Identification and Characterization of a New Type III Polyketide Synthase from a Marine Yeast, Naganishia uzbekistanensis

A putative Type III Polyketide synthase (PKSIII) encoding gene was identified from a marine yeast, Naganishia uzbekistanensis strain Mo29 (UBOCC-A-208024) (formerly named as Cryptococcus sp.) isolated from deep-sea hydrothermal vents. This gene is part of a distinct phylogenetic branch compared to all known terrestrial fungal sequences. This new gene encodes a C-terminus extension of 74 amino acids compared to other known PKSIII proteins like Neurospora crassa. Full-length and reduced versions of this PKSIII were successfully cloned and overexpressed in a bacterial host, Escherichia coli BL21 (DE3). Both proteins showed the same activity, suggesting that additional amino acid residues at the C-terminus are probably not required for biochemical functions. We demonstrated by LC-ESI-MS/MS that these two recombinant PKSIII proteins could only produce tri- and tetraketide pyrones and alkylresorcinols using only long fatty acid chain from C8 to C16 acyl-CoAs as starter units, in presence of malonyl-CoA. In addition, we showed that some of these molecules exhibit cytotoxic activities against several cancer cell lines.

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Alteration of reaction and substrate specificity of a bacterial type III polyketide synthase by site-directed mutagenesis

Biochemical Journal ◽

10.1042/bj20020953 ◽

2002 ◽

Vol 367 (3) ◽

pp. 781-789 ◽

Cited By ~ 31

Author(s):

Nobutaka FUNA ◽

Yasuo OHNISHI ◽

Yutaka EBIZUKA ◽

Sueharu HORINOUCHI

Keyword(s):

Amino Acid ◽

Active Site ◽

Polyketide Synthase ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Amino Acid Residues ◽

Type Iii Polyketide Synthase ◽

Melanin Biosynthesis ◽

Type Iii ◽

Malonyl Coa

RppA, which belongs to the type III polyketide synthase family, catalyses the synthesis of 1,3,6,8-tetrahydroxynaphthalene (THN), which is the key intermediate of melanin biosynthesis in the bacterium Streptomyces griseus. The reaction of THN synthesis catalysed by RppA is unique in the type III polyketide synthase family, in that it selects malonyl-CoA as a starter substrate. The Cys-His-Asn catalytic triad is also present in RppA, as in plant chalcone synthases, as revealed by analyses of active-site mutants having amino acid replacements at Cys138, His270 and Asn303 of RppA. Site-directed mutagenesis of the amino acid residues that are likely to form the active-site cavity revealed that the aromatic ring of Tyr224 is essential for RppA to select malonyl-CoA as a starter substrate, since substitution of Tyr224 by amino acids other than Phe and Trp abolished the ability of RppA to accept malonyl-CoA as a starter, whereas the mutant enzymes Y224F and Y224W were capable of synthesizing THN via the malonyl-CoA-primed reaction. Of the site-directed mutants generated, A305I was found to produce only a triketide pyrone from hexanoyl-CoA as starter substrate, although wild-type RppA synthesizes tetraketide and triketide pyrones in the hexanoyl-CoA-primed reaction. The kinetic parameters of Ala305 mutants and identification of their products showed that the substitution of Ala305 by bulky amino acid residues restricted the number of elongations of the growing polyketide chain. Both Tyr224 (important for starter substrate selection) and Ala305 (important for intermediate elongation) were found to be conserved in three other RppAs from Streptomyces antibioticus and Streptomyces lividans.

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Molecular analysis of a type III polyketide synthase gene in Fallopia multiflora

Biologia ◽

10.2478/s11756-010-0113-8 ◽

2010 ◽

Vol 65 (6) ◽

Cited By ~ 6

Author(s):

Shu-Jing Sheng ◽

Zhong-Yu Liu ◽

Wei Zhao ◽

Li Shao ◽

Shu-Jin Zhao

Keyword(s):

Polyketide Synthase ◽

Stilbene Synthase ◽

Northern Blotting ◽

Amino Acid Residues ◽

Type Iii Polyketide Synthase ◽

Reading Frame ◽

Type Iii ◽

Close Relationship ◽

Polyketide Synthase Gene ◽

Cloned Gene

AbstractA type III polyketide synthase (PKS) cDNA and the corresponding gene (FmPKS) were isolated from the rhizomes of Fallopia multiflora (Thunb.) (Polygonaceae). The full-length FmPKS cDNA was 1,228 bp, containing an open reading frame of 1,137 bp encoding a 378 amino acid residues long protein. The coding sequence of the gene was interrupted by three introns, a different gene structure from most of the type III PKS genes studied so far containing only one intron at a conserved site, except for the recently cloned gene PcPKS2 from Polygonum cuspidatum. Phylogenetic analysis revealed that it shares a close relationship with Rheum tataricum stilbene synthase and formes a group with functionally diverse chalcone synthase-like enzymes. Southern blotting showed that there are three to four copies of the FmPKS gene in the genome. The expression pattern of the FmPKS transcript was determined by Northern blotting and high expression levels were detected in rhizomes and old stems, while the lowest levels were found in old leaves, a pattern that strongly correlates with the accumulation of the major bioactive principles called 2,3,5,4’-tetra-hydroxy-stilbene-2-O-β-D-glucoside, suggesting that FmPKS might play an important role in its biosynthesis.

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Discovery of anti-infective adipostatins through bioactivity-guided isolation and heterologous expression of a type III polyketide synthase

Bioorganic Chemistry ◽

10.1016/j.bioorg.2021.104925 ◽

2021 ◽

pp. 104925

Author(s):

Lukuan Hou ◽

Ying Li ◽

Qihao Wu ◽

Miyang Li ◽

Ethan A. Older ◽

...

Keyword(s):

Heterologous Expression ◽

Polyketide Synthase ◽

Type Iii Polyketide Synthase ◽

Type Iii

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A Fine Balancing Act of Type III Polyketide Synthase

Chemistry & Biology ◽

10.1016/j.chembiol.2004.09.001 ◽

2004 ◽

Vol 11 (9) ◽

pp. 1177-1178 ◽

Cited By ~ 1

Author(s):

Shiou-Chuan Tsai

Keyword(s):

Polyketide Synthase ◽

Type Iii Polyketide Synthase ◽

Type Iii

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Biosynthesis of Dictyostelium discoideum differentiation-inducing factor by a hybrid type I fatty acid–type III polyketide synthase

Nature Chemical Biology ◽

10.1038/nchembio811 ◽

2006 ◽

Vol 2 (9) ◽

pp. 494-502 ◽

Cited By ~ 86

Author(s):

Michael B Austin ◽

Tamao Saito ◽

Marianne E Bowman ◽

Stephen Haydock ◽

Atsushi Kato ◽

...

Keyword(s):

Fatty Acid ◽

Dictyostelium Discoideum ◽

Polyketide Synthase ◽

Type I ◽

Type Iii Polyketide Synthase ◽

Type Iii ◽

Hybrid Type ◽

Acid Type ◽

Fatty Acid Type

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In silico identification of Capsicum type III polyketide synthase genes and expression patterns in Capsicum annuum

Open Life Sciences ◽

10.1515/biol-2020-0077 ◽

2020 ◽

Vol 15 (1) ◽

pp. 753-762

Author(s):

Delong Kan ◽

Di Zhao ◽

Pengfei Duan

Keyword(s):

Molecular Mechanisms ◽

Polyketide Synthase ◽

Expression Patterns ◽

Class Ii ◽

Chili Pepper ◽

Type Iii Polyketide Synthase ◽

Type Iii ◽

Genes Encoding ◽

Duplication Events ◽

Pepper Leaves

AbstractStudies have shown that abundant and various flavonoids accumulate in chili pepper (Capsicum), but there are few reports on the genes that govern chili pepper flavonoid biosynthesis. Here, we report the comprehensive identification of genes encoding type III polyketide synthase (PKS), an important enzyme catalyzing the generation of flavonoid backbones. In total, 13, 14 and 13 type III PKS genes were identified in each genome of C. annuum, C. chinense and C. baccatum, respectively. The phylogeny topology of Capsicum PKSs is similar to those in other plants, as it showed two classes of genes. Within each class, clades can be further identified. Class II genes likely encode chalcone synthase (CHS) as they are placed together with the Arabidopsis CHS gene, which experienced extensive expansions in the genomes of Capsicum. Interestingly, 8 of the 11 Class II genes form three clusters in the genome of C. annuum, which is likely the result of tandem duplication events. Four genes are not expressed in the tissues of C. annuum, three of which are located in the clusters, indicating that a portion of genes was pseudogenized after tandem duplications. Expression of two Class I genes was complementary to each other, and all the genes in Class II were not expressed in roots of C. annuum. Two Class II genes (CA00g90790 and CA05g17060) showed upregulated expression as the chili pepper leaves matured, and two Class II genes (CA05g17060 and CA12g20070) showed downregulated expression with the maturation of fruits, consistent with flavonoid accumulation trends in chili pepper as reported previously. The identified genes, sequences, phylogeny and expression information collected in this article lay the groundwork for future studies on the molecular mechanisms of chili pepper flavonoid metabolism.

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