Team:SZU-China/Engineering
Engineering
Success
Blue is the essence of jeans, and they all come from a long-standing dye called indigo, first extracted from plants, such as wood blue, isatis indica, indigo blue, etc. These days, jeans have been popular all over the world, and the annual output jeans have reached several billion pieces. Most of the jeans dyes are chemically synthetic indigo which brings many environmental problems. What's worse, a variety of toxic and harmful chemicals, such as hydrogen cyanide, formaldehyde, are involved in the dyeing processes. But many factories directly discharge sewage without treatment to reduce costs. That is why textile dyeing has become one of the most polluting industries in the world.
Fig.1 A river in Xintang, "Hometown of Jeans" (Image source: Google satellite)
Aiming at reducing the demand for the use of harmful chemicals, we are wondering if we can use synthetic biology methods to produce low-cost bio-indigo.
In the blue indigo plants, such as Polygonum tinctorium, Indigofera tinctoria, Baphicacanthus cusia, and Isatis tinctoria, cells of these plants contain the secondary metabolites indican (indoxyl-β-D-glucoside) and isatan B (1H-Indol-3-yl β-D-ribo-3-hexosulopyranoside) as indigo precursors. The mechanisms of indigo production in P. tinctorium showed that indican is stored in vacuoles and is degraded to indoxyl and β-D-glucose following the release of β-D-glucosidase from chloroplasts. Subsequently, indoxyl is oxidized by air to form the dimer indigo but only under conditions of cell lysis.
SZU-China 2020 are planning to produce bio-indigo in engineered bacteria. There are two key steps. First, we need indole, the precursor substance of indigo. Tryptophan can be transformed into indole by tryptophanase encoded by tnaA enzyme. Secondly, under the action of the flavin monooxygenase encoded by the gene FMO, the hydroxyl group is introduced into the indole to form the intermediate hydroxyindole. Finally, when the cell is lysed. Indole can spontaneously oxidize to indigo. Therefore, recombinant tryptophanase and recombinant flavin monooxygenase are expressed by engineered E.coli to achieve the purpose of biosynthesis of indigo.
When searching in the iGEM library, we dramatically found that iGEM13_Berkeley, iGEM19_GreatBay_SZ and iGEM19_Tongji_China have all tried to produce bio-indigo. Great minds think alike! At the same time, we contacted SHANGHAI_SFLS_SPBS, who also wants to produce bio-indigo this year.
We used the part of iGEM19_GreatBay_SZ to construct the recombinant tnaA-Fmo plasmid and express in DH5alpha.
Fig.2 Plasmid map of pSC101-TALEsp2-tnaA-Fmo
We used DMSO for resolubilization, and bio-indigo had an obvious absorption peak at 617nm, which was the same as that recorded in the literature. And the pigment is clearly blue in daylight.
Fig.3 Bioidigo absorption spectrum
During the experiment, we found that the production of bio-indigo was unsatisfactory. Literature shows that if we want to increase the production of bio-indigo, we need to add a large amount of tryptophan to the culture medium. However, the cost of tryptophan far exceeds synthetic indigo, which is contrary to the original purpose of reducing costs.
Fig.4 Indigo standard curve
In addition, we got the unexpected orange-red dye in the fermentation product. After we contacted with SHANGHAI_SFLS_SPBS, we found that this phenomenon also happened in their experiments. After consulting the literature, that may be due to the production of isatin, which is obtained by oxidation of indigo. It is preliminarily speculated that this is due to the overexpression of FMO, which caused the indigo to be further oxidized into isatin.
Fig.5 Unexpected orange-red dyeabsorption spectrum
Meanwhile, bio-indigo is hard to dye, for it is insoluble in water which makes it needs repeating dyeing many times to have an obvious blue colour. Therefore, we hope to develop a more environmentally friendly and lower cost blue pigment that can be more effective for denim dyeing.
Due to the shortcomings of bio-indigo, we hope to find a better blue dye to replace bio-indigo. After consulting a large number of literature, we found a more environmentally friendly, low-cost blue dye, Gardenia Blue.
Gardenia Blue is a blue dye from gardenia fruits, with advantages of stable physical and chemical properties, heat resistance, acid and alkali resistance. It is easily soluble in water, making the dyeing easy.
Besides, Gardenia Blue pigment has a wildly used, because, with the addition of different amino acids, it can be shown in different colours from green, blue to purple that meets basic needs of customers for jeans.
Two-step enzymatic synthesis of Gardenia Blue wildly uses in the industry. Firstly, the extracted gardenoside transformed into genipin via the activity of β-glucosidase. Then genipin combined with primary amino acids at a high temperature, a process which generates natural blue pigments.
The one-step chemoenzymatic reaction also used in the production of Gardenia Blue, in which the enzymatic hydrolysis of geniposide and colour reaction of amino acids proceed simultaneously.
However, the colour reaction carried out at a high temperature, where most industrial β-glucosidase can not tolerate, which makes it hard to implement such a simplified method.
We hope to produce β-glucosidase with high-temperature resistance. Directly adding the enzyme to the mixture of gardenoside and primary amino acids, instead of a step-by-step process, to obtain a higher quality of Gardenia Blue. It saves production costs, making it possible to obtain high-quality natural blue pigment industrially.
Fig.6 Blue pigment production from geniposide and amino acids
Through literature research, we found a very heat-resistant β-glucosidase (bglA) from Thermotoga Maritima, an enzyme with high solubility, high optimum reaction temperature, and good temperature stability. We hope to express bglA efficiently in E. coli, to achieve the goal of industrial production of Gardenia Blue.
To efficiently express heterologous proteins in E. coli, we optimized the bglA gene sequence for E. coli codons. And we added a GST tag to the N-terminus of bglA to improve the solubility of the protein to avoid the formation of inclusion bodies.
Fig.7 Plasmid map of pGEX-4T-1-H-beta-glucosidase
We tested the enzyme activity of β-glucosidase and set a temperature gradient to find the temperature at which it reached the maximum enzyme activity, and plotted the enzyme activity-temperature curve. We also tested the enzyme activity at different reaction times and plotted the enzyme activity-time curve.
Fig.8 a. standard curve of bata-glucosidase enzyme activity b. beta-glucosidase enzyme activity-temperature curve c. beta-glucosidase enzyme activity-time curve
The heat-resistant bglA enzyme produced by E.coli has such high activity surprised us. In the temperature of 80 degrees Celsius, it is obvious to see a blue pigment with the naked eye in five minutes reaction. The multi-salt bridge structure between the peptide chains of the thermophilic protein and the hydrogen bond on the main chain may count for its heat-resistance. To further enhance the enzyme activity, we analyzed the protein structure of bglA, found its active site, made mutations at the enzyme active site, and performed molecular docking to find mutant strains with higher enzyme activity.
After single factor analysis, we determined that our best formula is to add 440ul geniposide, 6ul glycine, 50ul enzyme to 1ml system, heating for 150 minutes in 80 seconds.
In the results, it's shown that the production of Gardenia Blue pigment using heat-resistant bglA has become very simple and efficient through the one-step chemoenzymatic reaction.
Fig.9 a. Gardenia blue pigment solution produced by different amino acids; b. OD595 of different amino acids; c. the absorption curve of gardenia blue with different amino acids
Fig.10 a. Gardenia blue yield - Geniposide Concentration curve; b. Gardenia blue yield-glycine concentration curve; c. Gardenia blue yield-temperature curve ; d. Gardenia Blue Yield-Time curve
After determined the best formula for the production of gardenia blue, we carried out mass production of gardenia blue to imitate the industrial dyeing process.
Glycine-Gardenia Blue has a better and darker coloration, which verified the superiority of glycine.
We also explored the effects of different metal mediators on dyeing. We have found that the dyeing effect is best after 40 minutes of iron pretreatment.
Fig.11 a.Influence of the dyeing time; b.the influence holding dyeing temperature; c.the influence of different metal mediators
In addition to full dyeing, there is another essential process: fraying. The traditional fraying process usually involves sand washing, water washing, sandblasting, chemical ageing and other steps. With the low cost of those processes, many small jeans factories wildly accepted those traditional fraying process. However, it consumes a lot of water, accompanied by the use of a large amount of toxic and harmful chemical reagents such as indigo, solsine, alkali and potassium manganate, which not only does harm to the health of workers in the jeans factory but also the environment. The remaining problems hurt the health of consumers who buy such jeans. The current environmental friendly fraying process, the laser, is too expensive for most factories.
The purpose of traditional methods such as sand washing is to destroy the structure of the cellulose on the surface of the jeans to achieve a worn-out effect. Therefore, we hope to adopt a more eco-friendly and low-cost method to complete the process of fraying.
We hope to use cellulase to achieve this effect. The cellulase hydrolyzes the cellulose, causing a part of the indigo dye to fall off the fabric to achieve the "worn feeling". Relevant literature points out that compared with stone washing, cellulase washing can obtain the same effect as stone washing. Besides, cellulase washing has unique advantages such as high efficiency, non-toxicity and harmlessness, and do little damage to machines and fabrics.
We expect to mass-produce this enzyme in E.coli. CL34 from Streptomyces and IARI-SP-2 endo-beta-1,4-glucanase from Bacillus subtilis is endo-β-glucanases.
Those enzymes randomly cut the amorphous region inside the cellulose polysaccharide chain, destroying the entire cellulose structure, which lines on our expectations. We connect the gene to the vector to construct an expression vector.
Fig.12 Plasmid maps
We first characterized the CL34 enzyme activity. We drew the enzyme activity-temperature curve as follows. It's shown that the optimum temperature for CL34 is at 45°C.
As for the enzyme activity-temperature curve of IARI-SP-2 endo-beta-1,4-glucanase, the result shows that the enzyme has the highest enzyme activity at 38°C with 28.6964u. Compared with the best enzyme activity of CL34, 29.65138u, it is slightly lower by 3.22%, but it has lower temperature requirements.
The traditional fraying process does not involve heating up. Therefore, if our target enzyme has the best enzyme activity at room temperature, we can save energy as much as possible and increase economic benefits, so we chose IARI- SP-2 endo-beta-1,4-glucanase as our best enzyme.
Separately, we measured the enzyme activity-time curves of the two enzymes. It shows that not only the initial enzyme activity of CL34 reaction is much lower than IARI-SP-2 endo-beta-1,4-glucanase, but also the decay rate is much faster than IARI-SP-2 endo-beta-1,4-glucanase. It convinces us that IARI- SP-2 endo-beta-1,4-glucanase is a better choice.
Fig.13 a.enzyme activity-temperature curve; b.enzyme activity-time curve
To test the actual effect of cellulase, we soaked four cloths of equal quality in IARI-SP-2 endo-beta-1,4-glucanase solution in PBS buffer. Finally, the surface was photographed with an electron microscope to observe the destruction of the cellulose structure on the surface.
In order to further improve our cellulase, we consulted Professor Liu Gang, an expert in cellulase research, and he informed us that the activity of recombinant cellulase is related to many factors. It may be that the protein is not folded correctly or the expression host is not compatible or other reasons.
However, the cellulase enzyme washing method has been used in the industry. The activity and efficiency of the enzyme are important issues. We must find a recombinant enzyme with better activity. Professor Liu Gang also suggested that we use a mixed enzyme system, which can better adapt to different working environments in the industrial system and achieve better results.
Fig.14 electron micrograph of cellulase treatment
Shintaro Inoue, Rihito Morita, Keiko Kuwata, Tadashi Kunieda, Haruko Ueda, Ikuko Hara-Nishimura, Yoshiko Minami, Tissue-specific and intracellular localization of indican synthase from Polygonum tinctorium, Plant Physiology and Biochemistry, Volume 132, 2018, Pages 138-144, ISSN 0981-9428,https://doi.org/10.1016/j.plaphy.2018.08.034
Hsu, T., Welner, D., Russ, Z. et al. Employing a biochemical protecting group for a sustainable indigo dyeing strategy. Nat Chem Biol 14, 256–261 (2018). https://doi.org/10.1038/nchembio.2552
M. Sadeghi, H. Naderi-Manesh, M. Zarrabi, B. Ranjbar, Effective factors in thermostability of thermophilic proteins, Biophysical Chemistry, Volume 119, Issue 3, 2006, Pages 256-270, ISSN 0301-4622, https://doi.org/10.1016/j.bpc.2005.09.018.
