Discovery of a non‐stereoselective cytochrome P450 catalyzing either 8α‐ or 8β‐hydroxylation of germacrene A acid from the Chinese medicinal plant, Inula hupehensis

Abstract

These authors contributed equally to this work. SUMMARYSesquiterpene lactones (STLs) are C15 terpenoid natural products with a-methylene c-lactone moiety. A large proportion of STLs in Asteraceae species is derived from the central precursor germacrene A acid (GAA). Formation of the lactone rings depends on the regio-(C6 or C8) and stereoselective (a-or b-)hydroxylations of GAA, producing STLs with four distinct stereo-configurations (12,6a-, 12,6b-, 12,8a-, and 12,8b-olide derivatives of GAA) in nature. Curiously, two configurations of STLs (C12,8a and C12,8b) are simultaneously present in the Chinese medicinal plant, Inula hupehensis. However, how these related yet distinct STL stereo-isomers are co-synthesized in I. hupehensis remains unknown. Here, we describe the functional identification of the I. hupehensis cytochrome P450 (CYP71BL6) that can catalyze the hydroxylation of GAA in either 8a-or 8b-configuration, resulting in the synthesis of both 8a-and 8b-hydroxyl GAAs. Of these two products, only 8a-hydroxyl GAA spontaneously lactonizes to the C12,8a-STL while the 8b-hydroxyl GAA remains stable without lactonization. Chemical structures of the C12,8a-STL, named inunolide, and 8b-hydroxyl GAA were fully elucidated by nuclear magnetic resonance analysis and mass spectrometry. The CYP71BL6 displays 63-66% amino acid identity to the previously reported CYP71BL1/2 catalyzing GAA 6a-or 8b-hydroxylation, indicating CYP71BL6 shares the same evolutionary lineage with other stereoselective cytochrome P450s, but catalyzes hydroxylation in a non-stereoselective manner. We observed that the CYP71BL6 transcript abundance correlates closely to the accumulation of C12,8-STLs in I. hupehensis. The identification of CYP71BL6 provides an insight into the biosynthesis of STLs in Asteraceae.

Mikania micrantha genome provides insights into the molecular mechanism of rapid growth

“…STLs, which are mainly derived from the precursor germacrene A acid (GAA), are known for their allelopathic effects and are characteristic of the family Asteraceae 19,33,34 . Several enzymes acting downstream of GAA have been elucidated recently, such as HaG8H, LsCOS, and Ih8H, that lead to the formation of different STLs with distinctive stereochemistry [35][36][37] . However, the biosynthetic pathways of STLs in Mikania remain unknown.…”

“…However, the biosynthetic pathways of STLs in Mikania remain unknown. Lactone ring formation in STLs is dependent upon the (C6 or C8) position and stereoselective hydroxylations of GAA, which can result in STLs with four unique configurations (12, 6α-, 12, 6β-, 12, 8α-, and 12, 8β-olides derivatives of GAA) 37 . Ih8H homolog is important enzyme convert to 7, 8-trans lactone from GAA.…”

Traps and Pitfalls—Unspecific Reactions in Metabolic Engineering of Sesquiterpenoid Pathways

“…Recently, it was shown that acid-induced rearrangement may also play a role in plant expression systems and may form rearrangement product combinations with Michael-type adducts [13]. To overcome acid-induced rearrangement in yeast culture, yeast cultures can be buffered [7,39]. The use of MOPS buffer has proven to be better compatible with yeast growth than HEPES buffer [7,39].…”

“…To overcome acid-induced rearrangement in yeast culture, yeast cultures can be buffered [7,39]. The use of MOPS buffer has proven to be better compatible with yeast growth than HEPES buffer [7,39]. If an acidified HPLC system is necessary, run times can be reduced and when preparative HPLC runs are performed, fractions can be collected in neutral buffer [10,39].…”

“…The use of MOPS buffer has proven to be better compatible with yeast growth than HEPES buffer [7,39]. If an acidified HPLC system is necessary, run times can be reduced and when preparative HPLC runs are performed, fractions can be collected in neutral buffer [10,39]. The right choice of SPME fibers reduces the acid-induced conversion of enzyme products in SPME-GC-MS analysis [32].…”