• 한국미생물·생명공학회지
• /
• 제23권2호
• /
• pp.178-186
• /
• 1995

Streptomyces albus wild type ATCC 21838 produced salinomycin, polyether antibiotic. To clone genes related salinomycin production, a genomic library was screened using actI as a DNA hybridization probe. pWHM 210 was isolated, which contained an approximately 24 kb of insert DNA. A 3.8 kb region in the 24 kb insert DNA was hybridized to actI and the nucleotide sequence of this region was determinied. Two open reading frames found in the same direction were homologous to genes for $\beta$-keto acyl synthase/acyl transferase and chain length determining factor in type II PKS (polyketide synthase). The genes were components of minimal type II PKS genes, highly conserved and showed the strong simiarity to other type II PKS genes known today.

• 한국미생물·생명공학회지
• /
• 제38권2호
• /
• pp.136-143
• /
• 2010

국내에서 분리한 9균주의 Sorangium cellulosum로부터 중합효소연쇄반응(PCR)을 통해 polyketide synthase(PKS)의 ketosynthase(KS) domain을 암호화하는 DNA를 증폭하고, 플라스미드 벡터에 클로닝한 후, 염기서열을 결정하였다. 전체 83개의 클로닝된 DNA 조각을 분석하여 유사한 조각을 배제한 결과, 43조각이 KS domain을 암호화하는 독립된 DNA 조각으로 판명되었다. 43조각 중 32조각의 아미노산 서열이 이미 클로닝된 PKS 유전자의 아미노산 서열과 70%-100% 유사하였으며, 나머지 11 조각은 알려진 서열과 67% 이하의 상동성을 가져 새로운 PKS 유전자일 가능성이 매우 높음을 보여주었다.

• Journal of Microbiology and Biotechnology
• /
• 제13권4호
• /
• pp.613-619
• /
• 2003

The polyketide backbone of rifamycin B is assembled by the type I rifamycin polyketide synthase (PKS) encoded by the rifA-rifE genes. In order to produce novel analogs of rifamycin via engineering of the PKS genes, inactivation of the ${\beta}-ketoacyl:acyl$ carrier protein reductase (KR) domain in module 8 of rifD, by site-specific mutagenesis of the NADPH binding site, was attempted. Module 8 contains a nonfunctional dehydratase (DH) domain and a functional KR domain that is involved in the reduction of the ${\beta}-carbonyl$ group, resulting in the C-21 hydroxyl of rifamycin B. This mutant strain produced linear polyketides, from tetraketide to octaketide, which were also produced by a rifD-disruption mutant as a consequence of premature termination of the polyketide assembly. Another attempt to replace the DH domain of module 7, which has been considered nonfunctional, with a functional homolog derived from module 7 of rapamycin-producing PKS also resulted in the production of linear polyketides, including the heptaketide intermediate and its precursors. Premature release of the carbon chain assembly intermediates is an unusual property of the rifamycin PKS. that is not seen in other PKSs such as the erythromycin PKS.

• Journal of Microbiology
• /
• 제35권1호
• /
• pp.40-46
• /
• 1997

The pWHM220 cosmid with a 24-kb insert cloned from Streptomyces albus ATCC 21838 induces the biosynthesis of a polysther antibiotic similar to salinomycin in Streptomyces invidans. We have analyzed this region by DNA sequencing as well as Southern blot hybridization with type I and type II polyketide synthase (PKS) probes. Surprisingly, we found another set of type II SKS genes only 10-kb from the original PKS genes, salABCDE. The DNA sequence revealed two complete open reading frames (ORFs) named salB2 and salC2, and one partial ORF that does not resemble any known DNA or deduced protein sequence. The salC2 should code for chain length determining factor while the deduced amino acid sequence encoded by salB2 exhibits high similarity to .betha.-ketoacyl synthase from different PKS gene clusters. The highest identity was found for .betha.-keetoacyl synthases from S. argillaceus (MtmP. 59.1% identity), the mithramycin producer and from S. venezuelae ISP5230 (JadA, 52.3% identity), the jadomycin producer. The SalC2 protein clearly resembles its counterparts in order aromatic PKS gene clusters that are believed to influence the length of the polyketide chain. The highest identities observed were to that of S. argillaceus (MtmK, 62.3%) and S. venezuelae ISP 5230 (JadB, 55.1%) proteins, Moreover, the deduced amino acid sequences of the salB2 and salC2 products were 29.0% identical.

• Molecules and Cells
• /
• 제38권12호
• /
• pp.1105-1110
• /
• 2015

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.

• Journal of Microbiology and Biotechnology
• /
• 제13권5호
• /
• pp.819-822
• /
• 2003

Polyketides are an extensive class of secondary metabolites with diverse molecular structures and biological activities. A plasmid-based minimal polyketide synthase (PKS) expression cassette was constructed using a subset of actinorhodin (act) biosynthetic genes (actI-orfl, actI-orf2, actI-orf3, actIII, actⅦ, and actIV) from Streptomyces coelicolor, which specify the construction of an orange-fluorescent anthraquinone product aloesaponarin II, a type II polyketide compound derived from one acetyl coenzyme A and 7 malonyl coenzyme A extender units. This system was designed as an indicator pathway in S. parvulus to generate a hybrid type II polyketide compound via gene-specific replacement. The act ${\beta}-ketoacyl$ synthase unit (actI-orfl and actI-orf2) in the expression cassette was specifically replaced with oxytetracycline ${\beta}-ketoacyl$ synthase otcY-orfl and otcY-orf2). This plasmid-based hybrid PKS cassette generated a novel orange-fluorescent compound structurally different from aloesaponarin II in both S. lividans and S. parvulus. In addition, several additional distinctive blue-fluorescent compounds were detected, when this hybrid PKS cassette was expressed in S. coelicolor B78 (actI-orf2 mutant), implying that the expression of plasmid-based hybrid PKS cassette in Streptomyces species should be an efficient way of generating hybrid type II polyketide compounds.

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

• Mycobiology
• /
• 제42권1호
• /
• pp.34-40
• /
• 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
• /
• 제16권1호
• /
• pp.153-157
• /
• 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.

• 한국동물학회:학술대회논문집
• /
• 한국동물학회 1994년도 한국생물과학협회 학술발표대회
• /
• pp.64-64
• /
• 1994

No Abstract, See Full Text