Can microorganisms also produce dyes? Synthetic biology technology to manufacture carminic acid

  There was a saying in the natural dye world that only crushed insects and forest plants can dye the most vivid colors.
  It can be seen that in the past, most natural dyes were obtained from animals or plants, such as vegetable dyes: comfrey (purple), hematoxylin (black), yellow gardenia (yellow) and Sufang (red), etc., animal dyes : Carminic acid and Tyrol violet, etc. In recent years, with the development of biotechnology, researchers are working to engineer microorganisms to produce dyes.
  ”It has the potential to change the paradigm for the production of this compound,” says synthetic biologist Rasmus JN Frandsen of the Technical University of Denmark of engineering microbes to produce dyes.
  Take carminic acid, for example, an aromatic polyketide found in cochineal insects (i.e., Dactylopius coccus, an oval scale insect about 0.2 inches long) that is often used to color food, textiles, and cosmetics. The use of carminic acid dates back thousands of years to the reign of the Mayan and Aztec empires, when scarlet merchandise that existed at the time was shown to be carminic acid-stained. From 1967 to 2009, the U.S. Food and Drug Administration approved the use of carminic acid in various foods such as yogurt, cakes, candy, beverages, and meat.

  It’s generally made like this: Workers first raise cochineal worms on a cactus of their choice (also known as pear cactus or cochineal), dry them and sell them to processors, who grind the worms into powder, Pair with salt to isolate carminic acid. Using current methods, an estimated 70,000 bugs are needed to produce 1 pound of dry insects and 0.2 lb of carminic acid.
  As global demand for carminic acid increases, along with rising labor costs, the carminic acid industry has become strained. In Peru, the largest cochineal producer, the price per ton of carminic acid dye rose by 40 percent between 2013 and 2019.
  Price isn’t its only problem, either. In 2018, a study from Japan also noted that a small percentage of people will develop an allergic reaction to cochineal dye due to residual insect molecules, even if the levels are not higher than other common allergens.
  Therefore, the researchers turned the new method of producing carminic acid to metabolic engineering, that is, manipulating the metabolic pathway inside the organism to produce carminic acid, but the challenge is that the complete biochemical pathway for making carminic acid is unknown. The aforementioned Frandsen team decided to start with the structure of carminic acid and figure out how to reverse engineer it with enzymes from known biochemical pathways.
  Frandsen’s team first predicted the required starting components, biochemical steps, and enzymes that catalyze these steps. They designed eight potential biochemical pathways that could produce carminic acid, and tested several hosts for genetic engineering, ultimately identifying a fungus, Aspergillus nidulans.

  Through trial and error, the team created a tricycle of carminic acid after deleting some genes from the fungus (to disable competing biochemical pathways) and adding several others that provide the appropriate enzymes (one from plants, two from bacteria) The core, which is processed by an unknown enzyme already present in Aspergillus nidulans, produces an intermediate structure called kermesic.
  Finally, by adding the gene for the enzyme that the cochineal itself contains that converts to carminic acid, it was found that the fungus could produce carminic acid. The research results were published in Scientific reports in August 2018 under the title “Heterologous production of the widely used natural food colorantcarminic acid in Aspergillus nidulans”. But at the time the reaction was far from efficient enough to consider large-scale production, and because one of the enzymes was still unknown, it was difficult to optimize production.
  In April 2021, Sang Yup Lee’s group from the Korea Advanced Institute of Science and Technology first reported at JACS the biosynthesis of carminic acid from glucose using engineered Escherichia coli, after simple metabolic engineering, followed by batch feeding Fermentation can produce 0.63±0.02mg/L of carminic acid from glucose.
  The experiment is still small, but the scientists say that if it were scaled up and assumed to produce 5 grams of carminic acid per liter, it would be possible to grow E. coli in a 100,000-liter fermenter for 5 days, which would require Consume a year of cochineal worms growing on one hectare of cacti.
  Frandsen said the two studies show the enormous potential of synthetic biology, but there is still a lot of work to be done before scaling up the process to an industrial level, such as adjusting the amount or efficiency of various enzymes in microbes to optimize rouge The production of red acid and reduce the amount of unwanted by-products.

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