Colorful GMOs could keep food fresh longer

If you’ve ever eaten a beet-filled meal you probably remember the pink urine and unsettlingly red stool you pass a bit later. The culprit is a molecule called beetroot red, and it’s used as a natural food dye in many products including candies, canned soups, and sausages (yikes). Previously I said that anthocyanins and carotenoids are the most-used vegetable-based pigments in processed foods. Beetroot red falls into neither of those categories.

https://commons.wikimedia.org/wiki/File:Rote_Bete_eine_Haelfte.jpg

Instead it is a member of another group of pigment called betalains that occur in fungi and plants. Whereas anthocyanins are fairly ubiquitous across many plant phyla, betalains are quite rare. They are found only in certain members of the order Caryophyllales, such as cacti, amaranths, and beets (the name betalain comes from the Latin root for beet). Whereas a PubMed search for ‘anthocyanins’ returns over 8700 results, a search for ‘betalains’ returns just 151. Fourteen of those are from this year, including one study describing the heterologous expression of betalains in non-Caryophyllales plants1. This could introduce intensely colorful new cultivars of many fruits and vegetables.

Biosynthesis of the betalains: betaxanthins and betacyanins

The story begins with the route plants use to make betalains, which you can follow along above. In the first step, the amino acid tyrosine is oxidized to DOPA. DOPA’s phenolic ring can be oxidatively cleaved to generate 4,5-seco-DOPA, which spontaneously cyclizes into betalamic acid. Betalamic acid condenses with a variety of amino acids to generate the first class of betalains: the yellow-orange betaxanthins. DOPA can also be oxidized to dopaquinone, which reacts with itself to form cyclo-DOPA. cyclo-DOPA condenses with the aforementioned betalamic acid to form the core of the second class of betalains: the red-violet betacyanins. Betacyanins bear in common with the anthocyanins that they garner further complexity by decoration with carbohydrates. This is the case with betanin…AKA beetroot red, the principal betalain in beets.

Identifying the enzyme that catalyzes each step can be a difficult task. By fishing from fungal cell lysates with DOPA as a lure, the enzyme that cleaves DOPA to make betalamic acid, 4,5-dioxygenase, was isolated and identified2. The enzyme that adds on the glucose molecule, glucosyltransferase, was similarly found in plant cell lysates by fishing with glucose3. These two enzymes, known since the 90s, account for the downstream steps of betalain synthesis.

The first step, tyrosine to DOPA, was finally solved last year4. This time, the researchers confronted the problem genetically rather than biochemically. They performed an analysis of all the RNA being translated in a betalain-producing species, looking for genes expressed similarly to the previously-identified enzymes. They found two related enzymes, CYP76AD1 and CYP76AD6, that both can convert tyrosine to DOPA. However, only CYP76AD1 can oxidize DOPA to dopaquinone – the branching-off point between betaxanthins and betacyanins. The redundancy and relatedness of the enzymes is probably what staved off their discovery until the advent of robust sequencing technologies.

Pigmented yeast expressing betalain biosynthetic genes (From Polturak et. al. 2016)

Since only CYP76AD1 can make cyclo-DOPA, it is necessary for the synthesis of red-violet betacyanins. The authors demonstrated this by making a DNA construct with all 4 genes (CYP76AD1/6, 4,5-dioxygenase, glucosyltransferase) transformed it into yeast – sure enough, resulting in orange-ish pigmented yeast. Removing the CYP76AD1 gene yielded yellow yeast (betaxanthin-only), and removing CYP76AD6 yielded purple yeast (presumably, betacyanin-dominated); removal of both resulted in complete loss of pigmentation, as was also observed without the downstream enzymes. This year, the same research group then extended their work by introducing the genes (in a vector called pX11) into plants that don’t normally produce betalains; namely, tobacco, potatoes, tomatoes, and eggplants1. As with the yeast, the pictures speak for themselves…very purple, very wild.

Wild-type or betalain expressing potato, eggplant, unripe & ripe tomatoes (From Polturak et. al. 2017)

Like anthocyanins, betalains are potent antioxidants. Unlike anthocyanins, it appears that betaxanthins and to a lesser extent betacyanins can enter your cells. The most common betaxanthin, indicaxanthin is 77-78% bioavailable, as opposed to 11-50% for betanin5. These results were found in vitro and would need to be established in vivo, but the numbers are impressive. For reference, anthocyanins were from 0.26-1.8% bioavailable in animal studies6. It remains to be shown how long absorbed betalains are circulate for—probably very short, judging by how quickly my pee turns pink after drinking some beet juice. But perhaps future scientists doping the betalain gene cluster into fruits and veggies can focus on incorporating yellow betaxanthins rather than violet betacyanins for a greater antioxidant punch?

There are no human clinical trials of betalains. In trying to find potential in vivo data on betalains, I came across a review on the seemingly numerous health benefits of beetroot supplements, many of which are credited to betalains7. I was starting to drink the kool-aid (beet-aid?) until I checked the conflicts of interest statement and saw that the authors are affiliated with a beetroot supplement company. Stay in your lane, Big Beet. Caveat: I do think beets are really good for you, but probably has less to do with betalains and more to do with their nitrate content. That’s a topic for another post, another time.

But even if betalains don’t do anything for you and me, they certainly help their plant hosts. Expressing betalains in tobacco plants made them more resistant to gray mold1. Necrotrophic mold grows on decaying plant tissue, so presumably the antioxidant function kept the tissue healthy and living and therefore mold-proof. Plants engineered to express betalains could be more resistant to spoiling, increasing shelf life and reducing food waste.

Once again, we have colorful plant pigments that are powerful antioxidants but nutritionally dubious. Maybe the best way to view betalains and anthocyanins is as markers of healthy food, since they occur in fresh fruits and vegetables. The most significant source for betalains, beets, have intriguing health benefits in small studies, so dig in. I like ‘em roasted and sliced, with some spicy mustard and greens…and I don’t even mind their colorful second act.

 

References

  1. Polturak, G. et al. Engineered gray mold resistance, antioxidant capacity, and pigmentation in betalain-producing crops and ornamentals. P Natl Acad Sci Usa 114, 9062–9067 (2017).
  2. Girod, P. A. & Zryd, J. P. Biogenesis of betalains: purification and partial characterization of DOPA 4, 5-dioxygenase from Amanita muscaria. Phytochemistry 30, 169–174 (1991).
  3. Vogt, T., Zimmermann, E., Grimm, R., Meyer, M. & Strack, D. Are the characteristics of betanidin glucosyltransferases from cell-suspension cultures of Dorotheanthus bellidiformis indicative of their phylogenetic relationship with flavonoid glucosyltransferases? Planta 203, 349–361 (1997).
  4. Polturak, G. et al. Elucidation of the first committed step in betalain biosynthesis enables the heterologous engineering of betalain pigments in plants. New Phytologist 210, 269–283 (2016).
  5. Tesoriere, L., Fazzari, M., Angileri, F., Gentile, C. & Livrea, M. A. In vitro digestion of betalainic foods. Stability and bioaccessibility of betaxanthins and betacyanins and antioxidative potential of food digesta. J. Agric. Food Chem. 56, 10487–10492 (2008).
  6. Fang, J. Bioavailability of anthocyanins. Drug Metabolism Reviews 46, 508–520 (2014).
  7. Clifford, T., Howatson, G., West, D. & Stevenson, E. The Potential Benefits of Red Beetroot Supplementation in Health and Disease. Nutrients 7, 2801–2822 (2015).