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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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Comparison of potential products produced by two or three decarboxylative condensation reactions. (A) Prediction of pyrone production from two decarboxylative condensations. (B) Expected product from three decarboxylative condensations.
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fig5: Comparison of potential products produced by two or three decarboxylative condensation reactions. (A) Prediction of pyrone production from two decarboxylative condensations. (B) Expected product from three decarboxylative condensations.

Mentions: It has been proposed that either olivetol or olivetolic acid are products of polyketide synthase in the THCA pathway (Raharjo et al., 2004b). However assays of plant extracts found that olivetol, the decarboxylation product of olivetolic acid, was not a substrate in the pathway (Fellermeier and Zenk, 1998). Products of the three PKS genes identified in this study were tested for olivetolic acid synthesis in vitro. CAN24 and CAN383 yielded identical products according to HPLC analysis (data not shown). Because CAN24 was more abundant, this PKS gene was analysed in detail, along with CAN1069, which had CHS activity as shown in Fig. 4. The size of the product produced by the CAN24-encoded enzyme was smaller than olivetolic acid (Fig. 4F). Further, the absorption spectrum did not match olivetol. A sequence identical to CAN24 has been deposited in the NCBI database (accession AB164375) and was annotated as having olivetol synthase activity but without supporting data. The product generated in our analyses possibly represents a derailment product in which the enzyme catalyses two decarboxylative condensations instead of three (Fig. 5). The failure of in vitro conditions to support the complete synthesis of native PKS products is well-documented (Springob et al., 2007). The artificial nature of in vitro reaction products is reflected by the failure to find these products in vivo (Austin and Noel, 2003). This may indicate that reaction conditions were insufficient or that accessory proteins may be needed to produce olivetolic acid. Similar results were obtained in a study that characterized a gene identical to CAN1069 where the enzyme could use hexanoyl-CoA as a starter molecule, but only yielded a possible derailment product (Raharjo et al., 2004b). That plant extracts used in in vitro assays have also not yielded olivetolic acid suggests that in vivo-like reaction conditions have yet to be imitated (Raharjo et al., 2004a; Flores-Sanchez and Verpoorte, 2008a).


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)

Comparison of potential products produced by two or three decarboxylative condensation reactions. (A) Prediction of pyrone production from two decarboxylative condensations. (B) Expected product from three decarboxylative condensations.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig5: Comparison of potential products produced by two or three decarboxylative condensation reactions. (A) Prediction of pyrone production from two decarboxylative condensations. (B) Expected product from three decarboxylative condensations.
Mentions: It has been proposed that either olivetol or olivetolic acid are products of polyketide synthase in the THCA pathway (Raharjo et al., 2004b). However assays of plant extracts found that olivetol, the decarboxylation product of olivetolic acid, was not a substrate in the pathway (Fellermeier and Zenk, 1998). Products of the three PKS genes identified in this study were tested for olivetolic acid synthesis in vitro. CAN24 and CAN383 yielded identical products according to HPLC analysis (data not shown). Because CAN24 was more abundant, this PKS gene was analysed in detail, along with CAN1069, which had CHS activity as shown in Fig. 4. The size of the product produced by the CAN24-encoded enzyme was smaller than olivetolic acid (Fig. 4F). Further, the absorption spectrum did not match olivetol. A sequence identical to CAN24 has been deposited in the NCBI database (accession AB164375) and was annotated as having olivetol synthase activity but without supporting data. The product generated in our analyses possibly represents a derailment product in which the enzyme catalyses two decarboxylative condensations instead of three (Fig. 5). The failure of in vitro conditions to support the complete synthesis of native PKS products is well-documented (Springob et al., 2007). The artificial nature of in vitro reaction products is reflected by the failure to find these products in vivo (Austin and Noel, 2003). This may indicate that reaction conditions were insufficient or that accessory proteins may be needed to produce olivetolic acid. Similar results were obtained in a study that characterized a gene identical to CAN1069 where the enzyme could use hexanoyl-CoA as a starter molecule, but only yielded a possible derailment product (Raharjo et al., 2004b). That plant extracts used in in vitro assays have also not yielded olivetolic acid suggests that in vivo-like reaction conditions have yet to be imitated (Raharjo et al., 2004a; Flores-Sanchez and Verpoorte, 2008a).

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