Biotransformation of indole to indigo and indirubin by engineered Komagataella phaffii expressing MaFMO from Methylophaga aminisulfidivorans SK1

For centuries, the deep blue of denim and the vibrant red of certain medicines have come from two distinct molecules: indigo and indirubin. Indigo is the classic textile dye that gives jeans their characteristic color, while its structural cousin, indirubin, is a bioactive compound found in traditional herbal remedies, known for influencing how cells grow and communicate. For a long time, producing these substances meant relying on chemical factories that use harsh solvents and generate significant waste. In recent years, scientists have turned to nature's own machinery—microbes—to make these compounds more cleanly. By engineering bacteria and yeast to act as tiny chemical factories, researchers can feed them simple starting materials and watch them transform those materials into valuable pigments. The key to this transformation often lies in a specific type of enzyme, a biological tool that uses oxygen to modify molecules, turning a colorless starting block into a colored product.

In a new study, researchers at Ajou University in South Korea explored whether a specific type of yeast, known as Komagataella phaffii, could be trained to perform this task. While bacteria have been used for this purpose before, yeast offers different advantages, such as the ability to handle complex protein folding and grow to very high densities in industrial settings. The team focused on an enzyme called MaFMO, which comes from a bacterium that lives in sulfur-rich environments. This enzyme is a single, self-contained unit that can take a molecule called indole and, using oxygen and energy from the cell, convert it into the reactive intermediates needed to form indigo and indirubin. The researchers wanted to see if they could successfully install this bacterial enzyme into the yeast genome and, if so, how different conditions like the amount of food, the speed of mixing, and the addition of certain chemicals would affect the final amount of blue and red pigment produced.

The team began by inserting the gene for the MaFMO enzyme into the yeast's DNA, creating a strain that could produce the enzyme when fed methanol. When they added indole to the culture, the result was immediate and visible. The yeast cells, which were normally a pale cream color, turned a distinct blue, particularly in the clumps of cells at the bottom of the flask. This visual change was the first sign that the yeast had successfully taken up the bacterial enzyme and was using it to create pigment. To confirm exactly what was being made, the researchers extracted the pigments from the cells and analyzed them using sophisticated instruments. They found that the yeast was indeed producing both indigo and indirubin. Indigo was the dominant product, appearing in much larger quantities, while indirubin was present in smaller amounts. This confirmed that the yeast host could support the complex chemical steps required to make both molecules, not just one.

With the basic system working, the researchers then tested how to make the process more efficient by changing various conditions. They started by adjusting the amount of indole fed to the yeast. When they increased the amount of indole from a low level up to 3 millimoles per liter, the amount of blue pigment produced increased, reaching a peak of nearly 20 milligrams per liter. However, when they pushed the concentration higher to 5 millimoles per liter, the production of both pigments dropped sharply. This suggested that too much of the starting material was actually harmful to the cells or was inhibiting the enzyme's work, a common issue in biological systems where high concentrations of a substance can stress the organism.

Next, they looked at the role of methanol, which serves as both food and the trigger to turn on the enzyme gene. They tested concentrations ranging from 0.5% to 2.0%. Surprisingly, changing the amount of methanol within this range had very little effect on how much pigment was made. This indicated that once the enzyme was being produced, the amount of methanol was not the main factor limiting the speed of the reaction. Instead, other factors like the availability of oxygen or the health of the cells were likely more important. The researchers also tested how long they should let the yeast work. Extending the production time from 24 hours to 72 hours resulted in a steady increase in pigment, with the highest amounts recorded at the three-day mark. This showed that the yeast could remain active and productive for several days, continuously converting the indole into pigment.

Oxygen was another critical variable. The researchers found that if the flasks were left completely still, with no shaking, no pigment was produced at all. This makes sense because the enzyme needs oxygen to function. However, once the flasks were shaken, increasing the speed from a moderate pace to a faster one did not significantly boost the yield. This suggested that in the small flasks used, the oxygen supply was already sufficient, and the bottleneck lay elsewhere. The team also experimented with adding specific chemicals to see if they could influence the balance between the blue indigo and the red indirubin. Adding a small amount of L-cysteine, a common amino acid, changed the ratio of the two pigments, shifting the production slightly toward more indirubin. This suggests that the chemical environment inside the cell can be tweaked to favor one product over the other. In contrast, adding a common surfactant called Tween 80 at high concentrations completely stopped pigment production, likely because it damaged the cell membranes or interfered with the enzyme's ability to work.

The study concludes that this yeast platform is a viable starting point for making these valuable pigments, but it is not yet a fully optimized industrial process. The researchers demonstrated that the yeast can produce both indigo and indirubin, but the amounts are currently lower than what has been achieved in highly optimized bacterial systems. The work highlights that to improve production, future efforts should focus on carefully feeding the indole to avoid toxicity, managing the oxygen supply, and fine-tuning the internal chemistry of the cell to favor the desired product. By proving that this specific yeast can host this specific enzyme, the study opens a new door for using eukaryotic cells to manufacture complex, value-added chemicals in a more sustainable way.