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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 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 cellulose. It is currently used to create materials for tissue engineering, 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. MICROBIOLOGY 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 | Published online May 31, 2016 | E3431–E3440 Downloaded by guest on September 23, 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 conditions and produce cel- other products (7), and in the laboratory has been used to create lulose at high yields, sequence its genome, and develop a syn- biodegradable tissue scaffolds (13), nanoreinforcements (19), and thetic biology toolkit for its genetic engineering. This toolkit artificial blood vessels (18), as well as sensors (20), flexible elec- provides the characterization data necessary for the engineering trodes (21), organic light-emitting diode (OLED) displays, and of K. rhaeticus iGEM, and enables transformation, controlled other materials (22). expression of constitutive and inducible transgenes, and control Functionalization or modification of bacterial cellulose has over endogenous gene expression of this strain. We use these mainly been achieved by chemical or mechanical modifications tools to engineer a system that allows tunable control over native of the cellulose matrix or via changing culturing conditions (7, cellulose production, and produce novel patterned and func- 22), whereas only a few attempts at genetic engineering have tionalized cellulose-based biomaterials. been made (13, 23). However, genetic engineering may allow a greater range of materials to be produced, by enabling fine Results control over cellulose synthesis genes and production of protein- Isolation, Characterization, and Genome Sequencing of K. rhaeticus cellulose composite biomaterials. Here we isolate a strain of iGEM. As part of the International Genetically Engineered Ma- Komagataeibacter rhaeticus (previously Gluconacetobacter rhaeticus) chine Competition (iGEM) (25), we evaluated Gluconacetobacter

E3432 | www.pnas.org/cgi/doi/10.1073/pnas.1522985113 Florea et al. Downloaded by guest on September 23, 2021 hansenii ATCC 53582 [one of the highest-reported cellulose- kpsS,andrfaB) and may play a role in cellulose productivity (35, 36). PNAS PLUS producing strains (26), recently reclassified as Komagataeibacter Finally, to determine whether the iGEM strain can fix atmospheric hansenii ATCC 53582 (27)] and a strain isolated from Kombucha nitrogen similar to G. diazotrophicus, we searched its genome for tea as potential new synthetic biology hosts (Fig. 1A). We chose genes associated with nitrogen fixation. We located five genes (ntrB, the latter strain (hereafter called “iGEM”) for further work, as -C, -X,and-Y and nifU)(SI Appendix,TableS1) associated with preliminary experiments showed that it can be transformed more nitrogen fixation in Acetobacteraceae (37); however, interestingly, we readily with plasmid DNA than G. hansenii ATCC 53582. Fur- did not find the genes homologous to nifHDK, which form the main thermore, the iGEM strain produced more cellulose than nitrogenase subunits in G. diazotrophicus. G. hansenii ATCC 53582 on sucrose in small-scale tests (Fig. 1B) and also produced cellulose at high yields on cheap, low-nitrogen Genetic Engineering Toolkit for Komagataeibacter. Very few genetic Kombucha tea medium (Fig. 1B). Surprisingly, it could also tools are available for the engineering of Acetobacteraceae.We grow on the defined nitrogen-free LGI medium (Fig. 1 C and D therefore developed a complete set of tools for its engineering, and SI Appendix,Figs.S1andS2). Because several species of consisting of protocols, modular plasmids, promoters, reporter Acetobacteraceae, notably Gluconacetobacter diazotrophicus, proteins, and inducible constructs that enable external control of have been confirmed to fix atmospheric nitrogen (28, 29), this gene expression (Fig. 3A). suggested possible nitrogen fixation (see below). Finally, because Protocols and plasmid backbones. We first used the plasmid pBla- cellulose has been reported to increase resistance toward UV Vhb-122 [previously described to replicate in Acetobacteraceae (38)] light and environmental stresses in closely related species (10), we to develop protocols for the preparation of electrocompetent cells, tested whether cellulose could also confer resistance toward transformation, plasmid purification, and genomic DNA extraction chemical stresses, which may occur in unrefined feedstocks or of K. rhaeticus iGEM (SI Appendix, Supplementary Protocols). during industrial production. We tested the susceptibility of the Using these protocols, we then assessed eight plasmids for iGEM strain to 70% (vol/vol) ethanol, 10% (vol/vol) bleach, 0.1 M propagation in K. rhaeticus iGEM: pSEVA311, pSEVA321, NaOH, and 0.1 M HCl (Fig. 1E), and found that when encased in pSEVA331, pSEVA341, pSEVA351, pBAV1K-T5-sfGFP, pSB1C3, cellulose it is highly tolerant to chemical stressors, being over and pBca1020 (see SI Appendix,TableS2for details). From these, 1,000-fold more resistant than E. coli to all treatments (Fig. pSEVA321, 331, 351, pBAV1K-T5-sfGFP, and pBla-Vhb-122 1E). Finally, scanning electron microscopy of a cellulose pelli- showed replication in iGEM (SI Appendix, Fig. S5), giving a total cle confirmed that, as with other closely related species, cells of of five different plasmids to act as vectors. We further engineered K. rhaeticus iGEM are rod-like, ∼2 μm in length, and heavily pSEVA321 and pSEVA331 into pSEVA321Bb and pSEVA331Bb, encased in cellulose during normal growth (Fig. 1F and SI Ap- making them compatible with the widely used BioBrick standard pendix,Fig.S3). (25), to enable rapid cloning of publicly available DNA parts. We To determine the genetic basis of high cellulose productivity then used pSEVA331Bb for all subsequent studies, due to its likely and to provide background information for genetic engineering higher copy number. of the iGEM strain, we sequenced its genome to 400× coverage and Reporter proteins and constitutive and inducible promoters. We next assembled the genome using the genome of Gluconacetobacter tested expression of seven reporter proteins (mRFP1, GFPmut3, xylinus NBRC 3288 (30) as the reference genome for scaffolding sfGFP, and chromoproteins tsPurple, aeBlue, gfasPurple, and (European Nucleotide Archive accession no. PRJEB10933). spisPink) (see SI Appendix,TablesS3andS4for details), of which Sequencing showed that the genome totals 3.87 Mbp with a mRFP1, GFPmut3, and sfGFP showed visually detectable ex- GC% of 62.7 and contains a predicted 3,573 genes, with an N50 pression. We then chose 10 promoters from an open-access col- (shortest sequence length at 50% of the genome) of 3.16 Mbp. lection of synthetic minimal E. coli promoters and, using mRFP1 The genome is divided between a chromosome of 3.16 Mbp, at as the reporter, characterized these in K. rhaeticus iGEM (Fig. 3B least two plasmids: pKRi01 (238 kbp) and pKRi02 (3 kbp), and 37 and SI Appendix,TableS4;alsoseeSI Appendix,Fig.S6for a unplaced contigs (in total 460 kbp), which may be part of the comparison with promoter strengths in E. coli). For inducible chromosome or additional plasmids, and could not be confidently promoters, we engineered four constructs allowing gene expres- assigned due to being flanked by repetitive sequences (Fig. 2A). sion to be induced externally by anhydrotetracycline (ATc) or N-acyl The genome sequence revealed several interesting aspects about homoserine lactone (AHL) (see SI Appendix,Fig.S7for an overview the biology of K. rhaeticus iGEM. First, a 16S rRNA phylogeny of constructs). From these, the AHL-inducible constructs (pLux01 suggests the iGEM strain to be a previously unidentified strain of and pLux02) showed higher induction and lower leakiness than K. rhaeticus (SI Appendix,Fig.S4), rather than G. xylinus,whichis the ATc-induced constructs (pTet01 and pTet02) (Fig. 3C) and, normally thought to be associated with Kombucha tea. Further- contrary to our initial expectations, they also gave robust induction more, the sequence shows the presence of four acs operons on of mRFP1 expression when cells were encased in the pellicle, the chromosome, sharing 40–65% amino acid identity (Fig. 2). showing visible fluorescence (Fig. 3 D and E and SI Appendix, Up to three acs operons have been reported in other bacterial Fig. S8). This is notable, as it shows that cells in the pellicle can cellulose-producing species [G. xylinus ATCC 23769 and G. hansenii effectively receive signals from their environment despite their ATCC 53582 (31, 32)], indicating that the high cellulose synthase cellulose encasing. Because K. rhaeticus is highly resistant to copy number may be a possible contributor to the high cellulose various environmental hazards within cellulose (Fig. 1E), the productivity observed here (Fig. 1B). These operons also differ in ability to receive signals while being protected by cellulose makes structure. The acs1 operon contains separate acsA and acsB, it a potentially suitable host for applications requiring tolerance to whereas they are fused in the other operons, and the only genomic toxic chemicals and long-term survival.

copy of acsD is found in acs1.Operonacs4 uniquely contains only MICROBIOLOGY acsAB, and phylogenetic analysis indicates that acs4 is most closely Engineering Control over Cellulose Production. Because wild-type related to the acs2 operon (Fig. 2B), and possibly arose via du- species produce cellulose constitutively, a major goal of genetic plication and subsequent translocation. From the genes flanking engineering of Acetobacteraceae has been to achieve control over acs operons, cmcAX, ccpAX, bglxA, bcsX,andbcsY have been cellulose production. Constitutive cellulose production complicates previously shown to contribute to cellulose production in closely genetic engineering techniques and is not always desirable for related species (26, 33, 34). We found two other, stand-alone industrial applications as it imparts a high metabolic cost, which in copies of bglxA from the genome (genomic positions 517401– well-aerated conditions typically leads to the emergence of cellu- 519440 and 3029825–3032221), and also identified genes close to lose-nonproducing mutants (39). It is therefore desirable to inhibit acs2 that are associated with extracellular matrix formation (kpsC, cellulose production during periods when it is not required, to

Florea et al. PNAS | Published online May 31, 2016 | E3433 Downloaded by guest on September 23, 2021 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 a predicted 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 least two 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. For the chromosome, consecutive rings show (from the outside in): (i) read coverage, (ii and iii), genes on (ii) forward and (iii) reverse strands, (iv) acs operons involved in cellulose synthesis, (v) GC%, and (vi) GC skew. Additionally, the genome contains 37 scaffolds (totaling 460 kbp) that could not be confidently placed due to repetitive sequences. These scaffolds may be part of the chromosome or plasmids, or may belong to additional plasmids (the closely related species G. xylinus NBRC 3288 and K. xylinus E25 contain five and seven plasmids, respectively). (B and C) Phylogenetic relationships (B) and amino acid sequence identity (C)ofacs operons. Phylogeny and sequence identity indicate that acs2 and acs4 operons are most closely related. Amino acid sequences were aligned and percent identity was calculated using MUSCLE (54) and the tree was generated using the neighbor-joining method (55). The tree is drawn to scale, with bootstrap values (56) from 1,000 replicates shown next to the branches. All positions containing gaps were eliminated from analysis. See Methods for further details on sequencing and analysis.

prevent the proliferation of these mutants. Furthermore, fine production was not related to toxicity, as growth rate did not decrease control over cellulose production levels may allow control over the compared with wild-type levels (Fig. 4C and SI Appendix,Fig.S10). density of cellulose fibrils, and thus the macroscale properties of This system was engineered to be a general platform for targeted the cellulose. To achieve controlled cellulose production, we engi- knockdowns in Komagataeibacter and other bacterial species, as ex- neered a system in which an E. coli Hfq and an sRNA targeting pression of E. coli Hfq makes it independent from the host Hfq, and UGPase mRNA (UDP-glucose pyrophosphorylase) are coexpressed the broad host range pSEVA331Bb backbone enables replication in from a plasmid in response to AHL (plasmid J-sRNA-331Bb) (Fig. a wide range of species. Furthermore, new sRNAs can be added to 4A;alsoseeSI Appendix,Fig.S9for a detailed overview). The the plasmid, and the 24-base sRNA region can be recoded rapidly by sRNA contains a 24-base region complementary to UGPase site-directed mutagenesis, making the construct easily modifiable for mRNA and an E. coli Hfq-binding region. When expressed, it other targets. binds to the target UGPase mRNA and recruits E. coli Hfq, inhibiting UGPase translation. We targeted the UGPase gene, as Genetic Engineering of Patterned and Functionalized Biomaterials. it catalyzes the production of UDP-glucose critical for cellulose Owing to the discovery that K. rhaeticus gene expression can synthesis (40) and is present in single copy in the genome, be induced even when inside a cellulose pellicle, we hypothesized allowing knockdown by a single sRNA. We found this system to that it may be possible to generate spatially and temporally be highly efficient, as cellulose production was suppressed com- patterned biomaterials that are controlled by the diffusion of the pletely upon full induction and could be fine-tuned using different inducer AHL and timing of exposure to induction during pellicle concentrations of AHL (Fig. 4B). The observed reduction in cellulose growth. To test this, we induced growing cellulose pellicles with

E3434 | www.pnas.org/cgi/doi/10.1073/pnas.1522985113 Florea et al. Downloaded by guest on September 23, 2021 A B PNAS PLUS K. rhaeticus synthetic biology toolkit v1.0

Plasmid backbones: pSEVA321, 331, 351 pBla-Vhb-122 pBAV1k

Reporter genes: GFPmut3 sfGFP mRFP1

Constitutive C promoters: 9 promoters of diﬀerent strength

Inducible 2x AHL-inducible promoters: promoters

2x ATc-inducible promoters

DEF Uninduced Microscope

1cm Induced in pellicle

1cm 1cm 200 μm

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)inK. rhaeticus iGEM, as measured by total mRFP1 fluorescence per cell (fluorescence at

630 nm divided by OD600). Promoter strengths in B and C were assayed in liquid HS with cellulase to remove formation of cellulose fibrils interfering with measurements. (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 or wild-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 were grown in HS (without cellulase), and fluorescence was quantified by fluorescence microscopy image analysis. (E and F) Induction in the pellicle results in visible mRFP1 production compared with uninduced cells (E) (indicated by the arrow; also see SI Appendix, Fig. S8), and results in granular fluorescence due to localization 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.

cells containing the AHL-inducible construct pLux01 with dif- and purified components to be used for functionalization. To test ferent concentrations of AHL, at different locations and time for the possibility of post hoc functionalization, we produced points (Fig. 5 A and B). We found that both spatial and temporal mRFP1 in E. coli, extracted and added it to bacterial cellulose, control were possible. When a limited amount of inducer was and compared it by fluorescence microscopy with cellulose pro- added to one side of the pellicle, cells produced mRFP1 following duced by K. rhaeticus with in vivo constitutive mRFP1 expression. the diffusion gradient of AHL (Fig. 5A). Furthermore, because We found that extracted proteins can diffuse well throughout the cells are active only in the top layer of the cellulose pellicle (7), pellicle and functionalize the cellulose evenly (Fig. 5D), whereas when inducer was added at different times midway through pel- the granular fluorescence exhibited by expression from pellicle- licle growth, only cells at the growing top layer produced mRFP1, based cells (Fig. 3E;alsoseeSI Appendix,Fig.S11for a full-size capturing the temporal difference between uninduced cells in the comparison) indicates that mRFP1 remains largely in the bottom layers and induced cells at the top (Fig. 5B). K. rhaeticus cells and would likely require active secretion or MICROBIOLOGY To produce functionalized cellulose materials where the nano- lysis of cell membranes to access the extracellular cellulose. cellulose matrix is coated by proteins of interest, we considered To further increase the efficiency of functionalization, we two strategies: genetic engineering of K. rhaeticus to produce these engineered expression vectors that allow easy fusion of proteins proteins in situ, or separate expression of proteins in E. coli, which to one of four different cellulose-binding domains (CBDs): are then purified and applied directly to bacterial cellulose (Fig. CBDclos (41), CBDCex (42), dCBD (43), and CBDcipA (44). 5C). Although the latter requires a three-step process (protein CBDs are short peptides that bind tightly to cellulose fibrils, thus and cellulose production separately, followed by combining the increasing protein adhesion to cellulose (45). In these constructs, two), it may be preferred for medical and other applications where proteins can be modularly fused to CBDs via restriction enzyme very high purity of material is required, as it would allow defined cloning. We assessed the cellulose binding strengths of these CBDs

Florea et al. PNAS | Published online May 31, 2016 | E3435 Downloaded by guest on September 23, 2021 Fig. 4. sRNA construct (J-sRNA-331Bb) for control of cellulose production. (A) Overview of the sRNA silencing construct. Constitutively produced LuxR binds to 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-base sequence 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 production 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 induction 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 cells

are not different from negative controls (adjusted P = 0.12 for uninduced vs. pSEVA331Bb 100 nM AHL). (C)OD600 of cultures 3 h postinoculation. Differences between OD600 are not significant for any comparisons (adjusted P > 0.05), showing that induction of the sRNA silencing construct does not reduce growth rate. 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 determined with one-way ANOVA and Tukey’s multiple comparisons tests for B and C. See Methods for further details of all assays.

by washing four different E. coli-extracted CBD-sfGFP fusion genes for nitrogen fixation, as alternative nitrogenases have been proteins with different solvents (dH2O, EtOH, BSA, and PBS) and isolated in other species (48). However, in either case, from the measuring the fluorescence that remained bound. Addition of perspective of manufacturing, low nitrogen tolerance allows the CBDs to sfGFP gave up to a fivefold increase in binding to cellu- use of cheap, low-nitrogen media, potentially reducing cellulose- lose compared with GFP alone (SI Appendix,Fig.S12). Finally, manufacturing costs. because bacterial cellulose is a candidate for new textile materials To use this strain for biotechnology applications, it was first and of high interest to the fashion industry, we used our approach necessary to develop methods and tools for its genetic manipu- to demonstrate production of functionalized garments. Using the lation. For a genetic toolkit to be useful, it should minimally allow CBDcipA-sfGFP fusion protein extract and dried pellicle material introduction of foreign DNA, plasmid propagation, and a degree from K. rhaeticus cultures, we created bacterial cellulose fashion of control over heterologous gene expression levels. Although the accessories by functionalization of cellulose fibrils with green required tools can differ between species and the applications in fluorescent protein (SI Appendix,Fig.S13), indicating that this which they are used, they should ideally also allow tunable regu- approach is scalable to produce macroscale objects. lation of transgene expression through inducible or repressible systems and regulation over native gene expression. A toolkit Discussion should further provide relevant characterization data about the Here we isolated a strain of bacterial cellulose-producing host, such as a genome sequence and growth and productivity K. rhaeticus (K. rhaeticus iGEM), which can grow on acidic and rates in different conditions. Indeed, many of these elements have low-nitrogen media, is highly resistant to damaging chemical been part of toolkits created for other species (5, 6, 49). The agents when encased in cellulose, and is notable for its high toolkit described here for K. rhaeticus iGEM contains all of these cellulose production. Although it is clear that K. rhaeticus iGEM features, providing protocols and DNA constructs to allow trans- can effectively produce cellulose in low-nitrogen media, whether formation, precisely controlled constitutive and inducible expression it fixes nitrogen is still an open question. Nitrogen fixation seems of heterologous genes, control over endogenous gene expression, to be prevalent in Acetobacteraceae, as multiple species (most and a genome sequence with characterization data. notably G. diazotrophicus) have been recorded to be nitrogen- Using this toolkit, we created two approaches for engineering fixing (46). However, in the case of K. rhaeticus iGEM, although new cellulose-based materials, first through genetic engineering it can grow in nitrogen-free LGI medium (Fig. 1 C and D and SI of K. rhaeticus itself, and second through application of extracted Appendix, Figs. S1 and S2) and its genome contains a set of genes proteins to the bacterial cellulose. Genetic engineering offers associated with nitrogen fixation (SI Appendix, Table S1), we simple, one-step in situ production of materials, and would be were unable to find genes homologous to G. diazotrophicus ideal for applications where the cellulose matrix is modified as it nifHDK, which form the structural subunits of the nitrogenase is being made. The physical and biochemical properties of cel- complex (47). This either suggests that despite careful handling, lulose are largely dependent on the microscale morphology and very low level nitrogen contamination may have been present, structure of cellulose fibrils, so modification of these during allowing K. rhaeticus to produce cellulose in LGI medium or, production by expressed heterologous enzymes could result in alternatively, that K. rhaeticus uses a different set of nitrogenase new material properties. For example, by engineering cells to

E3436 | www.pnas.org/cgi/doi/10.1073/pnas.1522985113 Florea et al. Downloaded by guest on September 23, 2021 PNAS PLUS ACSpaal Post-hoc D paerning funconalizaon

10 cm Transform E. coli

Extract protein

Apply to 10 cm cellulose 100 μm

B Temporal Induced Induced Induced Induced paerning Not induced on day 0 only on days 4 -9 on days 2-9 on days 0 - 9 i

3cm ii

1cm iii

1cm

Fig. 5. Engineering of patterned and functionalized cellulose materials on a macroscale. (A) Spatial patterning. Cells were induced for mRFP1 expression by addition 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) and imaged 9 d postinoculation, with overview (i), white light (ii), and fluorescence (iii) images of the pellicles and pellicle cross-sections shown. Note that pellicles induced on day 0 only show low fluorescence due to natural degradation of AHL in the media over time. (C) Overview of the cellulose functionalization strategy via post hoc addition of mRFP1 extracted from E. coli.(D) Fluorescence microscopy of a cross-section of cellulose functionalized with mRFP1 through addition of mRFP1 extracted from E. coli. Smooth fluorescence is seen throughout the pellicle cross-section, compared with the granular fluorescence seen 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- sectionisshown(Right). Images were cropped and contrast was adjusted to improve clarity for all images. See Methods for fluorescence imaging conditions and other details.

incorporate N-acetylglucosamine residues into cellulose, Yadav not significantly change growth rate, control over cellulose pro- et al. (13) created biodegradable, low-immunogenicity cellulose duction levels may also allow control of the specific cell-vs.-cel- tissue scaffolds. Our second approach (addition of purified pro- lulose and thus protein-vs.-cellulose concentrations in the teins to already-produced cellulose) is likely to be preferable for material. In the case of functionalization of cellulose with CBD applications where bulk material properties are not changed but fusion proteins, in principle any protein stable enough to func- the material is functionalized with new properties. In our experi- tion in the intended extracellular environment could be used ments, post hoc functionalization resulted in an even, thorough for functionalization. distribution of functionalizing proteins (Fig. 5D), and may be By providing genetic engineering tools that allow control of preferable in medical and tissue engineering applications where production of bacterial cellulose and modification of it as a highly pure materials are required, as both the cellulose and material, this work enables a variety of potential applications. functionalizing proteins can be purified before formation of the The physical properties and pore size of the cellulose might be composite material. Together, these two approaches complement tunable by altering gene expression of the K. rhaeticus acs op- each other, as the ability to modify cellulose via genetic engi- erons or by complexing cellulose in situ with secreted structural neering as well as functionalize it with purified proteins allows for proteins such as curli fimbriae (12). Cellulose can be function- a wider range of materials to be engineered. alized with specific proteins (Fig. 5C and SI Appendix, Figs. S12 MICROBIOLOGY Here, genetic engineering allowed us to control cellulose pro- and S13), which may be used to create advanced materials such duction by sRNA-mediated knockdown and create spatial and as bacterial cellulose wound dressings coated with antimicrobial temporal patterning through induction with N-acyl homoserine peptides or novel high-specificity water filters coated with pro- lactone. We used an AHL-inducible system here; however, for teins binding specific contaminants. In another area, the toolkit industrial applications, it is likely straightforward to exchange it allows creating patterned cellulose structures in three dimensions for a control system using cheaper chemical or physical inducers (Fig. 5 A and B). In combination with functionalization, this may if required. Although we did not test for this specifically, it is be used in tissue engineering for one-step production of cellulose- important to note that as knockdown of cellulose production did based tissue engineering scaffolds that contain specifically patterned

Florea et al. PNAS | Published online May 31, 2016 | E3437 Downloaded by guest on September 23, 2021 growth factors. Furthermore, theremarkablerobustnessof gestion with SpeI (R3133S; NEB), and subsequent religation. Constitutive K. rhaeticus within cellulose (Fig. 1E) offers potential uses in bio- promoter-mRFP1 constructs (BBa_J23100–Bba_J23117 by iGEM 2006 Berkeley) sensing. The growing pellicle can store changes in environmental were received from the iGEM Registry of Standard Biological Parts (52) and signals by writing the conditions into different cellulose layers as subcloned into pSEVA331Bb. ATc-inducible constructs were kindly provided by Francesca Ceroni at Imperial College London, and AHL-inducible constructs B they grow (Fig. 5 ), acting as a record akin to ice cores. Finally, BBa_J09855 (Jon Badalamenti, iGEM 2005) and BBa_F2620 (iGEM 2004 MIT) because cellulose synthesis and nitrogen fixation in closely related were received from the Registry of Standard Biological Parts. Inducible con- Acetobacteraceae are areas of active research, this toolkit is also a structs were then cloned into J23100-mRFP1-pSEVA331Bb, replacing the con- valuable resource for basic studies aiming to dissect the molecular stitutive J23100 promoter (thus controlling downstream mRFP1 expression) to mechanisms of these processes. As there are many possible appli- create pTet01, pTet02, pLux01, and pLux02. cations enabled by this work, we are making this strain and the CBDclos and CBDcex were received as BBa_K863111 and BBa_K863101 genetic engineering toolkit available through ATCC and AddGene. (iGEM 2012 Bielefeld) from the Registry of Standard Biological Parts, and CBDcipA and dCBD were synthesized as a GeneArt String (Life Technologies). Methods CBDs were then fused to sfGFP (BBa_I746909, iGEM 2007 Cambridge) and Isolation, Characterization, and Culturing of K. rhaeticus iGEM. K. rhaeticus cloned into an expression vector (BBa_J04500, Kristen DeCelle, iGEM 2005) iGEM was isolated from a Kombucha SCOBY (symbiotic colony of bacteria downstream of the pLacI promoter. An sRNA construct was also synthesized and yeast) of Czech origin (Happy Kombucha) by streaking homogenized as a GeneArt String (Life Technologies) based on descriptions of Na et al. (53) SCOBY material on Hestrin–Schramm (HS) agarose (SI Appendix, Table S5), and subcloned downstream of the pLux promoter in pLux01, replacing verifying cell morphology under a light microscope, and restreaking isolated mRFP1. All constructs were first transformed into E. coli Turbo, and colonies colonies on HS agarose twice. Two percent (wt/vol) glucose was used in HS were screened using colony PCR with GoTaq Green (M7122; Promega) and unless stated otherwise. Glycerol stocks were prepared by culturing the the primers i53 and i54 (SI Appendix, Table S6). Plasmid DNA from positive iGEM strain statically in HS medium for 6 d, followed by addition of 0.2% colonies was extracted with a QIAprep Spin Miniprep Kit (27104; Qiagen), (vol/vol) cellulase (Trichoderma reesei cellulase; C2730; Sigma), incubation DNA was sequenced (Source BioScience), and correct sequences were with 230-rpm shaking, 30 °C for 1 d, addition of glycerol to 25% (vol/vol), transformed into K. rhaeticus iGEM (SI Appendix, Supplementary Protocols). and storage at −80 °C. DNA sequences of all constructs created in this work are accessible through For cellulose production, seed cultures of K. rhaeticus iGEM were in- the Registry of Standard Biological Parts (25) (see SI Appendix, Table S4 oculated from glycerol stocks and grown statically at 30 °C in Kombucha tea, for accession numbers). Because many of the constructs characterized in HS, or LGI medium (see SI Appendix, Supplementary Protocols and Table S5 K. rhaeticus iGEM are widely used and accessible through the Registry of for details). When grown with shaking without cellulose production, cul- Standard Biological Parts, no constructs were codon-optimized, to allow tures were grown in HS cellulase [0.4% (vol/vol)] at 30 °C, 230-rpm shaking at them to be used in K. rhaeticus iGEM without modification by the user. 45° tube angle. Unless otherwise stated, 50-mL Corning tubes (CLS430829; Sigma) with 5–20 mL media were used for culturing. When grown on HS Characterization of Constitutive Promoters, Inducible Promoters, CBD Fusions, agar, plates were incubated inverted at 30 °C. For culturing of K. rhaeticus and sRNA Constructs. For characterization of constitutive promoters and in- transformed with plasmids encoding kanamycin or chloramphenicol resistance ducible promoters without cellulose formation, 1–5 mL liquid HS cellulase genes, kanamycin was added to 500 μg/mL for HS agar and 50–100 μg/mL for [8–10% (vol/vol)] containing 34 μg/mL chloramphenicol in 50-mL Corning liquid HS, and chloramphenicol was added to 340 μg/mL for HS agar and tubes was inoculated from seed cultures to OD600 0.02 and grown at 30 °C, 34–68 μg/mL for liquid HS. 230 rpm for 3 h, and 200 μL culture was then pipetted into 96-well plates Cellulose productivity on different media was measured by culturing in (Corning Costar). Inducer was added to inducible cultures to 1 μg/mL for ATc 20 mL HS glucose [2% (wt/vol)], HS sucrose [2% (wt/vol)], HS without a carbon and 1 μM for AHL. High concentrations of cellulase were used to remove any source (negative control), and Kombucha tea in 50-mL Corning tubes at 30 °C interference by cellulose in spectroscopy measurements. Plates were covered for 10 d, with loose caps for increased air diffusion, and kept at 4 °C until with Breathe-Easy membrane (Z380059; Sigma), and OD600 and mRFP1 in- measurement of cellulose weight (SI Appendix, Supplementary Protocols). tensity (excitation 590 nm, emission 630 nm) were measured every 15 min Productivity of K. rhaeticus iGEM was reported in comparison with pro- using a Synergy HT microplate reader (BioTek) at 29 °C and high shaking speed. ductivity of the high-producing G. hansenii ATCC 53582 instead of maximal For characterization of inducible promoters in pellicle form (Fig. 3 D and F cellulose yield per volume of media, as maximal total productivity is highly and SI Appendix, Fig. S8), K. rhaeticus iGEM containing pLux01 was independent on specific culturing conditions and may not be a good measure oculated from glycerol stocks into HS with 34 μg/mL chloramphenicol and of genetically determined production capabilities. To test cellulose pro- grown statically for 8 d at 30 °C. For cultures induced during growth, AHL

ductivity on nitrogen-free LGI medium, 80 μLOD600 1 E. coli or K. rhaeticus was added to 1 μM before inoculation, and for cultures induced in pellicle, iGEM seed culture was inoculated into 25 mL LGI medium in 50-mL glass AHL was added to 1 μM 6 d postinoculation. Pellicles were washed with PBS tubes (3119-0050; Thermo Fischer) and imaged 9 d postinoculation. 8 d after inoculation, microscopy samples were prepared with a sterile razor, For scanning electron micrographs, cellulose was gold-coated under and fluorescence was quantified using fluorescence microscopy (Nikon vacuum and imaged at 2,000–6,000× magnification and 20 kV. See SI Ap- Eclipse Ti) at mCherry preset (590-nm emission, 0.2-s exposure, 675 V EM pendix, Supplementary Methods for details. gain, 150× magnification). A single-layer cellulose sheet was placed on the sample and fluorescence intensity was determined as intensity/exposure Chemical Tolerance Assays. For both E. coli and K. rhaeticus, cells were treated time for quantitative measurements. For white-light images (Fig. 3E), 250-mL with 0.5 mL 0.1 M NaOH, 0.1 M HCl, 70% (vol/vol) ethanol, 10% (vol/vol) beakers (CLS1000250; Sigma) containing 100 mL HS media were inoculated with bleach, or PBS for 5, 30, or 90 min. Treatments were then plated; colonies 5mLOD600 1 seed culture, and the beakers were covered with Breathe-Easy were photographed in white light and counted to determine the fraction of membrane and incubated statically at 30 °C. For the induced pellicle, 1,000 μL surviving cells compared with PBS-treated cells. For details of the assay, see SI 100 mM AHL was pipetted daily 4 d after inoculation along the edges of the Appendix, Supplementary Methods. pellicle, and the pellicle was imaged 9 d postinoculation. For characterization of CBD binding strengths, 200 μL CBD-sfGFP fusion Genome Sequencing, Assembly, Bioinformatics, and Statistics. The K. rhaeticus proteins extracted from E. coli (SI Appendix, Supplementary Protocols) were iGEM genome was sequenced with an Illumina MiSeq using 250-bp paired- added to a 96-well plate containing homogenized bacterial cellulose, in- end reads, to a coverage of ∼400×. Reads were then downsampled to 100× cubated overnight at 4 °C, and washed thrice with treatment [dH2O, PBS, 5% coverage, assembled using the BugBuilder pipeline (50), quality-controlled, (vol/vol) BSA or 70% (vol/vol) EtOH] (SI Appendix, Supplementary Protocols). and annotated using Prokka with the full read set (51). All statistical tests GFP fluorescence was measured on a 96-well plate reader (Synergy HT; were performed with Prism 6 (GraphPad Software). For details of genome BioTek) (see SI Appendix, Supplementary Protocols for details). sequencing and bioinformatics, see SI Appendix, Supplementary Methods. For characterization of sRNA constructs, HS with 34 μg/mL chloramphenicol

was inoculated to OD600 0.04 with K. rhaeticus iGEM containing plasmid J09855- Engineering of Constitutive Promoters, Inducible Promoters, CBD Fusions, and sRNA-331Bbandinducedwith10–500 nM AHL. Forty hours after inoculation, sRNA Constructs. pSEVA331Bb and pSEVA321Bb were constructed from pellicles were washed, dried at 60 °C for 16 h, and weighed (see SI Appendix,

pSEVA331 and pSEVA321, respectively, via substituting the native polylinker Supplementary Protocols for details). For characterization of growth rate, OD600 with BioBrick prefix and suffix. This was done by PCR mutagenesis with Q5 was measured in [3% (vol/vol)] HS cellulase with 34 μg/mL chloramphenicol using polymerase (M0491S; NEB), primers i75 and i76 (SI Appendix, Table S6), di- the protocol used for characterization of constitutive promoters.

E3438 | www.pnas.org/cgi/doi/10.1073/pnas.1522985113 Florea et al. Downloaded by guest on September 23, 2021 Engineering of Functionalized Biomaterials. For spatial patterning, K. rhae- professional fashion designer. See SI Appendix, Supplementary Protocols PNAS PLUS ticus containing pLux01 was inoculated into 1 L HS chloramphenicol in 2-L for further details. Erlenmeyer flasks, 500 μL 100 nM AHL was added to one side of the pellicle 2 d later, and pellicle was imaged 4 d after induction. For tem- ACKNOWLEDGMENTS. We thank Dr. Cheng-Kang Lee from the National poral patterning, 250-mL beakers with 100 mL HS media were inoculated Taiwan University of Science and Technology and Dr. Jyh-Ming Wu from Chinese Culture University for sharing plasmid pBla-Vhb-122; Ms. Victoria with 5 mL seed culture at OD600 1, and the beakers were covered with Breathe-Easy membrane and incubated statically at 30 °C. After this, 1,000 μL Geaney from the Royal College of Art, London, for help with functionalized garments; Dr. Koon-Yang Lee (Imperial College London) for helpful advice 100 mM AHL was pipetted along the edges of the pellicle daily with and feedback on the manuscript; Dr. Takayuki Homma (Imperial College membrane replacement, starting at different days postinoculation as London) for help with genome library preparation; Dr. Laurence Game shown in Fig. 5B, and the pellicle was imaged 9 d postinoculation. For (Imperial College London) for help and advice with genome sequencing; functionalization of cellulose with mRFP1 and CBDcipA-sfGFP, proteins Mr. Geraint Barton (Imperial College London) for help and advice with were first produced in E. coli and extracted via sonication, and extracts genome assembly and analysis; Dr. Francesca Ceroni (Imperial College were applied to purified wet or dried bacterial cellulose, respectively. For London) for providing ATc-inducible constructs and advice; Ms. Catherine Ainsworth, Mr. Nicolas Kral, Dr. Geoff Baldwin, and Dr. Guy-Bart Stan of mRFP1-functionalized cellulose, fluorescence intensity was determined Imperial College London for instruction and helpful discussions through- from a single cellulose sheet using fluorescence microscopy. For CBDcipA- out the project; Dr. Mahmoud Ardakani (Imperial College London) for sfGFP, extracts were applied to dried bacterial cellulose with a paintbrush taking SEM images; and Dr. Carlos Bricio Garberi (Imperial College London) and dried. The resulting material was then crafted into accessories by a for taking pellicle photographs.

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