Cloning and Functional Characterization of Cycloartenol Synthase from the Red Seaweed Laurencia dendroidea

Abstract

The red seaweed Laurencia dendroidea belongs to the Rhodophyta, a phylum of eukaryotic algae that is widely distributed across the oceans and that constitute an important source of bioactive specialized metabolites. Laurencia species have been studied since 1950 and were found to contain a plethora of specialized metabolites, mainly halogenated sesquiterpenes, diterpenes and triterpenes that possess a broad spectrum of pharmacological and ecological activities. The first committed step in the biosynthesis of triterpenes is the cyclization of 2,3-oxidosqualene, an enzymatic reaction carried out by oxidosqualene cyclases (OSCs), giving rise to a broad range of different compounds, such as the sterol precursors cycloartenol and lanosterol, or triterpene precursors such as cucurbitadienol and β-amyrin. Here, we cloned and characterized the first OSC from a red seaweed. The OSC gene was identified through mining of a L. dendroidea transcriptome dataset and subsequently cloned and heterologously expressed in yeast for functional characterization, which indicated that the corresponding enzyme cyclizes 2,3-oxidosqualene to the sterol precursor cycloartenol. Accordingly, the gene was named L. dendroidea cycloartenol synthase (LdCAS). A phylogenetic analysis using OSCs genes from plants, fungi and algae revealed that LdCAS grouped together with OSCs from other red algae, suggesting that cycloartenol could be the common product of the OSC in red seaweeds. Furthermore, profiling of L. dendroidea revealed cholesterol as the major sterol accumulating in this species, implicating red seaweeds contain a ‘hybrid’ sterol synthesis pathway in which the phytosterol precursor cycloartenol is converted into the major animal sterol cholesterol.

Marine natural products

“…This report also presented rened NMR spectral assignments for palytoxin and claimed this as the rst isolation of the toxin from a marine plant. 381 Other studies included the diel variation of sesquiterpene elatol 382 production: a chemical defense mechanism in Laurencia dendroidea, 383 the geographic distribution along the SE Brazilian coast of natural products produced by L. dendroidea, 384 a study on the sterol biosynthesis pathway in L. dendroidea by the cloning and functional characterisation of a cycloartenol cyclase, 385 the biological activities of known compounds from three edible Gracilaria spp., 386 and the design and synthesis of analogues of galaxamide (Galaxaura lamentosa) 387 as potential antitumor agents. 388 Other synthetic studies included the rst total synthesis of an antibacterial polyhalogenated monoterpene, (À)-anverene (Plocamium cartilagineum 389 ), 390 and the total syntheses 391 of the Laurencia marilzae metabolites 12-epoxyobtusallene IV, 392 obtusallene X, 392 and marilzabicycloallenes C and D. 393…”

Inferring Biochemical Reactions and Metabolite Structures to Understand Metabolic Pathway Drift

“…We decided to use cycloartenol even if we did not find it by gas chromatography (GC)-MS for the two following reasons. First, a cycloartenol synthase from the red alga Laurencia dendroidea was cloned and expressed in yeast cells, where it is able to transform squalene into cycloartenol, even if the authors did not report cycloartenol identification in the whole alga by GC-MS (Calegario et al, 2016). Second, unambiguous cycloartenol derivatives are known in another florideophyte red alga, Tricleocarpa fragilis (Horgen et al, 2000).…”

Change in Bitterness, Accumulation of Cucurbitacin B and Expression Patterns of CuB Biosynthesis-related Genes in Melon During Fruit Development

“…Cucurbitacins synthesis starts with the cyclization of 2,3-oxidosqualene to cucurbitadienol, which is primarily determined by oxidosqualene cyclases (OSCs). Indeed, several sequences encoding for OSCs have been cloned and characterized in many plants (Andre et al, 2016;Calegario et al, 2016;Davidovich-Rikanati et al, 2015;Dhar et al, 2014;Pensec et al, 2016;Zheng et al, 2015). Three OSC cDNAs (CPX, CPQ and CPR) were isolated from pumpkin seedlings, and CPQ encoded cucurbitadienol synthase, which was the first committed enzyme for cucurbitacins biosynthesis in plants (Shibuya et al, 2004).…”