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Identification of candidate genes affecting Delta9-tetrahydrocannabinol biosynthesis in Cannabis sativa.

Marks MD, Tian L, Wenger JP, Omburo SN, Soto-Fuentes W, He J, Gang DR, Weiblen GD, Dixon RA - J. Exp. Bot. (2009)

Bottom Line: Sequencing of over 2000 clones from the library resulted in the identification of over 1000 unigenes.One of these was identical to a previously reported chalcone synthase (CHS) and was found to have CHS activity.All three could use malonyl-CoA and hexanoyl-CoA as substrates, including the CHS, but reaction conditions were not identified that allowed for the production of olivetolic acid (the proposed product of the PKS activity needed for THCA synthesis).

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Biology, University of Minnesota, St Paul, MN 55108, USA. marks004@umn.edu

ABSTRACT
RNA isolated from the glands of a Delta(9)-tetrahydrocannabinolic acid (THCA)-producing strain of Cannabis sativa was used to generate a cDNA library containing over 100 000 expressed sequence tags (ESTs). Sequencing of over 2000 clones from the library resulted in the identification of over 1000 unigenes. Candidate genes for almost every step in the biochemical pathways leading from primary metabolites to THCA were identified. Quantitative PCR analysis suggested that many of the pathway genes are preferentially expressed in the glands. Hexanoyl-CoA, one of the metabolites required for THCA synthesis, could be made via either de novo fatty acids synthesis or via the breakdown of existing lipids. qPCR analysis supported the de novo pathway. Many of the ESTs encode transcription factors and two putative MYB genes were identified that were preferentially expressed in glands. Given the similarity of the Cannabis MYB genes to those in other species with known functions, these Cannabis MYBs may play roles in regulating gland development and THCA synthesis. Three candidates for the polyketide synthase (PKS) gene responsible for the first committed step in the pathway to THCA were characterized in more detail. One of these was identical to a previously reported chalcone synthase (CHS) and was found to have CHS activity. All three could use malonyl-CoA and hexanoyl-CoA as substrates, including the CHS, but reaction conditions were not identified that allowed for the production of olivetolic acid (the proposed product of the PKS activity needed for THCA synthesis). One of the PKS candidates was highly and specifically expressed in glands (relative to whole leaves) and, on the basis of these expression data, it is proposed to be the most likely PKS responsible for olivetolic acid synthesis in Cannabis glands.

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Biochemical pathways leading from primary metabolites to THCA showing candidate Cannabis unigenes encoding enzymes in the pathway. (A) Production of THCA. (B) De novo fatty acid pathway leading to the formation of hexanol. (C) Breakdown of fatty acids leading to the formation of hexanol. (D) MEP pathway leading to geranyl pyrophosphate. Candidate Cannabis unigenes are shown, as well as the relative gland over leaf expression ratios for a subset of the candidates (see Table 2).
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fig2: Biochemical pathways leading from primary metabolites to THCA showing candidate Cannabis unigenes encoding enzymes in the pathway. (A) Production of THCA. (B) De novo fatty acid pathway leading to the formation of hexanol. (C) Breakdown of fatty acids leading to the formation of hexanol. (D) MEP pathway leading to geranyl pyrophosphate. Candidate Cannabis unigenes are shown, as well as the relative gland over leaf expression ratios for a subset of the candidates (see Table 2).

Mentions: The specific biochemical steps leading to THCA are proposed to begin with a reaction involving a type III PKS enzyme that catalyses the synthesis of olivetolic acid from hexanoyl-CoA and three molecules of malonyl-CoA (Fig. 2A; Fellermeier et al., 2001). Malonyl-CoA is derived from the carboxylation of acetyl-CoA. ESTs encoding acetyl-CoA carboxylase were identified. Hexanoyl-CoA could be produced by more than one pathway in the trichomes. One route to produce hexanoyl-CoA would involve the early termination of the fatty acid biosynthetic pathway, yielding hexanoyl-ACP (Fig. 2B). The hexanoyl moiety would then be transferred to CoA by the action of an ACP-CoA transacylase or it would be cleaved by the action of a thioesterase, yielding n-hexanol, which would then be converted into n-hexanoyl-CoA by the action of acyl-CoA synthase. Most of the enzymes needed for this route are represented in the EST database, except for the transacylase and 2,3-trans-enoyl-ACP reductase (Fig. 2B). A second route to hexanoyl-CoA would involve the production of hexanol from the breakdown of the fatty acid linoleic acid via the lipoxygenase (LOX) pathway (Fig. 2C; Hatanaka, 1999). A survey of the sequenced ESTs revealed candidate genes encoding the enzymes needed to synthesize linoleic acid from acetyl-CoA by the typical fatty acid biosynthetic pathway in plastids followed by the production of hexanol from linoleic acid via the LOX pathway. An third pathway related to the biosynthesis of branched chain amino acids has been proposed to be involved in the production of short-chain and medium-chain fatty acids (Kroumova et al., 1994). However, the enzymes in this pathway [2-isopropylmalate synthase, 3-isopropylmalate dehydratase, 3-isopropylmalate dehydrogenase, and 2-oxoisovalerate dehydrogenase (acylating)] were not represented in the Cannabis trichome EST library.


Identification of candidate genes affecting Delta9-tetrahydrocannabinol biosynthesis in Cannabis sativa.

Marks MD, Tian L, Wenger JP, Omburo SN, Soto-Fuentes W, He J, Gang DR, Weiblen GD, Dixon RA - J. Exp. Bot. (2009)

Biochemical pathways leading from primary metabolites to THCA showing candidate Cannabis unigenes encoding enzymes in the pathway. (A) Production of THCA. (B) De novo fatty acid pathway leading to the formation of hexanol. (C) Breakdown of fatty acids leading to the formation of hexanol. (D) MEP pathway leading to geranyl pyrophosphate. Candidate Cannabis unigenes are shown, as well as the relative gland over leaf expression ratios for a subset of the candidates (see Table 2).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2736886&req=5

fig2: Biochemical pathways leading from primary metabolites to THCA showing candidate Cannabis unigenes encoding enzymes in the pathway. (A) Production of THCA. (B) De novo fatty acid pathway leading to the formation of hexanol. (C) Breakdown of fatty acids leading to the formation of hexanol. (D) MEP pathway leading to geranyl pyrophosphate. Candidate Cannabis unigenes are shown, as well as the relative gland over leaf expression ratios for a subset of the candidates (see Table 2).
Mentions: The specific biochemical steps leading to THCA are proposed to begin with a reaction involving a type III PKS enzyme that catalyses the synthesis of olivetolic acid from hexanoyl-CoA and three molecules of malonyl-CoA (Fig. 2A; Fellermeier et al., 2001). Malonyl-CoA is derived from the carboxylation of acetyl-CoA. ESTs encoding acetyl-CoA carboxylase were identified. Hexanoyl-CoA could be produced by more than one pathway in the trichomes. One route to produce hexanoyl-CoA would involve the early termination of the fatty acid biosynthetic pathway, yielding hexanoyl-ACP (Fig. 2B). The hexanoyl moiety would then be transferred to CoA by the action of an ACP-CoA transacylase or it would be cleaved by the action of a thioesterase, yielding n-hexanol, which would then be converted into n-hexanoyl-CoA by the action of acyl-CoA synthase. Most of the enzymes needed for this route are represented in the EST database, except for the transacylase and 2,3-trans-enoyl-ACP reductase (Fig. 2B). A second route to hexanoyl-CoA would involve the production of hexanol from the breakdown of the fatty acid linoleic acid via the lipoxygenase (LOX) pathway (Fig. 2C; Hatanaka, 1999). A survey of the sequenced ESTs revealed candidate genes encoding the enzymes needed to synthesize linoleic acid from acetyl-CoA by the typical fatty acid biosynthetic pathway in plastids followed by the production of hexanol from linoleic acid via the LOX pathway. An third pathway related to the biosynthesis of branched chain amino acids has been proposed to be involved in the production of short-chain and medium-chain fatty acids (Kroumova et al., 1994). However, the enzymes in this pathway [2-isopropylmalate synthase, 3-isopropylmalate dehydratase, 3-isopropylmalate dehydrogenase, and 2-oxoisovalerate dehydrogenase (acylating)] were not represented in the Cannabis trichome EST library.

Bottom Line: Sequencing of over 2000 clones from the library resulted in the identification of over 1000 unigenes.One of these was identical to a previously reported chalcone synthase (CHS) and was found to have CHS activity.All three could use malonyl-CoA and hexanoyl-CoA as substrates, including the CHS, but reaction conditions were not identified that allowed for the production of olivetolic acid (the proposed product of the PKS activity needed for THCA synthesis).

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Biology, University of Minnesota, St Paul, MN 55108, USA. marks004@umn.edu

ABSTRACT
RNA isolated from the glands of a Delta(9)-tetrahydrocannabinolic acid (THCA)-producing strain of Cannabis sativa was used to generate a cDNA library containing over 100 000 expressed sequence tags (ESTs). Sequencing of over 2000 clones from the library resulted in the identification of over 1000 unigenes. Candidate genes for almost every step in the biochemical pathways leading from primary metabolites to THCA were identified. Quantitative PCR analysis suggested that many of the pathway genes are preferentially expressed in the glands. Hexanoyl-CoA, one of the metabolites required for THCA synthesis, could be made via either de novo fatty acids synthesis or via the breakdown of existing lipids. qPCR analysis supported the de novo pathway. Many of the ESTs encode transcription factors and two putative MYB genes were identified that were preferentially expressed in glands. Given the similarity of the Cannabis MYB genes to those in other species with known functions, these Cannabis MYBs may play roles in regulating gland development and THCA synthesis. Three candidates for the polyketide synthase (PKS) gene responsible for the first committed step in the pathway to THCA were characterized in more detail. One of these was identical to a previously reported chalcone synthase (CHS) and was found to have CHS activity. All three could use malonyl-CoA and hexanoyl-CoA as substrates, including the CHS, but reaction conditions were not identified that allowed for the production of olivetolic acid (the proposed product of the PKS activity needed for THCA synthesis). One of the PKS candidates was highly and specifically expressed in glands (relative to whole leaves) and, on the basis of these expression data, it is proposed to be the most likely PKS responsible for olivetolic acid synthesis in Cannabis glands.

Show MeSH
Related in: MedlinePlus