Limits...
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

LC-MS analysis of phleichrome in recombinant C. parasitica. (A) TLC analysis of the ethyl acetate extracted pigment is shown. Lanes 1–4 show sample preparations from wild-type C. parasitica and three representative transformants (TNF#44, #16, and #56). Lane 5 shows purified phleichrome from previous studies (Lee et al., 2007) as a control. The arrow indicates the expected spot for phleichrome. (B) LCMS analysis of the methanol extract of the corresponding spots on the TLC plate. LC-MS profile of the extract from the representative recombinant C. parasitica (TNF#16) expressing the Cppks1 gene (ii) was compared to the profile of the extract from wild-type C. parasitica (iii) and the phleichrome standard (i). Note that the new peak corresponding to the retention time of purified phleichrome is marked by an asterisk. Mass analysis of the corresponding peak (*) from the recombinant C. parasitica matched that of the purified phleichrome.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4697002&req=5

f4-molce-38-12-1105: LC-MS analysis of phleichrome in recombinant C. parasitica. (A) TLC analysis of the ethyl acetate extracted pigment is shown. Lanes 1–4 show sample preparations from wild-type C. parasitica and three representative transformants (TNF#44, #16, and #56). Lane 5 shows purified phleichrome from previous studies (Lee et al., 2007) as a control. The arrow indicates the expected spot for phleichrome. (B) LCMS analysis of the methanol extract of the corresponding spots on the TLC plate. LC-MS profile of the extract from the representative recombinant C. parasitica (TNF#16) expressing the Cppks1 gene (ii) was compared to the profile of the extract from wild-type C. parasitica (iii) and the phleichrome standard (i). Note that the new peak corresponding to the retention time of purified phleichrome is marked by an asterisk. Mass analysis of the corresponding peak (*) from the recombinant C. parasitica matched that of the purified phleichrome.

Mentions: Because phleichrome is responsible for the characteristic deep red pigmentation in the mycelia and culture medium, we looked for changes in the colour of colonies in single-spored transformants. As shown in Fig. 3, two colonies became pinkish over time, in contrast to the original orange colour, as culture aged. However, no discernible changes in any of the other characteristics, including growth rate and sporulation, were observed in these transformants. Therefore, we assessed the presence of phleichrome in these selected transformants. TLC analysis using an EtOAc extract of the mycelia revealed the presence of yellowish pigment at an Rf value of 0.24, the same Rf as for purified phleichrome (Fig. 4A). However, these pigments were also present in wild-type C. parasitica. These results indicated that TLC was not sensitive enough to differentiate phleichrome from other residual pigments, such as skyrin and oxyskyrin. The amount of heterologous phleichrome produced may also have been too low for detection by TLC.


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)

LC-MS analysis of phleichrome in recombinant C. parasitica. (A) TLC analysis of the ethyl acetate extracted pigment is shown. Lanes 1–4 show sample preparations from wild-type C. parasitica and three representative transformants (TNF#44, #16, and #56). Lane 5 shows purified phleichrome from previous studies (Lee et al., 2007) as a control. The arrow indicates the expected spot for phleichrome. (B) LCMS analysis of the methanol extract of the corresponding spots on the TLC plate. LC-MS profile of the extract from the representative recombinant C. parasitica (TNF#16) expressing the Cppks1 gene (ii) was compared to the profile of the extract from wild-type C. parasitica (iii) and the phleichrome standard (i). Note that the new peak corresponding to the retention time of purified phleichrome is marked by an asterisk. Mass analysis of the corresponding peak (*) from the recombinant C. parasitica matched that of the purified phleichrome.
© Copyright Policy
Related In: Results  -  Collection

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

f4-molce-38-12-1105: LC-MS analysis of phleichrome in recombinant C. parasitica. (A) TLC analysis of the ethyl acetate extracted pigment is shown. Lanes 1–4 show sample preparations from wild-type C. parasitica and three representative transformants (TNF#44, #16, and #56). Lane 5 shows purified phleichrome from previous studies (Lee et al., 2007) as a control. The arrow indicates the expected spot for phleichrome. (B) LCMS analysis of the methanol extract of the corresponding spots on the TLC plate. LC-MS profile of the extract from the representative recombinant C. parasitica (TNF#16) expressing the Cppks1 gene (ii) was compared to the profile of the extract from wild-type C. parasitica (iii) and the phleichrome standard (i). Note that the new peak corresponding to the retention time of purified phleichrome is marked by an asterisk. Mass analysis of the corresponding peak (*) from the recombinant C. parasitica matched that of the purified phleichrome.
Mentions: Because phleichrome is responsible for the characteristic deep red pigmentation in the mycelia and culture medium, we looked for changes in the colour of colonies in single-spored transformants. As shown in Fig. 3, two colonies became pinkish over time, in contrast to the original orange colour, as culture aged. However, no discernible changes in any of the other characteristics, including growth rate and sporulation, were observed in these transformants. Therefore, we assessed the presence of phleichrome in these selected transformants. TLC analysis using an EtOAc extract of the mycelia revealed the presence of yellowish pigment at an Rf value of 0.24, the same Rf as for purified phleichrome (Fig. 4A). However, these pigments were also present in wild-type C. parasitica. These results indicated that TLC was not sensitive enough to differentiate phleichrome from other residual pigments, such as skyrin and oxyskyrin. The amount of heterologous phleichrome produced may also have been too low for detection by TLC.

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