CRISPR/Cas9 targeted inactivation of the kauniolide synthase in chicory results in accumulation of costunolide and its conjugates in taproots

Abstract

Chicory taproots accumulate sesquiterpene lactones lactucin, lactucopicrin, and 8-deoxylactucin, predominantly in their oxalated forms. The biosynthetic pathway for chicory sesquiterpene lactones has only partly been elucidated; the enzymes that convert farnesyl pyrophosphate to costunolide have been described. The next biosynthetic step of the conversion of costunolide to the tricyclic structure, guaianolide kauniolide, has so far not been elucidated in chicory. In this work three putative kauniolide synthase genes were identified in chicory named CiKLS1, CiKLS2, and CiKLS3. Their activity to convert costunolide to kauniolide was demonstrated in vitro using yeast microsome assays. Next, introduction of CRISPR/Cas9 reagents into chicory protoplasts was used to inactivate multiple chicory KLS genes and several chicory lines were successfully regenerated. The inactivation of the kauniolide synthase genes in chicory by the CRISPR/Cas9 approach resulted in interruption of the sesquiterpene lactone biosynthesis in chicory leaves and taproots. In chicory taproots, but not in leaves, accumulation of costunolide and its conjugates was observed to high levels, namely 1.5 mg/g FW. These results confirmed that all three genes contribute to STL accumulation, albeit to different extent. These observations demonstrate that three genes oriented in tandem on the chicory genome encode kauniolide synthases that initiate the conversion of costunolide toward the sesquiterpene lactones in chicory.

Lactucin Synthase Inactivation Boosts the Accumulation of Anti-inflammatory 8-Deoxylactucin and Its Derivatives in Chicory (Cichorium intybus L.)

“…To study whether CYP71DD33, CYP71DD35, and CYP71DD20 indeed catalyze biosynthetic steps of chicory STL biosynthesis, the corresponding genes were next inactivated in chicory using CRISPR/Cas9-based genome editing. The CRISPR/Cas9 reagents were delivered to chicory protoplasts via transient transfection of two plasmids encoding the Cas9 protein and guide RNAs for CYP71DD33 or via transient transfection of the ribonucleoprotein complexes (RNPs) for CYP71DD35 and CYP71DD20 , as previously described. , …”

“…Multiple protein sequence alignments were performed using CLC genomics workbench 20.0.4 software (Qiagen). An unrooted phylogenetic tree was constructed to compare the protein sequences of CYP71DD20, CYP71DD33, and CYP71DD35 to other cytochrome P450 enzymes described to catalyze the hydroxylation of germacrene A, germacrene A acid, or costunolide, namely C. intybus germacrene A oxidase (CiGAO), 11 L. sativa germacrene A oxidase (LsGAO), 11 C. intybus costunolide synthase (CiCOS), 12 L. sativa costunolide synthase (LsCOS), 13 Helianthus annuus germacrene A acid 8β-hydroxylase (HaG8H), 17 Inula hupehensis germacrene A acid 8β-hydroxylase (IhG8H), 18 C. intybus kauniolide synthase 1 (CiKLS1), 14 Tanacetum parthenium kauniolide synthase (TpKLS), 15 H. annuus costunolide 14-hydroxylase (HaC14H), 16 T. parthenium 3beta hydroxylase (Tp3BH), 21 T. parthenium parthenolide synthase (TpPTS), 21 and H. annuus eupatolide synthase (HaES). 17 Bootstrap N-J trees were generated with 100 replicates of bootstrap analysis in CLC genomics workbench 20.0.4 software (Qiagen).…”

“…23 Inactivation of the three kauniolide synthase genes (CiKLS) in chicory led to the accumulation of costunolide and its conjugates in chicory taproots. 14 In this work, we describe the identification of the lactucin synthase in chicory and demonstrate that inactivation of the corresponding gene by CRISPR/Cas genome editing results in significant accumulation of 8-deoxylactucin and its derivatives that have anti-inflammatory properties in the taproot.…”

“…The biosynthetic pathway leading to chicory STLs has been partially elucidated (Figure ). The common terpene precursor FPP is first cyclized to germacrene A by the germacrene A synthase (CiGAS), and consequently, three cytochrome P450 enzymes, namely the germacrene A oxidase (CiGAO), costunolide synthase (CiCOS), , and kauniolide synthase (CiKLS) , catalyze the next steps in the STL biosynthesis to form kauniolide, with a specific tricyclic structure typical of guaianolide STLs. The following steps in the biosynthesis have not been elucidated but presumably involve further oxygenation of kauniolide on positions 2 and 15 by cytochrome P450 enzymes to yield 8-deoxylactucin.…”

“…The later steps in the STL biosynthesis, from kauniolide onward, appear to take place in the latex itself, as the gene encoding kauniolide synthase has latex-specific expression. 14 Several protocols for CRISPR/Cas9-based genome editing have been established for chicory. 22−24 Enzymes in the terpene biosynthetic pathway have been successfully inactivated in chicory using the CRISPR/Cas9 approach.…”