• Journal of Microbiology and Biotechnology
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• 제25권11호
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• pp.1787-1795
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• 2015

The transition from primary to secondary metabolism in antibiotic-producing Streptomyces correlates with expression of genes involved in stress responses. Consequently, regulatory pathways that regulate specific stress responses are potential targets to manipulate to increase antibiotic titers. In this study, genes encoding key proteins involved in regulation of the osmotic stress response in Streptomyces avermitilis, the industrial producer of avermectins, are investigated as targets. Disruption of either osaB_Sa, encoding a response regulator protein, or osaC_Sa, encoding a multidomain regulator of the alternative sigma factor SigB, led to increased production of both oligomycin, by up to 200%, and avermectin, by up to 37%. The mutations also conditionally affected morphological development; under osmotic stress, the mutants were unable to erect an aerial mycelium. In addition, we demonstrate the delivery of DNA into a streptomycete using biolistics. The data reveal that information on stress regulatory responses can be integrated in rational strain improvement to improve yields of bioactive secondary metabolites.

• Journal of Microbiology and Biotechnology
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• 제23권7호
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• pp.959-965
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• 2013

Monascus species have been used to produce fermented rice called Monascus-fermented rice (MFR). To improve a Monascus strain via activation of secondary metabolite (SM) gene clusters for use in the production of MFR, we overexpressed an ortholog of the laeA gene, which encodes a global positive regulator of secondary metabolism under the control of the strong heterologous Aspergillus nidulans alcA promoter in Monascus pilosus. The OE::laeA transformant produced more SMs, including those not detected under uninduced conditions. MFR produced using the M. pilosus OE::laeA strain contained 4 times more monacolin K, a cholesterol-lowering agent, than MFR produced using the wild-type strain. In addition, pigment production was remarkably increased, and the antioxidant activity was increased as well. The results from this study suggest that Monascus species, which are important industrial fermentative fungi in Asia, can be improved for the production of functional foods by overexpressing the laeA gene.

• Journal of Microbiology and Biotechnology
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• 제29권7호
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• pp.1144-1154
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• 2019

There have been several studies regarding lichen-associated bacteria obtained from diverse environments. Our screening process identified 49 bacterial species in two lichens from the Himalayas: 17 species of Actinobacteria, 19 species of Firmicutes, and 13 species of Proteobacteria. We discovered five types of strong antimicrobial agent-producing bacteria. Although some strains exhibited weak antimicrobial activity, NP088, NP131, NP132, NP134, and NP160 exhibited strong antimicrobial activity against all multidrug-resistant strains. Polyketide synthase (PKS) fingerprinting revealed results for 69 of 148 strains; these had similar genes, such as fatty acid-related PKS, adenylation domain genes, PfaA, and PksD. Although the association between antimicrobial activity and the PKS fingerprinting results is poorly resolved, NP160 had six types of PKS fingerprinting genes, as well as strong antimicrobial activity. Therefore, we sequenced the draft genome of strain NP160, and predicted its secondary metabolism using antiSMASH version 4.2. NP160 had 46 clusters and was predicted to produce similar secondary metabolites with similarities of 5-100%. Although NP160 had 100% similarity with the alkylresorcinol biosynthetic gene cluster, our results showed low similarity with existing members of this biosynthetic gene cluster, and most have not yet been revealed. In conclusion, we expect that lichen-associated bacteria from the Himalayas can produce new secondary metabolites, and we found several secondary metabolite-related biosynthetic gene clusters to support this hypothesis.

• Journal of Microbiology
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• 제37권2호
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• pp.102-110
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• 1999

Genetic determinant for the secondary metabolism was studied in heterologous expression in Streptomyces lividans TK-24 using Streptomyces griseus ATCC 10137 as a donor strain. Chromosomal DNA of S. griseus was ligated into the high-copy number Streptomyces shuttle plasmid, pWHM3, and introduced into S. lividans TK-24. A plasmid clone with 4.3-kb BamHI DNA of S. griseus (pMJJ201) was isolated by detecting for stimulatory effect on actinorhodin production by visual inspection. The 4.3-kb BamHI DNA was cloned into pWHM3 under the control of the strong constitutive ermEp promoter in both directions (pMJJ202); ermEp promoter-mediated transcription for coding sequence reading right to left: pMJJ203; ermEp promoter-mediated transcription for coding sequence reading left to right) and reintroduced into S. lividans TK-24. The production of actinorhodin was markedly stimulated due to introduction of pMJJ202 on regeneration agar. The introduction of pMJJ202 also stimulated production of actinorhodin and undecylproidigiosin in submerged culture employing the actinorhodin production medium. Introduction of pMJJ203 resulted in a marked decrease of production of the two pigments. Nucleotide sequence analysis of the 4.3-kb region revealed three coding sequences: two coding sequences reading left to right, ORF1 and ORF2, one coding sequence reading right to left, ORF3. Therefore, it was suggested that the ORF3 product was responsible for the stimulation of antibiotic production. The C-terminal region of ORF3 product showed a local alignment with Myb-related transcriptional factors, which implicated that the ORF3 product might be a novel DNA-binding protein related to the regulation of secondary metabolism in Streptomyces.

• Journal of Ginseng Research
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• 제43권4호
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• pp.645-653
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• 2019

Background: Cytochrome P450 enzymes catalyze a wide range of reactions in plant metabolism. Besides their physiological functions on primary and secondary metabolites, P450s are also involved in herbicide detoxification via hydroxylation or dealkylation. Ginseng as a perennial plant offers more sustainable solutions to herbicide resistance. Methods: Tissue-specific gene expression and differentially modulated transcripts were monitored by quantitative real-time polymerase chain reaction. As a tool to evaluate the function of PgCYP736A12, the 35S promoter was used to overexpress the gene in Arabidopsis. Protein localization was visualized using confocal microscopy by tagging the fluorescent protein. Tolerance to herbicides was analyzed by growing seeds and seedlings on Murashige and Skoog medium containing chlorotoluron. Results: The expression of PgCYP736A12 was three-fold more in leaves compared with other tissues from two-year-old ginseng plants. Transcript levels were similarly upregulated by treatment with abscisic acid, hydrogen peroxide, and NaCl, the highest being with salicylic acid. Jasmonic acid treatment did not alter the mRNA levels of PgCYP736A12. Transgenic lines displayed slightly reduced plant height and were able to tolerate the herbicide chlorotoluron. Reduced stem elongation might be correlated with increased expression of genes involved in bioconversion of gibberellin to inactive forms. PgCYP736A12 protein localized to the cytoplasm and nucleus. Conclusion: PgCYP736A12 does not respond to the well-known secondary metabolite elicitor jasmonic acid, which suggests that it may not function in ginsenoside biosynthesis. Heterologous overexpression of PgCYP736A12 reveals that this gene is actually involved in herbicide metabolism.

• Journal of Microbiology and Biotechnology
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• 제31권7호
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• pp.912-920
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• 2021

SOS response is a conserved response to DNA damage in prokaryotes and is negatively regulated by LexA protein, which recognizes specifically an "SOS-box" motif present in the promoter region of SOS genes. Myxococcus xanthus DK1622 possesses a lexA gene, and while the deletion of lexA had no significant effect on either bacterial morphology, UV-C resistance, or sporulation, it did delay growth. UV-C radiation resulted in 651 upregulated genes in M. xanthus, including the typical SOS genes lexA, recA, uvrA, recN and so on, mostly enriched in the pathways of DNA replication and repair, secondary metabolism, and signal transduction. The UV-irradiated lexA mutant also showed the induced expression of SOS genes and these SOS genes enriched into a similar pathway profile to that of wild-type strain. Without irradiation treatment, the absence of LexA enhanced the expression of 122 genes that were not enriched in any pathway. Further analysis of the promoter sequence revealed that in the 122 genes, only the promoters of recA2, lexA and an operon composed of three genes (pafB, pafC and cyaA) had SOS box sequence to which the LexA protein is bound directly. These results update our current understanding of SOS response in M. xanthus and show that UV induces more genes involved in secondary metabolism and signal transduction in addition to DNA replication and repair; and while the canonical LexA-dependent regulation on SOS response has shrunk, only 5 SOS genes are directly repressed by LexA.

• Journal of Plant Biotechnology
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• 제29권4호
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• pp.265-275
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• 2002

주요 농작물에서 건강-방어용 flavonoids 생성, phytoalexin (isoflavonoid, flavanol, proanthocyanidin)의 생성 및 소절을 통한 식물의 저항력 증대, 색소 (flavonol, anthocyanin)의 합성에 의한 자외선 방어, nod 유전자 inducer (flavones, isoflavones)의 대량 발현에 의한 혹 형성 (nodulation) 효율증대 등은 대사공학 적으로 향상 가능한 부분들이다. 파란 꽃을 개화하는 품종이 카네이션, 국화, 장미 등 중요 장식용 화훼작물들에는 결핍되어 있는데,이는 F3'5'H 유전자가 없어서 파란색 delphinidin 색소를 생산할 수 없기 때문으로 추정된다. 따라서 F3'5'H 유전자를 형질전환 하여 이러한 제한을 극복하고 delphinidin 유도체 생산이 가능하게 되면 파란색 꽃의 생산 가능성을 증대시킬 수 있게 된다. 또한 영양학적인 측면에서 이미 중요한 생리적 기능이 밝혀진 catechin을 비롯한 proanthocyanidin 과 anthocyanin은 의약품 및 식품첨가제 등 다양한 분야에서 크게 시장성을 넓히고 있어 상업적 측면에서 대사공학의 유망한 목표가 되고 있다. 최근의 대사공학 분야에서의 많은 성공에도 불구하고, flavonoid에 대한 고도의 대사공학 조절을 이용하여 원하는 flavonoid 화합물을 생성하거나, 원치 않는 flavonoid 화합물을 억제하도록 하는 데는 여전히 기술적 문제점들이 남아있다. 예를 들면 IFS와 FLS 등의 유전자 분리 그리고 조직 및 시기 특이적인 promoter 개발 등이 동시에 이루어져야 하며, co-pigmentation 및 액포 pH와 관련된 메카니즘에 대한 이해, 화훼작물들의 형질전환 기술 개발 등이 이루어져야 원하는 꽃의 착색 조절이 가능하게 될 것이다. 최근 나팔꽃에서 액포의 $Na^{＋}$H$^{＋}$ exchanger를 파괴하여 화색을 변경시킨 mutants 연구를 통하여 조만간 액포 pH의 조절을 이용한 식물 대사공학이 가능할 것으로 기대되고 있다 (Yamaguchi et al. 2001). 아직 자연계에서 기본적인 골격의 변경만으로 수천 종류의 flavonoid가 생성 가능한가는 여전히 의문점으로 남아 있으나, 분명한 것은 다양한 식물 체계에서의 노력으로 농업, 원예, 그리고 영양분 증대를 위한 flavonoid 대사를 어떻게 조절할 것인가에 대한 정보를 얻을 수 있고, 또한 flavonoid 생합성 연구로부터 얻어진 정보들을 통하여 세포질 대사와 기본적인 생물학적 현상에 대한 이해를 넓힐 수 있게 될 것이다.

• Molecular & Cellular Toxicology
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• 제3권1호
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• pp.11-18
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• 2007

Mechanisms of insecticide resistance found in insects may include three general categories. Modified behavioral mechanisms can let the insects avoid the exposure to toxic compounds. The second category is physiological mechanisms such as altered penetration, rapid excretion, lower rate transportation, or increased storage of insecticides by insects. The third category relies on biochemical mechanisms including the insensitivity of target sites to insecticides and enhanced detoxification rate by several detoxifying mechanisms. Insecticides metabolism usually results in the formation of more water-soluble and therefore more readily eliminated, and generally less toxic products to the host insects rather than the parent compounds. The representative detoxifying enzymes are general esterases and monooxygenases that catalyze the toxic compounds to be more water-soluble forms and then secondary metabolism is followed by conjugation reactions including those catalyzed by glutathione S-transferases (GSTs). However, a change in the resistant species is not easily determined and the levels of mRNAs do not necessarily predict the levels of the corresponding proteins in a cell. As genomics understands the expression of most of the genes in an organism after being stressed by toxic compounds, proteomics can determine the global protein changes in a cell. In this present review, it is suggested that the environmental proteomic application may be a good approach to understand the biochemical mechanisms of insecticide resistance in insects and to predict metabolomic changes leading to physiological changes of the resistant species.

• The Plant Pathology Journal
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• 제30권2호
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• pp.220-227
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• 2014

The GacS/GacA system in the root colonizer Pseudomonas chlororaphis O6 is a key regulator of many traits relevant to the biocontrol function of this bacterium. Proteomic analysis revealed 12 proteins were down-regulated in a gacS mutant of P. chlororaphis O6. These GacS-regulated proteins functioned in combating oxidative stress, cell signaling, biosynthesis of secondary metabolism, and secretion. The extent of regulation was shown by real-time RT-PCR to vary between the genes. Mutants of P. chlororaphis O6 were generated in two GacS-regulated genes, trpE, encoding a protein involved in tryptophan synthesis, and prnA, required for conversion of tryptophan to the antimicrobial compound, pyrrolitrin. Failure of the trpE mutant to induce systemic resistance in tobacco against a foliar pathogen causing soft rot, Pectobacterium carotovorum SCCI, correlated with reduced colonization of root surfaces implying an inadequate supply of tryptophan to support growth. Although colonization was not affected by mutation in the prnA gene, induction of systemic resistance was reduced, suggesting that pyrrolnitrin was an activator of plant resistance as well as an antifungal agent. Study of mutants in the other GacS-regulated proteins will indicate further the features required for biocontrol-activity in this rhizobacterium.

• Journal of Applied Biological Chemistry
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• 제49권3호
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• pp.110-113
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• 2006

Prolyl endopeptidase (PEP, EC 3.4.21.26, also referred to as prolyl oligopeptidase) degrades proline containing, biologically active neuropeptides such as vasopressin, substance P and thyrotropin-releasing hormone by cleaving peptide bonds on carboxyl side of prolyl residue within neuropeptides of less than 30 amino acids. Evaluation of PEP levels in postmortem brains of Alzheimer's disease patients revealed significant increases in PEP activity. Therefore, a specific PEP inhibitor can be a good candidate of drug against memory loss. Upon our examination for PEP inhibitory activity from micronutrients, ascorbic acid (vitamin C) showed small but significant PEP inhibition (13% PEP inhibition at $8{\mu}g{\cdot}ml^{-1}$). Palmitic acid showed almost no PEP inhibition. However, 6-O-palmitoyl ascorbic acid ($\underline{1}$) showed 70% PEP inhibition at $8{\mu}g{\cdot}ml^{-1}$ indicating that hydrophobic portion of the compound $\underline{1}$ may facilitate the inhibitory effect. $IC_{50}$ value of compound $\underline{1}$ was $12.6{\pm}0.2{\mu}M$. The primary and secondary Lineweaver Burk and Dixon plots for compound $\underline{1}$ indicated that it is a non-competitive inhibitor with inhibition constant (Ki) value of $23.7{\mu}M$.