Shotgun Proteomic Analysis of Yeast-Elicited California Poppy (Eschscholzia californica) Suspension Cultures Producing Enhanced Levels of Benzophenanthridine Alkaloids

Abstract

The California poppy, Eschscholzia californica, produces benzophenanthridine alkaloids (BPAs), an important class of biologically active compounds. Cell cultures of E. californica were investigated as an alternative and scalable method for producing these valuable compounds; treatment with yeast extract increased production from low levels to 23 mg/g dry weight (DW) of BPAs. A shotgun proteomic analysis of E. californica cell cultures was undertaken to explore changes in metabolism associated with enhanced BPA production. We implemented differential centrifugation and then shotgun proteomics based on nanoliquid chromatography/mass spectrometry (nano-LC-MS/MS) for peptide separation and analysis. A unigene database available for E. californica was translated and utilized for protein identification. Approximately 646 proteins (3% false discovery rate at the protein level) were identified. Differentially abundant proteins observed with elicitation included enzymes involved in (S)-adenosyl methionine (SAM) biosynthesis and BPA biosynthesis. These results demonstrate (1) the identification of proteins from a medicinal plant using shotgun proteomics combined with a well-annotated, translated unigene database and (2) the potential utility of proteomics for exploring changes in metabolism associated with enhanced secondary metabolite production.

Analysis of benzo[c]phenanthridine alkaloids inEschscholtzia californicacell culture using HPLC-DAD and HPLC-ESI-MS/MS

“…Fig. 1 represents the structures of eight target alkaloids together with their locations in the metabolic pathway proposed by Oldham et al 15,20) As can be seen, four dihydrobenzo[c]phenanthridines are the precursors of their relevant oxidized forms (end products) which have well known commercial values. The oxidized-forms of dihydrobenzo[c]phenanthridines could be easily separated using HPLC conditions described by Chen et al 8) However, the separation of dihydrobenzo[c]phenanthridines was extremely difficult, which might be caused by their subtle differences in hydrophobicity.…”

Proteins differentially expressed in elicited cell suspension culture of Podophyllum hexandrum with enhanced podophyllotoxin content

“…Proteomic studies in plant systems have primarily been performed in fully sequenced model systems such as Arabidopsis thaliana , Oryza sativa (rice), Populus trichocarpa (black cottonwood), and Vitis vinifera (grape vine) since mass spectrometry (MS)-based proteomics requires the availability of a protein database [25]. Relatively few studies have applied proteomics for investigating secondary metabolism of medicinal plants [26-28], and in particular have focused on applying proteomics for discovering new enzymes involved in secondary metabolism [29,30].…”

YeATSAM analysis of the walnut and chickpea transcriptome reveals key genes undetected by current annotation tools

“…Nt BBE was constitutively expressed in the Phytophthora infestans -resistant potato genotype SW93-1015 ( Ali et al , 2012). Benzophenanthridines are antimicrobial; the California poppy ( Eschscholzia californica ) is used as a traditional medicine ( Cheney, 1963; Oldham et al , 2010). Oral administration of the alkaloid berberine isolated from a Chinese herb lowered cholesterol in 32 hypercholesterolemic patients over three months ( Kong et al , 2004).…”