Environmental Impact

Constructs

We propose to produce biological pigments that can be used in ink at a smaller environmental impact than current sources. We have identified pigments that can be produced in Escherichia coli that correspond to the four main colors in ink cartridges: black, cyan, magenta, and yellow. They are melanin, indigoidine, anthocyanin, and zeaxanthin, respectively. We have decided to produce these pigments because there has been success in producing these pigments in the past ( Misawa et al., 1990, Cabrera-Valladares et al., 2006, Lagunas-Muñoz et al., 2006,Yan et al., 2008, Lim et al., 2015, Xu et al., 2015). Using synthetic biology techniques, genes corresponding to pigment biosynthetic pathways from a variety of organisms will be engineered for expression in E. coli. Pigment production will be followed by purification to separate the pigments from the bacteria, which will then allow the produced pigments to be incorporated into ink.

Standard Genetic Construct for Pigment Production

Each of the four pigment production constructs is designed in the same way. Each construct consists of a T7 promoter, the E. coli RBS B0034 (with the exception of the zeaxanthin construct, which uses Pantoea ananatis native RBSs), the coding sequences for pigment biosynthetic genes, and the double terminator B0015.

Use of a T7 promoter allows us to utilize the induction system of the E. coli strain BL21(DE3). In this strain, T7 RNA polymerase (RNAP) is expressed by the lacUV5 promoter, which can be induced by the addition of IPTG to the culture. T7 RNAP will then transcribe the pigment biosynthesis constructs(Fig 1.).

Fig 1. The T7 RNAP expression system in E. coli BL21(DE3)

Additionally, suing this system allows us to control exactly when the cultures will start to produce the pigment biosynthetic enzymes, and therefore the pigments themselves. This will help the bacteria to survive longer and produce more pigment in case the enzymes or pigments are toxic to the cells. We can choose to induce the system earlier or later, depending on the effects we see. Upregulated expression of the T7 RNAP will also lead to overexpression of the biosynthetic pathway, thus maximizing enzyme and pigment yield.

Melanin

We chose Melanin as our Key color, the black. It was selected because it has previously been produced in E. coli (Lagunas-Muñoz et al., 2006) . The gene we used was melA from the organism Rhizobium etli which had been previously submitted to the Parts Registry (BBa_K193600). We incorporated this gene into our new construct as described above (New Composite Part BBa_K2481108, Fig 2). melA encodes a tyrosinase that converts L-tyrosine to dopaquinone, which then undergoes an enzyme-independent polymerization into melanin (Fig 3).

Fig 2. melA epxression construct for use in E. coli BL21(DE3).

Fig 3. The pathway from L-tyrosine to Melanin with the use of the melA gene.

Indigoidine

The cyan pigment we chose was indigoidine and will be produced by proteins encoded by the indB and indC genes from Streptomyces chromofuscus (Fig 4). The indB gene codes for a putative phosphatase and the indC gene codes for indigoidine synthase. Together, these enzymes convert L-glutamine into indigoidine (Yu et al., 2012, Fig 5.) It has been previously shown that indC alone can produce indogoidine, but the inclusion of indB expression in the system will increase yields significantly (Xu et al.,2015). A previous part for IndC has been submitted to the iGEM Parts Registry (BBa_K1152013), though this part originated from Photorhabdus luminescens laumondii TT01.

Fig 4. indB and ibdC expression constructs for use in E. coli BL21(DE3).

Fig 5. The pathway from l-Glutamine to Indigoidine and its associated genes.

Anthocyanin

The Magenta pigment we decided on was Anthocyanin. The genes we used are; dfr from the organism Anthurium andraeanum, F3h and ans from Malus domestica, and 3gt from Petunia hybrid. We chose these genes from these organisms as they have been expressed in E. coli as well as the anthocyanin has been proved non-harmful to humans and the environment. These genes converted the initial molecule Eriodictyol into our final molecule Anthocyanin (Fig 3). Team Darmstadt 2014 made the molecule pelargonidin for their use in a bio solar panel.

Fig 3. The anthocyanin synthesis pathway, from our initial molecule Eriodictyol into our final molecule Anthocyanin.

Zeaxanthin

The yellow pigment we used is Zeaxanthin, which is a part of the Carotenoid synthesis pathway. The genes we used were CrtY and CrtZ from the organism P. ananatis. These two genes create enzymes that turned our initial molecule Lycopene into the molecule Zeaxanthin (Fig 4). We will supplementing our media with Lycopene because it allows us to skip three genes in the pathway and improve our chances of success. These genes were selected as they have been produced in E. coli as well as the safe final product.

Fig 4. The carotenoid synthesis pathway and our initial and final molecules. As well as the genes that we skipped by adding Lycopene to our media.

References

Xu, F., Gage, D., and Zhan, J. (2015) Efficient production of indigoidine in Escherichia coli. Journal of Industrial Microbiology & Biotechnology. 42, 1149–1155

Yu,D., Xu, F., Valiente, J., Wang, S., and Zhan, J. (2012) An indigoidine biosynthetic gene cluster from Streptomyces chromofuscus ATCC 49982 contains an unusual IndB homologue. Journal of Industrial Microbiology & Biotechnology. 40, 159–168

Cude, W. N., Mooney, J., Tavanaei, A. A., Hadden, M. K., Frank, A. M., Gulvik, C. A., May, A. L., and Buchan, A. (2012) Production of the Antimicrobial Secondary Metabolite Indigoidine Contributes to Competitive Surface Colonization by the Marine Roseobacter Phaeobacter sp. Strain Y4I. Applied and Environmental Microbiology. 78, 4771–4780

Brachmann, Alexander O., Ferdinand Kirchner, Carsten Kegler, Sebastian C. Kinski, Imke Schmitt, and Helge B. Bode. "Triggering the production of the cryptic blue pigment indigoidine from Photorhabdus luminescens." Journal of Biotechnology 157.1 (2012): 96-99. Web.

Yan, Y., Li, Z., and Koffas, M. A. (2008) High-yield anthocyanin biosynthesis in engineered Escherichia coli. Biotechnology and Bioengineering. 100, 126–140

Lim, C. G., Wong, L., Bhan, N., Dvora, H., Xu, P., Venkiteswaran, S., and Koffas, M. A. G. (2015) Development of a Recombinant Escherichia coli Strain for Overproduction of the Plant Pigment Anthocyanin. Applied and Environmental Microbiology. 81, 6276–6284

Sedkova, N., Tao, L., Rouviere, P. E., and Cheng, Q. (2005) Diversity of Carotenoid Synthesis Gene Clusters from Environmental Enterobacteriaceae Strains. Applied and Environmental Microbiology. 71, 8141–8146

Misawa, N., Nakagawa, M., Kobayashi, K., Yamano, S., Izawa, Y., Nakamura, K., and Harashima, K. (1990) Elucidation of the Erwinia uredovora carotenoid biosynthetic pathway by functional analysis of gene products expressed in Escherichia coli. Journal of Bacteriology. 172, 6704–6712

Lagunas-Muñoz, V., Cabrera-Valladares, N., Bolívar, F., Gosset, G., and Martínez, A. (2006) Optimum melanin production using recombinant Escherichia coli. Journal of Applied Microbiology. 101, 1002–1008

Cabrera-Valladares, N., Martínez, A., Piñero, S., Lagunas-Muñoz, V. H., Tinoco, R., Anda, R. D., Vázquez-Duhalt, R., Bolívar, F., and Gosset, G. (2006) Expression of the melA gene from Rhizobium etli CFN42 in Escherichia coli and characterization of the encoded tyrosinase. Enzyme and Microbial Technology. 38, 772–779