ABSTRACT
To develop an optimal bioprocess for secondary metabolite production and explain the bioprocess at the molecular level, we examine the synergistic effects of sequential treatment with methyl jasmonate (MJ), salicylic acid (SA) and yeast extract (YE) on benzophenanthridine alkaloid accumulation and protein expression in Eschscholtzia californica suspension cultures. Serial treatment of MJ, SA and YE at 24h intervals enhanced the accumulation of dihydrosanguinarine (2.5 times) and sanguinarine (5.5 times). This sequential treatment using different signal elicitors was more effective than single elicitor or simultaneous treatment of the elicitors; it induced benzophenanthridine alkaloid accumulation to 917.7+/-42.0mg/L. Also, (S)-methylcoclaurine-3'-hydroxylase (CYP80B1) and 3'-hydroxy-(S)-N-methylcoclaurine-4'-O-methyltransferase (4'OMT) expressions among enzymes in sanguinarine biosynthetic pathway explained the synergistic effects by sequential treatment of the elicitors. The sequential treatment strategy using elicitors related to different signal transduction pathways can be used to design better processes to increase accumulation of secondary metabolites in plant cell culture. Analysis of protein expression provides the detailed information about metabolite accumulation through the correlated results.
Subject(s)
Acetates/administration & dosage , Benzophenanthridines/metabolism , Cell Extracts/administration & dosage , Cyclopentanes/administration & dosage , Eschscholzia/metabolism , Oxylipins/administration & dosage , Plant Proteins/metabolism , Salicylic Acid/administration & dosage , Yeasts/chemistry , Alkaloids/metabolism , Cell Extracts/chemistry , Cells, Cultured , Dose-Response Relationship, Drug , Drug Combinations , Drug Synergism , Eschscholzia/drug effectsABSTRACT
Methyl jasmonate (MJ) and yeast extract (YE) induce protein expression and benzophenanthridine alkaloid accumulation in Eschscholtzia californica suspension cell cultures. One hundred microM MJ primarily induced dihydrosanguinarine 509.0+/-7.4 mg/l); 0.2 g/l YE induced sanguinarine (146.8+/- 3.8 mg/l) and an unknown compound. These results occur because dihydrobenzophenanthridine oxidase (DHBO) is induced by YE and not by MJ. YE and chitin (CHI) had similar effects on sanguinarine production and DHBO expression. Differential induction of secondary metabolites was shown in E. californica suspension cultures and the expression of proteins confirmed the metabolite results. Furthermore, treatment by various oligosaccharides helped us to understand the elicitation effect of YE in signal transduction pathways.
Subject(s)
Acetates/metabolism , Benzophenanthridines/biosynthesis , Cyclopentanes/metabolism , Eschscholzia/metabolism , Oxylipins/metabolism , Plant Proteins/metabolism , Yeasts/metabolism , Acetates/analysis , Benzophenanthridines/analysis , Biomass , Biosynthetic Pathways , Cells, Cultured , Cyclopentanes/analysis , Eschscholzia/chemistry , Eschscholzia/growth & development , Oxidoreductases/metabolism , Oxylipins/analysis , Plant Proteins/analysisABSTRACT
Production of the benzophenanthridine alkaloids in Eschscholtzia californica suspension cell cultures was optimized by adding 0.5 mg methyl jasmonate (MJ) and 0.02 mg salicylic acid (SA)/g FCW after 7 days cultivation. Sanguinarine reached 24 mg/g DCW by such treatment; 10 times higher than in control cell cultures. MJ and SA induced expression of berberine bridge enzyme and 3'-hydroxy-(S)-N-methylcoclaurine-4'-O-methyltransferase, respectively. MJ plus SA induced over-expression of both enzymes.
