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 ribosome binding site (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(Figure 1.).
Figure 1. The T7 RNA Polymerase expression system in E. coli BL21(DE3)
Additionally, using 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.
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, Figure 2). melA encodes a tyrosinase that converts L-tyrosine to dopaquinone, which then undergoes an enzyme-independent polymerization into melanin (Figure 3).
Figure 2. melA epxression construct for use in E. coli BL21(DE3).
Figure 3. The pathway from L-tyrosine to Melanin with the use of the melA gene.
The cyan pigment we chose was indigoidine and will be produced by proteins encoded by the indB and indC genes from Streptomyces chromofuscus (Figure 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, Figure 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.
Figure 4. indB and ibdC expression constructs for use in E. coli BL21(DE3).
Figure 5. The pathway from L-Glutamine to Indigoidine and its associated genes.
The magenta pigment will be anthocyanin, a pigment commonly found in plants. It is produced using the following genes: DFR, which codes for Dihydroflavenol 4-reductase (Anthurium andraeanum), F3H, which codes for 3B hydroxylase (Malus domestica), ANS, which codes for Anthocyanin synthase (Malus domestica), and 3GT, which codes for 3-o-glucosyltransferase (Petunia hybrid) (Figure 6). The E. coli membrane transporter yadH has also been shown to export anthocyanin from the cell (Yan et al., 2008, Lim et al., 2015). The DFR, F3H, ANS and 3GT genes constitute a pathway that converts eriodictyol into anthocyanin (Figure 7) and overexpressing the yadH transporter in E. coli has been shown to increase anthocyanin yields (Lim et al., 2015). A key reason for choosing to produce anthocyanin as opposed to a different pigment, is that it can change color according to the pH level of its environment (Yan et al., 2008). This may allow us to have more versatility in making ink of different colour and shades, simply by adjusting the pH of the solvent. The efficacy of this method has yet to be determined. Previous constructs for anthocyanin production have been submitted to the iGEM Parts Registry (BBa_K1497023).
Figure 6. Anthocyanin biosynthesis expression constructs for use in E. coli BL21(DE3)
Figure 7. The anthocyanin synthesis pathway, from the initial molecule Eriodictyol into the final molecule Anthocyanin.
The yellow will be zeaxanthin, and will be produce by a modified carotenoid biosynthesis pathway. The genes used will be crtY and crtZ, which come from the organism Pantoea ananatis (Figure 8). The crtY gene encode a lycopene cyclase and crtZ and codes for beta-carotene hydroxylase (Sedkova et al., 2005, Misawa et al., 2007). We will attempt to skip the first steps of the carotenoid biosynthesis pathway to reduce the number of foreign proteins introduced into E. coli by supplementing the media with lycopene (Figure 9). We have used previously made parts for lycopene cyclase and the Beta-carotene hydroxylase have been submitted to the iGEM Parts Registry (BBa_ I742155 and BBa_I742158).
Figure 8. Anthocyanin biosynthesis expression constructs for use in E. coli BL21(DE3).
Figure 9. The carotenoid synthesis pathway and our initial and final molecules. As well as the genes that we skipped by adding Lycopene to our media.
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