• Journal of Microbiology and Biotechnology
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• 제15권3호
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• pp.656-659
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• 2005

Small peptides synthesized by nonribosomal peptide synthetases (NRPSs) genes are found in bacteria and fungi. While some microbial taxa have few, others make a large number and variety. However, biochemical characterization of the products synthesized by NPRS demands a great deal of efforts. Since the completion of genome projects of numerous microorganisms, the numbers of available NRPSs genes are being expanded. Prediction of the peptides encoded by NRPS could save time and efforts. We chose the NRPS gene from Bradyrhizobium japonicum as a model to predict the peptide structure encoded by NRPS genes. Using computational analyses, the domain structure of this gene was defined, and the structure of a peptide synthesized by this NRPS was deduced. It was found that it encoded a tripeptide consisting of proline-serine-phenylalanine. This method would be helpful to predict the structure of small peptides with various NPRS genes from the genome sequence.

• 한국미생물·생명공학회지
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• 제24권1호
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• pp.67-71
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• 1996

The plasmid pK8 was constructed to verify the existence of an adenylate domain in peptide synthetase by using pGC12. 1.2 kb fragment, coding tyrocidine synthetase 1 (123 kDa) was deleted, and 79.6 kDa one was expressed in Escherichia coli XL1-blue. The truncated multienzyme activated phenylalanine and substrate analogues with comparable kinetics as the over expressed synthetase. ATP-[$^{32}P$]PPi exchange reaction was measured for the enzyme assay.

• Journal of Microbiology and Biotechnology
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• 제8권4호
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• pp.310-317
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• 1998

Peptide synthetases are large multifunctional enzyme complexes that catalyze the nonribosomal synthesis of a structurally diverse family of peptide antibiotics. These enzymes are composed of functionally independent domains with independent enzymatic activities. Their specific linkage order of domains forms the protein template that defines the sequence of the incorporated amino acids. Within each domain, several motifs of highly conserved sequences have been identified from the sequence alignment of the various peptide synthetases ［30］. Taking advantage of the conserved nucleotide sequence of Core 1 and Core 2, we designed PCR primers to amplify the peptide synthetase genes from three different gram-positive bacterial strains. Nucleotide sequence analysis of the amplified PCR products from those three strains showed significant homology to various peptide synthetase genes, suggesting that the PCR products are parts of peptide synthetase genes. Therefore, this rapid and efficient PCR technique can be used for the isolation of peptide synthetase genes from various strains.

• Journal of Microbiology and Biotechnology
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• 제27권3호
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• pp.439-449
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• 2017

Beauveria bassiana infects a number of pest species and is known to produce insecticidal substances, such as the nonribosomal peptides (NRPs) beauvericin and bassianolide. However, most NRPs and their biological roles in B. bassiana remain undiscovered. To identify NRPs that potentially contribute to pathogenesis, the 21 predicted NRP synthetases (NRPSs) or NRPS-like proteins of B. bassiana ARSEF 2860 were primarily ranked into three functional groups: basic metabolism (7 NRPSs), pathogenicity (12 NRPSs), and unknown function (2 NRPSs). Based on the transcript levels during in vivo growth on diamondback moth (Plutella xylostella (Linnaeus)), half of the Group II NRPSs were likely to be involved in infection. Given that the metabolites biosynthesized by these NRPSs remain to be determined, our result underlines the importance of the NRPSome in fungal pathogenesis, and will serve as a guide for future genomic mining projects to discover functionally essential and structurally diverse NRPs in fungal genomes.

• Journal of Microbiology and Biotechnology
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• 제25권7호
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• pp.1015-1025
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• 2015

The development of diverse polymyxin derivatives is needed to solve the toxicity and resistance problems of polymyxins. However, no platform has generated polymyxin derivatives by genetically engineering a polymyxin synthetase, which is a nonribosomal peptide synthetase. In this study, we present a two-step approach for the construction of engineered polymyxin synthetases by substituting the adenylation (A) domains of polymyxin A synthetase, which is encoded by the pmxABCDE gene cluster of Paenibacillus polymyxa E681. First, the seventh L-threonine-specific A-domain region in pmxA was substituted with the L-leucine-specific A-domain region obtained from P. polymyxa ATCC21830 to make polymyxin E synthetase, and then the sixth D-leucine-specific A-domain region (A_6-D-Leu-domain) was substituted with the D-phenylalanine-specific A-domain region (A_6-D-Phe-domain) obtained from P. polymyxa F4 to make polymyxin B synthetase. This step was performed in Escherichia coli on a pmxA-containing fosmid, using the lambda Red recombination system and the sacB gene as a counter-selectable marker. Next, the modified pmxA gene was fused to pmxBCDE on the chromosome of Bacillus subtilis BSK4dA, and the resulting recombinant strains BSK4-PB and BSK4-PE were confirmed to produce polymyxins B and E, respectively. We also succeeded in constructing the B. subtilis BSK4-PP strain, which produces polymyxin P, by singly substituting the A_6-D-Leu-domain with the A_6-D-Phe-domain. This is the first report in which polymyxin derivatives were generated by genetically engineering polymyxin synthetases. The two recombinant B. subtilis strains will be useful for improving the commercial production of polymyxins B and E, and they will facilitate the generation of novel polymyxin derivatives.

• Journal of Microbiology and Biotechnology
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• 제21권4호
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• pp.359-365
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• 2011

Cyanobacterial strains of the genus Spirulina have recently been identified as an excellent source of sulfolipids, some of which possess anti-HIV properties. Thus, to investigate the distribution of sufolipid biosynthesis pathways in Spirulina, a genetic screening/phylogentic study was performed. Five different strains of Spirulina [Spirulina (Jiangmen), Spirulina sp., S. platensis, S. maxima, and Spirulina seawater] sourced from different locations were initially classified via 16S rDNA sequencing, and then screened for the presence of the sulfolipid biosynthesis genes sqdB and sqdX via a PCR. To assess the suitability of these strains for human consumption and safe therapeutic use, the strains were also screened for the presence of genes encoding nonribosomal peptide synthases (NRPSs) and polyketide synthases (PKSs), which are often associated with toxin pathways in cyanobacteria. The results of the 16S rDNA analysis and phylogenetic study indicated that Spirulina sp. is closely related to Halospirulina, whereas the other four Spirulina strains are closely related to Arthrospira. Homologs of sqdB and sqdX were identified in Spirulina (Jiangmen), Spirulina sp., S. platensis, and the Spirulina seawater. None of the Spirulina strains screened in this study tested positive for NRPS or PKS genes, suggesting that these strains do not produce NRP or PK toxins.

• The Plant Pathology Journal
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• 제40권3호
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• pp.322-328
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• 2024

Soybean (Glycine max), a crucial global crop, experiences yearly yield reduction due to diseases such as anthracnose (Colletotrichum truncatum) and root rot (Fusarium spp.). The use of fungicides, which have traditionally been employed to control these phytopathogens, is now facing challenges due to the emergence of fungicide-resistant strains. Streptomyces bacillaris S8 strain S8 is previously known to produce valinomycin t through a nonribosomal peptide synthetase (NRPS) pathway. The objective of this study was to evaluate the antifungal activity of S. bacillaris S8 against C. truncatum and Fusarium sp., assessing its efficacy against soybean pathogens. The results indicate that strain S8 effectively controlled both above-ground and underground soybean diseases, using the NRPS and NRPS-related compound, suggesting its potential as a biological control in plant-microbe interactions. These findings underscore the pivotal role of the stain S8 in fostering healthy soybean microbial communities and emphasize the significance of microbiota structure studies in unveiling potent biocontrol agents.

• 한국산학기술학회논문지
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• 제14권8호
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• pp.4086-4092
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• 2013

Mycosporine-like 아미노산(MAAs)은 UV 흡수물질이며, 다양한 해양생물들은 MAAs의 합성과 축적을 통하여 환경 자외선의 직 간접적인 영향을 감소시키는 기능을 진화시켜 왔다. 이 연구에서 우리는 radiofrequency(RF) 발생장치를 제작하였고, 이를 미세조류 배양에 적용하였다. $0.35{\pm}0.05$ mHz의 RF를 Chlamydomonas sp. 배양기에 공급하였고, 정해진 시간(0, 0.5, 1 및 2 시간)에 시료를 채취하였다. MAAs 생합성 관련 유전자인 dehydroquinate synthase homolog (DHQS-like)와 nonribosomal peptide synthetase homolog (NRPS-like) 유전자를 Chlamydomonas sp.로부터 클로닝하였고, RF 노출에 대한 유전자 발현을 qRT-PCR을 이용하여 분석하였다. 연구결과 RF에 노출된 Chlamydomonas sp.의 DHQS-like와 NRPS-like 유전자의 발현은 노출 1시간에 각각 1.46 배 및 1.19 배 증가하였다. 이러한 결과는 DHQS-like와 NRPS-like 유전자가 Chlaydomonas sp.의 MAAs 생합성을 진단할 수 있는 좋은 바이오마커 후보가 될 수 있음을 의미한다.

• Journal of Microbiology and Biotechnology
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• 제19권3호
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• pp.229-237
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• 2009

Among 12 marine bacterial strains from the China coast that exhibited interesting bioactivity (positive for both antimicrobial and cytotoxic activities), only four strains, namely, NJ6-3-1, NJ6-3-2, NB-6, and YTHM-17, had a KS domain or A domain when screened for PKS and NRPS genes using a PCR. Interestingly, two of these strains belonging to Pseudoalteromonas and associated with the marine sponge Hymeniacidon perleve were positive for both PKS and NRPS, whereas the other two strains of Pseudoalteromonas did not have a PKS or NRPS gene. A molecular phylogeny analysis and DGGE analysis of the Pseudoalteromonas sp. indicated that they had a specific affinity with the host marine sponge Hymeniacidon perleve. Furthermore, an analysis of a partial sequence of Pseudoalteromonas sp. NJ6-3-2 isolated from the marine sponge Hymeniacidon perleve obtained from genomic walking using a computational approach indicated a relatively complete PKS module including auxiliary domains (DH, KR, and Cy).

• The Plant Pathology Journal
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• 제34권4호
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• pp.327-334
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• 2018

Northern corn leaf spot and southern corn leaf blight caused by Cochliobolus carbonum (anamorph, Bipolaris zeicola) and Cochliobolus heterostrophus (anamorph, Bipolaris maydis), respectively, are common maize diseases in Korea. Accurate detection of plant pathogens is necessary for effective disease management. Based on the polyketide synthase gene (PKS) of Cochliobolus carbonum and the nonribosomal peptide synthetase gene (NRPS) of Cochliobolus heterostrophus, primer pairs were designed for PCR to simultaneously detect the two fungal pathogens and were specific and sensitive enough to be used for duplex PCR analysis. This duplex PCR-based method was found to be effective for diagnosing simultaneous infections from the two Cochliobolus species that display similar morphological and mycological characteristics. With this method, it is possible to prevent infections in maize by detecting infected seeds or maize and discarding them. Besides saving time and effort, early diagnosis can help to prevent infections, establish comprehensive management systems, and secure healthy seeds.