• Mycobiology
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• 제42권1호
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• pp.34-40
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• 2014

Usnea longissima has a long history of use as a traditional medicine. Several bioactive compounds, primarily belonging to the polyketide family, have been isolated from U. longissima. However, the genes for the biosynthesis of these compounds are yet to be identified. In the present study, three different types of non-reducing polyketide synthases (UlPKS2, UlPKS4, and UlPKS6) were identified from a cultured lichen-forming fungus of U. longissima. Phylogenetic analysis of product template domains showed that UlPKS2 and UlPKS4 belong to group IV, which includes the non-reducing polyketide synthases with an methyltransferase (MeT) domain that are involved in methylorcinol-based compound synthesis; UlPKS6 was found to belong to group I, which includes the non-reducing polyketide synthases that synthesize single aromatic ring polyketides, such as orsellinic acid. Reverse transcriptase-PCR analysis demonstrated that UlPKS2 and UlPKS4 were upregulated by sucrose; UlPKS6 was downregulated by asparagine, glycine, and alanine.

• Journal of Microbiology and Biotechnology
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• 제13권5호
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• pp.823-827
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• 2003

Streptomyces venezuelae ATCC 15439 is notable in its ability to produce two distinct groups of macrolactones. It has been reported that the generation of two macrolactone structures results from alternative expression of pikromycin (Pik) polyketide synthase (PKS). It was previously reported that the hybrid pikromycin-tylosin PKS can also produce two different macrolactones but its mechanistic basis remains unclear. In order to address this question, a series of site-directed mutagenesis of tentative alternative ribosome binding site and translation start codons in tylGV were performed. The results suggest that macrolactone ring size is not determined by the alternative expression of TylGV but through other mechanism(s) involving direct interaction between the PikAIII and TE domain or skipping of the final chain elongation step. This provides new insight into the mechanism of macrolactone ring size determination in hybrid PKS as well as an opportunity to develop novel termination activities for combinatorial biosynthesis.

• Journal of Microbiology
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• 제44권6호
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• pp.649-654
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• 2006

A standard type II polyketide synthase (PKS) gene cluster was isolated while attempting to clone the biosynthetic gene for lipstatin from Streptomyces toxytricini NRRL 15,443. This result was observed using a Southern blot of a PstI-digested S. toxytricini chromosomal DNA library with a 444 bp amplified probe of a ketosynthase (KS) gene fragment. Four open reading frames [thioesterase (TE), $\beta$-ketoacyl systhase (KAS), chain length factor (CLF), and acyl carrier protein (ACP)], were identified through the nucleotide sequence determination and analysis of a 4.5 kb cloned DNA fragment. In order to confirm the involvement of a cloned gene in lipstatin biosynthesis, a gene disruption experiment for the KS gene was performed. However, the resulting gene disruptant did not show any significant difference in lipstatin production when compared to wild-type S. toxytricini. This result suggests that lipstatin may not be synthesized by a type II PKS.

• Journal of Applied Biological Chemistry
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• 제64권3호
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• pp.225-236
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• 2021

The epipyrone (EPN) biosynthetic gene cluster of Epicoccum nigrum is composed of epnC, epnB, and epnA, which encode cytochrome P450 oxidase, glycosyltransferase, and highly reducing polyketide synthase, respectively. Gene inactivation mutants for epnA, epnB, and epnC were previously generated, and it was found that all of them were incapable of producing EPN and any of its related compounds. It was also reported that epnB inactivation abolished epnA transcription, generating ΔepnAB. This study shows that the introduction of native epnC readily restored EPN production in ΔepnC, suggesting that epnC is essential for polyketide release from EpnA and implies that EpnC works during the polyketide chain assembly of EpnA. Introduction of epnC promoter-epnA restored EPN production in ΔepnA. The ΔepnB genotype was prepared by introducing the epnA expression vector into ΔepnAB, and it was found that the resulting recombinant strain did not produce any EPN-related compounds. A canonical epnB inactivation strain was also generated by deleting its 5'-end. At the deletion point, an Aspergllus nidulans gpdA promoter was inserted to ensure the transcription of epnA, which is located downstream of epnB. Examination of the metabolite profile of the resulting ΔepnB mutant via LC-mass spectrometry verified that no EPN-related compound was produced in this strain. This substantiates that C-glycosylation by EpnB is a prerequisite for the release of EpnA-tethered product. In conclusion, it is proposed that cytochrome P450 oxidase and glycosyltransferase work in concert with polyketide synthase to generate EPN without the occurrence of any free intermediates.

• Journal of Microbiology and Biotechnology
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• 제25권11호
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• pp.1796-1800
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• 2015

Sporopollenin is a poorly characterized mixed aliphatic and aromatic polymer with ester and ether linkages. Recent studies have reported that α-pyrone polyketide compounds generated by Arabidopsis thaliana, polyketide synthase A (PKSA) and tetraketide α-pyrone reductase 1 (TKPR1), are previously unknown sporopollenin precursors. Here, the yeast Saccharomyces cerevisiae was introduced to test potential sporopollenin biosynthetic pathways in vivo. A PKSA/TKPR1 dual expressor was generated and various chain-length alkyl α-pyrones were identified by GC-MS. The growth rate of the strain containing PKSA/TKPR1 appeared normal. These results indicate that PKSA/TKPR1-expressing yeast would be a starting platform to investigate in vivo sporopollenin metabolism.

• Journal of Microbiology and Biotechnology
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• 제16권1호
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• pp.153-157
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• 2006

A soil metagenomic library was constructed using an E. coli-fosmid cloning system with environmental DNAs extracted from Kwangreung forest topsoil. We targeted the genes involved in the biosynthesis of bacterial polyketides. Initially, a total of 36 clone pools (10,800 clones) were explored by the PCR-based method using the metagenomic DNAs from each pool and a degenerate primer set, which has been designed based on the highly conserved regions among ketoacyl synthase (KS) domains in actinomycete type I polyketide synthases (PKS Is). Six clone pools were tentatively selected as positive and further examined through a hybridization-based method for selecting a fosmid clone containing PKS I genes. Colony hybridization was performed against fosmid clones from the 6 positive pools, and finally 4 clones were picked out and confirmed to contain the conserved DNA fragment of KS domains. In this study, we present a simple and feasible sorting method for a desired clone from metagenomic libraries.

• Journal of Microbiology and Biotechnology
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• 제25권10호
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• pp.1648-1652
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• 2015

Azaphilone polyketides are synthesized by iterative non-reducing fungal polyketide synthases (NR-fPKSs) with a C-terminus reductive domain (-R). Several azaphilone biosynthetic gene clusters contain a putative serine hydrolase gene; the Monascus azaphilone pigment (MAzP) gene cluster harbors mppD. The MAzP productivity was significantly reduced by a knockout of mppD, and the MAzP NR-fPKS-R gene (MpPKS5) generated its product in yeast only when co-expressed with mppD. Site-directed mutations of mppD for conserved Ser/Asp/His residues abolished the product formation from the MpPKS5/mppD co-expression. MppD and its homologs are thus proposed as a new protein factor involved in the product formation of NR-fPKS-R.

• Journal of Applied Biological Chemistry
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• 제42권3호
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• pp.119-124
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• 1999

About two thirds of the naturally occurring antibiotics have been discovered from actinomycetes. Therefore, the probability of discovering further new antibiotics from actinomycetes is declining as many known metabolites are isolated repeatedly. However, various efforts leave been made in order to enhance the probability of discovering novel compounds. In the present study, we have developed new screening strategies based on the antibiotic biosynthetic pathway, and the genetic information, utilizing polymerase chain reaction. We have selected macrolide type polyketides. In order to divide the ansamycin group antibotic of macrolide type polyketides, we have selected 3-amino-5-hydroxybenzoic acid (AHBA) moiety which contains a biosynthetically unique structural element in the group as a target molecules. Oligonucleotide primers were designed to amplify DNA fragments of macrolide type polyketide synthase and AHBA synthase genes from fourteen actinomycetes species. This method was successfully applied to all three of the known macrolide type polyketide produccing actinomycetes tested. In addition, it also identified the presence of potential macrolide type polyketide producing genes from seven actinomycetes that were known to produce none of macrolide type polyketides, and AHBA biosynthetic genes in one actinomycetes. This technique is potentially useful for the screening of new antibiotices and cloning of their biosynthetic genes.

• Journal of Microbiology and Biotechnology
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• 제17권5호
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• pp.830-839
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• 2007

Pradimicins are potent antifungal antibiotics having an unusual dihydrobenzo[$\alpha$]naphthacenequinone aglycone substituted with D-alanine and sugars. Pradimicins are polyketide antibiotics produced by Actinomadura hibisca P157-2. The gene cluster involved in the biosynthesis of pradimicins was cloned and sequenced. The pradimicin gene cluster was localized to a 39-kb DNA segment and its involvement in the biosynthesis of pradimicin was proven by gene inactivation of prmA and prmB(ketosynthases $\alpha\;and\;\beta$). The pradimicin gene cluster consists of 28 open reading frames(ORFs), encoding a type II polyketide synthase(PKS), the enzymes involved in sugar biosynthesis and tailoring enzymes as well as two resistance proteins. The deduced proteins showed strong similarities to the previously validated gene clusters of angucyclic polyketides such as rubromycin, griseorhodin, and fredericamycin. From the pradimicin gene cluster, prmP3 encoding a component of the acetyl-CoA carboxylase complex was disrupted. The production levels of pradimicins of the resulting mutants decreased to 62% of the level produced by the wild-type strain, which indicate that the acetyl-CoA carboxylase gene would have a significant role in the production of pradimicins through supplying the extender unit precursor, malonyl-CoA.

• Journal of Microbiology and Biotechnology
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• 제18권3호
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• pp.427-433
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• 2008

The cephabacins produced by Lysobacter lactamgenus are ${\beta}$-lactam antibiotics composed of a cephem nucleus, an acetate residue, and an oligopeptide side chain. In order to understand the precise implication of the polyketide synthase (PKS) module in the biosynthesis of cephabacin, the genes for its core domains, ${\beta}$-ketoacyl synthase (KS), acyltransferase (AT), and acyl carrier protein (ACP), were amplified and cloned into the pET-32b(+) expression vector. The sfp gene encoding a protein that can modify apo-ACP to its active holo-form was also amplified. The recombinant KS, AT, apo-ACP, and Sfp overproduced in the form of $His_6$-tagged fusion proteins in E. coli BL21(DE3) were purified by nickel-affinity chromatography. Formation of stable peptidyl-S-KS was observed by in vitro acylation of the KS domain with the substrate [L-Ala-L-Ala-L-Ala-L-$^3H$-Arg] tetrapeptide-S-N-acetylcysteamine, which is the evidence for the selective recognition of tetrapeptide produced by nonribosomal peptide synthetase (NRPS) in the NRPS/PKS hybrid. In order to confirm whether malonyl CoA is the extender unit for acetylation of the peptidyl moiety, the AT domain, ACP domain, and Sfp protein were treated with $^{14}C$-malonyl-CoA. The results clearly show that the AT domain is able to recognize the extender unit and decarboxylatively acetylated for the elongation of the tetrapeptide. However, the transfer of the activated acetyl group to the ACP domain was not observed, probably attributed to the improper capability of Sfp to activate apo-ACP to the holo-ACP form.