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A Novel Class of Plant Type III Polyketide Synthase Involved in Orsellinic Acid Biosynthesis from Rhododendron dauricum

View Article: PubMed Central - PubMed

ABSTRACT

Rhododendron dauricum L. produces daurichromenic acid, the anti-HIV meroterpenoid consisting of sesquiterpene and orsellinic acid (OSA) moieties. To characterize the enzyme responsible for OSA biosynthesis, a cDNA encoding a novel polyketide synthase (PKS), orcinol synthase (ORS), was cloned from young leaves of R. dauricum. The primary structure of ORS shared relatively low identities to those of PKSs from other plants, and the active site of ORS had a unique amino acid composition. The bacterially expressed, recombinant ORS accepted acetyl-CoA as the preferable starter substrate, and produced orcinol as the major reaction product, along with four minor products including OSA. The ORS identified in this study is the first plant PKS that generates acetate-derived aromatic tetraketides, such as orcinol and OSA. Interestingly, OSA production was clearly enhanced in the presence of Cannabis sativa olivetolic acid cyclase, suggesting that the ORS is involved in OSA biosynthesis together with an unidentified cyclase in R. dauricum.

No MeSH data available.


Related in: MedlinePlus

Secreted production of polyketides by the transgenic P. pastoris harboring the ORS gene. Induction time indicates the period after the onset of ORS expression by feeding media containing methanol. The polyketide data are means ± SD of triplicate determinations by HPLC.
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Figure 8: Secreted production of polyketides by the transgenic P. pastoris harboring the ORS gene. Induction time indicates the period after the onset of ORS expression by feeding media containing methanol. The polyketide data are means ± SD of triplicate determinations by HPLC.

Mentions: In this study, we also tried to heterologously express ORS in the methylotrophic yeast P. pastoris, in order to partially mimic the situation in the plant cells, and to determine whether the catalytic properties of ORS are altered in vivo. The liquid culture of transgenic P. pastoris secreted all of the expected polyketide metabolites into the culture medium (Figure 8), and negligible amounts of products were detected in the cellular extracts throughout the culture period. In contrast, no polyketide products accumulated in the culture of the control P. pastoris transformed with an empty vector. Therefore, ORS is functionally expressed in Pichia cells, and it synthesized polyketides from endogenous substrates and effectively secreted the products into the medium. The secreted polyketides from the transgenic Pichia reached the maximum at 96 h after the onset of protein expression, with product proportions similar to those of the in vitro ORS reaction (Figure 8). This result suggested that orcinol (or methyl tetra-β-ketide CoA) is the major product of ORS even in an in vivo environment.


A Novel Class of Plant Type III Polyketide Synthase Involved in Orsellinic Acid Biosynthesis from Rhododendron dauricum
Secreted production of polyketides by the transgenic P. pastoris harboring the ORS gene. Induction time indicates the period after the onset of ORS expression by feeding media containing methanol. The polyketide data are means ± SD of triplicate determinations by HPLC.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037138&req=5

Figure 8: Secreted production of polyketides by the transgenic P. pastoris harboring the ORS gene. Induction time indicates the period after the onset of ORS expression by feeding media containing methanol. The polyketide data are means ± SD of triplicate determinations by HPLC.
Mentions: In this study, we also tried to heterologously express ORS in the methylotrophic yeast P. pastoris, in order to partially mimic the situation in the plant cells, and to determine whether the catalytic properties of ORS are altered in vivo. The liquid culture of transgenic P. pastoris secreted all of the expected polyketide metabolites into the culture medium (Figure 8), and negligible amounts of products were detected in the cellular extracts throughout the culture period. In contrast, no polyketide products accumulated in the culture of the control P. pastoris transformed with an empty vector. Therefore, ORS is functionally expressed in Pichia cells, and it synthesized polyketides from endogenous substrates and effectively secreted the products into the medium. The secreted polyketides from the transgenic Pichia reached the maximum at 96 h after the onset of protein expression, with product proportions similar to those of the in vitro ORS reaction (Figure 8). This result suggested that orcinol (or methyl tetra-β-ketide CoA) is the major product of ORS even in an in vivo environment.

View Article: PubMed Central - PubMed

ABSTRACT

Rhododendron dauricum L. produces daurichromenic acid, the anti-HIV meroterpenoid consisting of sesquiterpene and orsellinic acid (OSA) moieties. To characterize the enzyme responsible for OSA biosynthesis, a cDNA encoding a novel polyketide synthase (PKS), orcinol synthase (ORS), was cloned from young leaves of R. dauricum. The primary structure of ORS shared relatively low identities to those of PKSs from other plants, and the active site of ORS had a unique amino acid composition. The bacterially expressed, recombinant ORS accepted acetyl-CoA as the preferable starter substrate, and produced orcinol as the major reaction product, along with four minor products including OSA. The ORS identified in this study is the first plant PKS that generates acetate-derived aromatic tetraketides, such as orcinol and OSA. Interestingly, OSA production was clearly enhanced in the presence of Cannabis sativa olivetolic acid cyclase, suggesting that the ORS is involved in OSA biosynthesis together with an unidentified cyclase in R. dauricum.

No MeSH data available.


Related in: MedlinePlus