Scientists engineer microbe that mass-produces camouflage pigment

A remarkable advancement in microbial engineering has put a previously obscure natural pigment in the spotlight. If you've kept up with research on camouflage, optical material, or bioinspired chemistry, you have no doubt been intrigued for many years about xanthommatin, the color-changing pigment seen in insects and cephalopods.

However, researchers have struggled with obtaining enough xanthommatin to study, despite their intrigue. Natural extraction of sufficient quantities from an animal is impractical, and chemical synthesis has not yet yielded enough xanthommatin to study. Engineered microbes have tended to only produce minuscule amounts, incapable of being scaled up efficiently.

By pushing their microbiology to shift the relationship between survival and pigment production, a group assembled by researchers at UC San Diego has now changed the scale of study altogether. By making a bacterium's survival reliant on producing this pigment, the investigators developed a novel strategy to push the bacterium to levels of xanthommatin production not considered possible previously. Their novel efforts not only allow for pigment material but also natural materials made from hydrogen and carbon, instead of fossil fuels.

At the core of the discovery is a biological trick. In almost all natural product pathways, any extra produced carbon is lost as a waste fragment, like formate. The UC San Diego researchers had an idea that, if they could somehow render the waste fragment, a survival requirement of the cell, pigment production and growth would be linked. With more pigment comes more formate. More formate leads to more growth, and more growth leads to more pigment production and so on.

To initiate this process, the researchers utilized a soil bacterium characterized by its versatile metabolism and capacity to tolerate severe compounds. The scientists deleted the genes utilized by the cell to make a prerequisite one-carbon cofactor MTHF. In the absence of the cofactor, the synthetic strain could no longer create vital cellular building blocks, such as purines and methionine. It had become dependent on an external source of one-carbon units.

The next stage was to develop a bacterium to produce MTHF solely from formate. The scientists utilized a three-enzyme module to produce MTHF from formate (the three enzymes were derived from another species). The modified cell produced the missing compound from formate. With this module, the new bacteriophage could proliferate only when formate was present. The more formate the bacteriophage encountered, the faster their growth rate increased.

To connect this carbon-hungry cell to a pigment pathway, the researchers turned to the pigmented branch of metabolism that catabolizes the amino acid tryptophan. During the first few steps of this pathway, C2 carbon of tryptophan is released as formate, so the scientists incorporated a module that consists of two genes, which drive this reaction.

Upon introduction of glucose, glycine, and tryptophan to the derived bacteriophage of the overall pathway, the engineered cell emerged. In this case, the cell could only grow because the introduced pathway that catabolized tryptophan was incorporated into its metabolism, releasing large quantities of formate.

The derived cells produced massive quantities of kynurenine and kynurinic acid, far surpassing the amount produced by the parent strain. This surge in cellular metabolic odyssey illustrates the benefit of linking maintenance of growth to the production of an exotic compound.

To get the cells closer to making xanthommatin, the researchers added a third enzyme that converts kynurenine to 3-hydroxykynurenine, which is the primary building block of the pigment. The cultures with this three-gene mix changed color as they grew, changing from yellow to orange. In the analyses of the chemistry, it was evident that the cells with these enzymes were making two xanthommatin dimers and several monomers.

For the first time, the team was able to produce a pigment, xanthommatin, that was previously only derived from butterflies, dragonflies, and cephalopods.

While these engineered strains were producing enough pigment, they were not able to go solo and would need amino acids added. They were not ready to just be put into rich sole carbon source media and produce the pigment at the same time. The goal of the team was to have cells that would grow using glucose as a single carbon source and produce pigment amounts sufficient to analyze.

The researchers applied adaptive laboratory evolution, which is a system of guiding microbes through many generations of selective pressure. Over many transfers of the cells, the team gradually decreased the amino acid supplements until the cells were able to grow without amino acids. A mutation occurred in one of the important pathway enzymes, MetK, which altered the balance of glycine and methionine in the cell.

This round, the engineered strains could grow without the addition of glycine supplementation. A second round of evolution allowed the cells to grow without the need for adding additional tryptophan. In this case, the bacteria evolved a new promoter alteration that fine-tuned the expression of pigment-pathway genes based on the cellular demands. By the end of this project, the scientists had completely adapted a strain to use glucose as its only source of carbon and to still produce large quantities of pigment using the same pathway.

When tested in bioreactor conditions, this completely evolved strain demonstrated that indeed this would work. The culture became red as the pigment accumulated - it became dark, red by the third day of a single-run bioreactor experiment where pigment yields were over 0.5 grams of crude pigment from 0.91 L of culture, measuring less than a cup. Instead of mg being measured, the titers for this yield were called over 2 grams per liter, which is on the order of hundreds of times higher than the traditional methodology.

Some follow-up tests also showed that the microbially made pigment behaved mechanically similarly to the optoelectronic behavior of synthetic xanthommatin. The absorbance profile varied slightly, but both the synthetic and biological xanthommatin were reversibly color-changing and similar in electronic properties. What this means is that when it comes to compounds produced by biology, it can make a functional pigment and provides a sizable advancement in a compound that has been limited with tedious chemistry for far too long.

This is a rethink of what you might consider biomanufacturing. Rather than just forcing a microbe to make an unnatural compound, the survival of the microbe is inherently connected to steps that generate the unnatural compound. Effectively, you are selecting better and better producers with natural pressures on the cell.

"This is the natural pigment that provides an octopus or squid its camouflage superpower. And our achievement to advance the production of this material is only the tip of the iceberg," said senior author, Bradley Moore. Leah Bushin, main author of the study, clarified, "We thought we had come up with a clever way to trick the bacteria into making more of the material we were asking of it."

The team feels confident that the same method will carry over to many other pathways that decouple one-carbon fragments. Flavors, precursors to drugs, antibiotics, and much more, can all come out of these pathways. If enough friction is used, the growth-coupled biosynthesis will displace entire sectors of resource that use fossil resources and shift to renewable biological production.

Research findings are available online in the journal Nature Biotechnology.

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