Scientists from Osaka University, in collaboration with the National Agriculture and Food Research Organization (NARO), RIKEN, and Chiba University, report the discovery of a vital link in the complex biochemical pathway for saponin synthesis.

The researchers believe their finding will help pave the way for improving the commercial production of these high-value products, which have shown anticarcinogenic and antioxidant properties as well as antiviral and other pharmacological activities.

The team published its study “A cellulose synthase-derived enzyme catalyzes 3-O-glucuronosylation in saponin biosynthesis” in Nature Communications.

“Triterpenoid saponins are specialized metabolites distributed widely in the plant kingdom that consist of one or more sugar moieties attached to triterpenoid aglycones. Despite the widely accepted view that glycosylation is catalyzed by UDP-dependent glycosyltransferase (UGT), the UGT which catalyzes the transfer of the conserved glucuronic acid moiety at the C-3 position of glycyrrhizin and various soyasaponins has not been determined,” write the investigators.

Engineered biosynthetic pathway for production of glycyrrhizin in yeast
Engineered biosynthetic pathway for production of glycyrrhizin in yeast. The complete glycyrrhizin biosynthetic pathway was constructed by expressing β-amyrin synthase (βAS), two cytochrome P450s (CYP88D6 and CYP72A63), CSyGT, UGT73P12 and UDP-glucose dehydrogenase (UGD) in yeast. The engineered yeast utilizes endogenous metabolites (2,3-oxidosqualene and UDP-glucose) as substrates, and thus able to produce glycyrrhizin from simple sugars. [Osaka University]
“Here, we report that a cellulose synthase superfamily-derived glycosyltransferase (CSyGT) catalyzes 3-O-glucuronosylation of triterpenoid aglycones. Gene co-expression analyses of three legume species (Glycyrrhiza uralensis, Glycine max, and Lotus japonicus) reveal the involvement of CSyGTs in saponin biosynthesis, and we characterize CSyGTs in vivo using Saccharomyces cerevisiae.

CSyGT mutants of L. japonicus do not accumulate soyasaponin, but the ectopic expression of endoplasmic reticulum membrane–localized CSyGTs in a L. japonicus mutant background successfully complement soyasaponin biosynthesis. Finally, we produced glycyrrhizin de novo in yeast, paving the way for sustainable production of high-value saponins.”

The researchers studied the co-expression gene network of saponin synthesis using gene cloning and sequence comparisons, coupled with biochemical analyses in mutants and genetically modified plants of a model legume species. They discovered a new enzyme in the CSyGT family that is similar in structure to the enzymes producing cellulose in plant cell walls.

Unexpectedly, they showed that the new member of the family was responsible for a key step in saponin synthesis, where a sugar molecule is attached to the triterpenoid backbone. This discovery challenged the generally accepted view that a different class of enzyme was probably involved in this step, according to the scientists.

They went on to insert the gene for the newly discovered CSyGT enzyme, along with genes for other steps in the biochemical pathway, into yeast cells. The engineered cells successfully produced glycyrrhizin from simple sugars, indicating a potential route for industrial manufacture of saponins by growing yeast cells on a large scale.

“Our multi-disciplinary team showed, for the first time, that this type of enzyme is important in saponin synthesis,” says corresponding author Toshiya Muranaka, PhD, in the department of biotechnology, graduate school of engineering, Osaka University. “Our results fill a gap in previous knowledge and also challenge the accepted view of how this pathway for biosynthesis operates.”

“We showed that yeast cells can make glycyrrhizin when we insert the necessary plant genes,” explains Soo Yeon Chung, also in Osaka’s department of biotechnology. “This offers the prospect of new ways to produce these valuable substances commercially, and to generate completely novel types of saponin that might have further beneficial applications in medicine or the food industry.

Team members add that producing the chemicals in cell cultures would also reduce the need to deplete natural plant resources and help to meet sustainable development goals.

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