1. Successfully optimized the cultivation conditions for K.xylinus

K.xylinus, unlike engineered strains such as Escherichia coli or Saccharomyces cerevisiae, are not extensively studied in terms of their growth conditions, culture media, and methods for competent bacteria preparation. Therefore, our initial focus was on determining the growth curve of K.xylinus.

We have observed that K.xylinus enter a plateau phase approximately 48 hours after cultivation. Based on the growth curve of K.xylinus, we can roughly determine that they are in the logarithmic growth phase between 15 to 25 hours.This information is valuable in determining the optimal timing for preparing competent K.xylinus, among other applications.

Initially, during cultivation, we noticed that both the growth rate and membrane production efficiency were significantly low. After conducting extensive literature research, we made attempts to adjust the glucose and ethanol concentrations in the HS culture medium. By measuring the dry weight of the BC membrane produced by K.xylinus, we analyzed the impact of these two culture parameters and their interaction. Furthermore, we collaborated with the dry lab to establish a model and delve deeper into determining the optimal culture conditions. Our model revealed that the highest membrane yield was achieved when the glucose concentration ranged between 30g/L and 35g/L, while the ethanol concentration remained within 0% to 0.5%. These findings align with the report by S.A. Hutchens et al.

We provide the following explanation:

The presence of ethanol or glycerol as alternative electron sources has been reported to repress glucose oxidation in K.xylinus. This repression allows for an increased yield of cellulose production, as glucose synthesis competes with glucose oxidation by the membrane-bound glucose dehydrogenase enzyme. Additionally, K.xylinus possess membrane-bound alcohol and aldehyde dehydrogenases, which can oxidize alcohol to acetic acid to generate electrons.

2. Successfully constructed a indigoidine biosynthetic pathway in C. glutamicum

SDS-PAGE showing resolve of Marker,

1: cell lysate of wild type C. glutamicum,

2: cell lysate of IPTG-induced C. glutamicum expressing bpsA,

3: supernatant of 1,

4: supernatant of 2.

Stained using Coomassie Brilliant Blue. bpsA predicted to be 140 kDa

Based on the NFLS_Nanjing 2022 project, we utilized their composite part BBa_K4376003 as a reference and introduced our composite part BBa_K4605010 into C. glutamicum. The successful expression of indigoidine synthetase in C. glutamicum was confirmed through SDS-PAGE analysis. After induction with 1 mM IPTG for 12 hours, we observed a blue-colored bacterial pellet upon centrifugation, as depicted in Figure X. In contrast, when we introduced the empty vector pEKEX2, the fermentation broth appeared yellow, indicating the establishment of a metabolic pathway for indigoidine synthesis in C. glutamicum. Notably, the supernatant obtained after centrifugation in Figure X exhibited a light blue color, suggesting that C. glutamicum secretes a portion of the dye into the culture medium following indigoidine synthesis. Unfortunately, due to limitations, we were unable to perform transmission electron microscopy on the genetically modified C. glutamicum to visualize how the pigment is secreted out. However, this conclusion is supported by the electron microscopy images reported by Mohammad Ghiffary et al.

To further confirm the synthesis of indigoidine, we conducted scans and obtained the maximum absorption peak of the product, which coincided with the reported maximum absorption wavelength of indigoidine at 590nm. This indicates that our genetic modification and metabolic pathway construction in C. glutamicum were successful. We explored various organic solvents for extracting indigoidine and ultimately found that DMSO yielded the best results. By suspending the bacterial cells in DMSO, followed by sonication for cell disruption, we obtained the supernatant, resulting in the production of indigoidine microbial dye, as shown in Figure 5.

(A) A small amount of indigoidine was secreted into the culture medium, with the majority still remaining inside C. glutamicum.

(B) IPTG-induced/pTac-ind C. glutamicum with pEKEX2 (right) and IPTG-induced /pTac-ind C. glutamicum with pEKEX2-bpsA (left)

(C) bsorption spectrum of our product

(D) DMSO extraction of indigoidine

(E) Extraction of indigoidine produced by C. glutamicum using different organic solvents.

3. Successfully obtained a colored BC membrane that binds to liposoluble dyes through co-cultivation

In a 20mL cultivation medium for BC membrane, different concentrations of carmine/lemon yellow dye were added. The mixture was incubated for 6 days, and then the BC membrane was removed and washed several times with water until no more color was observed. A 0.5g portion of the membrane was taken and added to 3mL of water. Then, a 30μL concentration of 3000U/mL cellulase solution was added for membrane degradation, resulting in a suspension. The suspension obtained from the colorless BC after lysis was used as a blank control. The absorbance value at 428nm (the maximum absorption wavelength of lemon yellow) was measured.

In order to validate the binding performance of BC membrane with dyes, we first added different concentrations of carmine and lemon yellow into the cultivation medium of K.xylinus to obtain the relationship between water-soluble dye concentration and OD value. As shown in Figure 4, this method allowed us to obtain uniformly stained BC membrane. However, the results were not as expected, as the color of the BC membrane did not become darker with increasing dye concentration. On the contrary, there was no clear linear relationship observed, and the relationship between the two followed a bell-shaped curve with a peak. This indicates that higher concentrations of water-soluble dyes can exhibit side effects, such as the inhibitory effect on the growth and membrane production of K.xylinus, as observed when adding 10mg/ml of carmine to the cultivation medium. This indicates that even with water-soluble dyes, direct staining can still pose issues with high dye concentrations affecting the growth of K.xylinus.

The effect of indigoidine (DMSO extracting) dyeing is not very good either, uneven and the color is lighter. Therefore, after reviewing the literature and brainstorming, we decided to obtain the target product by co-culture method, that is, to culture indigoidine-expressing and BCM-expressing bacteria together, in order to make the fat-soluble dye dyeing effect better.

Co-culture results of the first step: We conducted shaking cultivation of the genetically modified C. glutamicum and BC membrane-producing K.xylinus as described above. We obtained the production of relatively uniform blue BC particles. To further illustrate the interaction between the BC membrane and C. glutamicum during co-cultivation, scanning electron microscopy (SEM) was employed. The SEM images provide visual evidence of the successful co-cultivation process, demonstrating the binding and integration of BC membranes with C. glutamicum.

These findings support the successful co-cultivation of the modified C. glutamicum and K.xylinus for the production of BC membranes. Microfibers produced by K.xylinus intertwine with each other to form a mesh-like structure, in which the indigoidine-secreting C. glutamicum are encapsulated. Due to the blue color of the genetically modified C. glutamicum, the BC particles that encapsulate them also appear blue. However, this deviates from our initial goal of producing a solid BC membrane that displays a distinct blue color and is applicable in the fashion industry. To make it suitable for production purposes, further processing is required. For instance, we may need to compress the BC particles into a membrane using a high-pressure filtration method.

Co-culture results of the second step: Based on extensive literature research, we have discovered that BC membranes are highly biocompatible scaffold materials used for culturing cells. Initially, we successfully obtained a complete BC membrane by cultivating K.xylinus. Subsequently, we placed the BC membrane on a culture medium containing C. glutamicum and conducted line cultivation, which resulted in the production of indigoidine. As a result, we successfully obtained blue BC membranes with custom-designed patterns. This method, in comparison to the co-culture results of the first step, offers a complete membrane structure and allows for independent creativity based on the producer's ideas. Most importantly, it accomplishes our initial goal of obtaining indigoidine-dyed BC membranes without the need for optimizing the culture medium.

4. Successfully constructed plasmids for expressing chromoproteins or synthesizing indigoidine in BC-producing strains—— K.xylinus

What we are working right now:

The fluorescence per cell would be measured using flow cytometry in K.xylinus strains that expressed EGFP/YFP under the control of three different heterologous promoters J23100, J23104, J23119. We can also use the plasmids we built to see how cellulose wraps up K. xylinus. Most importantly, chromoproteins can be used to characterize promoter strength so that we could add more information to K. xylinus genetic toolkit. We are currently working on expressing chromoproteins in K.xylinus to obtain cellulose membranes that emit fluorescence. This could potentially be used in the fashion industry in the future.

Meanwhile, we are also attempting to reconstruct the metabolic pathway for indigoidine synthesis in K.xylinus. We have referenced the existing knowledge on indigoidine synthesis in other bacteria, such as C. glutamicum. We would also take into consideration the expression results of chromoproteins in K.xylinus as a reference to fine-tune our promoter.

Future work

Once we have finished the construction of the color fiber system, the metabolic pathway for other liposoluble dyes, such as violacein, Tyrian purple, melanin and indigo can also be introduced into K.xylinus. We have conducted research on the synthesis pathways of these dyes (Yang, D. et al. 2021)and found that the process requires relatively few enzymes, making the construction of a metabolic system feasible. This approach would provide a paradigm for effectively addressing pollution issue in fashion industry and bring synthetic biology and its concepts to the public. Moreover, it holds immense potential for future production and application.

References

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