• Journal of Microbiology and Biotechnology
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• v.15 no.1
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• pp.66-71
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• 2005

Doxorubicin is a clinically important anticancer polyketide compound that is typically produced by Streptomyces peucetius var. caesius. To improve doxorubicin productivity by S. peucetius, a doxorubicin pathway-specific regulatory gene, dnrI, was cloned into a high-copy-number plasmid containing a catechol promoter system. The S. peucetius containing the recombinant plasmid exhibited approximately 9.5-fold higher doxorubicin productivity compared with the wild-type S. peucetius. The doxorubicin productivity by this recombinant S. peucetius strain was further improved through the optimization of culture media composition. Based on the Fractional Factorial Design (FFD), cornstarch, $K_2HPO_4$, and $MgSO_4$ were identified to be the key factors influencing doxorubicin productivity. The Response Surface Method (RSM) results based on 20 independent culture conditions with varying amounts of key factors predicted the highest theoretical doxorubicin productivity of 11.1 mg/l with corn starch of 46.33 g/l, $K_2HPO_4$ of 4.63 g/l, and $MgSO_4$ of 9.26 g/l. The doxorubicin productivity of the recombinant S. peucetius strain with the RSM-based optimized culture condition was experimentally verified to be 11.46 mg/l, which was approximately 30.8-fold higher productivity compared with the wild-type S. peucetius without culture media optimization.

• Journal of Microbiology and Biotechnology
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• v.14 no.2
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• pp.356-365
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• 2004

In analyzing the region of the Amycolatopsis mediterranei S699 chromosome responsible for the biosynthesis of the ansamycin antibiotic rifamycin, we identified a gene, designated orj0, which is located immediately upstream of the rifamycin polyketide synthase (PKS). Orj0 encodes a protein, on the basis of sequence-comparative analysis, that is similar to several cytochrome P450 monooxygenases from different sources. The rifamycin producer, A. mediterranei, predominantly produces rifamycin B from its macrocyclic intermediate, proansamycin X, through dehydrogenation and hydroxylation steps. However, an A. mediterranei strain, deleted in orj0 by gene replacement, no longer produced rifamycin B. Furthermore, a versatile replicative vector in A. mediterranei was constructed and rifamycin B production was restored in a complementation experiment of orj0 using this novel vector. These consecutive results verified that the arf0 protein, which is a P450 hydroxylase, is required for the production of rifamycin B in A. mediterranei.

• The Plant Pathology Journal
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• v.34 no.4
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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.

• Journal of Applied Biological Chemistry
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• v.61 no.4
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• pp.327-334
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• 2018

Monascus is a source of food colorant with high productivity of the pigment azaphilone. Monascus azaphilone (MAz) is biosynthesized through a single non-reducing polyketide pathway, the major components of which are ankaflavin (1), monascin (2), rubropunctatin (3) and monascorubrin (4); valuable biological activities have been reported for these compounds. Thus, various culture conditions were explored to reduce the cost of culture ingredients, enhance productivity and modulate compound composition. In the present study, we examined an extractive fermentation (EF) method with Diaion HP-20 resin (HP20) in direct comparison to a previously explored method involving Triton X-100 (TX100) to explore the modulated production of the major MAzs. We employed wild-type Monascus purpureus as well as two derivative recombinant strains (${\Delta}mppG$ and ${\Delta}mppE$) that are known to have differential MAz profiles as that of the wild-type strain. The HP20 resin was capable of modulating the MAz profile in favor of orange MAzs 3 and 4, oxidized congeners in this class, as was TX100-a phenomenon not previously observed for TX100 EF with Monascus anka. These finding substantiate that HP20 can be employed for the selective production of oxidized MAz and for diversifying the culture conditions used for Az production.

• Journal of Microbiology and Biotechnology
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• v.25 no.5
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• pp.579-588
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• 2015

Fungi of the genus Pestalotiopsis have drawn attention for their capability to produce an array of bioactive secondary metabolites that have potential for drug development. Here, we report the determination of a polyketide derivative compound, pestalotiollide B, in the culture of the saprophytic fungus Pestalotiopsis microspora NK17. Structural information acquired by analyses with a set of spectroscopic and chromatographic techniques suggests that pestalotiollide B has the same skeleton as the penicillide derivatives, dibenzodioxocinones, which are inhibitors of cholesterol ester transfer protein (CETP), and as purpactins A and C', inhibitors of acyl-CoA:cholesterol acyltransferase (ACAT). Strain NK17 can make a fairly high yield of pestalotiollide B (i.e., up to 7.22 mg/l) in a constitutive manner in liquid culture. Moreover, we found that a putative histone deacetylase gene, designated as hid1, played a role in the biosynthesis of pestalotiollide B. In the hid1 null mutant, the yield of pestalotiollide B increased approximately 2-fold to 15.90 mg/l. In contrast, deletion of gene hid1 led to a dramatic decrease of conidia production of the fungus. These results suggest that hid1 is a modulator, concerting secondary metabolism and development such as conidiation in P. microspora. Our work may help with the investigation into the biosynthesis of pestalotiollide B and the development for new CETP and ACAT inhibitors.

• Journal of Applied Biological Chemistry
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• v.52 no.3
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• pp.93-102
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• 2009

Polyunsaturated fatty acids (PUFA), especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have significantly beneficial effects on health in relation to cardiovascular, immune, and inflammatory conditions and they are involved in determining the biophysical properties of membranes as well as being precursors for signaling molecules. PUFA biosynthesis is catalyzed by sequential desaturation and fatty acyl elongation reactions. This aerobic biosynthetic pathway was thought to be taxonomically conserved, but an alternative anaerobic pathway for the biosynthesis of PUFA is now known to contain analogous polyketide synthases (PKS). Certain fish oil can be a rich source of PUFA although processed marine oil is generally undesirable as food ingredients because of the associated objectionable flavors that are difficult and cost-prohibitive to remove. Oil-seed plants contain only the 18-carbon polyunsaturated fatty acid alpha-linolenic acid, which is not converted in the human body to EPA and DHA. It is now possible to engineer common oilseeds which can produce EPA and DHA and this has been the focus of a number of academic and industrial research groups. Recent advances and future prospects in the production of EPA and DHA in oilseed crops are discussed here.

• Microbiology and Biotechnology Letters
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• v.35 no.1
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• pp.26-29
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• 2007

Tautomycetin (TMC), which is produced by Streptomyces sp. CK4412, is a novel activated T cell-specific immunosuppressive compound with an ester bond linkage between a terminal cyclic anhydride moiety and a linear polyketide chain bearing an unusual terminal alkene. Antifungal activity against Aspergillus niger and TMC productivity assayed by HPLC using culture extracts from Streptomyces sp. CK4412 grown on solid medium adjusted at various pH were measured. The cells cultured at acidic pH (pH 4-5) medium exhibited much stronger antifungal activity as well as higher TMC productivity than those cultured at neutral pH medium, implying that the acidic pH-shock should be an efficient strategy to induce the productivity of secondary metabolites in Streptomyces culture.

• Journal of Microbiology and Biotechnology
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• v.24 no.8
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• pp.1081-1087
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• 2014

Aspergillus oryzae is generally recognized as safe, but it is closely related to A. flavus in morphology and genetic characteristics. In this study, we tested the aflatoxigenicity and genetic analysis of nine commercial A. oryzae strains that were used in Korean soybean fermented products. Cultural and HPLC analyses showed that none of the commercial strains produced detectable amount of aflatoxins. According to the molecular analysis of 17 genes in the aflatoxin (AF) biosynthetic pathway, the commercial strains could be classified into three groups. The group I strains contained all the 17 AF biosynthetic genes tested in this study; the group II strains deleted nine AF biosynthetic genes and possessed eight genes, including aflG, aflI, aflK, aflL, aflM, aflO, aflP, and aflQ; the group III strains only had six AF biosynthetic genes, including aflG, aflI, aflK, aflO, aflP, and aflQ. With the reverse transcription polymerase chain reaction, the group I A. oryzae strains showed no expression of aflG, aflQ and/or aflM genes, which resulted in the lack of AF-producing ability. Group II and group III strains could not produce AF owing to the deletion of more than half of the AF biosynthetic genes. In addition, the sequence data of polyketide synthase A (pksA) of group I strains of A. oryzae showed that there were three point mutations (two silent mutations and one missense mutation) compared with aflatoxigenic A. flavus used as the positive control in this study.

• Journal of Microbiology and Biotechnology
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• v.24 no.12
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• pp.1609-1621
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• 2014

The plant pathogen Burkholderia gladioli, which has a broad host range that includes rice and onion, causes bacterial panicle blight and sheath rot. Based on the complete genome sequence of B. gladioli BSR3 isolated from infected rice sheaths, the genome of B. gladioli BSR3 contains the luxI/luxR family of genes. Members of this family encode N-acyl-homoserine lactone (AHL) quorum sensing (QS) signal synthase and the LuxR-family AHL signal receptor, which are similar to B. glumae BGR1. In B. glumae, QS has been shown to play pivotal roles in many bacterial behaviors. In this study, we compared the QS-dependent gene expression between B. gladioli BSR3 and a QS-defective B. gladioli BSR3 mutant in two different culture states (10 and 24 h after incubation, corresponding to an exponential phase and a stationary phase) using RNA sequencing (RNA-seq). RNA-seq analyses including gene ontology and pathway enrichment revealed that the B. gladioli BSR3 QS system regulates genes related to motility, toxin production, and oxalogenesis, which were previously reported in B. glumae. Moreover, the uncharacterized polyketide biosynthesis is activated by QS, which was not detected in B. glumae. Thus, we observed not only common QS-dependent genes between B. glumae BGR1 and B. gladioli BSR3, but also unique QS-dependent genes in B. gladioli BSR3.

• The Korean Journal of Physiology and Pharmacology
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• v.18 no.2
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• pp.109-120
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• 2014

The epothilones are a class of microtubule inhibitors that exhibit a strong antitumor activity. UTD2 is a novel epothilone analog generated by genetic manipulation of the polyketide biosynthetic gene cluster. This study investigated the effects of UTD2 on the actin cytoskeleton and its critical regulators, and the signaling pathways which are essential for cell motility, growth and survival in MCF-7 breast cancer cells. Results showed that UTD2 inhibited the cellular functions of actin cytoskeleton, such as wound-closure, migration and invasion, as well as adhesion. Our study further demonstrated that UTD2 suppressed Rac1 GTPase activation and reduced the activity of PAK1, which is a downstream effector of Rac1, while the activity of Cdc42 was not affected. Additionally, the phosphorylation of p38 and ERK were significantly inhibited, but the phosphorylation of JNK remained the same after UTD2 treatment. Moreover, UTD2 inhibited the activity and mRNA expression of MMP-2, which plays a key role in cell motility. UTD2 also reduced the phosphorylation of Akt, which is an important signaling kinase regulating the cell survival through Rac1. Furthermore, UTD2 interrupted the synergy between Rac1 and Raf in focus formation assays. Taken together, these results indicated that UTD2 exerted multiple effects on the actin cytoskeleton and signaling pathways associated with Rac1. This study provided novel insights into the molecular mechanism of the antineoplastic and antimetastatic activities of epothilones. Our findings also suggest that the signaling pathways regulated by Rac1 may be evaluated as biomarkers for the response to therapy in clinical trials of epothilones.