There is a saying in the natural dye world that only crushed insects and forest plants can be soaked in the most vivid colors.
It can be seen that in the past, most natural dyes were obtained from animals or plants, such as plant dyes: comfrey (purple), sumac (black), yellow gardenia (yellow) and su fang (red), etc., animal dyes: carmine acid and Tyros violet. In recent years, with the development of biotechnology, researchers are working to engineer microorganisms to produce dyes.
"It has the potential to change the production paradigm of the compound." Rasmus JN Frandsen, a synthetic biologist at the Technical University of Denmark, commented on the engineering modification of microbial dyes.
Take carmine, for example, an aromatic polyketone compound found in cochineal worms (i.e., Actylopius coccus, an oval mesenchymal about 0.2 inches long) and is often used to color food, textiles and cosmetics. The use of carmine acid dates back thousands of years to the reign of the Maya and Aztec Empires, when scarlet goods proved to be dyed with carmine acid. From 1967 to 2009, the U.S. Food and Drug Administration approved the use of carmine acid for a variety of foods such as yogurt, cakes, candy, beverages, and meat.
Its manufacturing process generally goes like this: workers first raise cochineals on their chosen cacti (also known as pear cacti or cochineal), dry them and sell them to processors, who grind the bugs into powders and pair the powder with salt to separate carmine acid. Using current methods, it is estimated that 70,000 bugs are needed to produce 1 pound of dried insects and 0.2 pounds of carmine acid.
Picture 丨 Traditional harvesting method of cochineal (Source: Smithsonian Magazine)
As global demand for carmine acid increases, along with rising labor costs, the carmine acid industry has become strained. In Peru, the largest producer of cochineal, the price per ton of carmine dye rose by 40% between 2013 and 2019.
Price isn't the only problem with it, either. A 2018 study from Japan also reported that a small percentage of people developed allergic reactions to cochineal dyes due to residual insect molecules, even if the level was no higher than that of other common allergens.
As a result, researchers have shifted the new method of producing carmine acid to metabolic engineering, that is, manipulating the metabolic pathways within organisms to produce carmine acid, but the challenge is that the complete biochemical pathway for making carmine acid is unknown. The Frandsen team mentioned above decided to start with the structure of carmine acid and figure out how to reverse engineer it with enzymes of known biochemical pathways.
Frandsen's team first predicted the required starting ingredients, biochemical steps, and enzymes that catalyze those steps. They designed eight potential biochemical pathways that could produce carmine acid and tested several hosts for genetic engineering, ultimately identifying a fungus, Aspergillus nidulans.
Through repeated experiments, the team created a tricyclic core of carmineic acid after removing some genes from the fungus (to disable competing biochemical pathways) and adding several other genes that provide appropriate enzymes (one from plants and two from bacteria), which was processed by an unknown enzyme already present in Aspergillus nidulans to produce an intermediate structure called kermesic.
Finally, adding genes that contain enzymes contained in the cochineal worm itself that can be converted into carmine acid was found to produce carmine acid. The findings were published in The Scientific reports in August 2018 under the title "Heterologous production of the widely used natural food colorant carminic acid in Aspergillus nidulans." But the efficiency of the reaction at the time was far from sufficient to account for large-scale production, and since one of the enzymes was still unknown, it was difficult to optimize production.
(Source: Research Paper 1)
In April 2021, Sang Yup Lee's research group from the Korea Advanced Institute of Science and Technology reported for the first time at JACS the work of biosynthesis of carmine acid from glucose with engineered E. coli, after simple metabolic engineering, followed by batch feed fermentation, and the production of 0.63± 0.02 mg/L of carmine acid from glucose.
(Source: Research Paper 2)
Although the scale of the experiment is still small, the scientists said that if the scale is expanded and it is assumed that 5 grams of carmine acid are produced per liter, then cultivating E. coli in a 100,000 liter fermentation tank can be produced in 5 days, which will require consuming one hectare of cochine grown on cacti for a year.
Frandsen said the two studies show great potential for synthetic biology, but there is still a lot of work to be done before expanding the process to an industrial level, such as adjusting the number or efficiency of various enzymes in microbes to optimize the production of carmine acid and reduce the number of unwanted by-products.
Resources:
1、https://www.smithsonianmag.com/innovation/scientists-are-making-cochineal-a-red-dye-from-bugs-in-the-lab-180979828/
3、https://pubs.acs.org/doi/10.1021/jacs.0c12406
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