Department of Energy’s Joint BioEnergy Institute (JBEI) has developed a new eco-friendly production platform for producing blue pigment which may provide a sustainable alternative to synthetic indigo, a dye used to colour denim and many other items. This opens the door for next-generation bioproduction of dyes replacing environmentally unfriendly process.

The team was investigating microbe-based bio-manufacturing when it came across the eco-friendly production platform for a blue pigment called indigoidine. With a similar vividly saturated hue as synthetic indigo, a dye used around the world to color denim and many other items, the team’s fungi-produced indigoidine could provide an alternative to a largely environmentally unfriendly process.

“Originally extracted from plants, most indigo used today is synthesised,” said lead researcher Aindrila Mukhopadhyay, who directs the Host Engineering team at JBEI. “These processes are efficient and inexpensive, but they often require toxic chemicals and generate a lot of dangerous waste. With our work we now have a way to efficiently produce a blue pigment that uses inexpensive, sustainable carbon sources instead of harsh precursors. And so far, the platform checks many of the boxes in its promise to be scaled-up for commercial markets.”

Importantly, these commercial markets already have considerable demand for what the scientists hope to supply. After meeting with many key stakeholders in the textile industry, the team found that many companies are eager for more sustainably sourced pigments because customers are increasingly aware of the impacts of conventional dyes. “There seems to be a shift in society toward wanting better processes for creating everyday products,” said Maren Wehrs, a graduate student at JBEI and first author of the paper describing the discovery, now published in Green Chemistry. “That’s exactly what JBEI is trying to do, using tools derived from biological systems – it just so happens that our engineered biological platform worked very well.”

The story began when the team set out to test how well a hardy fungi species called Rhodosporidium toruloides could express nonribosomal peptide synthetases (NRPSs) – large enzymes that bacteria and fungi use to assemble important compounds. The scientists examined this fungi’s NRPS expression capability by inserting a bacterial NRPS into its genome. They chose an NRPS that converts two amino acid molecules into indigoidine – a blue pigment – to make it easy to tell if the strain engineering had worked. Quite simply, when it did, the culture would turn blue.

Going into this experiment, indigoidine itself was not the main interest for the team. Instead, they were focused on the larger picture: exploring how the assembly line functionality of these enzymes could be harnessed to create biosynthetic manufacturing pathways for valuable organic compounds, such as biofuels, and assessing whether or not the fungi represented a good host species for the production of these compounds. But when they cultivated their engineered strain, they discovered the culture was blue.

With an average titer of 86 grams of indigoidine per liter of bioreactor culture, the yield of the strain – which they named Bluebelle – is by far the highest that has ever been reported. (Other research groups, including the JBEI team, have synthesised indigoidine using different host microbes.) Adding to the weight of the achievement, the record-breaking yield was obtained from a culture process that uses nutrient and precursor inputs sourced from sustainable plant material. Previous pathways required considerably more expensive inputs yet made about one-tenth the amount of indigoidine.

Beyond the potential applications of indigoidine, the study succeeded in its original goal of providing a potential production pathway for other NRPSs – something that is much more valuable than any single product. These complex enzymes have multiple subunits that each perform a distinct and predictable action in assembling a compound out of smaller molecules. Scientists at JBEI and beyond are keen to engineer enzymes that use NRPSs’ Lego block-like features to produce advanced bioproducts that are currently hard to make.

“A big challenge is to get a microbe to efficiently express such enzymes. This host has huge potential to fulfill that need,” said Mukhopadhyay. The next steps will be to characterise how indigoidine could be used as a dye and to dig deeper into the capabilities of R toruloides.