Self-pigmenting textiles grown from cellulose-producing bacteria with engineered tyrosinase expression

Abstract

Environmental concerns are driving interest in postpetroleum synthetic textiles produced from microbial and fungal sources. Bacterial cellulose (BC) is a promising sustainable leather alternative, on account of its material properties, low infrastructure needs and biodegradability. However, for alternative textiles like BC to be fully sustainable, alternative ways to dye textiles need to be developed alongside alternative production methods. To address this, we genetically engineer Komagataeibacter rhaeticus to create a bacterial strain that grows self-pigmenting BC. Melanin biosynthesis in the bacteria from recombinant tyrosinase expression achieves dark black coloration robust to material use. Melanated BC production can be scaled up for the construction of prototype fashion products, and we illustrate the potential of combining engineered self-pigmentation with tools from synthetic biology, through the optogenetic patterning of gene expression in cellulose-producing bacteria. With this study, we demonstrate that combining genetic engineering with current and future methods of textile biofabrication has the potential to create a new class of textiles.

Fig. 1. Eumelanin production from K. rhaeticus tyrosinase expression.

a, Chemical pathway of eumelanin production from l-tyrosine. The first step involves the hydroxylation of l-tyrosine to l-DOPA that is catalyzed by tyrosinase—here acting as a monophenol mono-oxygenase. This step is then followed by the catalysis of l-DOPA to dopaquinone, which is catalyzed by the diphenolase activity of Tyr1. The remaining steps in the pathway occur spontaneously in the presence of oxygen, leading to the generation of eumelanin. b, Genetic construct maps for the following two K. rhaeticus tyrosinase expression strains: K. rhaeticus ptyr1 and K. rhaeticus ctyr1. Both constructs use the same constitutive promoter (pJ23104), RBS (B0034) and terminator (L3S1P00). K. rhaeticus ptyr1 uses a plasmid with a pBBR1 origin of replication and a chloramphenicol resistance cassette. c, A two-step process for eumelanin production from K. rhaeticus grown in shaking conditions. Strains are grown in HS-glucose media, washed and resuspended with PBS to remove spent media before being mixed with melanin development buffer. d, Tyr1-producing strains are assayed for eumelanin production. Eumelanin production per initial cell was determined by measuring OD₄₀₅ over 12 h, divided by the initial OD₆₀₀ of each well at time point 0. Points show the mean of three biological replicates. Error bars are the s.d. of three biological replicates. e, Initial reaction rate per initial cell was determined by measuring the gradient of eumelanin accumulation per initial cell from 50 to 170 min after the start of measurement. Bars show the means of three biological replicates of each strain, while error bars show the s.d. f, Optical microscopy images of K. rhaeticus ptyr1 and K. rhaeticus ctyr1 before (mel−) and after melanin development (mel+). A zoomed-in example of a single cell is shown with a cyan outline. Images shown are representative of four random images taken for each strain and treatment. Source data

Fig. 2. Using tyr1 expressing K. rhaeticus to produce melanated BC.

a, The production process for melanated BC involves two steps. Tyrosinase-expressing K. rhaeticus are grown in static conditions to produce a pellicle. Once grown, this is collected and placed in development buffer and incubated with agitation between 30 °C and 50 °C until the material reaches the desired shade. b, Images show a time lapse of the progression of eumelanin accumulation over 24 h at 30 °C for K. rhaeticus ptyr1 and K. rhaeticus ctyr1 pellicles placed in development buffer. c, Pellicle production can be conducted in standardized containers to produce sheets of BC from which pattern pieces can be cut out and assembled. d, A K. rhaeticus ctyr1 pellicle, grown in a 300 × 200 mm container, after eumelanin development step. e, A finalized wallet prototype, cut and assembled from two pressed and dried melanated BC sheets. f, Pellicle production can also occur in shaped containers, producing BC preshaped to the 2D pattern of the final pattern piece. g, A K. rhaeticus ptyr1 pellicle grown in a shaped container, after eumelanin development. Metal pins seen within the perimeter of the pellicle hold a network of woven thread that becomes integrated into the pellicle during growth. h, A finalized shoe upper prototype produced from a melanated shaped BC sheet with integrated yarn that has been wrapped around a foot-shaped last and placed on a shoe sole.

Fig. 3. Properties of melanated BC.

a, A melanated (mel+) and unmelanated (mel−) swatch, from the same original K. rhaeticus ptyr1 pellicle. These swatches had been dried and used as demonstration pieces. b, SEM of mel+ and mel− K. rhaeticus ptyr1 BC. The top and bottom surfaces pertain to the air-facing and media-facing pellicle surfaces, respectively. Images shown are representative of at least five images per condition. c, The sessile drop method was used to measure the contact angle on K. rhaeticus ptyr1 mel− (beige) and mel+ (black) pellicles. An unpaired t test result gave a value of P < 0.005 and error bars represent s.d. from eight mel− and nine mel+ drop measurements. Representative water drop shapes for mel+ and mel− pellicles are shown above the graph. d, Comparative tensile tests of melanated and unmelanated BC sheets were conducted using BC sheets prepared from halves of the same K. rhaeticus ptyr1 pellicle. Representative images of BC breaks are given for mel− and mel+ BC as well as stress–strain curves of the technical repeats from three biological replicates of mel+ and mel− pellicles. e–g, Tensile strength (e), Young’s modulus (f) and strain at break (g) for mel+ and mel− pellicles—the P values from paired t tests between mel+ and mel− BC were 0.17, 0.92 and 0.85, respectively. Error bars show s.d. from three biological replicates and each biological replicate is the average of three or more technical replicates. Source data

Fig. 4. Functional optogenetics in K. rhaeticus.

a, Proposed procedure to make patterned BC through optogenetics. b, The Opto-T7RNAP system uses a split T7-RNA polymerase, made blue light activatable via fusion with photo-sensitive magnet proteins. c, Genetic arrangements of K. rhaeticus optogenetic strains. Expression of the split T7RNAP genes is induced with arabinose. d, Red fluorescence scan of the top surface of a blue light exposed wet K. rhaeticus pOpto-T7RNAP*(563-F2)-mCherry pellicle (diameter = 150 mm). Graphic on top right shows the image projected onto the pellicle during growth. Pellicle shown is representative of two patterned pellicle repeats. e, The right of the projected image contained a gradated strip, from minimum to maximum light let through. Data show the intensity of red fluorescence seen in the pellicle against this gradated strip. The s.d. of pixel intensity for each horizontal slice is shown in pink. Black dotted line represents the intensity of unexposed pellicle regions. f, Smallest projected mark on the exposed pellicle. g, Characterization of optogenetics constructs with mCherry target gene under differing arabinose percentage (wt/vol) concentration. Bars (blue, exposed and gray, unexposed) show mean increase in red fluorescence after 6 h normalized by OD₆₀₀. Error bars show s.d. of three biological replicates. Fold difference between exposed and unexposed cells is shown above, except in instances of poor growth. h, Comparison between projection video and the resulting wet K. rhaeticus Opto-T7RNAP(563-F1)-tyr1 pellicle after eumelanin development (dimensions = 300 × 170 mm). Rectangles (black and blue) at top of projection video are timed to appear to aid in calculating minimum light exposure time. Densitometry scan and photograph of top surface of pellicle are shown. i, Zoomed-in sections of a densitometry scan of the K. rhaeticus Opto-T7RNAP(563-F1)-tyr1 pellicle. Black triangle points to the sixth rectangle, indicating a 40-h required exposure time. j, Optogenetic construct characterization with tyr1 under differing arabinose induction. The bars (blue, exposed and gray, unexposed) show the mean and s.d. of three biological replicates of initial (0–100 min) reaction rate of eumelanin production measured at OD₄₀₅, normalized to the number of initial cells at OD₆₀₀ at time point 0. Source data

Extended Data Fig. 1. Post-pellicle growth media pH of K. rhaeticus ptyr1.

a, Pellicles produced after 7 days growth at 30 °C in a 24-well deep well plate. Each plate column contains 4 replicated media compositions. Buffered HS-glucose media pH values before growth are noted beneath, as well as additional components required for melanin production. b, pH values of media underneath the pellicle after 7 days growth at 30 °C. Data shown are average and standard deviation of 4 replicates, except for media set to pH 7 with L-tyrosine and Cu, where only 3 replicates produced pellicles.

Extended Data Fig. 2. Characterization of melanin production rates in differing melanin development buffer formulations.

a, Time course experiment of eumelanin accumulation at OD₄₀₅ from K. rhaeticus tyr1 in a range of pH values. Phosphate buffered saline (PBS) set to 7.4 containing 0.5 g/L L-tyrosine and 10 μM CuSO₄ is shown in gray. Points represent the mean while error bars represent the SD of 6 replicates. b, Average initial reaction rates of melanin accumulation from K. rhaeticus tyr1 in buffers of differing pH values. Reaction rates are derived from the gradient of OD₄₀₅ over the first 140 minutes of the experiment. Colors match those in panel a. Bars represent the mean rate while error bars represent the SD of 6 replicates. c,d, Time course experiment of melanin accumulation and initial reaction rates of melanin accumulation from K. rhaeticus WT. Points and bars represent the mean while error bars represent SD of 6 technical replicates. e, Time course experiment of melanin accumulation at OD₄₀₅ from K. rhaeticus tyr1 in differing PBS concentrations. A PBS concentration of 0x corresponds to ddH₂O containing only melanin development substrates (L-tyrosine and CuSO₄) while a PBS concentration of 1× corresponds to 137 mM NaCl, 2.7 mM KCL, 10 mM Na₂HPO₄ and 1.8 mM KH₂PO₄. Melanin development substrates are maintained at 0.5 g/L tyrosine and 10 μM CuSO₄ for all PBS concentrations. Points represent the mean while error bars represent SD of 6 replicates. f, Average initial reaction rates of melanin accumulation from K. rhaeticus tyr1 in differing PBS concentrations. Reaction rates are derived from the gradient of OD₄₀₅ over the first 140 minutes of the experiment. Error bars represent the SD of 6 replicates. g, Average initial reaction rates of melanin accumulation from K. rhaeticus tyr1 in buffers containing different metal (II) ions. Ions present in solution at 20 μM. Error bars represent the SD of 6 replicates. h, Average initial reaction rates of melanin accumulation from K. rhaeticus tyr1 in buffers containing different copper (II) concentration. Error bars represent the SD of 6 replicates.

Extended Data Fig. 3. Assaying cell supernatant for tyrosinase activity.

A 12-hour time course experiment of eumelanin accumulation at OD₄₀₅ from K. rhaeticus tyr1 and K. rhaeticus culture, centrifuged cell pellets and remaining supernatant. Presence or absence of L-tyrosine at 0.5 g/L in each assay sample is shown below each bar. Average initial reaction rates shown as bars were derived from the gradient of OD₄₀₅ from 50–190 minutes after the start of the assay. Error bars represent the standard deviation of 6 replicates.

Extended Data Fig. 4. Timelapse of pellicle melanin production.

a, Timelapse images of K. rhaeticus ptyr1 and ctyr1 pellicles. Pellicles labeled under HS, were incubated in HS media, while those pellicles labeled under Mel+ were incubated in PBS with 0.5 g/L L-tyrosine and 10 μM CuSO₄. b, Graph displays the average brightness value of the pellicle images over 24 hours as melanin production occurs. Brightness value is derived from converting images from the RGB (red, green, blue) color space into the HSB (hue, saturation, brightness) color space, where the brightness value represents the value of a color as a percentage between black and peak luminosity, 0–100% respectively. Points represent the means while error bars represent the standard deviation of brightness values from 3 separate pellicles from K. rhaeticus ctyr1 and 4 separate pellicles from K. rhaeticus ptyr1.

Extended Data Fig. 5. Melanin pigmentation strength and stability during sterilization.

a, Average initial reaction rates of melanin accumulation from K. rhaeticus tyr1 in PBS buffer with 10 μM CuSO₄ with differing concentrations of L-tyrosine. An L-tyrosine concentration of 1 g/L is above the maximum solubility of tyrosine in water at 25 °C—OD₄₀₅ readings at this concentration and above become inaccurate due to suspended undissolved L-tyrosine. Error bars represent the standard deviation of 6 replicates and mean reaction rates shown as bars are derived from the first 140 minutes of the reaction. b, K. rhaeticus ptyr1 pellicle pigmentation after 24 hours in a shaking melanin development bath at 30 °C containing differing L-tyrosine concentration. The top row shows pellicles from K. rhaeticus ptyr1 grown in HS-glucose media without L-tyrosine and CuSO₄ while the bottom row shows pellicles grown in HS-glucose media with L-tyrosine and CuSO₄. c, Images of melanated and unmelanated pellicle cross-sections from K. rhaeticus WT, ptyr1 and ctyr1. Top and side views are shown with backlighting to clarify differences in melanin pigmentation. d, Effects of various sterilization methods on melanin stability in melanated pellicles from K. rhaeticus ptyr1. Pellicles had been bathed in development buffer containing 0.5 g/L L-tyrosine and 10 μM CuSO₄ for 24 hours. Pellicles were exposed to sterilizing conditions for 2 hours, except gel doc UV light, to which the pellicle was exposed to for 20 minutes.

Extended Data Fig. 6. Testing melanin BC color fastness with water spotting test.

The top illustration demonstrates the water spotting process. Photographs of dried melanated BC from both K. rhaeticus strains, with and without glycerol treatment are shown at three time points: before water spotting, immediately after water spotting and 16 hours after water spotting. Images are representative of 3 water spotting tests for each pellicle and treatment.

Extended Data Fig. 7. Additional optogenetic data.

a, Five variants of the opto-T7RNAPs system were integrated into the K. rhaeticus chromosome. Only Opto-T7RNAP*(563-F2) was used to create the plasmid-based arrangement. b, A rig for projecting light onto a growing pellicle. Blue light from LED floodlight passes through an acetate transparency, on which is a printed image. The light that passes through this image is then focused and projected beneath onto the growing pellicle. Overall assembly is 620 mm tall. c, Blue-light response from all tested integrated constructs that were transformed with an mCherry target gene plasmid. Bars (blue = exposed, gray = unexposed) show average increase in red fluorescence after 6 hours normalized by OD₆₀₀. Bars show mean rate while error bars show standard deviation of 3 biological replicates placed on the same plate. Fold difference between exposed and unexposed cells is shown at top. d, Comparison of K. rhaeticus _pOpto-T7RNAP*(563-F2)-mCherry pellicle fluorescence scan and _pOpto-T7RNAP*(563-F2)-tyr1 pellicle photograph. Both pellicles are produced using identical methods. e, Commercial projector-based assembly for patterning blue light on to a growing pellicle. f, Characterization data from tested genome-integrated constructs that were transformed with a tyr1 target gene plasmid. Fold difference is given above the bars, but not shown when rates are 0 or below. Bars show mean rate while error bars show standard deviation of 3 biological replicates placed on the same plate.