulose-based tissue engineering scaffolds that contain specifically patterned

Post-hoc func�onaliza�on

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Fig. 5. Engineering of patterned and functionalized cellulose materials on a macroscale. (A) Spatial patterning. Cells were induced for mRFP1 expression byaddition of 100 nM AHL to one side of a 1-L culture and the pellicle was imaged for red fluorescence 3 d postinduction. (Inset) Pellicle imaged in white light.(B) Temporal patterning. Cells were induced with AHL daily at different times through pellicle growth (0 d only, or starting at 0, 2, or 4 d postinoculation) andimaged 9 d postinoculation, with overview (i), white light (ii), and fluorescence (iii) images of the pellicles and pellicle cross-sections shown. Note that pelliclesinduced on day 0 only show low fluorescence due to natural degradation of AHL in the media over time. (C) Overview of the cellulose functionalizationstrategy via post hoc addition of mRFP1 extracted from E. coli. (D) Fluorescence microscopy of a cross-section of cellulose functionalized with mRFP1 throughaddition of mRFP1 extracted from E. coli. Smooth fluorescence is seen throughout the pellicle cross-section, compared with the granular fluorescenceseen in Fig. 3E (also see SI Appendix, Fig. S11). For A and B, iii, computationally determined and averaged brightness (gray value) along the pellicle cross-section is shown (Right). Images were cropped and contrast was adjusted to improve clarity for all images. See Methods for fluorescence imaging con-ditions and other details.

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growth factors. Furthermore, the remarkable robustness ofK. rhaeticus within cellulose (Fig. 1E) offers potential uses in bio-sensing. The growing pellicle can store changes in environmentalsignals by writing the conditions into different cellulose layers asthey grow (Fig. 5B), acting as a record akin to ice cores. Finally,because cellulose synthesis and nitrogen fixation in closely relatedAcetobacteraceae are areas of active research, this toolkit is also avaluable resource for basic studies aiming to dissect the molecularmechanisms of these processes. As there are many possible appli-cations enabled by this work, we are making this strain and thegenetic engineering toolkit available through ATCC and AddGene.

MethodsIsolation, Characterization, and Culturing of K. rhaeticus iGEM. K. rhaeticusiGEM was isolated from a Kombucha SCOBY (symbiotic colony of bacteriaand yeast) of Czech origin (Happy Kombucha) by streaking homogenizedSCOBY material on Hestrin–Schramm (HS) agarose (SI Appendix, Table S5),verifying cell morphology under a light microscope, and restreaking isolatedcolonies on HS agarose twice. Two percent (wt/vol) glucose was used in HSunless stated otherwise. Glycerol stocks were prepared by culturing theiGEM strain statically in HS medium for 6 d, followed by addition of 0.2%(vol/vol) cellulase (Trichoderma reesei cellulase; C2730; Sigma), incubationwith 230-rpm shaking, 30 °C for 1 d, addition of glycerol to 25% (vol/vol),and storage at −80 °C.

For cellulose production, seed cultures of K. rhaeticus iGEM were in-oculated from glycerol stocks and grown statically at 30 °C in Kombucha tea,HS, or LGI medium (see SI Appendix, Supplementary Protocols and Table S5for details). When grown with shaking without cellulose production, cul-tures were grown in HS cellulase [0.4% (vol/vol)] at 30 °C, 230-rpm shaking at45° tube angle. Unless otherwise stated, 50-mL Corning tubes (CLS430829;Sigma) with 5–20 mL media were used for culturing. When grown on HSagar, plates were incubated inverted at 30 °C. For culturing of K. rhaeticustransformed with plasmids encoding kanamycin or chloramphenicol resistancegenes, kanamycin was added to 500 μg/mL for HS agar and 50–100 μg/mL forliquid HS, and chloramphenicol was added to 340 μg/mL for HS agar and34–68 μg/mL for liquid HS.

Cellulose productivity on different media was measured by culturing in20mL HS glucose [2% (wt/vol)], HS sucrose [2% (wt/vol)], HS without a carbonsource (negative control), and Kombucha tea in 50-mL Corning tubes at 30 °Cfor 10 d, with loose caps for increased air diffusion, and kept at 4 °C untilmeasurement of cellulose weight (SI Appendix, Supplementary Protocols).Productivity of K. rhaeticus iGEM was reported in comparison with pro-ductivity of the high-producing G. hansenii ATCC 53582 instead of maximalcellulose yield per volume of media, as maximal total productivity is highlydependent on specific culturing conditions and may not be a good measureof genetically determined production capabilities. To test cellulose pro-ductivity on nitrogen-free LGI medium, 80 μL OD600 1 E. coli or K. rhaeticusiGEM seed culture was inoculated into 25 mL LGI medium in 50-mL glasstubes (3119-0050; Thermo Fischer) and imaged 9 d postinoculation.

For scanning electron micrographs, cellulose was gold-coated undervacuum and imaged at 2,000–6,000× magnification and 20 kV. See SI Ap-pendix, Supplementary Methods for details.

Chemical Tolerance Assays. For both E. coli and K. rhaeticus, cells were treatedwith 0.5 mL 0.1 M NaOH, 0.1 M HCl, 70% (vol/vol) ethanol, 10% (vol/vol)bleach, or PBS for 5, 30, or 90 min. Treatments were then plated; colonieswere photographed in white light and counted to determine the fraction ofsurviving cells compared with PBS-treated cells. For details of the assay, see SIAppendix, Supplementary Methods.

Genome Sequencing, Assembly, Bioinformatics, and Statistics. The K. rhaeticusiGEM genome was sequenced with an Illumina MiSeq using 250-bp paired-end reads, to a coverage of ∼400×. Reads were then downsampled to 100×coverage, assembled using the BugBuilder pipeline (50), quality-controlled,and annotated using Prokka with the full read set (51). All statistical testswere performed with Prism 6 (GraphPad Software). For details of genomesequencing and bioinformatics, see SI Appendix, Supplementary Methods.

Engineering of Constitutive Promoters, Inducible Promoters, CBD Fusions, andsRNA Constructs. pSEVA331Bb and pSEVA321Bb were constructed frompSEVA331 and pSEVA321, respectively, via substituting the native polylinkerwith BioBrick prefix and suffix. This was done by PCR mutagenesis with Q5polymerase (M0491S; NEB), primers i75 and i76 (SI Appendix, Table S6), di-

gestion with SpeI (R3133S; NEB), and subsequent religation. Constitutivepromoter-mRFP1 constructs (BBa_J23100–Bba_J23117 by iGEM 2006 Berkeley)were received from the iGEM Registry of Standard Biological Parts (52) andsubcloned into pSEVA331Bb. ATc-inducible constructs were kindly provided byFrancesca Ceroni at Imperial College London, and AHL-inducible constructsBBa_J09855 (Jon Badalamenti, iGEM 2005) and BBa_F2620 (iGEM 2004 MIT)were received from the Registry of Standard Biological Parts. Inducible con-structs were then cloned into J23100-mRFP1-pSEVA331Bb, replacing the con-stitutive J23100 promoter (thus controlling downstream mRFP1 expression) tocreate pTet01, pTet02, pLux01, and pLux02.

CBDclos and CBDcex were received as BBa_K863111 and BBa_K863101(iGEM 2012 Bielefeld) from the Registry of Standard Biological Parts, andCBDcipA and dCBD were synthesized as a GeneArt String (Life Technologies).CBDs were then fused to sfGFP (BBa_I746909, iGEM 2007 Cambridge) andcloned into an expression vector (BBa_J04500, Kristen DeCelle, iGEM 2005)downstream of the pLacI promoter. An sRNA construct was also synthesizedas a GeneArt String (Life Technologies) based on descriptions of Na et al. (53)and subcloned downstream of the pLux promoter in pLux01, replacingmRFP1. All constructs were first transformed into E. coli Turbo, and colonieswere screened using colony PCR with GoTaq Green (M7122; Promega) andthe primers i53 and i54 (SI Appendix, Table S6). Plasmid DNA from positivecolonies was extracted with a QIAprep Spin Miniprep Kit (27104; Qiagen),DNA was sequenced (Source BioScience), and correct sequences weretransformed into K. rhaeticus iGEM (SI Appendix, Supplementary Protocols).DNA sequences of all constructs created in this work are accessible throughthe Registry of Standard Biological Parts (25) (see SI Appendix, Table S4for accession numbers). Because many of the constructs characterized inK. rhaeticus iGEM are widely used and accessible through the Registry ofStandard Biological Parts, no constructs were codon-optimized, to allowthem to be used in K. rhaeticus iGEM without modification by the user.

Characterization of Constitutive Promoters, Inducible Promoters, CBD Fusions,and sRNA Constructs. For characterization of constitutive promoters and in-ducible promoters without cellulose formation, 1–5 mL liquid HS cellulase[8–10% (vol/vol)] containing 34 μg/mL chloramphenicol in 50-mL Corningtubes was inoculated from seed cultures to OD600 0.02 and grown at 30 °C,230 rpm for 3 h, and 200 μL culture was then pipetted into 96-well plates(Corning Costar). Inducer was added to inducible cultures to 1 μg/mL for ATcand 1 μM for AHL. High concentrations of cellulase were used to remove anyinterference by cellulose in spectroscopy measurements. Plates were coveredwith Breathe-Easy membrane (Z380059; Sigma), and OD600 and mRFP1 in-tensity (excitation 590 nm, emission 630 nm) were measured every 15 minusing a Synergy HTmicroplate reader (BioTek) at 29 °C and high shaking speed.

For characterization of inducible promoters in pellicle form (Fig. 3 D and Fand SI Appendix, Fig. S8), K. rhaeticus iGEM containing pLux01 was in-oculated from glycerol stocks into HS with 34 μg/mL chloramphenicol andgrown statically for 8 d at 30 °C. For cultures induced during growth, AHLwas added to 1 μM before inoculation, and for cultures induced in pellicle,AHL was added to 1 μM 6 d postinoculation. Pellicles were washed with PBS8 d after inoculation, microscopy samples were prepared with a sterile razor,and fluorescence was quantified using fluorescence microscopy (NikonEclipse Ti) at mCherry preset (590-nm emission, 0.2-s exposure, 675 V EMgain, 150× magnification). A single-layer cellulose sheet was placed on thesample and fluorescence intensity was determined as intensity/exposuretime for quantitative measurements. For white-light images (Fig. 3E), 250-mLbeakers (CLS1000250; Sigma) containing 100 mL HS media were inoculated with5 mL OD600 1 seed culture, and the beakers were covered with Breathe-Easymembrane and incubated statically at 30 °C. For the induced pellicle, 1,000 μL100 mM AHL was pipetted daily 4 d after inoculation along the edges of thepellicle, and the pellicle was imaged 9 d postinoculation.

For characterization of CBD binding strengths, 200 μL CBD-sfGFP fusionproteins extracted from E. coli (SI Appendix, Supplementary Protocols) wereadded to a 96-well plate containing homogenized bacterial cellulose, in-cubated overnight at 4 °C, and washed thrice with treatment [dH2O, PBS, 5%(vol/vol) BSA or 70% (vol/vol) EtOH] (SI Appendix, Supplementary Protocols).GFP fluorescence was measured on a 96-well plate reader (Synergy HT;BioTek) (see SI Appendix, Supplementary Protocols for details).

For characterization of sRNA constructs, HS with 34 μg/mL chloramphenicolwas inoculated to OD600 0.04 with K. rhaeticus iGEM containing plasmid J09855-sRNA-331Bb and induced with 10–500 nM AHL. Forty hours after inoculation,pellicles were washed, dried at 60 °C for 16 h, and weighed (see SI Appendix,Supplementary Protocols for details). For characterization of growth rate, OD600

was measured in [3% (vol/vol)] HS cellulase with 34 μg/mL chloramphenicol usingthe protocol used for characterization of constitutive promoters.

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Engineering of Functionalized Biomaterials. For spatial patterning, K. rhae-ticus containing pLux01 was inoculated into 1 L HS chloramphenicol in 2-LErlenmeyer flasks, 500 μL 100 nM AHL was added to one side of thepellicle 2 d later, and pellicle was imaged 4 d after induction. For tem-poral patterning, 250-mL beakers with 100 mL HS media were inoculatedwith 5 mL seed culture at OD600 1, and the beakers were covered withBreathe-Easy membrane and incubated statically at 30 °C. After this, 1,000 μL100 mM AHL was pipetted along the edges of the pellicle daily withmembrane replacement, starting at different days postinoculation asshown in Fig. 5B, and the pellicle was imaged 9 d postinoculation. Forfunctionalization of cellulose with mRFP1 and CBDcipA-sfGFP, proteinswere first produced in E. coli and extracted via sonication, and extractswere applied to purified wet or dried bacterial cellulose, respectively. FormRFP1-functionalized cellulose, fluorescence intensity was determinedfrom a single cellulose sheet using fluorescence microscopy. For CBDcipA-sfGFP, extracts were applied to dried bacterial cellulose with a paintbrushand dried. The resulting material was then crafted into accessories by a

professional fashion designer. See SI Appendix, Supplementary Protocolsfor further details.

ACKNOWLEDGMENTS. We thank Dr. Cheng-Kang Lee from the NationalTaiwan University of Science and Technology and Dr. Jyh-Ming Wu fromChinese Culture University for sharing plasmid pBla-Vhb-122; Ms. VictoriaGeaney from the Royal College of Art, London, for help with functionalizedgarments; Dr. Koon-Yang Lee (Imperial College London) for helpful adviceand feedback on the manuscript; Dr. Takayuki Homma (Imperial CollegeLondon) for help with genome library preparation; Dr. Laurence Game(Imperial College London) for help and advice with genome sequencing;Mr. Geraint Barton (Imperial College London) for help and advice withgenome assembly and analysis; Dr. Francesca Ceroni (Imperial CollegeLondon) for providing ATc-inducible constructs and advice; Ms. CatherineAinsworth, Mr. Nicolas Kral, Dr. Geoff Baldwin, and Dr. Guy-Bart Stan ofImperial College London for instruction and helpful discussions through-out the project; Dr. Mahmoud Ardakani (Imperial College London) fortaking SEM images; and Dr. Carlos Bricio Garberi (Imperial College London)for taking pellicle photographs.

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