Hernandulcin Production in Cell Suspensions of Phyla Scaberrima: Exploring Hernandulcin Accumulation through Physical and Chemical Stimuli

Abstract

Hernandulcin (HE) is a non‐caloric sweetener synthesized by the Mexican medicinal plant Phyla scaberrima. Herein we present the results of HE production through cell suspensions of P. scaberrima as well as the influence of pH, temperature, biosynthetic precursors and potential elicitors to enhance HE accumulation. The incorporation of mevalonolactone (30–400 mg L−1) farnesol (30–400 mg L−1), AgNO3 (0.025–0.175 M), cellulase (5–60 mg L−1; 0.3 units/mg), chitin (20–140 mg L−1) and (+)‐epi‐α‐bisabolol (300‐210 mg L−1) to the cell suspensions, resulted in a differential accumulation of HE and biomass. Among elicitors assayed, chitin, cellulase and farnesol increased HE production from 93.2 to ∼160 mg L−1 but, (+)‐epi‐α‐bisabolol (obtained by a synthetic biology approach) increased HE accumulation up to 182.7 mg L−1. HE produced by the cell suspensions was evaluated against nine strains from six species of gastrointestinal bacteria revealing moderate antibacterial activity (MIC, 214–465 μg mL−1) against Staphylococcus aureus, Escherichia coli and Helicobacter pylori. Similarly, HE showed weak toxicity against Lactobacillus sp. and Bifidobacterium bifidum (>1 mg mL−1), suggesting a selective antimicrobial activity on some species of gut microbiota. According to our results, chitin and (+)‐epi‐α‐bisabolol were the most effective molecules to enhance HE accumulation in cell suspensions of P. scaberrima.

Controlled Production of Carnosic Acid and Carnosol in Cell Suspensions of Lepechinia meyenii Treated with Different Elicitors and Biosynthetic Precursors

“…The addition of high concentrations of mevalonolactone (>300 mg L −1 ) did not exert a noteworthy change in the concentration of CA and CS nor in biomass accumulation ( Figure 4 C ). This result coincided with that reported in the cell suspension system of Phylla scaberrima feed with the same precursor [12] . Since mevalonate can be distributed in the biosynthesis of several terpenes including mono‐, sesqui‐, and triterpenes, small quantities of the precursor are probably destined for the biosynthesis of CA [12] .…”

“…Concentrations of 50–150 mg L −1 , produced a significant accumulation of CA and CS but concentrations over 200 mg L −1 were not linked with a clear increase of these diterpenes. This effect was also coincident with that observed in cell suspension of P. scaberrima in which the acyclic precursor farnesol was transformed into hernandulcin [12] . The absorption of these substances may be facilitated by the amphipathic character of geranylgeraniol and its ability to cross cell membranes [33] …”

“…On the other hand, pH over 8 caused a significant inhibition of cell accumulation. The toxic effect of acidic and alkaline pH has been previously demonstrated in the cells suspensions of S. officinalis and Phyla scaberrima [12,20] . This effect is probably associated with the oxidation of vitamins and other nutrients found in the culture media [20] .…”

“…Mevalonolactone is a water‐soluble form of mevalonic acid which is tagged as a general precursor of terpenes [12] . The addition of high concentrations of mevalonolactone (>300 mg L −1 ) did not exert a noteworthy change in the concentration of CA and CS nor in biomass accumulation ( Figure 4 C ).…”

“…This fact facilitates recovering bioactive metabolites with high purity avoiding the use of complex and expensive protocols of purification. Since cell suspensions are comprised of independent cells, elicitation can be efficiently applied in this system and the responses are usually quicker than those observed in the whole plant [12] . To the best of our knowledge, little is known about the ecological role of CA and possible elicitors able to stimulate its accumulation.…”