WO2017203281A1 - Production of functionalised cellulose - Google Patents

Abstract

Functionalised cellulose material and the processes for preparing such material are provided. The process may comprise the steps of (i) providing a microorganism capable of producing cellulose; (ii) providing culture conditions that enable the production of cellulose by the microorganism; (iii) expressing at least one functionalising agent within the microorganism, wherein the expression is under the control of at least one inducible or repressible promoter operably linked thereto;(iv) controlling expression of the at least one functionalising agent within the microorganism such that the at least one functionalising agent is produced;(v) producing cellulose by the microorganism with concurrent production of the at least one functionalising agent; and (vi) producing a composite cellulose material that has been functionalized with the at least one functionalising agent. Functionalising agents may be distributed, isolated or otherwise localised within the matrix of the material.

Description

PRODUCTION OF FUNCTIONALISED CELLULOSE

FIELD OF THE INVENTION The present invention is directed towards the genetic engineering of microorganisms to produce cellulose-based materials, with applications in composite materials engineering and biotechnology.

BACKGROUND OF THE INVENTION Acetobacteraceae demonstrate a characteristic ability to produce bacterially produced nanocellulose in significant quantities (1). Although it is still unclear why these organisms produce cellulose, it has been shown to confer the host a high resistance to UV light and a competitive advantage in colonization over other microorganisms (2). Komagataeibacter is one genus from the Acetobacteraceae family of which multiple species produce bacterially produced nanocellulose. In Komagataeibacter cellulose nanofibers are synthesized from UDP-glucose by the acs (Acetobacter cellulose synthase) operon proteins AcsA and AcsB (9) and secreted by AcsC and AcsD, forming an interconnected cellulose 'pellicle' around cells (1).

In materials science, genetic engineering has been used to create several novel biomaterials, such as strong underwater protein-based adhesives (3), self-assembling, functionalized amyloid-based biofilms (4), biodegradable bacterially produced nanocellulose based tissue engineering scaffolds (5), and many others. Bacterially produced nanocellulose has long been a focus of research because, unlike plant-based cellulose, it is free from other chemical species (lignin and pectin) and is synthesized as a continuous highly interconnected lattice-like matrix (6). This makes the material mechanically strong (nanocellulose fibres possess tensile stiffness of between 100-160 GPa and tensile strength of at least 1 GPa (7, 8)), while still flexible, biocompatible and highly hydrophilic, capable of storing water over 90% of total weight (9, 10). Due to these properties, bacterially produced nanocellulose is commercially used in medical wound-dressings, high-end acoustics and many other products (1), and in the laboratory, has been used to create biodegradable tissue scaffolds (5), nano-reinforcements (1 1), artificial blood vessels (10), as well as sensors (12), flexible electrodes (13), OLED displays and other materials (14).

Functionalization or modification of bacterially produced nanocellulose has mainly been achieved after formation of the matrix by chemical or mechanical modifications of the cellulose matrix or via changing culturing conditions (1 , 14), while only a few attempts at genetic engineering have been made (5, 15). When functionalisation occurs after matrix formation this is referred to as occurring post-hoc.

Hence, there exists a need to create a greater range of cellulosic biomaterials such as tailored composites that bring added functionality in addition to the industrially useful mechanical properties of bacterially produced nanocellulose by enabling fine control over cellulose synthesis, functionalisation and production of protein-cellulose composite biomaterials.

Here, the Inventors have utilised a strain of Komagataeibacter rhaeticus (previously classified as Gluconaceteobacter rhaeticus) (16) that can grow in low nitrogen conditions while producing cellulose at high yields, and have developed a synthetic biology toolkit for its genetic engineering. This toolkit provides an exemplary organism, K. rhaeticus iGEM, which enables transformation and controlled expression of constitutive and inducible transgenes, as well as control over endogenous gene expression of this strain.

An aim of the present invention is to improve the functionality of bacterially-produced nanocellulose, in order to yield a versatile high-value composite material. Further objectives may include low cost and environmentally friendly production of bacterially-produced nanocellulose composites. These and other uses, features and advantages of the invention should be apparent to those skilled in the art from the teachings provided herein.

SUMMARY OF THE INVENTION The present inventors have provided a process that allows tunable control over native cellulose production in microorganisms, and production of novel patterned and functionalized cellulose- based biomaterials. This means that the cellulose composite materials may be functionalised whilst they are being synthesised rather than after the matrix has been made, thereby affording considerable advantages in terms of the creation of novel composite products.

In a first aspect the invention comprises a process for preparing a functionalised cellulose material, the process comprising the steps of: (i) providing a microorganism capable of producing cellulose; (ii) expressing at least one functionalising agent within the microorganism, wherein the expression is under the control of at least one inducible or repressible promoter operably linked thereto; (iii) providing culture conditions that enable the production of cellulose by the microorganism (iv) controlling expression of the at least one functionalising agent within the microorganism; and (v) producing a functionalised cellulose material.

The functionalised cellulose material may comprise a cellulose matrix comprising the at least one functionalising agent. Suitably the cellulose matrix comprises nanocellulose.

The functionalising agent may be bonded to the cellulose matrix. In particular, the functionalising agent may be bonded to the cellulose matrix via a covalent bond. Alternatively, the functionalising agent may be bonded to the cellulose matrix via an electrostatic interaction. The functionalising agent may be comprised within the cellulose matrix. The functionalising agent may be comprised within one or more cells embedded within the cellulose matrix.

Alternatively, the functionalising agent may catalyse covalent modification of the cellulose material, without necessarily binding to the material itself. In one embodiment this functionalising agent is an enzyme, which may function as a kinase, transaminase, transacetylase or glycosyltransferase, catalysing for example the production of phosphocellulose, cellulose amide, cellulose acetate or glycosylated cellulose within all or some of the material. The functionalising agent may be selected from the group consisting of: chelating agents; fluorescent agents; catalysing agents, antimicrobial agents, anti-infection agents, probiotic agents, fertilising agents, reactive agents, sensing agents, pigmentation agents, molecular binding agents, agents that reconfigure the native cellulose material, or any combination of agents from the mentioned groups, where these agents can either be proteins, RNA, DNA, peptides or small molecules whose synthesis is catalysed by proteins or RNA.

The microorganism may be selected from the group consisting of: Escherichia coli, Komagataeibacter (including Komagataeibacter rhaeticus and Komagataeibacter rhaeticus iGEM), Gluconacetobacter (Including Gluconacetobacter hansenii and Gluconacetobacter xylinus), Acetobacter (including Acetobacter. hansenii), Sarcina ventriculi, Agrobacterium (including Agrobacterium pasteurianus), Azotobacter, Rhizobium, Pseudomonas, Salmonella, Alcaligene, fungi and algae.

The at least one inducible or repressible promoter may be selected from the group consisting of: anhydrotetracycline (aTc) and N-acyl homoserine lactone (AHL) inducible or repressible promoters.

The functionalisation of the functionalised cellulose material may be controlled temporally and/or spatially. In a second aspect the invention provides a functionalised cellulose material prepared according to the method comprising the steps of: (i) providing a microorganism capable of producing cellulose; (ii) expressing at least one functionalising agent within the microorganism, wherein the expression is under the control of at least one inducible or repressible promoter operably linked thereto; (iii) providing culture conditions that enable the production of cellulose by the microorganism (iv) controlling expression of the at least one functionalising agent within the microorganism; and (v) producing a functionalised cellulose material.

In such a functionalised cellulose material, at least one functionalising agent may be homogenously distributed throughout the cellulose material. In such a functionalised cellulose material, at least one functionalising agent may be localised to the interior of the cellulose material.

In such a functionalised cellulose material, at least one functionalising agent may be distributed up to a depth of greater than or equal to 50% of the total cellulose material.

The functionalised cellulose material may be in the form of a pellicle. The functionalised cellulose material may comprise bacterially produced nanocellulose. A functionalised bacterially produced nanocellulose material as described above may be used as a membrane for filtration.

In a third aspect the invention may also provide a process for controlling nanocellulose production levels in a microorganism, the process comprising the steps of: (i) providing a microorganism capable of producing nanocellulose, (ii) providing culture conditions that enable the production of nanocellulose by the microorganism, (iii) providing the microorganism with an expression construct that encodes a regulatory factor, wherein production of the regulatory factor leads to decreased or increased nanocellulose production by the organism, and wherein the production of the regulatory factor is under the control of at least one inducible or repressible promoter operably linked thereto, (v) decreasing, increasing, or blocking nanocellulose production in the microorganism with one or more agents that cause induction or repression of the aforementioned promoter.

In a fourth aspect the invention may also provide a process for preparing a functionalised cellulose material, the process comprising the steps of: (i) providing a first microorganism capable of producing cellulose, (ii) providing culture conditions that enable the production of cellulose by the first microorganism, (iii) providing a second microorganism that is capable of surviving in culture conditions that enable the production of cellulose by the first microorganism, (iv) expressing at least one functionalising agent within the second microorganism, wherein the expression is under the control of at least one inducible or repressible promoter operably linked thereto, and controlling expression of the at least one functionalising agent within the second microorganism such that the at least one functionalising agent is produced, (v) producing cellulose by the first microorganism with concurrent production of the at least one functionalising agent by the second microorganism, and (vi) producing a composite cellulose material that has been functionalised with the at least one functionalising agent.

In a fifth aspect the invention may also provide a process for preparing a functionalised cellulose material, the process comprising the steps of: (i) providing a first microorganism capable of producing cellulose, (ii) providing culture conditions that enable the production of cellulose by the first microorganism, (iii) expressing at least a first functionalising agent within the first microorganism, wherein the expression is under the control of at least one inducible or repressible promoter operably linked thereto, and controlling expression of the first functionalising agent within the microorganism such that the first functionalising agent is produced, (iv) providing a second microorganism that is capable of surviving in culture conditions that enable the production of cellulose by the first microorganism, (v) expressing at least a second functionalising agent within the second microorganism, wherein the expression of the second functionalising agent is under the control of at least one inducible or repressible promoter operably linked thereto, (vi) controlling expression of the second functionalising agent within the second microorganism such that the second functionalising agent is produced, (vii) producing cellulose by the first microorganism with concurrent production of the at least first and second functionalising agents by the first and second microorganisms, and (viii) producing a composite cellulose material that has been functionalised.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 depicts a K. rhaeticus synthetic biology toolkit, (a) Overview of the toolkit contents, (b) Constitutive promoter strengths and (c) AHL and ATc inducible construct expression strengths in K. rhaeticus iGEM, as measured by total mRFP1 fluorescence per cell (fluorescence at 630nm divided by OD600). (d) Total mRFP1 fluorescence expressed from pLuxOI construct, when K. rhaeticus cells were induced with AHL inside cellulose pellicle (induced in pellicle), compared to uninduced or wild-type (WT) cells, (e) Induction in pellicle results in visible mRFP1 production compared to uninduced cells (indicated with the white arrow, also see Figure 2), and (f) results in granular fluorescence due to localization within cells. N=3 for all experiments, error bars=SD. For (e and f), images were cropped and contrast was adjusted to improve clarity. See Figure 3 for detailed overview of constructs and Methods for details of characterization assays. Figure 2 shows mRFP1 production by induced K. rhaeticus cells. Induction in pellicle results in visible mRFP1 production compared to uninduced cells. Images were taken 48 h after induction with AHL. Images were cropped and contrast adjusted to improve clarity.

Figure 3 depicts an overview of ATc and AHL-inducible constructs and experimental setup for measuring promoter strength (a) Design of constructs - see legend for descriptions of symbols and different elements used (b) Overview of experimental set-up. To measure mRFP1 production rate and the promoter inducible range, constructs were transformed into K. rhaeticus, cultured in the presence of high concentrations of cellulase (to prevent formation of cellulose fibrils that inhibit measurement). Constructs were then induced with ATc or AHL and mRFP1 production measured subsequently on a plate reader. See Supplementary Methods for details of experimental design.

Figure 4 shows a characterization of Komagataeibacter rhaeticus iGEM. (a) Morphology of a typical cellulose pellicle produced by K. rhaeticus iGEM. (b) Cellulose productivity of K. rhaeticus iGEM and G. hansenii ATCC 53582 on different growth media, shown as pellicle dry weight after 10-day incubation in 20 mL liquid HS media, (c-d) Growth and production of a cellulose pellicle (denoted by arrow) by K. rhaeticus iGEM in nitrogen-free LGI medium, (e) Comparison of K. rhaeticus and E. coli survival after incubation for 5, 30 or 90 minutes with toxic chemicals (0.1 M HCI, 0.1 M NaOH, 70% EtOH, 10% bleach). Survival is defined as fraction of survived cells compared to PBS-treated cells, (f) Scanning electron micrographs of K. rhaeticus iGEM encased in bacterially produced nanocellulose, taken after 8 days of growth at 6000x magnification. N=3 biological replicates for all experiments, error bars=SD. Statistical significance determined for (b) with 2-way ANOVA and Bonferroni's multiple comparisons test and for (d) 1 -way ANOVA with Tukey's multiple comparisons tests. For (a) and (c), images were taken 9 days post-inoculation. Images were cropped and contrast was adjusted to improve clarity, without affecting details.

Figure 5 shows engineering of patterned and functionalized cellulose materials on macroscale. (a) Spatial patterning. Inset shows the pellicle imaged in white light, (b) Temporal patterning. Cells were induced with AHL daily at different times through pellicle growth (0 days only, or starting at 0, 2, or 4 days post-inoculation), and imaged 9 days post-inoculation, with overview (i), white-light (ii) and fluorescence (iii) images of the pellicles and pellicle cross-sections shown, (c) Overview of 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. For (a) and (b iii), computationally determined and averaged brightness (Grey Value) along the pellicle cross-section is shown correspondingly to the right of the image. Images were cropped and contrast was adjusted to improve clarity for all images.

Figure 6 depicts mRFP1 functionalization with genetic engineering vs addition of protein extracts. Granular fluorescence in strains expressing mRFP1 , compared to smooth fluorescence in cellulose functionalized with extracted mRFP1 , indicates that mRFP1 is localizing within cells, and may require active transport for efficient release. Corresponding bright-field images are added below for comparison.

Figure 7 depicts K. rhaeticus iGEM and E. coli Turbo streaked or plated from glycerol stocks or overnight culture onto semi solid LGI-agar medium. K. rhaeticus iGEM growth can be seen as white, faint colonies with a clearly distinguishable morphology, whereas no colonies were present on E. coli Turbo plates (noted with dashed lines). Note that due to being transparent, the colonies are difficult to distinguish when imaged, however are readily apparent upon visual inspection due to their morphologies and effects on surrounding agar. In experiment 1 , plates were streaked from glycerol stocks. To rule out accidental nitrogen contamination, fresh media were prepared and the experiment repeated (experiment 2), where plates were streaked from glycerol stocks and plated (100 μΙ) from overnight seed culture. Images are taken 12 days post-inoculation. Contrast was edited without affecting important details in all images to improved clarity. Figure 8 shows cellulose produced by K. rhaeticus iGEM when grown in liquid nitrogen-free LGI medium. K. rhaeticus iGEM produces a visible white pellicle. Images ta ken 9 days post-inoculation and cellulose pellicles are indicated with arrows. Cultures were inoculated with overnight seed culture to OD600 = 0.003. Inoculum volume did not exceed 80 μΙ_ to avoid excessive nitrogen carry- over from the inoculum media.

Figure 9 shows scanning electron microscopy images of K. rhaeticus iGEM encased in bacterially produced nanocellulose. Figures were taken after 8 days of growth, a t 6000x magnification, 20kV (left) and 2000x magnification, 20kV (right).

Figure 10 depicts K. rhaeticus iGEM genome, (a) Overview of K. rhaeticus iGEM genome. For the chromosome, consecutive rings show (from outside in): (1) read coverage, (2) genes on forward and (3) reverse strands, (4) acs operons involved in cellulose synthesis, (5) GC% and (6) GC skew, (b) Phylogenetic relationships and (c) amino acid sequence identity of acs cellulose synthase operons. Phylogeny and sequence identity indicate that acs2 and acs4 operons are most closely related. Amino acid sequences were aligned and percent identity calculated using MUSCLE (54) and the tree was generated using the Neighbour-Joining method. The tree is drawn to scale, with Bootstrap values from 1000 replicates shown next to the branches. All positions containing gaps were eliminated from analysis.

Figure 1 1 shows an sRNA construct (J-sRNA-331 Bb) for control of cellulose production, (a) Overview of the sRNA silencing construct. Constitutively produced LuxR binds to pLux in the presence of AHL and upregulates production of E. coli Hfq and an sRNA targeting the UGPase mRNA. The 5' end of the sRNA contains a 24 bp sequence complementary to UGPase mRNA, and binds to it in the presence of E. coli Hfq, leading to silencing of the UGPase gene, (b) Cellulose production of induced and uninduced cultures shown as cellulose dry weight measured 40 h post- inoculation. "No cellulose" = empty weighing boats, "pSEVA331 Bb 100 nM AHL" and "pSEVA331 Bb" = iGEM strain with empty pSEVA331 Bb vector with and without 100 nM AHL, respectively. Full induction with 100 nM and 500 nm AHL results in complete suppression of cellulose synthesis (adjusted p<0.0001 both "Uninduced" vs "100 nM AHL" and "500 nM AHL"). Addition of AHL itself does not decrease cellulose productivity (adjusted p>0.999 for "pSEVA331 Bb" vs "pSEVA331 Bb with 100nM AHL") and uninduced cells are not different from negative controls (adjusted p=0.12 for "Uninduced" vs "pSEVA331 Bb 100nM AHL"). (c) OD600 of cultures 3 h post-inoculation. 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 (b) pSEVA331 -Bb and pSEVA331 -Bb with 100 nM AHL), Statistical significance determined with 1 -way ANOVA and Tukey's multiple comparison tests for (b) and (c). Figure 12 depicts plasmid vectors capable of replication in K. rhaeticus iGEM (a) pSEVA321 -Bb, pSEVA331 -Bb, pSEVA351 and (b) pBla-Vhb-122 transformed into K. rhaeticus and tested for propagation in these cells using colony PCR. Lanes 1 -3: colony PCR replicates from transformed K. rhaeticus, 4: Positive control (pure plasmid DNA), 5: colony PCR of untransformed K. rhaeticus 6: negative control (no DNA). (c) pBAVI K transformed into K. rhaeticus, extracted by plasmid purification and confirmed by PCR amplification. Lanes 1 -5: extracted plasmid DNA replicates, 6: positive control (pure plasmid DNA), 7: negative control (no DNA). Ladder - NEB 2log Ladder. Images were cropped to remove empty lanes for improved clarity. Figure 13 shows constitutive promoter average strengths in K. rhaeticus iGEM and E. coli, normalized against J23104. Although all promoters are functional, their relative strengths differ between K. rhaeticus and E. coli. For K. rhaeticus, data is shown as grey bars, with standard deviation of N=3 biological replicates, characterized in liquid HS-medium containing cellulase, measured 3 h post-inoculation. Relative promoter strengths in E. coli are superimposed as black stripes, and taken from existing online data (Anderson J. 2006, Constitutive promoter family. Available at: http://parts.igem.Org/Part:BBa_J23100) where promoter strengths were also characterized using mRFP1 .

Figure 14 depicts an overview of the insert of plasmid J-sRNA-331 Bb. The annotated sequence of J-s RNA-331 Bb is uploaded as part BBa_K1321328 to Registry of Standard Biological Parts (24) along with all other sequences used in this study. See Supplementary Table S4 for accessions of all parts.

Figure 15 shows sRNA construct (J-sRNA-331 Bb) for control of cellulose production. Growth curves of induced and uninduced cultures, measured as OD600 in HS-cellulase medium. No difference in growth curves indicates that expression of the s RNA construct doesn't reduce growth rate, and therefore that the reduction in cellulose production is specifically caused by the sRNA action, not toxicity or burden. Error bars=SD, N=3 biological replicates. See Methods for experimental details.

Figure 16 shows graphs of a characterization of binding strength of four different cellulose binding domains (CBDs) to cellulose. Error bars = SD, N=3 technical replicates. *=p<0.05, **=p<0.01 , ***=p<0.001 , ****=p<0.0001 , ns = not significantly different from GFP. Statistical significance was determined by one-way ANOVA, followed by Dunnet's multiple comparisons tests with adjusted p- values.

Figure 17 shows bacterially produced nanocellulose functionalized with extracted CBDci pA-sfGFP fusion protein to create sfGFP-cellulose garments, trans-illuminated with blue light (a) or taken as a white light image (b). Figure 18 is a graph that depicts that the membrane of one embodiment slots modularly into existing filtration systems improving their contaminant targeting abilities.

Figure 19 is a graph that shows performance of a water filter functionalized with heavy-metal binding phytochelatin-dCBD fusion proteins.

Figure 20 is a plasmid map showing an expression construct produced for AHL inducible expression of the P. syringae WssFGHI operon in K. rhaeticus. The 4 genes are believed to form a complex that enzymatically acetylates bacterial cellulose fibres and this, in turn, can cause a morphological change in the bacterial cellulose material.

Figure 21 shows bright field microscopy pictures of bacterial cellulose pellicles from liquid cultures. The pellicles show a morphological difference between an unmodified cellulose pellicle (a) and the pellicle produced by a modified strain expressing the WssFGHI operon (b). The operon produces a protein complex that enzymatically modifies the cellulose producing a textured or "wrinkly" phenotype.

DETAILED DESCRIPTION OF THE INVENTION Unless otherwise indicated, the practice of the present invention employs conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA technology, and chemical methods, which are within the capabilities of a person of ordinary skill in the art. Such techniques are also explained in the literature, for example, M.R. Green, J. Sambrook, 2012, Molecular Cloning: A Laboratory Manual, Fourth Edition, Books 1 -3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel, F. M. et al. (1995 and periodic supplements; Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York, N. Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; J. M. Polak and James O'D. McGee, 1990, In Situ Hybridisation: Principles and Practice, Oxford University Press; M. J. Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach, IRL Press; and D. M. J. Lilley and J. E. Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press. Each of these general texts is herein incorporated by reference.

Prior to setting forth the invention, a number of definitions are provided that will assist in the understanding of the invention. All references cited herein are incorporated by reference in their entirety. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, the term 'comprising' means any of the recited elements are necessarily included and other elements may optionally be included as well. 'Consisting essentially of means any recited elements are necessarily included, elements that would materially affect the basic and novel characteristics of the listed elements are excluded, and other elements may optionally be included. 'Consisting of means that all elements other than those listed are excluded. Embodiments defined by each of these terms are within the scope of this invention.

The term 'expression construct' is used to denote a DNA molecule that is either linear or circular, into which another DNA sequence fragment of appropriate size can be integrated. Such DNA fragment(s) can include additional segments that provide for transcription of a gene encoded by the DNA sequence fragment. The additional segments can include and are not limited to: promoters, transcription terminators, enhancers, internal ribosome entry sites, untranslated regions, polyadenylation signals, selectable markers, origins of replication and such like. Expression constructs may include expression vectors and are often derived from plasmids, cosmids, viral vectors and yeast artificial chromosomes; vectors are often recombinant molecules containing DNA sequences from several sources. Genes comprised within expression vectors of the present invention may encode proteins, polypeptides, and functional RNAs (such as microRNAs, siRNAs and ribozymes) as products. In specific embodiments of the present invention the gene comprised within the expression construct may encode a regulatory factor, such as a transcription factor or an siRNA, that is capable of regulating expression of endogenous nanocellulose production of the host microorganism.

The term 'isolated', when applied to a polynucleotide sequence, denotes that the sequence has been removed from its natural organism of origin and is, thus, free of extraneous or unwanted coding or regulatory sequences. The isolated sequence is suitable for use in recombinant DNA processes and within genetically engineered protein synthesis systems. Such isolated sequences include cDNAs and genomic clones. The isolated sequences may be limited to a protein encoding sequence only, or can also include 5' and 3' regulatory sequences such as promoters and transcriptional terminators.

The term 'isolated', when applied to a polypeptide is a polypeptide that has been removed from its natural organism of origin. It is preferred that the isolated polypeptide is substantially free of other polypeptides native to the proteome of the originating organism. It is most preferred that the isolated polypeptide be in a form that is at least 95% pure, more preferably greater than 99% pure. In the present context, the term 'isolated' is intended to include the same polypeptide in alternative physical forms whether it is in the native form, denatured form, dimeric/multimeric, glycosylated, crystallised, or in derivatized forms.

The term 'operably linked', when applied to DNA sequences, for example in an expression construct, indicates that the sequences are arranged so that they function cooperatively in order to achieve their intended purposes, i.e. in a typical vector a promoter sequence allows for initiation of transcription that proceeds through one or more linked coding sequences as far as the termination sequence.

A 'polynucleotide' is a single or double stranded covalently-linked sequence of nucleotides in which the 3' and 5' ends on each nucleotide are joined by phosphodiester bonds. The polynucleotide may be made up of deoxyribonucleotide bases or ribonucleotide bases. Polynucleotides include DNA and RNA, and may be manufactured synthetically in vitro or isolated from natural sources. Sizes of polynucleotides are typically expressed as the number of base pairs (bp) for double stranded polynucleotides, or in the case of single stranded polynucleotides as the number of nucleotides (nt). One thousand bp or nt equal a kilobase (kb). Polynucleotides of less than around 40 nucleotides in length are typically called Oligonucleotides'.

The term 'nucleic acid' as used herein, is a single or double stranded covalently-linked sequence of nucleotides in which the 3' and 5' ends on each nucleotide are joined by phosphodiester bonds. The polynucleotide may be made up of deoxyribonucleotide bases or ribonucleotide bases. Nucleic acids may include DNA and RNA, and may be manufactured synthetically in vitro or isolated from natural sources. Nucleic acids may further include modified DNA or RNA, for example DNA or RNA that has been methylated, or RNA that has been subject to post-translational modification, for example 5'-capping with 7-methylguanosine, 3'-processing such as cleavage and polyadenylation, and splicing. Nucleic acids may also include synthetic nucleic acids (XNA), such as hexitol nucleic acid (HNA), cyclohexene nucleic acid (CeNA), threose nucleic acid (TNA), glycerol nucleic acid (GNA), locked nucleic acid (LNA) and peptide nucleic acid (PNA). Sizes of nucleic acids, also referred to herein as "polynucleotides" are typically expressed as the number of base pairs (bp) for double stranded polynucleotides, or in the case of single stranded polynucleotides as the number of nucleotides (nt). One thousand bp or nt equal a kilobase (kb). Polynucleotides of less than around l OOnucleotides in length are typically called "oligonucleotides" and may comprise primers for use in manipulation of DNA such as via polymerase chain reaction (PCR).

A 'polypeptide' is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or in vitro by synthetic means. Polypeptide of less than around 12 amino acid residues in length is typically referred to as a 'peptide'. The term 'polypeptide' as used herein denotes the product of a naturally occurring polypeptide, precursor form or proprotein. Polypeptides also undergo maturation or post-translational modification processes that may include, but are not limited to: glycosylation, proteolytic cleavage, lipidization, signal peptide cleavage, propeptide cleavage, phosphorylation, and such like. A 'protein' is a macromolecule comprising one or more polypeptide chains.

The term 'promoter' as used herein denotes a site on DNA to which RNA polymerase will bind and initiate transcription. Promoters are commonly, but not always, located in the 5' non-coding regions of genes. The terms 'inducible promoter' and 'repressive promoter' are used to describe a promoter whose activity is controlled, typically activated, up-regulated or 'induced'; or decreased, down-regulated or 'repressed' by the presence or absence of biotic or abiotic factors respectively. The activity of such promoters can be regulated by either chemical or environmental factors. Chemically-regulated promoters include promoters whose transcriptional activity is regulated by the presence or absence of one or more regulatory factors that may be selected from: alcohol, tetracycline, steroids, metals, small molecules, metabolites and other compounds. Chemically-regulated promoters are well- known by the skilled person and are for example described in Terpe (2006), Appl. Microbiol. Biotechnol. 72:21 1 -222. Non-limiting examples of chemically-regulated inducible promoters that can be used in cellulose producing microorganisms may include the isopropyl β-D-l - thiogalactopyranoside (IPTG) - dependent lac promoter, the anhydrotetracycline-dependent tet promoter or the L- arabinose-dependent araBAD promoter. Other non-limiting examples of inducible promoters include the lac promoter, the /acUV5 promoter, the tac promoter, the trc promoter, the T5 promoter, the T7 promoter, the l-lac promoter, the araBAD promoter, the rha promoter or the tet promoter.

Physically-regulated promoters include promoters whose transcriptional activity is regulated by the environment to which the host organism exposed, such as the presence or absence of light, low or high temperatures, or changes in pH to name but a few. Physically-regulated promoters are well- known to the skilled person and are for example described in Terpe (2006), Appl. Microbiol. Biotechnol. 72:21 1 -222, Milias- Argeitis et al. (201 1 ), Nat. Biotechnol. 29:1 1 14-1 1 16 and Levskaya et al. (2005), Nature 438:441 -442. Examples of physically-regulated promoters include the temperature- dependent pL promoter, the light-responsive Phy/PIF system in yeast, or the fusion of the phytochrome Cph1 from Synechocystis PCC6803 to the E. coli histidine kinase EnvZ.

The term 'constitutive promoter' is used to describe a promoter that is in a permanent state of activity allowing for gene expression in the absence of any activating biotic or abiotic regulatory factors.

The term 'pellicle' as used herein refers to a piece of microorganism, typically bacterially, produced nanocellulose, that has not been pulverized or otherwise chemically or physically degraded to yield reconstituted cellulose, and that thus retains its high tensile stiffness, tensile strength, water-to- cellulose ratio, and other properties characteristic of unmodified bacterially produced nanocellulose. A pellicle of bacterially produced nanocellulose according to the present invention can provide a three-dimensional scaffold which can be functionalised accordingly. The pellicle may comprise a pellet, a disk or even a sheet of bacterially produced nanocellulose.

The terms 'bacterial cellulose', 'microbial cellulose', 'nanocellulose', 'bacterially produced cellulose' and 'bacterially produced nanocellulose' as used herein are equivalent and refer to cellulose produced by bacteria or microorganisms, such as species from the genera of Gluconacetobacter, Acetobacter, Komagataeibacter and others, that is characterised by high tensile strength, high tensile stiffness, high chemical purity, biocompatibility and high water-to-cellulose ratio as described in detail in (7). Suitably such bacterial nanocellulose will be substantially pure of associated molecules typically present in plant-derived cellulose such as lignin.

The term 'functionalisation' as used herein, is intended to refer to the addition of a factor that has a specified or pre-determined functionality beyond that already present in native unmodified bacterially produced nanocellulose to a cellulose matrix. As mentioned previously, functionalisation factors may be added after the cellulose matrix has been formed, such as to a pellicle or later to a derivative cellulose containing product. However, it is an advantage of one embodiment of the present invention that functionality can be added at any phase during the synthesis of the cellulose matrix, thereby allowing for both temporal and spatial control of functionalisation of the cellulose matrix.

The term 'functionalising agents' as used herein refer to polypeptides, proteins, RNA, DNA or small molecules that can be used to modify the chemical properties (such as biodegradability in the presence of cellulases, proteases or other proteins; adsorption; chelation; anti-microbial activity; or catalysis of chemical reactions) or physical properties (such as tensile strength; tensile stiffness; colour; fluorescence; hydrophobicity; hyrophilicity; and water content). Functionalizing agents may be introduced into bacterially produced nanocellulose either by production by genetically engineered microorganisms or via addition in their purified form to bacterially produced nanocellulose. Since bacterially produced nanocellulose is characterised by, for example, a nanoporous structure and a high number or free hydroxyl groups there exist multiple routes to matrix functionalisation according to the present invention. In one embodiment, functionalisation is achieved by co- expression of a functionalising factor at the same time as the host organism is synthesising nanocellulose. Optionally the functionalising factor may remain within the cells of the host organism which are subsequently embedded within the matrix of the pellicle thereby facilitating matrix functionalisation by virtue of straightforward localisation within the nanocellulose scaffold. In alternative embodiments it may be advantageous to effect direct linkage of the functionalising agent to the matrix via covalent bonding or via non-covalent interactions. Covalent bonding may suitably occur via chemical reaction with at least one free hydroxyl group within the cellulose molecule. Non- covalent interactions can be mediated via moieties capable of adsorption to the cellulose matrix such as via cellulose binding domains (CBDs; including CBD_CLOS and CBD_CEX), antibodies - including antigen binding fragments thereof such as scFv and Fab' units - other antigen binding polypeptides such as aptamers or MHC Class II proteins. It will be appreciated that modification of functionalising agents in order to render them compatible for adsorption to the cellulose matrix may be achieved via a variety of methods, including biosynthetic methods that may be incorporated into a genetically engineered host organism. By way of non-limiting example where functionalising agents comprise polypeptides, these polypeptides may be modified by processes, such as post-translational processing (e.g., phosphorylation, acylation, etc.), or by chemical modification techniques. Modifications may occur anywhere in the polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide functionalising agent. In addition a given polypeptide may have a plurality of modifications. Modifications suitably include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of a phosphatidylinositol, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, cross-linking cyclization, disulfide bond formation, demethylation, formylation, gamma-carboxylation, GPI anchor formation, hydroxylation, iodination, methylation, myristolyation, oxidation, pegylation, glycosylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, and transfer-RNA mediated addition of amino acids to protein such as arginylation. See, e.g., Creighton, T.E., Proteins - Structure and Molecular Properties 2nd Ed., W.H. Freeman and Company, New York (1993); Posttranslational Covalent Modification of Proteins, B.C. Johnson, Ed., Academic Press, New York, pp. 1 -12 (1983).

It is also possible, within specific embodiments of the invention, for functionalising agents to be expressed within the cellulose matrix that chemically modify the cellulose matrix itself. Hence, covalent modification by one or more functionalising agents may occur where any of the functionalising agents comprises an enzymatic activity that works on bacterial cellulose. By way of non-limiting example, the functionalising agent may comprise an enzyme activity that functions as a kinase (for example E. coli glucokinase GIK), transaminase, transacetylase (for example the Pseudomonas syringae WssFGHI complex) or glycosyltransferase. The reaction products of such activities would include, for instance, phosphocellulose, amino cellulose esters, and cellulose acetates.

For example, a bacterial hexokinase enzyme sequence with an added peptide secretion tag may be cloned and expressed within the cellulose producing organism. The hexokinase enzyme may then be produced and secreted by the organism into the surrounded media. With supplementation of the media to include adenosine triphosphate (ATP), the hexokinase enzyme may then phosphorylate the newly synthesized cellulose matrix also within the media to produce phosphocellulose in a single biological process. In a further embodiment of the invention bacterial cellulose is acetylated. Bacterial cellulose represents a promising material for use as an adsorbent with high surface area and chemistry that is amenable to simple chemical modification. However, a key limitation for use of cellulose based adsorbents is susceptibility to biological degradation. Acetylation of the cellulose fibrils, even at low degrees of substitution, protects them from degradation. Genetic operons believed to produce protein complexes that enzymatically acetylate bacterial cellulose have been identified in two bacterial cellulose producing species Pseudomonas species, P. synringae, and P. fluorescens (17, 18). In particular, P. synringae strain DC300 has the shortest operon capable of acetylating cellulose, the four gene WssFGHI operon; the 4 genes are believed to form a complex that enzymatically acetylates the bacterial cellulose fibres and this causes a morphological change. These protein complexes had never previously been expressed outside of their original host. Heterologous expression of multi gene operons in high cellulose productivity Acetobacter species was not previously possible due to their burden and large size. The present invention utilises a compact plasmid expression system with inducible promoters to make this possible (detailed in examples 4 and 5 below). Acetylation of the bacterial cellulose in the natural host has been shown to give a rough textured, ruffled or "wrinkly" phenotype to the biofilms whereas bacterial cellulose produced without the expression of the acetylation complex appears superficially smooth or planar (18). Functional expression of the WssFGHI complex in a heterologous cellulose producing host also replicates a similar morphological phenotype. In this way, chemical modification of the cellulose matrix can effect morphological and structural changes to the cellulose that can alter the fundamental properties of the material. By way of example, it is shown that acetylation of the cellulose leads to a textured or wrinkled appearance that may increase the available surface area of the material, which may be useful for certain applications. In alternative embodiments of the invention a combination of functionalisation during synthesis of the matrix together with post hoc functionalisation may be employed. In this embodiment of the invention additional chemical modification of the cellulose matrix may be utilised by generating aldehyde groups through mild periodic acid oxidation followed by biotinylation of the carbonyls. In this way avidin/streptavidin labelled functionalising agents may be incorporated into the matrix.

The term 'composite' as used herein refers to a material that comprises a nanocellulose matrix that has been functionalised, and therefore comprises at least one functionalising agent. Hence, the combination of nanocellulose matrix and functionalising agent together forms a composite material. The composite material may be comprised within a larger un-functionalised nanocellulose material in certain embodiments of the invention.

The term 'scaffold' as used herein refers to a three dimensional porous or matrix-like structure comprising a nanocellulose composite material that provides a surface suitable for adherence of biomaterials, including cells. A scaffold may further provide mechanical stability and support as well as providing an environment that facilitates proliferation. In accordance with an embodiment of the invention the scaffold may be entirely functionalised or only functionalised in discrete regions.

In one embodiment of the present invention a process is provided in which bacterially produced nanocellulose can be functionalised such that the functionality is controllable spatially and/or temporally in order to produce novel composite biomaterials.

Filtration/Adsorption Substrates and Membranes The present invention, therefore, provides uses related to functionalised bacterially produced nanocellulose, for example, membranes including the functionalised bacterially produced nanocellulose material. Such membranes have many uses, including filtration, adsorption and water treatment. In particular, such membranes may be used to filter a liquid to remove micro-pollutants, such as heavy metals, environmental pollutants, toxins or oestrogen-disrupting hormones. In addition, such membranes could be used for remediation of high value products, such as gold or heavy metals, in waste streams or in the environment. Alternatively, the membranes can be used for specific capture of high value products in the biotechnology industry, such as pharmaceutical products, biological products or recapture of catalytic compounds for reuse. In such embodiments of the invention the cellulose matrix may be functionalised with one or more agents having a specific target binding activity, such as an antibody (or a fragment thereof), an antibody mimetic compound (e.g. affilins, affibodies, affimers and Kunitz domains), an aptamer or any other selective-binding polypeptide.

The bacterially produced nanocellulose of the present invention can also be used for kidney or liver dialysis applications, removing toxins from the blood or other body fluids either ex vivo or in vitro.

In one embodiment of the invention, sensor function could be incorporated via a scaffold attached to, or adsorbed onto the nanocellulose material, for example, to detect nucleic acids, viruses, micropollutants or high value products existing in environment or waste streams. Sensors may include one or more biological pores having a trans-membrane protein structure defining a channel or hole that allows the translocation of molecules and ions from one side of a membrane to the other. The translocation of ionic species through the pore may be driven by an electrical potential difference applied to either side of the pore. Typically such a biological pores are referred to as "nanopores", in which the minimum diameter of the channel through which molecules or ions pass is in the order of nanometres (10^~9 metres). Nanopore sensors may be embedded within lipid membrane containing structures or cells within the composite biomaterials of the present invention. Furthermore, the bacterially produced nanocellulose composite material of the present invention may be formed into a bead, or otherwise pelletized, shredded or flaked to form smaller particles. These particulate forms of nanocellulose composite material can be immersed in large vessels containing wastewater to be treated. The particulate material will have high surface contact to the water due to the high surface area to volume ratio of the nanocellulosic material, and the adsorbing agent within the composite removes the micropollutant of interest. The benefit of these particulate materials is that gravity feed pressure can drive the process and so that it is possible to treat the water (or other fluids) in batches or even continuously. Surface functionalised materials may be provided in the shape of rods, particles, fibres and other forms, where the functionalised element brings new functionality to the product that the existing nanocellulose material would not have on its own.

Particulate forms of nanocellulose composite material can be manufactured in bulk and may be substituted like-for-like with current particulate purification materials, such as activated carbon, within existing fluid purification systems. A significant advantage of this approach is that re- engineering of existing fluid purification systems to accommodate the materials of the invention is not required. Another embodiment provides for use as functionalised microbeads for blood treatment as a polishing step to existing blood dialysis to specifically remove micropollutants expressed when patients have certain diseases. The micropollutants could include nucleic acids, ammonia and others. This would not necessarily be replacing existing blood dialysis, but provide a solution for some of the microtoxins that existing dialysis cannot tackle.

In a further specific embodiment of the invention, there is provided a disposable cartridge element that comprises the functionalised composite materials of the invention either in unitary or bead form. The cartridge may be configured to be compatible with existing fluid filtration and purification systems.

Medical and Cosmetic Dressings

The present invention can also find use in wound dressing, including dressings with moisture retaining properties, with anti-inflammation or pro-healing ligands attached as functionalising agents that can serve to improve the body's own immune and healing response. Other embodiments include patches that release, for example, anti-acne compounds, anti-microbial, anti-biotic, antineoplastic or other medications in a time-controlled release dosage.

The composite materials of the present invention may also be used in cosmetic products, for example face masks, compresses, or other forms of applique including those with dermal smoothing or acne deterring properties, such as antibacterial compounds, or with high moisture retention and delivery capacity. Those with sensitive skin could benefit from the purity and biocompatibility of the composite materials of the invention. In one embodiment of the invention, a nanocellulose material composite is comprised within a facemask which is intended to address the allergy and problem skin market by infusing high-grade nutrient and minerals in the one-step or two step production process. In one example the high-grade nutrient comprises alpha-tocopherol (vitamin E). Facemasks can also be grown in contour (thicker at some parts and thinner in others) and infused with differing levels of other compounds. This would allow customisation to the face according to the differing moisture and nutrition needs of different parts of the face, and would be achieved by the genetic stop/start control over the cellulose production and composite formation within the host organism. Finally, problem skin market may include those affected by scarring (medical and beauty market). With the composite production capacity, it is possible to cater shape and incorporate enzymes or proteins that can be embedded to improve skin elasticity, delay scar formation and improve circulation to a specific treatment zone or area.

Advanced Biomaterials and Scaffolds

Due to cellulose biocompatibility, the bacterially produced nanocellulose of the present invention is an appropriate material to be used as a medical extracellular matrix scaffold for stem cell organ or new tissue production. Accordingly, it may find use in regenerative medicine where factors are often required to regenerate organ tissue correctly

The bacterially produced nanocellulose of the present invention may also find use in encapsulating a vitamin or nutrient, for example, in food and drink products. The presently described technology would allow the nutrients / compounds to be embedded in the material, meaning there would be slower release and better absorption in the body. There is also the capacity for being a carrier material of microbiome, i.e. faecal transplant, to transport gut bacteria to patients that lack a proper microbiome, which may be useful in treatment of chronic inflammatory conditions such as IBS or even in obesity.

The bacterially produced nanocellulose of the present invention may be used in immunoprecipitation columns or membranes (western blot or HPLC columns).

Since the material of the present invention is shows high levels of tensile strength, it also finds utility in construction or personal protection.

Bioprocess control of bacterially produced nanocellulose production

Genetically unmodified microorganisms produce cellulose constitutively, regardless of environmental conditions, which can hinder bioprocess of bacterial nanocellulose production. Constitutive cellulose production can also lead to emergence of mutant cells incapable of cellulose production with a culture of microorganisms, and thus lead to a decrease in the culture's cellulose productivity over time (19). The present invention provides development of a genetic toolkit that enables inducible or repressible control over production of nanocellulose material in a cellulose- producing microorganism, and can be used to control nanocellulose production by microorganisms to improve nanocellulose production bioprocess. For example, cellulose production can be decreased or increased depending on production capabilities, and can be shut off during periods when it is necessary to expand a culture of microorganisms without cellulose production, to avoid the appearance of cellulose-nonproducing mutants. Inducible or repressible control over nanocellulose production by microorganisms may also be used to increase the quality (for example spatial homogeneity) of produced cellulose by fine-tuning the level of nanocellulose production by microorgansims.

Production of nanocellulose-based sensors

The present invention provides development of a genetic toolkit that enables functionalization of bacterially produced nanocellulose with agents that are sensitive to environmental chemical or physical stimuli and report this as a detectable output of colour, pH, light, or other means. Such cellulose sensors can be used to detect environmental toxins, heavy metals, pathogens (such as bacteria and viruses), specific DNA or RNA, high value compounds, or other chemicial or physical signals of interest, and thus provide low-cost and environmentally friendly sensors. Alternatively, the genetic toolkit enables engineering of microorganisms that remain encapsulated within the nanocellulose and can similarly detect and report the presence of any applied chemical or physical stimuli.

In accordance with an embodiment of the invention, there is provided a process for applying a desired functionality to a bacterial nanocellulose matrix during its synthesis by expressing an agent that determines or contributes to the desired functionality within the host organism that is synthesising the matrix. The expression of the agent may occur throughout the synthesis of the matrix thereby allowing a relatively homogenous distribution of the agent throughout the matrix. Alternatively, the expression of the agent may be placed under a degree of regulatory operable control allowing for temporal and spatial localisation of the agent. In this way, complex functionalities can be applied to a nanocellulose material during is manufacture, including but not limited to lamellar functionalities, or even focussed 'hotspots' of functionality within an otherwise unfunctionalised matrix. The ability to localise the functionalising agent provides hitherto unimagined benefits, including - for example - the capability to concentrate adsorbed compounds and molecules to particular regions within a nanocellulose material when acting as a filter.

In an embodiment of the invention a K. rhaeticus strain is engineered to express a chimaeric fusion protein under the operable control of an inducible promoter. The expression construct may comprise a plurality of genetic elements that allow for inducible expression of the chimaeric product. The construct may be in the form of a plasmid, cosmid, artificial chromosome or inserted heterologously into the genome of the host organism. In one embodiment the chimaeric fusion protein comprises at least a first element that acts as a cellular export signal, a second element that comprises a cellulose binding activity, and at least a third element that exhibits a desired functionality (e.g. ligand binding function or enzymatic activity). Chimaeric gene expression is induced at defined time points during cellulose matrix biosynthesis in order to provide a final composite biomaterial product in which the desired functionality is located within discrete zones. In an alternative embodiment, zonal control of chimaeric gene expression is obtained by applying the appropriate inducing signal to particular regions of the growing cellulose matrix during biosynthesis.

The invention is further illustrated by the following non-limiting examples.

EXAMPLES

Example 1 - Isolation, characterization and culturing of K. rhaeticus iGEM

K. rhaeticus iGEM was isolated from a Kombucha symbiotic colony of bacteria and yeast (SCOBY) of Czech origin (Happy Kombucha, Eastbourne, UK) by streaking homogenized SCOBY material on HS-agarose (Table 1), verifying cell morphology under light microscope, and re-streaking isolated colonies on HS-agarose twice. Two percent (w/v) glucose was used in HS unless stated otherwise. Glycerol stocks were prepared by culturing the iGEM strain statically in HS medium for 6 days, followed by addition of 0.2% (v/v) cellulase (7. reesei cellulase, cat. no. C2730; Sigma, St. Louis, USA), incubation at 230 rpm shaking, 30°C for 1 day, addition of glycerol to 25% (v/v) and storage at -80°C.

Table 1 - Media used in the study. For cellulose production, seed cultures of K. rhaeticus iGEM were inoculated from glycerol stocks and grown statically at 30°C in Kombucha tea, HS or LGI medium (see Table 1). When grown with shaking without cellulose production, cultures were grown in HS-cellulase (0.4% v/v) at 30°C, 230 rpm shaking at 45° tube angle. Unless otherwise stated, 50 ml_ Corning tubes (cat. no. CLS430829 SIGMA; Sigma) with 5-20 ml_ of media were used for culturing. When grown on HS agar, plates were incubated inverted at 30°C. For culturing of K. rhaeticus transformed with plasmids encoding kanamycin or chloramphenicol resistance genes, kanamycin was added to 500 μg/mL for HS-agar and 50-100 μg/mL for liquid HS, and chloramphenicol added to 340 μg/mL for HS-agar and 34-68 μg/mL for liquid HS.

Cellulose productivity on different media was measured by culturing in 20 ml_ of HS-glucose (2% w/v), HS-sucrose (2% w/v), HS without a carbon source (negative control) and Kombucha tea in 50 mL Corning tubes at 30°C for 10 days, with loose caps for increased air diffusion, and kept at 4°C until measurement of cellulose weight.

Productivity of K. rhaeticus iGEM was reported in comparison to productivity of the high-producing G. hansenii ATCC 53582 instead of maximal cellulose yield per volume of media, as maximal total productivity is highly dependent on specific culturing conditions, and may not be a good measure of genetically determined production capabilities. To test cellulose productivity on nitrogen-free LGI medium, 80 μΙ_ of OD600=1 E. coli or K. rhaeticus iGEM seed culture was inoculated into 25 mL LGI medium in 50 mL glass tubes (cat. no. 31 19-0050; Thermo Fischer, Waltham, USA), and imaged 9 days post-inoculation.

For scanning electron micrographs, cellulose was gold-coated under vacuum and imaged at 2000- 6000x magnification, 20kV. A 1 cm x 1 cm square was cut from a pellicle formed after 8 days of growth in HS media at 30 °C. 1 1 was soaked in 40 mL of FAA

fixative (50% ethanol, 5% acetic acid, 3.7% formalin) in a 9 mm petri dish container for 14 hours and dehydrated by soaking for 10 minutes at a time in a graded series of water-ethanol solutions from 50% ethanol to 100% ethanol, increasing 10% (v/v) each step. It was then washed twice in absolute ethanol, immersed in 40 mL of hexamethyldisilazane (cat. no. 440191 ; Sigma) for 5 minutes and air dried for 15 minutes. The top surface of the sample was then coated with gold under vacuum in preparation for SEM. SEM images were taken under vacuum using a JEOL JSM- 5610LV SEM with magnifications of 2000x-6000x, a 1 20 kV. Results

Gluconacetobacter hansenii ATCC 53582 (one of the highest reported cellulose producing strains (20), recently reclassified as Komagataeibacter hansenii ATCC 53582) was evaluated (21), and a new strain isolated from Kombucha tea as potential new synthetic biology hosts (Figure 4(a)). The latter strain (hereafter called 'iGEM') was chosen for further work, as preliminary experiments showed that it can be transformed more readily with plasmid DNA than G. hansenii ATCC 53582. Furthermore, the iGEM strain produced more cellulose than G. hansenii ATCC 53582 on sucrose in small-scale tests. As shown in Figure 4(b) 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). Hence, the iGem strain produced cellulose at high yields on low cost, low-nitrogen Kombucha tea medium (. Surprisingly, it could also grow on the defined nitrogen-free LGI medium (Figure 4(c), (d), Figure 7, Figure 8). Figure 4(c) shows K. rhaeticus iGEM grows significantly compared to negative controls G. hansenii and E. coli (adjusted p=0.026 and p=0.01 1 respectively), while G. hansenii and E. coli do not differ (adjusted p=0.742). As several species of Acetobacteraceae, notably Gluconacetobacter diazotrophicus have been confirmed to fix atmospheric nitrogen (22, 23), this suggested possible nitrogen fixation (see below). Finally, as cellulose has been reported to increase resistance towards UV and environmental stresses in closely related species (2), it was tested whether cellulose could also confer resistance towards chemical stresses, which may occur in unrefined feedstocks or during industrial production. The susceptibility of the iGEM strain to 70% ethanol, 10% bleach, 0.1 M NaOH and 0.1 M HCI was tested (Figure 4(e)), and found that when encased in cellulose it is highly tolerant to chemical stressors, being over 1000-fold more resistant than E. coli to all treatments). Finally, scanning electron microscopy of a cellulose pellicle confirmed that as with other closely related species, cells of K. rhaeticus iGEM are rod-like, approximately 2μηι in length, and are heavily encased in cellulose during normal growth (Figure 4(f), Figure 9).

To determine the genetic basis of high cellulose productivity and to provide background information for genetic engineering of the iGEM strain, its genome was sequenced to 400x coverage, and assembled the genome using the genome of Gluconacetobacter xylinus NBRC 3288 (24) as the reference genome for scaffolding (ENA project ID PRJEB10933). Sequencing showed that genome totals 3.87 Mbp with a GC% of 62.7 and contains a predicted 3572 genes, with an N50 of 3.16 Mbp. The genome is divided between a chromosome of 3.16 Mbp, at least two plasmids - pKRi01 (238 kbp) and pKRi02 (3 kbp), and 37 unplaced contigs (in total 460 kbp) which may be part of the chromosome or additional plasmids, and could not be confidently assigned due to being flanked by repetitive sequences (Figure 10(a)). The iGEM genome totals 3.87 Mbp with a GC% of 62.7, and contains a predicted 3505 protein coding genes, 3 rRNAs, 52 tRNAs and 13 other non-coding RNAs. The genome consists of a chromosome of approximately 3.16 Mbp and at least two plasmids - pKRi01 (238 kbp) and pKRi02 (3 kbp). The chromosome contains 2899 predicted genes with 63% GC content and contains 4 copies of acs (cellulose synthase) operons. Additionally the genome contains 37 scaffolds (totalling 460 kbp) that could not be confidently placed due to repetitive sequences. These scaffolds may be part of the chromosome, plasmids, or may belong to additional plasmids (closely related species G. xylinus NBRC 3288 and K. xylinus E25 contain 5 and 7 plasmids respectively). The genome sequence revealed several interesting aspects about the biology of K. rhaeticus iGEM. Firstly, a 16s rRNA phylogeny suggests the iGEM strain to be a new strain of Komagataeibacter rhaeticus (Figure 1 1), rather than Gluconacetobacter xylinus which is normally thought to be associated with Kombucha tea. Furthermore, the sequence shows the presence of 4 acs cellulose synthesis operons on the chromosome, sharing 40-65% amino acid identity (Figure 10(a), (b), (c)). Up to 3 acs operons have been reported in other bacterially produced nanocellulose producing species (in G. xylinus ATCC 23769 and G. hansenii ATCC 53582 (25, 26)), indicating that the high cellulose synthase copy number may be a possible contributor to the high cellulose productivity observed here (Figure 4(b). These operons also differ in structure. The acsl operon contains separate acsA and acsB genes, while they are fused in the other operons, and the only genomic copy of acsD is found in acsl . Operon acs4 uniquely contains only acsAB genes, and phylogenetic analysis indicates that acs4 is most closely related to the acs2 operon (Figure 10(b)), and possibly arose via duplication and subsequent translocation. From the genes flanking acs operons, cmcAX, ccpAX, bglxA, bcsX and bcsY have been previously shown to contribute to cellulose production in closely related species (20, 27, 28). Two other, standalone copies of bglxA were found from the genome (genomic position 517401 - 519440 and 3029825 - 3032221), and also identified genes close to acs2 that are associated with extracellular matrix formation (kpsC, kpsS and rfaB) and may play a role in cellulose productivity (29, 30). Finally, to determine whether the iGEM strain can fix atmospheric nitrogen similarly to G. diazotrophicus, its genome was searched for genes associated with nitrogen fixation. 5 genes (ntrB, C, X, Y and nifU - SI Appendix Table S1) were located associated with nitrogen fixation in Acetobacteraceae (31), however interestingly the genes homologous to nifHDK, which form the main nitrogenase subunits in G. diazotrophicus, were not found.

Example 2 - Chemical tolerance assays

For both E. coli and K. rhaeticus, cells were treated with 0.5 mL of 0.1 M NaOH, 0.1 M HCI, 70% ethanol, 10% bleach or PBS for 5, 30 or 90 minutes. Treatments were then plated; colonies photographed in white light and counted to determine the fraction of surviving cells compared to PBS-treated cells.

For E. coli Turbo, liquid LB was inoculated from glycerol stocks and grown to mid-log phase (OD600 = 0.4-0.6). Cells were normalized to 1 mL of OD600 = 0.5, centrifuged at 20,000 g for 2 minutes, and supernatant removed. Cells were treated by adding 0.5 mL of 0.1 M NaOH, 0.1 M HCI, 70% ethanol, 10% bleach or PBS, 2 s vortexing and incubation for 5, 30 or 90 minutes. Cells were then washed twice by centrifugation and resuspension with LB, incubated in 1 mL LB at 37 °C, 230 rpm shaking for 1 .5 h, plated in LB-agar plates in 10x serial dilutions and incubated 14 h. Plates were imaged with BioRad Gel Doc transilluminator with Epi white light (180 ms exposure), colonies counted using Quantity One (BioRad - Hertfordshire, UK) at sensitivity 10, and results manually corrected. For K. rhaeticus iGEM, 10 mL HS was inoculated from glycerol stocks, grown statically at 30 °C and harvested 6 days later. Pellicles were cut into 4 identical pieces with a sterile razor and treated with 10 mL of O.1 M NaOH, 0.1 M HCI , 70% ethanol, 10% bleach or PBS for 5, 30 or 90 minutes, followed by 2x washing in 10 mL HS for 10 mi n. After washing, pieces were placed in 10 mL HS-2% (v/v) cellulase, incubated for 3 hours a t 230 rpm, 30 °C, plated on HS-agar in 20x serial dilutions, and incubated for 48 hours a t 30 °C. Colonies were counted similarly to E. coli.

Example 3 - Genome sequencing, assembly, bioinformatics and statistics

K. rhaeticus iGEM genome was sequenced with an lllumina MiSeq (lllumina) using 250 bp paired- end reads, to a coverage of approximately 400x. Reads were then downsampled to 10Ox coverage, assembled using the BugBuilder pipeline (32), quality controlled and annotated using Prokka (33). All statistical tests were performed with Prism 6 (GraphPad Software, Inc., La Jolla, USA). gDNA for sequencing was extracted with Qiagen Blood and Tissue kit (cat. no. 69504; Qiagen - Venlo, Netherlands) and gDNA sequencing library prepared with Nextera DNA Library Preparation Kit (cat. no. FC-121 -1031 ; lllumina - San Diego, USA) according to the manufacturer's protocol. Library was sequenced with an lllumina MiSeq (lllumina) using 250 bp paired-end reads, to a coverage of approximately 400x. Reads were then downsampled to approximately 100x coverage and assembled using the BugBuilder pipeline (32), using Sickle (34) for read trimming (with read areas of quality score below 20 trimmed), Spades (35) for assembly and SIS (36) for scaffolding, with G. xylinus NBRC 3288 (37) as the reference genome. Resulting scaffolds were then further scaffolded using SSPACE (38) with the complete set of reads at default settings. This was followed by quality control of scaffolds, by aligning scaffolds using Mummerplot (39) and WebACT (40) and checking for mis-assemblies using Quast (41), using NBRC 3288 as reference genome. Mis- assemblies were then manually corrected, scaffolds manually linked, and edited genome checked again using Quast. Gapfiller (42) was then used to fill gaps within scaffolds. Origin of replication was located using DoriC (43) and scaffolds manually reorganized to position the origin at the beginning of the genome. The genome was then annotated using Prokka (33), all cellulose synthesis related genes were manually checked by BLASTP and BLASTN against the non- redundant database (44) and re-annotated as necessary in Artemis genome browser (45). Nitrogen fixation and cellulose synthesis genes were searched from the genome using BLAST+ (46) by converting the finished assembly and raw reads to BLAST databases and subjecting them to BLASTN or TBLASTX searches with genes of interest. 16s rRNA phylogeny was created by generating a multiple sequence alignment with MUSCLE (47) and a Neighbour-Joining tree using MEGA6 package (48) at default settings. Reads were mapped onto the genome using BWA (49) and genome was visualized using Circleator (50).

Example 4 - Engineering of constitutive promoters, inducible promoters, CBD-fusions and sRNA construct pSEVA331 Bb and pSEVA321 Bb were constructed from pSEVA331 and pSEVA321 respectively via substituting the native polylinker with BioBrick prefix and suffix. This was done by PCR mutagenesis with Q5 polymerase (cat. no. M0491 S; NEB, Hertz, UK), primers i75, i76 (Table 2), digestion with Spel (cat. no. R3133S; NEB) and subsequent re-ligation. Constitutive promoter- mRFP1 constructs (BBa_J23100 - Bba_J231 17 by iGEM 2006 Berkeley) were received from the iGEM Registry of Standard Biological Parts (51) and subcloned into pSEVA331 Bb. ATc inducible constructs were kindly provided by Dr Francesca Ceroni at Imperial College, and AHL inducible constructs BBa_J09855 (Jon Badalamenti, iGEM 2005) and BBa_F2620 (iGEM 2004 MIT) were received from Registry of Standard Biological Parts. Inducible constructs were then cloned into J23100-mRFP1 -pSEVA331 Bb, replacing the constitutive J23100 promoter (thus controlling downstream mRFP1 expression) to create pTet01 , pTet02, pLux01 and pLux02.

Table 2 - Primers (a) and PCR thermocycler protocols (b) used in the study. For i75 and i76, the doubly underlined section shows areas annealing to pSEVA331 or pSEVA321 , singly underlined sections show areas of BioBrick prefix and suffix, and the dashed underlined section shows Spel restriction site.

CBDCLOS (a monoclonal antibody CBD) and CBD_CEX (an exoglucanase derived CBD) were received as BBa_K8631 1 1 and BBa_K863101 (iGEM 2012 Bielefeld) from the Registry of Standard Biological Parts and CBDcipA and dCBD were synthesized as a GeneStrings (Life Technologies, Carlsbad, USA). CBDs were then fused to sfGFP (BBa_l746909, iGEM 2007 Cambridge) and cloned into an expression vector (BBa_J04500, Kristen DeCelle iGEM 2005) downstream of pLacl promoter. sRNA construct was also synthesized as a GeneString (Life Technologies) based on descriptions of Na. et al. (52) and subcloned downstream of the pLux promoter in pLuxOI , replacing mRFP1 . All constructs were first transformed into E. coli Turbo and colonies screened using colony PCR with GoTaq Green (cat. no. M7122; Promega, Madison USA) and primers i53, i54 (see Table 2). Plasmid DNA from positive colonies was extracted with Qiagen QIAprep Spin Miniprep Kit (cat. no. 27104; Qiagen, Venlo, Netherlands), DNA sequenced (Source Biosciences, Nottingham, UK), and correct sequences transformed into K. rhaeticus iGEM. DNA sequences of all constructs created in this work are accessible in the Registry of Standard Biological Parts (53) (see Table 3 for accession numbers). As many of the constructs characterized in K. rhaeticus iGEM are widely used and accessible through the Registry of Standard Biological Parts, no constructs were codon optimized, in order to allow them to be used in K. rhaeticus iGEM without modification by the user.

Accession

Plasmid backbones Description

number

pSEVA321 Bb Broad host-range plasmid in Biobrick format BBa_K1321301 pSEVA331 Bb Broad host-range plasmid in Biobrick format BBa_K1321300

Constitutive

promoters

J23100-mRFP1 - mRFP1 expressed from constitutive promoter J23100

BBa_K1321302 331 Bb in pSEVA331 Bb

J23101 -mRFP1 - mRFP1 expressed from constitutive promoter J23101

BBa_K1321310 331 Bb in pSEVA331 Bb

J23103-mRFP1 - mRFP1 expressed from constitutive promoter J23103

BBa_K1321312 331 Bb in pSEVA331 Bb

J23104-mRFP1 - mRFP1 expressed from constitutive promoter J23104

BBa_K1321313 331 Bb in pSEVA331 Bb

J23105-mRFP1 - mRFP1 expressed from constitutive promoter J23105

BBa_K1321314 331 Bb in pSEVA331 Bb

J23106-mRFP1 - mRFP1 expressed from constitutive promoter J23106

BBa_K1321315 331 Bb in pSEVA331 Bb

J231 10-mRFP1 - mRFP1 expressed from constitutive promoter J231 10

BBa_K1321316 331 Bb in pSEVA331 Bb

J231 12-mRFP1 - mRFP1 expressed from constitutive promoter J231 12

BBa_K1321317 331 Bb in pSEVA331 Bb

J231 14-mRFP1 - mRFP1 expressed from constitutive promoter J231 14

BBa_K1321318 331 Bb in pSEVA331 Bb

J231 17-mRFP1 - mRFP1 expressed from constitutive promoter J231 17

BBa_K1321318 331 Bb in pSEVA331 Bb

Table 3 - Descriptions and accession numbers of Registry of Standard Biological Parts for all sequences created in this study. Example 5 - Characterization of constitutive promoters, inducible promoters, CBD-fusions and sRNA construct

For characterization of constitutive promoters and inducible promoters without cellulose formation, 1 -5 mL of liquid HS-cellulase (8-10% v/v) containing 34 μg/mL chloramphenicol in 50 mL Corning tubes were inoculated from seed cultures to OD600=0.02, grown at 30°C, 230 rpm for 3 h and 200 μί of culture was then pipetted into 96-well plates (Corning Costar, New York USA). Inducer was added to inducible cultures to 1 μg/mL for ATc and 1 μΜ for AHL. High concentrations of cellulase were used to remove any interference by cellulose on spectroscopy measurements. Plates were covered with Breathe-Easy membrane (cat. no. Z380059; Sigma), and OD600 and mRFP1 intensity (excitation 590 nm, emission 630 nm) were measured every 15 minutes using Synergy HT microplate reader (BioTek, Winooski USA) at 29°C, high shaking speed. Promoter strengths in Figure 1 (b) and (c) were assayed in liquid HS-cellulase, to remove formation of cellulose fibrils interfering with measurements For characterization of inducible promoters in pellicle form (Figure 1 (d),(f) and Figure 2), K. rhaeticus iGEM containing pLuxOI was inoculation from glycerol stocks into HS with 34 μg/mL chloramphenicol and grown statically for 8 days at 30°C. Induction caused a significant increase in fluorescence compared to non-induced or wild-type cells (p<0.001 for both induced vs non-induced, and induced vs wild-type, determined with 1 -way ANOVA and Tukey's post-hoc tests). Cells were grown in HS (without cellulase) and fluorescence quantified by fluorescence microscopy image analysis. For cultures induced during growth, AHL was added to 1 μΜ before inoculation, and for cultures induced in pellicle, AHL was added to 1 μΜ 6-days post inoculation. Pellicles were washed with PBS 8 days after inoculation, microscopy samples prepared with a sterile razor, and fluorescence quantified using fluorescence microscopy (Nikon Eclipse Ti; Nikon, Tokyo, Japan) at mCherry pre-set (590nm emission, 0.2 s exposure, 675V EM gain, 150x magnification). A single layer cellulose sheet was located on the sample and fluorescence intensity determined as intensity/exposure time for quantitative measurements. For white-light images (Figure 1 (e)), 250 mL beakers (cat. no. CLS1000250; Sigma) containing 100 mL of HS media were inoculated with 5 mL of OD600=1 seed culture, the beakers covered with Breathe-Easy membrane, and beakers incubated statically at 30°C. For the induced pellicle, 1000 μί of 100 mM AHL was pipetted daily 4 days after inoculation along the edges for the pellicle, and pellicle imaged 9 days post-inoculation.

Bacterially produced nanocellulose was first purified and homogenized. 4 different cellulose binding domains fused to sfGFP were then added onto cellulose (for CBDclos, both N-terminal and C- terminal sfGFP fusions were created) and allowed to bind to cellulose overnight. 3 washes with either dH20, 70% EtOH, PBS or 5% BSA were carried out, and remaining sfGFP fluorescence was quantified on a plate reader. Data is presented in Figure 16 as percentage of initial fluorescence before washes. Negative control - GFPmut3 without a CBD fusion, which was used due to availability. Although GFPmut3 differs slightly from sfGFP, it does not bias the results here, as binding strength is calculated as a fraction of fluorescence remained after wash, not the total fluorescence, and later experiments with sfGFP showed similar results. CBDcipA showed good performance in all washes and was chosen for further experiments. Note that the purpose of this assay was to determine the relative strengths of CBDs, and whether CBDs and sfGFP retained their functionalities when fused - for detailed characterization of suitable CBDs, see (54) for CBDclos, (55) for CBDCex, (56) for dCBD and (57) for CBDci pA. For characterization of CBD binding strengths, 200 μί of CBD-sfGFP fusion proteins extracted from E. coli were added to a 96-well plate containing homogenized bacterially produced nanocellulose, incubated overnight at 4°C and washed thrice with treatment (dH₂0, PBS, 5% BSA or 70% EtOH). GFP fluorescence was measured on a 96-well plate reader (Synergy HT, Biotek). For characterization of sRNA construct, HS with 34 μg/mL chloramphenicol was inoculated to OD600=0.04 with K. rhaeticus iGEM containing plasmid J09855-SRNA-331 Bb, and induced with 10-500 nM AHL. 40 h after inoculation, pellicles were washed, dried at 60°C for 16 h and weighed. For characterization of growth rate, OD600 was measured in (3% v/v) HS-cellulase with 34 μg/mL chloramphenicol using the protocol use for characterization of constitutive promoters.

Example 6 - Engineering of functionalized biomaterials

For spatial patterning, K. rhaeticus containing pLux01 was inoculated into 1 L HS chloramphenicol in 2 L Erlenmeyer flasks, 500 of 100 nM AHL was added to one side of the pellicle 2 days later, and pellicle was imaged 4 days after induction. For temporal patterning, 250 ml_ beakers with 100 ml_ of HS media were inoculated with 5 ml_ of seed culture at OD600=1 , the beakers covered with Breathe-Easy membrane, and incubated statically at 30 °C. After this, 1000 μΙ_ of 100 mM AHL was pipetted along the edges of the pellicle daily with membrane replacement, starting at different days post-inoculation as shown in Figure 5(b), and pellicle imaged 9 days post-inoculation. For functionalization of cellulose with mRFP1 and CBDcipA-sfGFP, proteins were first produced in E. coli, extracted via sonication, and extracts applied to purified wet or dried bacterially produced nanocellulose respectively. For mRFP1 - functionalized cellulose, fluorescence intensity was determined from a single cellulose sheet using fluorescence microscopy. For CBDcipA-sfGFP, extracts were applied to dried bacterially produced nanocellulose with a paintbrush and further dried.

Genetic engineering toolkit for Komagataeibacter

Very few genetic tools, with little characterization data are available for engineering of Acetobacteraceae. The Applicant has therefore developed a complete set of tools for its engineering, consisting of protocols, modular plasmids, promoters, reporter proteins and inducible constructs that enable external control of gene expression (Figure 1 (a)).

Protocols and plasmid backbones - First, the plasmid pBla-Vhb-122 (previously described to replicate in Acetobacteraceae) (58) was used to develop protocols for the preparation of electrocompetent cells, transformation, plasmid purification and genomic DNA extraction of K. rhaeticus iGEM. Using these protocols, 8 plasmids were then assessed for propagation in K. rhaeticus iGEM - pSEVA31 1 , pSEVA321 , pSEVA331 , pSEVA341 , pSEVA351 ; pBAV1 K-T5-sfGFP, pSB1 C3 and pBca1020. From these, pSEVA321 , 331 , 351 ; pBAV1 K-T5-sfGFP and pBla-Vhb-122 showed replication in iGEM (Figure 12) giving a total of 5 different plasmids to act as vectors. pSEVA321 and pSEVA331 were further engineered into pSEVA321 Bb and pSEVA331 Bb, making them compatible with the widely-used BioBrick standard (59), to enable rapid cloning of publically- available DNA parts. pSEVA331 Bb was used for all subsequent studies, due to its likely higher copy number.

Reporter proteins, constitutive and inducible promoters - expression of 7 reporter proteins (mRFP1 , GFPmut3, sfGFP, and chromoproteins tsPurple, aeBlue, gfasPurple and spisPink) was tested, from which mRFP1 , GFPmut3 and sfGFP showed visually detectable expression. 10 promoters were chosen from an open-access collection of synthetic minimal E. coli promoters and using mRFP1 as the reporter, characterized these in K. rhaeticus iGEM (Figure 1 (b) and Table 3, also see Figure 13 for a comparison to promoter strengths in E. coli). Although all promoters are functional, their relative strengths may differ between host organisms. For inducible promoters, 4 constructs were engineered allowing gene expression to be induced externally by anhydrotetracycline (ATc) or N- acyl homoserine lactone (AHL) (see Figure 3 for overview of constructs). From these, the AHL- inducible constructs (pLux01 and pLux02) showed higher induction and lower leakiness than the ATc-induced constructs (pTetOI , pTet02) (Figure 1 (c)), and contrary to our initial expectations, they also gave robust induction of mRFP1 expression when cells were encased in the pellicle, showing visible fluorescence (Figure 1 (d), (e), Figure 2). This is notable as it shows that cells in the pellicle can effectively receive signals from their environment despite their cellulose encasing. As K. rhaeticus is highly resistant to various environmental hazards within cellulose (Figure 4(e)), the ability to receive signals while protected by cellulose makes it a potentially suitable host for applications requiring tolerance to toxic chemicals and long-term survival.

Engineering control over cellulose production

As wild-type species produce cellulose constitutively, a major goal of genetic engineering of Acetobacteraceae has been to achieve control over cellulose production. Constitutive cellulose production complicates genetic engineering techniques and is not always desirable for industrial applications as it is imparts a high metabolic cost, which in well-aerated conditions typically leads to the emergence of cellulose-nonproducing mutants (60). It is therefore desirable to inhibit cellulose production during periods when it is not required, in order to prevent the proliferation of these mutants. Furthermore, fine control over cellulose production levels may allow control over the density of cellulose fibrils, and thus the macroscale properties of cellulose. To achieve controlled cellulose production, a system in which an E. coli Hfq, and an sRNA targeting UGPase mRNA (UDP-glucose pyrophosphorylase) are co-expressed from a plasmid in response to AHL was engineered (plasmid J-sRNA-331 Bb; Figure 1 1 (a), also see Figure 15 for a detailed overview). The sRNA contains a 24 base region complementary to UGPase mRNA and an E. coli Hfq binding region. When expressed, it binds to the target UGPase mRNA and recruits E. coli Hfq, inhibiting UGPase translation. UGPase gene was targeted as it catalyses the production of UDP-glucose critical for cellulose synthesis (61) and is present in single-copy in the genome, allowing knockdown by a single sRNA. This system is highly efficient, as cellulose production was suppressed completely upon full induction and could be fine-tuned using different concentrations of AHL (Figure 1 1 (b)). The observed reduction in cellulose production was not related to toxicity, as growth rate did not decrease compared to wild type levels (Figure 1 1 (c), Figure 16). This system was engineered to be a general platform for targeted knockdowns in Komagataeibacter and other bacterial species, as expression of E. coli Hfq makes it independent from the host Hfq and the broad host range pSEVA331 Bb backbone enables replication in a wide range of species. Furthermore, new sRNAs can be added to the plasmid, and the 24-base sRNA region can be recoded rapidly by site-directed mutagenesis, making the construct easily modifiable for other targets.

Genetic engineering of patterned and functionalized biomaterials

Owing to the finding that K. rhaeticus gene expression can be induced even when inside a cellulose pellicle, the Applicant hypothesized that it may be possible to generate spatially and temporally patterned biomaterials that are controlled by the diffusion of the inducer AHL and timing of exposure to induction during pellicle growth. To test this, growing cellulose pellicles were induced with cells containing the AHL-inducible construct pLuxOI with different concentrations of AHL, at different locations and time points (Figure 5(a), (b)). Cells were induced for mRFP1 expression by addition of 100 nM AHL to one side of a 1 L culture and the pellicle imaged for red fluorescence 3 days post-induction. Note that pellicles induced on day 0 only show low fluorescence due to natural degradation of AHL in the media over time. Both spatial and temporal control was shown to be possible. When a limited amount of inducer was added to one side of the pellicle, cells produced mRFP1 following the diffusion gradient of AHL (Figure 5(a)). Furthermore, as cells are active only in the top layer of the cellulose pellicle (1), when inducer was added at different times mid-way through pellicle growth, only cells at the growing top layer produced mRFP1 , capturing the temporal difference between uninduced cells in the bottom layers and induced cells at the top (Figure 5(b)).

To produce functionalized cellulose materials where the nanocellulose matrix is coated by proteins of interest, two strategies were considered: genetic engineering of K. rhaeticus to produce these proteins in situ, or separate expression of proteins in E. coli which are then purified and applied directly to bacterially produced nanocellulose (Figure 5(c)). While the latter requires a three-step process (protein and cellulose production separately, followed by combining the two), it may be preferred for medical and other applications where very high purity of the material is required, as it would allow defined and purified components to be used for functionalization. To test for the possibility of post-hoc functionalization, mRFP1 was produced in E. coli, extracted and added it to bacterially produced nanocellulose, and compared it by fluorescence microscopy to cellulose produced by K. rhaeticus with in vivo constitutive mRFP1 expression. Smooth fluorescence is seen throughout the pellicle cross-section, compared to granular fluorescence seen in Fig 1 (e) (also see Figure 6). Extracted proteins can diffuse well throughout the pellicle and functionalize the cellulose evenly (Figure 5(d)), while the granular fluorescence exhibited by expression from pellicle-based cells (see Figure 1 (e), also Figure 6 for full-size comparison) indicates that mRFP1 remains largely in the K. rhaeticus cells and would likely require active secretion or lysis of cell membranes to access the extracellular cellulose. To further increase efficiency of functionalization, expression vectors that allow easy fusion of proteins to one of 4 different cellulose binding domains (CBDs) - CBDclos (54), CBDCex (55), dCBD (55) and CBDcipA were engineered (57). CBDs are short peptides that bind tightly to cellulose fibrils, thus increasing protein adhesion to cellulose (62). In these constructs, proteins can be modularly fused to CBDs via restriction enzyme cloning. The cellulose binding strengths of these CBDs were assessed by washing four different E. co//^'-extracted CBD-sfGFP fusion proteins with different solvents (dH20, EtOH, BSA and PBS) and measuring the fluorescence that remained bound. Addition of CBDs to sfGFP gave up to a 5-fold increase in binding to cellulose when compared to GFP alone (Figure 16). Finally, as bacterially produced nanocellulose is a candidate for new textile materials and of high interest to the fashion industry, this approach was used to demonstrate production of functionalized garments. Using the CBDcipA-sfGFP fusion protein extract and dried pellicle material from K. rhaeticus cultures, bacterially produced nanocellulose fashion accessories were created by functionalization of cellulose fibrils with green fluorescent protein (Figure 18), indicating that this approach is scalable to produce macroscale objects. Other functionalising agents may include any of the chromoproteins, for example, aeBlue from the biobrick registry and any adsorbing agent, for example phytochelatin.

According the methodology described in detail above, a phytochelatin-CBD functionalised bacterially produced nanocellulose membrane was synthesised. The resultant membrane was used to filter nickel ions from contaminated water proving that a composite functionalised membrane of the invention is better than raw cellulose. Due to the high tensile strength and small pore size of bacterially produced nanocellulose, which naturally make bacterially produced nanocellulose a good material for water filters, tested bacterial-cellulose functionalized with heavy-metal binding phytochelatin-dCBD fusion protein show unexpectedly good performance as heavy-metal specific water filters. Bacterially produced nanocellulose can successfully be used for water filtration, and when functionalized with heavy-metal binding phytochelatin-dCBD, effectively reduces nickel concentrations from 32000ppm to 1 .5ppm (Fig. 6D) in a single filtration step. Functionalization with phytochelatin-dCBD improved filter performance two-fold, in total reducing nickel concentration over 20000-fold on one filtration (see Figure 19). It is also possible to functionalise with multiple constructs at once, more specifically an adsorbing agent and a detecting agent, to enable the system to adsorb and detect the level of adsorption. Detection may be based on using markers that rely on Forster resonance energy transfer (FRET). This would be advantageous as it would give an indication of the saturation level of the material. Example 7 - Engineered Production of Morphologically Distinct, Acetylated Cellulose

Proteins that chemically modify the cellulose material to change its properties can be expressed from an inducible promoter. Genetic operons believed to produce protein complexes that act as transacetylase enzymes, enzymatically acetylating bacterial cellulose, have been identified in two Pseudomonas species, P. synringae, and P. fluorescens (17, 18). These protein complexes had never previously been expressed outside of their original host organism.

P. synringae strain DC300 has the shortest operon believed to acetylate cellulose, the four gene WssFGHI operon. This operon was amplified from a genomic DNA preparation from P. synringae by PCR. The operon was then cloned by Gibson assembly into the plasmid vector pSEVA331 and placed downstream of the AHL inducible promoter, pLux01 detailed above (see Figure 3a). Figure 20 shows the plasmid map for the construct to express WssFGHI from the AHL inducible pLux01 promoter used to produce acetylated cellulose product. These test plasmids were transformed into K. rhaeticus alongside an empty pSEVA331 control plasmid by electroporation. Single transformant colonies were picked and grown in 5 ml static liquid cultures with 100 nM AHL at 30°C for five days to observe pellicle growth.

Acetylation of the bacterial cellulose in the natural host has been shown to give a textured, ruffled or "wrinkly" phenotype to the biofilms whereas bacterial cellulose produced without the expression of the acetylation complex typically has a smooth or planar appearance (18). This phenotype is seen in the K. rhaeticus strains expressing the acetylation complex (Figure 21 ). The bright field microscopy images of unprocessed pellicles show the clear morphological differences in the pellicle of the transformed strain containing the pSEVA plasmid with the WssFGHI operon expressed from the pLux01 promoter compared to the control strain with the empty pSEVA331 vector. This shows enzymatic modification (acetylation) of the bacterial cellulose is taking place in the test strain during production.

Although particular embodiments of the invention have been disclosed herein in detail, this has been done by way of example and for the purposes of illustration only. The aforementioned embodiments are not intended to be limiting with respect to the scope of the appended claims, which follow. The choice of nucleic acid starting material, the clone of interest, or type of library used is believed to be a routine matter for the person of skill in the art with knowledge of the presently described embodiments. It is contemplated by the inventors that various substitutions, alterations, and modifications may be made to the invention without departing from the spirit and scope of the invention as defined by the claims.

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Claims

WHAT IS CLAIMED IS:

1 . A process for preparing a functionalised cellulose material, the process comprising the steps of:

(i) providing a microorganism capable of producing cellulose;

(ii) providing culture conditions that enable the production of cellulose by the microorganism;

(iii) expressing at least one functionalising agent within the microorganism, wherein the expression is under the control of at least one inducible or repressible promoter operably linked thereto;

(iv) controlling expression of the at least one functionalising agent within the microorganism such that the at least one functionalising agent is produced;

(v) producing cellulose by the microorganism with concurrent production of the at least one functionalising agent; and

(vi) producing a composite cellulose material that has been functionalized with the at least one functionalising agent.

2. The process of Claim 1 , wherein the functionalised cellulose material comprises a cellulose matrix comprising the at least one functionalising agent.

3. The process of Claim 2, wherein the functionalising agent is linked to the cellulose matrix.

4. The process of Claim 3, wherein the functionalising agent is bonded to the cellulose matrix.

5. The process of Claim 4, wherein the functionalising agent is bonded to the cellulose matrix either via a covalent bond or via a hydrogen bond.

6. The process of Claim 3, wherein the functionalising agent is linked to the cellulose matrix via an electrostatic interaction.

7. The process of Claim 3, wherein the functionalising agent comprises a peptide that binds selectively to the cellulose matrix.

8. The process of Claim 2, wherein the functionalising agent is comprised within the cellulose matrix.

9. The process of Claim 8, wherein the functionalising agent is comprised within one or more cells embedded within the cellulose matrix.

10. The process of Claim 1 , wherein the cellulose material is covalently modified by the at least one functionalising agent.

1 1 . The process of Claim 10, wherein the functionalising agent is an enzyme.

12. The process of Claim 1 1 , wherein the enzyme functions as a kinase, transaminase, transacetylase or glycosyltransferase.

13. The process of claim 12, wherein the enzyme functions as a transacetylase and is derived from the Wss operon of Pseudomonas syringae or Pseudomonas fluorescens.

14. The process of any preceding claim, wherein the functionalising agent comprises a polypeptide that is selected from the group consisting of: chelating proteins; fluorescent proteins; catalysing proteins, antimicrobial proteins, anti-infection proteins, probiotic proteins, fertilising proteins, reactive proteins, proteins that sense extracellular chemical or physical signals, pigmentation proteins, molecular binding proteins, proteins that reconfigure the native cellulose matrix, amyloid fibrillary proteins, or any combination thereof.

15. The process of any preceding claim, wherein the functionalising agent comprises a functional nucleic acid, suitably a functional RNA.

16. The process of any preceding claim, wherein the functionalising agent comprises a small molecule compound.

17. A process of controlling cellulose production, the process comprising the steps of:

(i) providing a microorganism capable of producing cellulose;

(ii) providing culture conditions that enable the production of cellulose by the microorganism;

(iii) transforming the microorganism with at least one expression vector , wherein the at least one expression vector comprises at least one gene that encodes a polypeptide or RNA, whose expression leads to decreased or increased cellulose production by the microorganism, wherein the expression is under the control of at least one inducible or repressible promoter operably linked thereto;

(iv) decreasing, increasing, or suppressing cellulose production of the microorganism with at least one agent that causes induction or repression of the aforementioned promoters

18. The process of any preceding claim, wherein the microorganism is selected from the group consisting of: Escherichia coli, Komagataeibacter rhaeticus, Komagataeibacter rhaeticus iGEM, Gluconacetobacter xylinus, Acetobacter, Sarcina ventriculi, Agrobacterium, A. pasteurianus, A. hansenii, Azotobacter, Rhizobium, Pseudomonas, Salmonella, Alcaligenes, fungi and algae.

19. The process of any preceding claim, wherein the at least one inducible promoter system is selected from the group consisting of: anhydrotetracycline (aTc) and N-acyl homoserine lactone (AHL) inducible promoter systems.

20. The process of any preceding claim, wherein the functionalisation of the functionalised cellulose material is controlled temporally.

21 . The process of any preceding claim, wherein the functionalisation of the functionalised cellulose material is controlled spatially.

22. A functionalised bacterial cellulose material prepared according to the method of any one of Claims 1 to 21 .

23. A functionalised bacterial cellulose material, wherein at least one functionalising agent is homogenously distributed throughout the bacterial cellulose material.

24. A functionalised bacterial cellulose material, wherein at least one functionalising agent is localised to the interior of the bacterial cellulose material.

25. A functionalised bacterial cellulose material, wherein at least one functionalising agent is distributed up to a depth of greater than or equal to 50% of the total bacterial cellulose material.

26. The functionalised bacterial cellulose material of Claim 22 to 25, wherein the functionalised bacterial cellulose material is in the form of a pellicle.

27. The functionalised bacterial cellulose material of Claim 22 to 25, wherein the functionalised cellulose material is in particulate form.

28. Use of a functionalised bacterial cellulose material of any of Claims 22 to 27 within a membrane for fluid filtration.

29. The use of claim 27, wherein the fluid filtration is selected from the group consisting of: waste water remediation; bodily fluid filtration; and water purification.

30. A process for preparing a functionalised cellulose material, the process comprising the steps of:

(i) providing a first microorganism capable of producing cellulose;

(ii) providing culture conditions that enable the production of cellulose by the first microorganism;

(iii) providing a second microorganism that is capable of surviving in culture conditions that enable the production of cellulose by the first microorganism; (iv) expressing at least one functionalising agent within the second microorganism, wherein the expression is under the control of at least one inducible or repressible promoter operably linked thereto, and controlling expression of the at least one functionalising agent within the second microorganism such that the at least one functionalising agent is produced;

(v) producing cellulose by the first microorganism with concurrent production of the at least one functionalising agent by the second microorganism; and

(vi) producing a composite cellulose material that has been functionalised with the at least one functionalising agent.

31 . A process for preparing a functionalised cellulose material, the process comprising the steps of:

(i) providing a first microorganism capable of producing cellulose;

(ii) providing culture conditions that enable the production of cellulose by the first microorganism;

(iii) expressing at least a first functionalising agent within the first microorganism, wherein the expression is under the control of at least one inducible or repressible promoter operably linked thereto, and controlling expression of the first functionalising agent within the microorganism such that the first functionalising agent is produced;

(iv) providing a second microorganism that is capable of surviving in culture conditions that enable the production of cellulose by the first microorganism;

(v) expressing at least a second functionalising agent within the second microorganism, wherein the expression of the second functionalising agent is under the control of at least one inducible or repressible promoter operably linked thereto;

(vi) controlling expression of the second functionalising agent within the second microorganism such that the second functionalising agent is produced;

(vii) producing cellulose by the first microorganism with concurrent production of the at least first and second functionalising agents by the first and second microorganisms; and

(viii) producing a composite cellulose material that has been functionalised.

32. The process of Claim 30 or Claim 31 , wherein the cellulose material is covalently modified by any of the functionalising agents.

33. The process of Claim 32, wherein any of the functionalising agents is an enzyme.

34. The process of Claim 33, wherein the enzyme functions as a kinase, transaminase, transacetylase or glycosyltransferase.

35. The process of claim 34, wherein the enzyme functions as a transacetylase and is derived from the Wss operon of Pseudomonas syringae or Pseudomonas fluorescens.