Scientists Ramp Up Licorice Sweetener Glycyrrhizin With Elicitation and CRISPR

Licorice has been prized for millennia as a flavoring and a medicine, but the molecule behind much of its therapeutic reputation, glycyrrhizin, is in growing global demand and stubbornly slow to produce. A comprehensive new review published in Discover Industrial Chemistry and Materials surveys the full arsenal of biotechnological strategies now being deployed to coax plants and microbes into making far more of this triterpenoid saponin glycoside, from stress-inducing chemical elicitors to CRISPR gene editing and fully synthetic production in brewer’s yeast. The assessment, led by Shriniwas P. Patil and colleagues at PCET’s School of Pharmacy, Pimpri Chinchwad University, argues that no single approach is sufficient on its own, but that their intelligent combination could transform how the world obtains one of its most pharmacologically versatile natural products.

Glycyrrhizin, also known as glycyrrhizinic or glycyrrhizic acid, accumulates in the roots and rhizomes of Glycyrrhiza glabra, the classic liquorice plant, as well as related species such as G. uralensis and G. inflata. On hydrolysis it yields 18β-glycyrrhetinic acid, also called glycyrrhetic acid or enoxolone, which is itself biologically active. The molecule’s commercial and clinical portfolio is remarkably broad. Its monoammonium salts serve as high-intensity sweeteners, and it is the chemical precursor for carbenoxolone, a hemisuccinyl ester derivative with mineralocorticoid activity that is marketed as an antiulcer drug. The review emphasizes that pharmacological studies have documented antiviral activity against hepatitis B and influenza A/H1N1 viruses, anti-inflammatory effects demonstrated through albumin denaturation assays and in ulcerative colitis, anticancer activity in colorectal cancer cell lines SW620 and HT29, and growth suppression of A549 lung adenocarcinoma cells through inhibition of thromboxane synthase.

Clinical evidence further strengthens the case for scaling up production. Glycyrrhizin has been tested alone and in combination therapies for chronic hepatitis C, including in interferon-resistant patients, where injection therapy has been associated with reduced hepatocellular carcinogenesis. A randomized placebo-controlled trial explored glycyrrhizic acid as an adjunctive treatment for depression through anti-inflammatory mechanisms. Against this backdrop of expanding medical relevance, the natural content of glycyrrhizin in even the richest licorice roots remains very low, and wild plants typically need three to four years of growth before harvest, creating a pressing supply problem that the review’s authors set out to address systematically.

Understanding the biosynthetic pathway is the foundation of every enhancement strategy. Glycyrrhetinic acid is an oleanane-type triterpenoid built through the mevalonate pathway from repeated units of isopentenyl pyrophosphate and dimethylallyl pyrophosphate. The pivotal first committed step is the cyclization of 2,3-oxidosqualene into β-amyrin, catalyzed by β-amyrin synthase. From there, sequential oxidation reactions driven mainly by the cytochrome P450 enzymes CYP88D6 and CYP72A154 generate a series of intermediates that culminate in glycyrrhetinic acid. The glycoside is then assembled by glycosyltransferases: UDP-glucose dehydrogenase converts UDP-glucose to UDP-glucuronic acid, and the enzyme UGT73P12 transfers a glucuronic acid moiety to the third carbon of glycyrrhetinic acid, forming glycyrrhetic acid 3-O-mono-β-D-glucuronide, or GAMG. A second glucuronosylation event on GAMG completes glycyrrhizin, with UDP-glucose pyrophosphorylase, known as UGP1, regenerating the UDP-glucose donor.

The most extensively explored strategy for boosting glycyrrhizin is elicitation, in which cultured plant tissues are deliberately stressed with biotic or abiotic agents to switch on secondary metabolism. The earliest in vitro attempt, by Shabani and colleagues in 2009, treated Glycyrrhiza glabra cultures with methyl jasmonate and salicylic acid at concentrations from 0.01 to 2.0 millimolar. Glycyrrhizin peaked at 0.1 millimolar methyl jasmonate after eight hours, while salicylic acid raised content up to 1 millimolar but depressed it at 2 millimolar, an early demonstration that elicitor dose and timing follow narrow windows of benefit. In 2010, Karwasara and colleagues extended the approach to cell cultures of Abrus precatorius, a related legume, finding that culture filtrate of Aspergillus niger at 7.5 percent and dried cell powder of Rhizopus stolonifer at 0.5 percent, together with yeast extract and 50 micromolar ascorbic acid, maximized both biomass and glycyrrhizin accumulation.

Subsequent work has diversified the elicitor toolkit considerably. Srivastava and colleagues subjected Agrobacterium rhizogenes-induced hairy roots of G. glabra to drought-mimicking PEG 6000, the heavy metal cadmium chloride, and the biotic elicitors cellulase and mannan. Polyethylene glycol at 1 percent produced the highest glycyrrhizin after 24 hours, cellulase at 200 micrograms per milliliter progressively raised content over seven days, and remarkably the lowest mannan dose of 10 milligrams per liter yielded the peak concentration of 3.3089 micrograms per milligram after ten days. In Taverniera cuneifolia root cultures, Awad and colleagues screened six fungal and five bacterial elicitors and found that Rhizobium leguminosarum drove glycyrrhizic acid to 6 milligrams per gram, while methyl jasmonate at 100 micromolar delivered a 2.5-fold increase. Jaiswal and colleagues, meanwhile, showed that adenine sulphate outperformed biotin, salicylic acid and polyamines in G. glabra callus, reaching 35.44 micrograms per gram, likely because sulphate assimilation supports amino acid and metabolite synthesis.

Microbial partners and physical stimuli have added further dimensions. Li and colleagues raised glycyrrhizin in adventitious roots of G. uralensis with low-dose salicylic acid, optimal sucrose at 4 percent and one-strength MS salts, and later showed that Aspergillus niger treatments in 5-liter balloon-type bubble bioreactors increased glycyrrhizin up to 200 milligrams per liter of elicitor, though higher doses reversed the effect. Xie and colleagues reported that the plant growth-promoting bacterium Bacillus pumilus, inoculated into drought-stressed G. uralensis plants, reduced antinutritional factors, improved protein digestibility and boosted antioxidants, cumulatively enhancing glycyrrhizin biosynthesis. In G. inflata hairy roots, methyl jasmonate at 100 micromolar produced 5.7 times more glycyrrhizin than controls after five days, whereas chitosan proved ineffective. Allahdou and colleagues found cellulase from Aspergillus niger at 200 micrograms per milliliter optimal for both glycyrrhizin and glycyrrhetinic acid in G. glabra hairy roots, and Afsharzadeh and colleagues combined hairy root transformation with red and blue LED light, recording antioxidant capacity gains over 55 days of exposure. Most strikingly, Yamamoto and colleagues documented overwhelming glycyrrhizin induction through symbiosis with the nitrogen-fixing rhizobium Mesorhizobium sp. J8, which elevated chlorophyll, nitrogenase activity and the expression of genes for glycyrrhizin and jasmonic acid synthesis.

Beyond elicitation, the review highlights genome editing as a fundamentally different lever. Chiyo and colleagues in 2023 used CRISPR/Cas9 in G. uralensis hairy roots to knock out the genes CYP93E3 and CYP72A566, which divert flux toward soyasaponins, along with CYP716A179 for oleanolic acid and LUS1 for betulinic acid. By eliminating these competing branches of β-amyrin metabolism, more precursor was channeled toward glycyrrhetinic acid and ultimately glycyrrhizin. When pathway blocking was paired with overexpression of CYP88D6, the oxidation step toward glycyrrhetinic acid, glycyrrhizin production rose again. Although the absolute gains were modest, the authors note that the results were achieved within a single month of culture, a dramatic contrast to the three to four years required by wild licorice, illustrating how pathway rationalization could compress production timelines.

The third pillar is heterologous biosynthesis in microbes, which began when Zhu and colleagues engineered 11-oxo-β-amyrin and glycyrrhetinic acid synthesis into Saccharomyces cerevisiae in 2017. In 2019, Wang and colleagues integrated codon-optimized CYP88D6 and CYP72A154, together with β-amyrin synthase and an Arabidopsis thaliana NADPH-cytochrome P450 reductase, into the yeast chromosome, producing a strain that yielded 2.5 milligrams per liter of β-amyrin and 14 micrograms per liter of glycyrrhetinic acid. Introducing a cytochrome b5 from G. uralensis multiplied glycyrrhetinic acid output eightfold, and combining both strategies achieved a 40-fold improvement to 0.5 milligrams per liter in batch fermentation, extended to a 630-fold improvement reaching 8.78 milligrams per liter in fed-batch mode. The review cautions that plant P450 enzymes often show reduced catalytic activity in yeast and can interact poorly with reductases, generating reactive oxygen species that impair cell growth and product yield.

Taken together, the review paints a picture of a field converging on integrated solutions. Every elicitation regime, whether chemical, microbial or physical, works by provoking the plant’s in vitro defense machinery, and every approach shows a concentration ceiling beyond which toxicity erases the gains. The glycyrrhizin pathway is multifaceted and compartmentalized, limiting metabolite flux, and many of its genes and regulators remain unknown, while gene editing can sometimes merely redirect accumulation into other unintended metabolites. Even so, the authors conclude that the enhancements already demonstrated carry genuine commercial value, both for glycyrrhizin and its carbenoxolone derivative, and that the same elicitation, editing and engineering playbook is readily transferable to other plant secondary metabolites, positioning licorice biotechnology as a template for the sustainable production of high-value natural products worldwide.

Subject of Research: Biotechnological strategies for enhancing glycyrrhizin biosynthesis in licorice and engineered microbes

Article Title: An overview of strategies used for increasing Glycyrrhizin biosynthesis

Article References: Patil, S. P., Patil, R. R., Kore, S. D., & Raut, M. K. (2026). An overview of strategies used for increasing Glycyrrhizin biosynthesis. Discover Industrial Chemistry and Materials, 1(1), Article 7. https://doi.org/10.1007/s44508-026-00008-9

Image Credits: AI Generated

DOI: 10.1007/s44508-026-00008-9

Keywords: glycyrrhizin, licorice, Glycyrrhiza glabra, elicitation, CRISPR, Saccharomyces cerevisiae, secondary metabolites, metabolic engineering, hairy root cultures, triterpenoid saponin, glycyrrhetinic acid, plant tissue culture