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Fungal polyketide synthase product chain-length control by partnering thiohydrolase.

Zabala AO, Chooi YH, Choi MS, Lin HC, Tang Y - ACS Chem. Biol. (2014)

Bottom Line: Fungal highly reducing polyketide synthases (HRPKSs) are an enigmatic group of multidomain enzymes that catalyze the biosynthesis of structurally diverse compounds.Bref-PKS demonstrated an NADPH-dependent reductive tailoring specificity that led to the synthesis of four different octaketide products with varying degrees of reduction.Furthermore, contrary to what is expected from the structure of BFA, Bref-PKS is found to be a nonaketide synthase in the absence of an associated thiohydrolase Bref-TH.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Biomolecular Engineering and ‡Department of Chemistry and Biochemistry, University of California , Los Angeles, California 90095, United States.

ABSTRACT
Fungal highly reducing polyketide synthases (HRPKSs) are an enigmatic group of multidomain enzymes that catalyze the biosynthesis of structurally diverse compounds. This variety stems from their intrinsic programming rules, which permutate the use of tailoring domains and determine the overall number of iterative cycles. From genome sequencing and mining of the producing strain Eupenicillium brefeldianum ATCC 58665, we identified an HRPKS involved in the biosynthesis of an important protein transport-inhibitor Brefeldin A (BFA), followed by reconstitution of its activity in Saccharomyces cerevisiae and in vitro. Bref-PKS demonstrated an NADPH-dependent reductive tailoring specificity that led to the synthesis of four different octaketide products with varying degrees of reduction. Furthermore, contrary to what is expected from the structure of BFA, Bref-PKS is found to be a nonaketide synthase in the absence of an associated thiohydrolase Bref-TH. Such chain-length control by the partner thiohydrolase was found to be present in other HRPKS systems and highlights the importance of including tailoring enzyme activities in predicting fungal HRPKS functions and their products.

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Related in: MedlinePlus

Transcriptional analysis of genes in Contig_286 determinestheputative boundary of the bref cluster. (A) Arrangementof genes in Contig_286. (B) RT-PCR analysis on the annotated geneswithin the contig. The template mRNA was extracted from a Day2 BFA-producingculture of E. brefeldianum in the optimized productionmedia, MEM.
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fig2: Transcriptional analysis of genes in Contig_286 determinestheputative boundary of the bref cluster. (A) Arrangementof genes in Contig_286. (B) RT-PCR analysis on the annotated geneswithin the contig. The template mRNA was extracted from a Day2 BFA-producingculture of E. brefeldianum in the optimized productionmedia, MEM.

Mentions: The genomic DNA of the BFA-producing strain E. brefeldianum ATCC 58665 was sequenced using Roche (454)GS FLX Titanium series and Illumina HiSeq 2000. The resulting GS FLXTitanium reads were first assembled using GS de novo assembler; theoutput contigs in FASTA format were then combined with the supplementaryHiSeq 2000 reads in a hybrid assembly using the Geneious Assemblerembedded in the Geneious software suite.19 The hybrid assembly generated 708 scaffolds consisting of nearly36 Mbases of nonredundant reads that roughly reflect the E.brefeldianum genome size. A local BLAST database was createdusing the scaffolds. Using the KS domain of the nonaketide synthaseLovB as a query sequence, 24 putative PKSs were identified: 11 HRPKSs;eight nonreducing PKSs (NRPKSs); two partially reducing PKSs (PRPKS);and two HRPKS-NRPS hybrids (Supplementary FigureS1). The lack of Cα-methylation in BFA excludedthe MT-containing HRPKSs, narrowing down the search to five HRPKS-containinggene clusters. Subsequently, RT-PCR was performed on the mRNA of theBFA-producing culture to determine the transcription of the HRPKSgenes, of which only Contig_286 PKS was highly transcribed at thetime point that coincided with BFA production (Figure 2 and Supplementary Figure S2),indicating the high likelihood of this HRPKS being involved in BFAbiosynthesis.


Fungal polyketide synthase product chain-length control by partnering thiohydrolase.

Zabala AO, Chooi YH, Choi MS, Lin HC, Tang Y - ACS Chem. Biol. (2014)

Transcriptional analysis of genes in Contig_286 determinestheputative boundary of the bref cluster. (A) Arrangementof genes in Contig_286. (B) RT-PCR analysis on the annotated geneswithin the contig. The template mRNA was extracted from a Day2 BFA-producingculture of E. brefeldianum in the optimized productionmedia, MEM.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215887&req=5

fig2: Transcriptional analysis of genes in Contig_286 determinestheputative boundary of the bref cluster. (A) Arrangementof genes in Contig_286. (B) RT-PCR analysis on the annotated geneswithin the contig. The template mRNA was extracted from a Day2 BFA-producingculture of E. brefeldianum in the optimized productionmedia, MEM.
Mentions: The genomic DNA of the BFA-producing strain E. brefeldianum ATCC 58665 was sequenced using Roche (454)GS FLX Titanium series and Illumina HiSeq 2000. The resulting GS FLXTitanium reads were first assembled using GS de novo assembler; theoutput contigs in FASTA format were then combined with the supplementaryHiSeq 2000 reads in a hybrid assembly using the Geneious Assemblerembedded in the Geneious software suite.19 The hybrid assembly generated 708 scaffolds consisting of nearly36 Mbases of nonredundant reads that roughly reflect the E.brefeldianum genome size. A local BLAST database was createdusing the scaffolds. Using the KS domain of the nonaketide synthaseLovB as a query sequence, 24 putative PKSs were identified: 11 HRPKSs;eight nonreducing PKSs (NRPKSs); two partially reducing PKSs (PRPKS);and two HRPKS-NRPS hybrids (Supplementary FigureS1). The lack of Cα-methylation in BFA excludedthe MT-containing HRPKSs, narrowing down the search to five HRPKS-containinggene clusters. Subsequently, RT-PCR was performed on the mRNA of theBFA-producing culture to determine the transcription of the HRPKSgenes, of which only Contig_286 PKS was highly transcribed at thetime point that coincided with BFA production (Figure 2 and Supplementary Figure S2),indicating the high likelihood of this HRPKS being involved in BFAbiosynthesis.

Bottom Line: Fungal highly reducing polyketide synthases (HRPKSs) are an enigmatic group of multidomain enzymes that catalyze the biosynthesis of structurally diverse compounds.Bref-PKS demonstrated an NADPH-dependent reductive tailoring specificity that led to the synthesis of four different octaketide products with varying degrees of reduction.Furthermore, contrary to what is expected from the structure of BFA, Bref-PKS is found to be a nonaketide synthase in the absence of an associated thiohydrolase Bref-TH.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Biomolecular Engineering and ‡Department of Chemistry and Biochemistry, University of California , Los Angeles, California 90095, United States.

ABSTRACT
Fungal highly reducing polyketide synthases (HRPKSs) are an enigmatic group of multidomain enzymes that catalyze the biosynthesis of structurally diverse compounds. This variety stems from their intrinsic programming rules, which permutate the use of tailoring domains and determine the overall number of iterative cycles. From genome sequencing and mining of the producing strain Eupenicillium brefeldianum ATCC 58665, we identified an HRPKS involved in the biosynthesis of an important protein transport-inhibitor Brefeldin A (BFA), followed by reconstitution of its activity in Saccharomyces cerevisiae and in vitro. Bref-PKS demonstrated an NADPH-dependent reductive tailoring specificity that led to the synthesis of four different octaketide products with varying degrees of reduction. Furthermore, contrary to what is expected from the structure of BFA, Bref-PKS is found to be a nonaketide synthase in the absence of an associated thiohydrolase Bref-TH. Such chain-length control by the partner thiohydrolase was found to be present in other HRPKS systems and highlights the importance of including tailoring enzyme activities in predicting fungal HRPKS functions and their products.

Show MeSH
Related in: MedlinePlus