Molecular Evolution of Fern Squalene Cyclases

Abstract

Triterpenes, a diverse group of natural products comprising six isoprene units, are distributed across various organisms from bacteria to higher plants. Ferns are sporophytes that produce triterpenes and are lower on the evolutionary scale than higher plants. Among ferns that produce triterpenes analogous to bacterial hopanoids, Polypodiodes niponica produces migrated dammaranes and oleananes, which are also widely found in higher plants. Because the study of terpene-producing ferns could help us to understand the molecular basis of triterpene biosynthesis, cDNA cloning of squalene cyclases (SCs) from P. niponica was carried out. Two SCs (PNT and PNG) were obtained. The heterologously expressed PNT produces tirucalla-7,21-diene (67% major), and PNG produces germanicene (69%). Phylogenetic analysis revealed that PNT and PNG, which produce higher-plant-type migrated dammaranes and oleananes, are closely related to bacterial-type SCs. Furthermore, analysis of the minor products indicated that fern SCs gained the ability to directly form dammarenyl cations, which are key intermediates in oleanane formation during molecular evolution.

Cloning and Functional Analysis of Three Chalcone Synthases from the Flowers of Safflowers Carthamus tinctorius

“…To obtain the ORF of three CtCHS cDNAs, nested PCR was carried out using clone specific primers (Supplemental Table 1) with the same method as the authors' previous paper [22]. The 1.2-kb PCR product was digested with SacI and XhoI and ligated into the corresponding sites of pET-32a(+) (Novagen) to construct the expression plasmids.…”

Migrated Hopene Synthases from Colysis pothifolia and Identification of a Migration Switch Controlling the Number of 1,2‐Hydride and Methyl Shifts

“…[11] For the GS115 recombinant strains, the transformant was grown in yeast extract peptone dextrose (YPD) medium (50 mL;1 %y east extract, 2% peptone, and 2% glucose) at 30 8Cf or 24 hw ith shaking. [11] For the GS115 recombinant strains, the transformant was grown in yeast extract peptone dextrose (YPD) medium (50 mL;1 %y east extract, 2% peptone, and 2% glucose) at 30 8Cf or 24 hw ith shaking.…”

“…[11] Again, the triterpene hydrocarbon fractions were loaded onto am inicolumn and eluted with n-hexane. These fractions were analyzed by GC, which was performed on aH itachi G-6000 instrument with aD B-5HT column (30 m0.25 mm, Agilent Te chnologies) under the same conditions as in our previous paper.…”

“…The SCs from ferns characterizedt hus far produce 22hydroxyhopane, [9] dammara-18(28),21-diene, [10] germanicene, [11] and tirucalla-7,21-diene, [11] whereas all the functionally characterized SCs from bacteria are SHCs, the product of whichi s hop-22(29)-ene (7). Ferns have also been shown to be as ource of SCs with more variablec atalytic properties than those derived from bacteria.…”

“…No genetic information on the migration reaction responsible for skeletal rearrangements in SC-catalyzed reactions is available, despite rigorous efforts to isolate and biochemically characterize SCs.T he tirucalladiene synthase from the fern Polypodiodes niponica (PNT), [11] which produces the migrated dammarane tirucalla-7,21-diene, is the only SC. SCs yielding migrated hopane triterpenes, which are characteristicm etabolites of ferns, remain to be isolated.…”