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Identification of a Polyketide Synthase Gene in the Synthesis of Phleichrome of the Phytopathogenic Fungus Cladosporium phlei.

So KK, Chung YJ, Kim JM, Kim BT, Park SM, Kim DH - Mol. Cells (2015)

Bottom Line: Based on in silico analysis of cloned genes, we hypothesized that the non-reducing PKS gene, Cppks1, is involved in phleichrome biosynthesis.In addition, heterologous expression of the Cppks1 gene in Cryphonectria parasitica resulted in the production of phleichrome.These results provide convincing evidence that the Cppks1 gene is responsible for the biosynthesis of phleichrome.

View Article: PubMed Central - PubMed

Affiliation: Institute for Molecular Biology and Genetics, Chonbuk National University, Jeonju 561-756, Korea.

ABSTRACT
Phleichrome, a pigment produced by the phytopathogenic fungus Cladosporium phlei, is a fungal perylenequinone whose photodynamic activity has been studied intensively. To determine the biological function of phleichrome and to engineer a strain with enhanced production of phleichrome, we identified the gene responsible for the synthesis of phleichrome. Structural comparison of phleichrome with other fungal perylenequinones suggested that phleichrome is synthesized via polyketide pathway. We recently identified four different polyketide synthase (PKS) genes encompassing three major clades of fungal PKSs that differ with respect to reducing conditions for the polyketide product. Based on in silico analysis of cloned genes, we hypothesized that the non-reducing PKS gene, Cppks1, is involved in phleichrome biosynthesis. Increased accumulation of Cppks1 transcript was observed in response to supplementation with the application of synthetic inducer cyclo-(l-Pro-l-Phe). In addition, heterologous expression of the Cppks1 gene in Cryphonectria parasitica resulted in the production of phleichrome. These results provide convincing evidence that the Cppks1 gene is responsible for the biosynthesis of phleichrome.

No MeSH data available.


Related in: MedlinePlus

Semi-quantitative RT-PCR analysis of Cppks1 transcript levels relative to levels of β-tubulin (β-tub). Total RNA was extracted 6- and 18-days after induction. Accumulation of Cppks1 transcript was compared with and without an induction. Experimental results were normalized to β-tub gene and the PKS gene Cppks3, a member of the HR-PKS subclass, was analyzed as an internal control. Note that equal amounts of RNA samples were loaded as shown in the bottom panel by the expression level of β-tub gene showing similar band intensities among samples and a representative ethidium bromide-stained rRNA bands from one of three independent experiments.
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f2-molce-38-12-1105: Semi-quantitative RT-PCR analysis of Cppks1 transcript levels relative to levels of β-tubulin (β-tub). Total RNA was extracted 6- and 18-days after induction. Accumulation of Cppks1 transcript was compared with and without an induction. Experimental results were normalized to β-tub gene and the PKS gene Cppks3, a member of the HR-PKS subclass, was analyzed as an internal control. Note that equal amounts of RNA samples were loaded as shown in the bottom panel by the expression level of β-tub gene showing similar band intensities among samples and a representative ethidium bromide-stained rRNA bands from one of three independent experiments.

Mentions: In our previous studies, we found that phleichrome production was significantly increased by addition of 150 μM cyclo-(l-Pro-l-Phe) synthetic inducer into the culture media (Fig. 1). Thus, we analyzed levels of the Cppks1 transcript in response to the inducer using semi-quantitative RT-PCR. Total RNA was extracted from cultures at 6 and 18 days postinoculation. These timepoints were chosen because discernable pigmentation was observed on the PDA plates by 6 days postinoculation and the maximum amount of phleichrome was produced by 18 days postinoculation. As shown in Fig. 2, supplementation of the synthetic inducer into culture significantly increased the accumulation of Cppks1 transcript. The level of Cppks1 transcript increased along with incubation time, similar to the temporal pattern of phleichrome production under induction conditions. However, the expression of Cppks3, which is an HR-PKS, did not change in response to the synthetic inducer supplementation. The coordinated response of Cppks1 transcript to the inducer suggests that Cppks1 gene is associated with phleichrome biosynthesis.


Identification of a Polyketide Synthase Gene in the Synthesis of Phleichrome of the Phytopathogenic Fungus Cladosporium phlei.

So KK, Chung YJ, Kim JM, Kim BT, Park SM, Kim DH - Mol. Cells (2015)

Semi-quantitative RT-PCR analysis of Cppks1 transcript levels relative to levels of β-tubulin (β-tub). Total RNA was extracted 6- and 18-days after induction. Accumulation of Cppks1 transcript was compared with and without an induction. Experimental results were normalized to β-tub gene and the PKS gene Cppks3, a member of the HR-PKS subclass, was analyzed as an internal control. Note that equal amounts of RNA samples were loaded as shown in the bottom panel by the expression level of β-tub gene showing similar band intensities among samples and a representative ethidium bromide-stained rRNA bands from one of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4697002&req=5

f2-molce-38-12-1105: Semi-quantitative RT-PCR analysis of Cppks1 transcript levels relative to levels of β-tubulin (β-tub). Total RNA was extracted 6- and 18-days after induction. Accumulation of Cppks1 transcript was compared with and without an induction. Experimental results were normalized to β-tub gene and the PKS gene Cppks3, a member of the HR-PKS subclass, was analyzed as an internal control. Note that equal amounts of RNA samples were loaded as shown in the bottom panel by the expression level of β-tub gene showing similar band intensities among samples and a representative ethidium bromide-stained rRNA bands from one of three independent experiments.
Mentions: In our previous studies, we found that phleichrome production was significantly increased by addition of 150 μM cyclo-(l-Pro-l-Phe) synthetic inducer into the culture media (Fig. 1). Thus, we analyzed levels of the Cppks1 transcript in response to the inducer using semi-quantitative RT-PCR. Total RNA was extracted from cultures at 6 and 18 days postinoculation. These timepoints were chosen because discernable pigmentation was observed on the PDA plates by 6 days postinoculation and the maximum amount of phleichrome was produced by 18 days postinoculation. As shown in Fig. 2, supplementation of the synthetic inducer into culture significantly increased the accumulation of Cppks1 transcript. The level of Cppks1 transcript increased along with incubation time, similar to the temporal pattern of phleichrome production under induction conditions. However, the expression of Cppks3, which is an HR-PKS, did not change in response to the synthetic inducer supplementation. The coordinated response of Cppks1 transcript to the inducer suggests that Cppks1 gene is associated with phleichrome biosynthesis.

Bottom Line: Based on in silico analysis of cloned genes, we hypothesized that the non-reducing PKS gene, Cppks1, is involved in phleichrome biosynthesis.In addition, heterologous expression of the Cppks1 gene in Cryphonectria parasitica resulted in the production of phleichrome.These results provide convincing evidence that the Cppks1 gene is responsible for the biosynthesis of phleichrome.

View Article: PubMed Central - PubMed

Affiliation: Institute for Molecular Biology and Genetics, Chonbuk National University, Jeonju 561-756, Korea.

ABSTRACT
Phleichrome, a pigment produced by the phytopathogenic fungus Cladosporium phlei, is a fungal perylenequinone whose photodynamic activity has been studied intensively. To determine the biological function of phleichrome and to engineer a strain with enhanced production of phleichrome, we identified the gene responsible for the synthesis of phleichrome. Structural comparison of phleichrome with other fungal perylenequinones suggested that phleichrome is synthesized via polyketide pathway. We recently identified four different polyketide synthase (PKS) genes encompassing three major clades of fungal PKSs that differ with respect to reducing conditions for the polyketide product. Based on in silico analysis of cloned genes, we hypothesized that the non-reducing PKS gene, Cppks1, is involved in phleichrome biosynthesis. Increased accumulation of Cppks1 transcript was observed in response to supplementation with the application of synthetic inducer cyclo-(l-Pro-l-Phe). In addition, heterologous expression of the Cppks1 gene in Cryphonectria parasitica resulted in the production of phleichrome. These results provide convincing evidence that the Cppks1 gene is responsible for the biosynthesis of phleichrome.

No MeSH data available.


Related in: MedlinePlus