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- 주제어 : Type III polyketide synthase (검색결과 8 건)
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Molecular characterization of two alkylresorcylic acid synthases from <i>Sordariomycetes</i> fungi
Ramakrishnan, Dhivya,Tiwari, Manish K.,Manoharan, Gomathi,Sairam, Thiagarajan,Thangamani, Rajesh,Lee, Jung-Kul,Marimuthu, Jeya Elsevier 2018 Enzyme and microbial technology Vol.115 No.-
<P><B>Abstract</B></P> <P>Two putative type III polyketide synthase genes (PKS) were identified from Sordariomycetes fungi. These two type III PKS genes from <I>Sordaria macrospora</I> (SmPKS) and <I>Chaetomium thermophilum</I> (CtPKS), shared 59.8% sequence identity. Both, full-length and truncated versions of type III PKSs were successfully cloned and overexpressed in a bacterial host, <I>Escherichia Coli</I> BL21 (DE3) using a N-terminus hexa-histidine tag. The full-length and the truncated construct of PKSs showed similar activity profiles, suggesting that additional amino acid residues at the C-terminal of both SmPKS and CtPKS may not be involved in catalytic functions. We demonstrate that these two recombinant polyketide synthases could efficiently synthesize tri- and tetraketide pyrones, resorcinols and resorcylic acids using various acyl-CoAs (C<SUB>4</SUB>–C<SUB>20</SUB>) as starter units. The truncated <I>S. macrospora</I> polyketide synthases (TrSmPKS) showed a maximum of 7.0 × 10<SUP>4</SUP> s<SUP>−1</SUP> M<SUP>−1</SUP> catalytic efficiency towards stearoyl-CoA.Whereas, truncated <I>C. thermophilum</I> polyketide synthases (TrCtPKS) preferred the long-chain acyl-CoA starter arachidoyl-CoA, to produce pentaketide and hexaketide resorcinols with a high catalytic efficiency of 6.2 × 10<SUP>4</SUP> s<SUP>−1</SUP> M<SUP>−1</SUP>. Homology model and substrate docking analyses suggest a shorter distance between sulfur of catalytic Cys152 and thioester carbonyl group of arachidoyl-CoA as well as stronger imidazolium–thiolate ion pair distance in TrCtPKS between catalytic Cys152-His309 compared to TrSmPKS- arachidoyl CoA complex. Enhanced binding interactions of CtPKS residues forming intermolecular contacts at the active site could be attributed to its high specificity towards arachidoyl-CoA. This study reports the functional characterization of two fungal type III polyketide synthases, SmPKS and CtPKS with high catalytic efficiency from S. <I>macrospora</I> and C. <I>thermophilum</I> respectively. Furthermore, the results suggested that the both SmPKS and CtPKS could be attractive targets for protein engineering to discern the unique substrate specificity and catalytic efficiency.</P> <P><B>Highlights</B></P> <P> <UL> <LI> The study reports the functional characterization of two fungal type III polyketide synthases, SmPKS and CtPKS with high catalytic efficiency. </LI> <LI> Both the recombinant PKSs efficiently synthesize pyrones, resorcylic acids and resorcinols using various acyl- CoA (C<SUB>4</SUB>–C<SUB>20</SUB>) as starter units. </LI> <LI> TrCtPKS produces pentaketide and hexaketide resorcinols from arachidoyl-CoA with a high catalytic efficiency of 6.2 × 10<SUP>4</SUP> s<SUP>−1</SUP> M<SUP>−1</SUP>. </LI> <LI> Enhanced binding interactions of CtPKS residues forming intermolecular contacts at the active site could be attributed to its high specificity. </LI> </UL> </P>
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Lan-Qing Ma,Yan-Wu Guo,Hui-Li Guo,Xing Li,Li-Li Huang,Bo-Ning Zhang,Xiao-Bin Pang,Ben-Ye Liu,Hong Wang 한국식물생명공학회 2013 Plant biotechnology reports Vol.7 No.3
In our recent work (Ma et al., in Planta229(3):457–469, 2009a and 229(4):1077–1086, 2009b),two three-intron type III PKS genes, PcPKS1 and PcPKS2,were isolated from Polygonum cuspidatum Sieb. et Zucc. Phylogenetic and functional analyses revealed PcPKS1 is athree-intron chalcone synthase (CHS) gene, and PcPKS2 isfound to be a three-intron benzalacetone synthase (BAS)gene. The regular CHS encoded by a single intron genehave not been isolated and characterized from P. cuspidatum. In this work a further CHS with one intron (PcPKS3)and a stilbene synthase (STS) gene with three-intron(PcPKS5) were isolated and characterized by functionaland phylogenetic analyses. In comparison with PcPKS1, abifunctional enzyme with both CHS and BAS activity, theenzymatic product of recombinant PcPKS3 was naringenin,bis-noryangonin (BNY) and 4-coumaroyltriacetic acidlactone (CTAL) occurred as side products. The PcPKS5synthesized resveratrol and a trace amount of naringeninfrom p-coumaroyl-CoA. To our knowledge, PcPKS5 is thefirst reported three-intron STS gene in flowering plants. Inthis work, we speculated that this involved a possibleevolutionary route of plant-specific type III PKS superfamilyin P. cuspidatum.
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Guo, Yan-Wu,Guo, Hui-Li,Li, Xing,Huang, Li-Li,Zhang, Bo-Ning,Pang, Xiao-Bin,Liu, Ben-Ye,Ma, Lan-Qing,Wang, Hong 한국식물생명공학회 2013 Plant biotechnology reports Vol.7 No.3
In our recent work (Ma et al., in Planta 229(3):457-469, 2009a and 229(4):1077-1086, 2009b), two three-intron type III PKS genes, PcPKS1 and PcPKS2, were isolated from Polygonum cuspidatum Sieb. et Zucc. Phylogenetic and functional analyses revealed PcPKS1 is a three-intron chalcone synthase (CHS) gene, and PcPKS2 is found to be a three-intron benzalacetone synthase (BAS) gene. The regular CHS encoded by a single intron gene have not been isolated and characterized from P. cuspidatum. In this work a further CHS with one intron (PcPKS3) and a stilbene synthase (STS) gene with three-intron (PcPKS5) were isolated and characterized by functional and phylogenetic analyses. In comparison with PcPKS1, a bifunctional enzyme with both CHS and BAS activity, the enzymatic product of recombinant PcPKS3 was naringenin, bis-noryangonin (BNY) and 4-coumaroyltriacetic acid lactone (CTAL) occurred as side products. The PcPKS5 synthesized resveratrol and a trace amount of naringenin from p-coumaroyl-CoA. To our knowledge, PcPKS5 is the first reported three-intron STS gene in flowering plants. In this work, we speculated that this involved a possible evolutionary route of plant-specific type III PKS superfamily in P. cuspidatum.
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Manoharan, Gomathi,Sairam, Thiagarajan,Thangamani, Rajesh,Ramakrishnan, Dhivya,K.Tiwari, Manish,Lee, Jung-Kul,Marimuthu, Jeya IPC Science and Technology Press 2019 Enzyme and microbial technology Vol.131 No.-
<P><B>Abstract</B></P> <P>Endophytic fungi provide benefits to host plants by producing a diverse class of secondary metabolites (natural products). Arrays of polyketide natural products are synthesized by specific classes of polyketide synthases (PKS I, II and III) in host organisms. In the present study, we attempt to screen and identify type III PKSs in culturable fungal endophytes isolated from the ethno medicinal plants including <I>Arbus precatorius</I>, <I>Bacopa monnieri,Citrus aurantifolia</I> and <I>Datura metel</I> to detect the genetic potential of endophytic fungi in producing bioactive compounds. A total of seventeen endophytic fungal strains belonging to eight genera were identified using fungal morphology and rDNA-ITS phylogenetic analyses. A CODEHOP-PCR based strategy was followed to design degenerate primers for the screening of type III PKS genes from fungal endophytes. We had successfully amplified partial PKS genes from eight endophytes. The amplified PKS sequences showed 60–99% identity to already characterized/putative PKS genes. From the partial sequence of FiPKS from <I>Fusarium incarnatum</I> BMER1, a full-length gene was amplified, cloned and characterized. FiPKScDNA was cloned and expressed in <I>E. coli</I> Lemo21 (DE3) and the purified protein was shown to produce pyrones and resorcinols using acyl-CoA thioesters as substrates. FiPKS showed the highest catalytic efficiency of 7.6 × 10<SUP>4</SUP> s<SUP>−1</SUP> M<SUP>-1</SUP> with stearoyl CoA as a starter unit. This study reports the identification and characterization of type III PKS from endophytes of medicinal plants by CODEHOP PCR.</P> <P><B>Highlights</B></P> <P> <UL> <LI> A CODEHOP PCR based screening method was employed for type III polyketide synthase gene identification in fungal endophytes. </LI> <LI> By this approach, partial type III PKS genes from eight fungal endophytes were amplified and sequenced. </LI> <LI> FiPKS gene from Fusarium incarnatum BMER1, an endophyte of Bacopa monnieri was cloned and functionally characterized. </LI> <LI> FiPKS produced pyrones and resorcinols with the highest catalytic efficiency of 7.6 x 104 s-1 M-1towards stearoyl CoA. </LI> </UL> </P>
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Huiwen Yan,Lei Sun,Jinge Huang,Yixing Qiu,Fuchao Xu,Riming Yan,Du Zhu,Wei Wang,Jixun Zhan 한국미생물학회 2018 The journal of microbiology Vol.56 No.11
A new type III polyketide synthase gene (Ssars) was discovered from the genome of Shiraia sp. Slf14, an endophytic fungal strain from Huperzia serrata. The intron-free gene was cloned from the cDNA and ligated to two expression vectors pET28a and YEpADH2p-URA3 for expression in Escherichia coli BL21(DE3) and Saccharomyces cerevisiae BJ5464, respectively. SsARS was efficiently expressed in E. coli BL21(DE3), leading to the synthesis of a series of polyketide products. Six major products were isolated from the engineered E. coli and characterized as 1,3-dihydroxyphenyl- 5-undecane, 1,3-dihydroxyphenyl-5-cis-6 -tridecene,1,3-dihydroxyphenyl- 5-tridecane, 1,3-dihydroxyphenyl-5-cis-8 - pentadecene, 1,3-dihydroxyphenyl-5-pentadecane, and 1,3- dihydroxyphenyl-5-cis-10 -heptadecene, respectively, based on the spectral data and biosynthetic origin. Expression of SsARS in the yeast also led to the synthesis of the same polyketide products, indicating that this enzyme can be reconstituted in both heterologous hosts. Supplementation of soybean oil into the culture of E. coli BL21(DE3)/SsARS increased the production titers of 1–6 and led to the synthesis of an additional product, which was identified as 5-(8 Z,11 Z-heptadecadienyl) resorcinol. This work thus allowed the identification of SsARS as a 5-alk(en)ylresorcinol synthase with flexible substrate specificity toward endogenous and exogenous fatty acids. Desired resorcinol derivatives may be synthesized by supplying corresponding fatty acids into the culture medium.
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Juvvadi Praveen Rao,Seshime Yasuyo,Kitamoto Katsuhiko The Microbiological Society of Korea 2005 The journal of microbiology Vol.43 No.6
Fungal secondary metabolites constitute a wide variety of compounds which either playa vital role in agricultural, pharmaceutical and industrial contexts, or have devastating effects on agriculture, animal and human affairs by virtue of their toxigenicity. Owing to their beneficial and deleterious characteristics, these complex compounds and the genes responsible for their synthesis have been the subjects of extensive investigation by microbiologists and pharmacologists. A majority of the fungal secondary metabolic genes are classified as type I polyketide synthases (PKS) which are often clustered with other secondary metabolism related genes. In this review we discuss on the significance of our recent discovery of chalcone synthase (CHS) genes belonging to the type III PKS superfamily in an industrially important fungus, Aspergillus oryzae. CHS genes are known to playa vital role in the biosynthesis of flavonoids in plants. A comparative genome analyses revealed the unique character of A. oryzae with four CHS-like genes (csyA, csyB, csyC and csyD) amongst other Aspergilli (Aspergillus nidulans and Aspergillus fumigatus) which contained none of the CHS-like genes. Some other fungi such as Neurospora crassa, Fusarium graminearum, Magnaporthe grisea, Podospora anserina and Phanerochaete chrysosporium also contained putative type III PKSs, with a phylogenic distinction from bacteria and plants. The enzymatically active nature of these newly discovered homologues is expected owing to the conservation in the catalytic residues across the different species of plants and fungi, and also by the fact that a majority of these genes (csyA, csyB and csyD) were expressed in A. oryzae. While this finding brings filamentous fungi closer to plants and bacteria which until recently were the only ones considered to possess the type III PKSs, the presence of putative genes encoding other principal enzymes involved in the phenylpropanoid and flavonoid biosynthesis (viz., phenylalanine ammonia-lyase, cinnamic acid hydroxylase and p-coumarate CoA ligase) in the A. oryzae genome undoubtedly prove the extent of its metabolic diversity. Since many of these genes have not been identified earlier, knowledge on their corresponding products or activities remain undeciphered. In future, it is anticipated that these enzymes may be reasonable targets for metabolic engineering in fungi to produce agriculturally and nutritionally important metabolites.
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