Lettuce Costunolide Synthase (CYP71BL2) and Its Homolog (CYP71BL1) from Sunflower Catalyze Distinct Regio- and Stereoselective Hydroxylations in Sesquiterpene Lactone Metabolism

Abstract

Sesquiterpene lactones (STLs) are terpenoid natural products possessing the ␥-lactone, well known for their diverse biological and medicinal activities. The occurrence of STLs is sporadic in nature, but most STLs have been isolated from plants in the Asteraceae family. Despite the implication of the ␥-lactone group in many reported bioactivities of STLs, the biosynthetic origins of the ␥-lactone ring remains elusive. Germacrene A acid (GAA) has been suggested as a central precursor of diverse STLs. The regioselective (C6 or C8) and stereoselective (␣ or ␤) hydroxylation on a carbon of GAA adjacent to its carboxylic acid at C12 is responsible for the ␥-lactone formation. Here, we report two cytochrome P450 monooxygenases (P450s) capable of catalyzing 6␣-and 8␤-hydroxylation of GAA from lettuce and sunflower, respectively. To identify these P450s, sunflower trichomes were isolated to generate a trichome-specific transcript library, from which 10 P450 clones were retrieved. Expression of these clones in a yeast strain metabolically engineered to synthesize substrate GAA identified a P450 catalyzing 8␤-hydroxylation of GAA, but the STL was not formed by spontaneous lactonization. Subsequently, we identified the closest homolog of the GAA 8␤-hydroxylase from lettuce and discovered 6␣-hydroxylation of GAA by the recombinant enzyme. The resulting 6␣-hydroxy-GAA spontaneously undergoes a lactonization to yield the simplest form of STL, costunolide. Furthermore, we demonstrate the milligram per liter scale de novo synthesis of costunolide using the lettuce P450 in an engineered yeast strain, an important advance that will enable exploitation of STLs. Evolution and homology models of these two P450s are discussed.

De Novo Synthesis of High-Value Plant Sesquiterpenoids in Yeast

“…Another simple change that could improve the yield of sesquiterpenoids in yeast is the medium. With its high stability in yeast, pESC-Leu2d can be used to reconstitute one-gene (STS alone) and three-gene (STS, P450, and CPR) biosynthetic pathways as described here and elsewhere (Nguyen et al, 2010;Ro et al, 2008), or even a four-gene (STS, two P450s, and CPR) pathway (Ikezawa et al, 2011) in both selection SC and rich YPA media. However, there is no selection pressure when YPA medium is used, and whether the transgenic yeast maintains the Leu2d plasmid or not will primarily depend on the cytotoxicity of the sesquiterpenoids endogenously produced in yeast.…”

“…In this case, we often reduce medium acidification (to pH 6 after the same period of culture) by adding HEPES buffer to synthetic complete (SC) medium to a final concentration of 100 mM (pH 7.0-7.5) (Ikezawa et al, 2011;Nguyen et al, 2010). Tris-HCl and phosphate buffers are found to interfere with yeast growth.…”

“…To compensate for the weak promoter activity, the yeast increases the copy number of the Leu2d plasmid, and the cDNA coded in the plasmid thus has a higher level of expression than the standard Leu2 selectable marker. We have routinely used EPY300 and the Leu2d selectable marker to produce complex sesquiterpenoids with a yield of 10-500 mg L -1 yeast culture in shaken flasks without much optimization (Ikezawa et al, 2011;Nguyen et al, 2010;Ro et al, 2008).…”

Plant P450s as versatile drivers for evolution of species-specific chemical diversity

“…The same group of DaeKyun Ro identified the subsequent biosynthetic steps in the routes to lettuce (Lactuca sativa) and sunflower (Helianthus annus) sesquiterpene lactones, catalysed by the founding members of the CYP71BL subfamily. It was shown that lettuce CYP71BL1 and its sunflower homologue CYP71BL2 catalyse the distinct 6a -and 8b-hydroxylation of germacrene A acid, and the authors suggest that the sunflower P450 has evolved from an ancestral 6a-hydroxylase in certain Asteraceae lineages [42] (figure 3). The first P450 function to be detected in plants was described in Echinocystis macrocalpa (Cucurbitaceae), where the successive oxidative conversion of (2)-kaur-16-ene (ent-kaurene) to the corresponding 19-alcohol, 19-aldehyde and 19-acid was observed, which required oxygen and NADPH, and showed inhibition by carbon monoxide [43].…”

Molecular traits to elucidate the ancestry of Helianthus x multiflorus

“…Subsequently, key enzymes of the STL biosynthesis in Helianthus that are responsible for the transformation of farnesyldiphosphate into germacrene A (Fig. 1) and subsequent oxidation to germacrene A acid and its hydroxylated derivatives, have been identified and characterized from the transcriptome of glandular trichomes in the secretory stage (G€ opfert et al, 2005, 2009Nguyen et al, 2010;Ikezawa et al, 2011). This has now made it possible for the first time to test the conclusions from the chemical studies on H. x multiflorus with genomic sequences of genes participating in the STL synthesis of the taxa putatively involved in the origin of the hybrid.…”