Indigoidine is a common jeans dye. The process of industrial manufacturing and jeans dyeing can cause serious problems such as water pollution and air pollution. In order to alleviate the environmental pollution caused by textile, Nanjing-China 2023 tries to use microbial cultivation technology to produce bacterial cellulose and dyes at the same time, and make the two into new colored textile fibers to replace traditional textile materials. Since K. xylinus can produce cellulose itself, we plan to obtain colored bacterial cellulose directly by cultivating modified K. xylinus. The first pigment our team has chosen to try is indigoidine, and if the experiment is successful and we have enough time, we will expand the pigment library and build a color fiber system. Making K. xylinus a cell factory for colored fibers provides a new paradigm for the textile industry.

Circle 1

Target 1: Obtain indigoidine dye

Why indigoidine: Among the natural pigments, indigoidine is a natural blue pigment encoded by a single module-type nonribosomal peptide synthetase (Panchanawaporn et al.2022). Also, indigoidine has a simple biosynthetic pathway, involving the condensation of two moles of L-glutamine, and is amenable to microbial overproduction. So we chose indigoidine as the first dye for the experiment.

Pre-experiment: , LINKS_China 2021 produced indigo dye and Tharian purple dye from Escherichia coli and used these two dyes to stain BCM. Through their results, we found that the coloring effect of indigoidine on BCM is not very good, which may be related to the fat-soluble properties of these dyes. To verify this idea, we used the water-soluble dyes lemon yellow, carmine (these dyes have also been used by the igem team before, and we verify here to ensure the accuracy and repeatability of the experiment) and the liposoluble dye indigoidine to stain BCM, and the results are as follows. It can be seen from the results that the water-soluble dyes dye clearly and evenly. In contrast, the effect of indigoidine (DMSO extracting) dyeing is not very good, 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.

Design: At the beginning of the project, we plan to achieve the target product through co-cultivation. First we need to get indigoidine dye. Indigoidine synthase catalyzes the condensation of two L-glutamine molecules to form indigoidine. 4′-phosphingolinetransferase (PPTase) is required to activate indigoidine synthase, which is excited by introducing coenzyme A into the peptide carrier domain (PCP) of indigoidine synthase(Hui et al.2021). Therefore, co-expression of indigoidine synthase and PPTase is often required for indigoidine biosynthesis.
As the pigment is biosynthesized via condensation of two L-glutamine residues, Corynebacterium glutamicum (C. glutamicum) was considered as an ideal production host because it carries strong fluxes for the biosynthesis of L-glutamate, a precursor of L-glutamine. C. glutamicum can also heterologously express indigoidine synthase and has the native pcpS gene (which expresses PPTase), which is a good carrier for synthetic dyes (Mohammad R et al.2021).

Build: On the basis of Nanjing-NFLS, we constructed a plasmid system containing bpsA. C. glutamicum can change its metabolism and synthesize indigoidine by expressing this enzyme. This is the most basic part, and we have also optimized the codon of bpsA gene to increase indigoidine production. We will use pEKEX2 plasmid backbone, ligated with bpsA sequences to try to express bpsA in C. glutamicum. In order to control the start and stop of dye synthesis, we added lactose operons in the design of parts, so that the dye synthesis can be induced by lactose. So, we built BBa_K437001, BBa_K4376002 parts and verified the Nanjing_NFLS’s parts BBa_K4376003.

Test: After cultivating the modified C. glutamicum, we obtained the dye that meets the experimental requirements. We then measure the absorbance of the product dye, confirm that the product we have is indigoidine, and characterize the yield.

Learn: Through consulting the information, we have obtained many methods for fixing color and improving the stability of indigoidine. This provides a good knowledge base for our follow-up experiments.

Target 2: Co-culture

Pre-design: Because K. xylinus can produce cellulose itself, it is a relatively intuitive and groundbreaking idea to culture K. xylinus with C. glutamicum. At the beginning, our idea was to create a culture environment suitable for the growth of both bacteria, directly co-culture, so that it can be naturally colored. However, because indigoidine is fat-soluble and denser than water, it is secreted and sinks to the bottom of the test tube. K. xylinus are aerobic bacteria that produce cellulose membranes on the surface of culture medium. In this case, insufficient contact between the two will lead to poor dyeing effect. But shaking cultivation would prevent the cellulose from forming a film, so we came up with the next idea.

Pre-test: Our second model requires a complete cellulose membrane. After conducting extensive literature research and discussions, we learned that bacterial culture medium is crucial for cultivating bacteria and increasing BCM production. Therefore, we performed numerous experiments to adjust the concentrations of glucose and ethanol to determine the optimal cultivation conditions. We collaborated with the team members in the dry lab to establish a model that simulated the interaction between glucose and ethanol, and determined the best cultivation conditions: glucose concentration ranging from 30g/L to 35g/L and ethanol concentration ranging from 0% to 0.5%.

Improved-design: We intend to utilize BCM as a framework to construct a growth scaffold for C. glutamicum (Gilbert et al.2021). Essentially, we will first culture K. xylinus to produce BCM, and then introduce C. glutamicum to the existing BCM. In the culture device designed by us, the components added from top to bottom are inoculated C. glutamicum, BCM, and C. glutamicum medium. This approach effectively addresses the issue of dye contact with BCM and significantly enhances the likelihood of successful coloring. Moreover, it holds immense potential for future production and application. For instance, we can employ advanced techniques to design BCM in various shapes, making it easier to achieve products with diverse colors using our method. This approach offers flexibility to cater to the needs of the masses and pushes the boundaries of synthetic biology to new heights.
During the co-cultivation process, the rotation speed of the shaker played a crucial role. If the rotation speed was too high, the cellulose would turn granular and fail to form a film. Conversely, if the rotation speed was too low, the dyeing could be uneven.

Test: Practice our ideas. The C. glutamicum was inoculated on BCM and the staining results were observed. After dyeing for a period of time, we washed the BCM and observed the surface structure of the film under electron microscope. The mechanism of pigment coloring was studied by electron microscopy.

In addition, we measured the growth curve of K. xylinus to understand the growth status of the bacteria and lay a good foundation for subsequent experiments.

Learn: Through this experiment, we found that the experimental results were affected by a variety of environmental factors, and it was difficult to meet the culture conditions of the two bacteria at the same time. The final staining results did also not match our expectations. So through discussion and exploration, we came up with a novel idea. That is, the bpsA gene is induced to express in K. xylinus, so that it directly produces BCM containing dye.

Cycle 1. Co-cultures

Circle 2

Target 3: Modifying K. xylinus

Design: There were some shortcomings in the co-culture experiment, so we decided to introduce the indigoidine synthesis plasmid into K. xylinus. The strain itself can synthesize glutamine, so we only need to introduce bpsA and pcpS. K. xylinus synthesizes glutamine by utilizing nitrogen and carbon sources in the medium. Glutamine can synthesize indigoidine under the action of bpsA activated by PPTase. This approach reduces bacterial usage, resulting in cost savings and promoting environmentally friendly practices. Moreover, only the growth conditions of single species of bacteria need to be adjusted, and the operation is simpler. Most importantly, this is the first time that dye plasmids have been introduced into K. xylinus, which is a very innovative and challenging idea.

Build: We constructed two plasmids. EGFP(YFP) gene and bpsA-pcpS gene fragments were independently connected to plasmids by homologous recombination. The former was designed to verify whether the plasmid was electrocuted into K. xylinus allowing the results to be visualized. At the same time, we can also use it to see how cellulose wraps up K. xylinus. Most importantly, EGFP can be used to characterize promoter strength. When the plasmid with gene EGFP(YFP) is successfully expressed, we then electrically transfer the plasmid containing bpsA-pcpS into bacteria to produce our target product. We will use a K. xylinus compatible PSB1A2 plasmid backbone, ligated with promoters such as strong promoters (J23100, J23104, etc.), and bpsA-pcpS sequences to try to express bpsA and PPTase in K. xylinus (Hui et al.2021).

Test: Detect fluorescence expression intensity and product expression. Due to time, we only tested whether the plasmid was successfully constructed and introduced.
Improve and rebuild: The first results showed that the plasmid we inserted was not well expressed. After discussion, we reconstructed a plasmid and changed the promoter on the basis of the original plasmid to improve the intensity of gene expression. After reviewing the literature, we selected J23100/J23104/J23119 as the promoter of this experiment. This promoter has been verified to be reliable by many teams.
Further exploration: If the project is successful, we can get different colors of cellulose by introducing different dye plasmids into K. xylinus . Thus, the application range of BCM can be expanded. In addition, we can also use the dyes produced by the previous team, such as spiruloviolet produced by BJU_China 2012, curcumin and lycopene produced by HUST_China 2021, and so on. The genes of pigments they use can be directly loaded into the plasmid we constructed to implement “Rainboweaver”, creating the color fiber system.

Cycle 2. Modifying K. xylinus

Finally, we completed the cycle of modification of K. xylinus. We believe that this transformation will play an important role in the BCM industry, and if we can successfully express colored cellulose, it will greatly reduce the cost of time and money, and also alleviate environmental pollution. We hope that this project can continue to develop and we will continue to study it.

Reference

[1] Panchanawaporn S, Chutrakul C, Jeennor S, Anantayanon J, Rattanaphan N, Laoteng K. Potential of Aspergillus oryzae as a biosynthetic platform for indigoidineidine, a non-ribosomal peptide pigment with antioxidant activity. PLoS One. 2022 Jun 23;17(6):e0270359. doi: 10.1371/journal.pone.0270359. PMID: 35737654; PMCID: PMC9223385.

[2] Hui, Cy., Guo, Y., Li, Lm. et al. indigoidineidine biosynthesis triggered by the heavy metal-responsive transcription regulator: a visual whole-cell biosensor. Appl Microbiol Biotechnol 105, 6087–6102 (2021).

[3] High-Level Production of the Natural Blue Pigment indigoidineidine from Metabolically Engineered Corynebacterium glutamicum for Sustainable Fabric Dyes.

[4] Gilbert C, Tang TC, Ott W, Dorr BA, Shaw WM, Sun GL, Lu TK, Ellis T. Living materials with programmable functionalities grown from engineered microbial co-cultures. Nat Mater. 2021 May;20(5):691-700. doi: 10.1038/s41563-020-00857-5. Epub 2021 Jan 11. PMID: 33432140.