• Journal of Microbiology and Biotechnology
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• v.29 no.12
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• pp.1931-1937
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• 2019

The heterologous expression of the Streptomyces natural product (NP) biosynthetic gene cluster (BGC) has become an attractive strategy for the activation, titer improvement, and refactoring of valuable and cryptic NP BGCs. Previously, a Streptomyces artificial chromosomal vector system, pSBAC, was applied successfully to the precise cloning of large-sized polyketide BGCs, including immunosuppressant tautomycetin and antibiotic pikromycin, which led to stable and comparable production in several heterologous hosts. To further validate the pSBAC system as a generally applicable heterologous expression system, the daptomycin BGC of S. roseosporus was cloned and expressed heterologously in a model Streptomyces cell factory. A 65-kb daptomycin BGC, which belongs to a non-ribosomal polypeptide synthetase (NRPS) family, was cloned precisely into the pSBAC which resulted in 28.9 mg/l of daptomycin and its derivatives in S. coelicolor M511(a daptomycin non-producing heterologous host). These results suggest that a pSBAC-driven heterologous expression strategy is an ideal approach for producing low and inconsistent Streptomyces NRPS-family NPs, such as daptomycin, which are produced low and inconsistent in native host.

• Journal of Microbiology and Biotechnology
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• v.3 no.2
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• pp.65-72
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• 1993

A heterologous gene expression and secretion system using a methylotrophic yeast, Hansenula polymorpha was developed for the production of anticoagulant hirudin. Hirudin gene was expressed under the control of a strong and inducible methanol oxidase (MOX or AOX) promoter. The mating factor a pre-pro leader sequence of Saccharomyces cerevisiae was employed for hirudin to be secreted into the extracellular medium. Hirudin expression cassette was introduced into three strains of H. polymorpha, A16, HPBl and DLl which have different genetic backgrounds. This expression cassette was stably integrated into the host chromosomal DNA. Biologically active and mature hirudin was efficiently expressed and secreted into the extracellular medium. About 19 mg/L of hirudin was found in the culture supernatant in the case of a two-copy integrant of the strain HPBl under suboptimal culture conditions.

• Journal of Microbiology and Biotechnology
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• v.25 no.7
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• pp.988-998
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• 2015

The filamentous fungus Aspergillus oryzae is a well-known expression host used to express homologous and heterologous proteins in a number of industrial applications. To facilitate higher yields of proteins of interest, we constructed the pAsOP vector to express heterologous proteins in A. oryzae. pAsOP carries a selectable marker, pyrG, derived from Aspergillus nidulans, and a strong promoter and a terminator of the amyB gene derived from A. oryzae. pAsOP transformed A. oryzae efficiently via the PEG-CaCl₂-mediated transformation method. As proof of concept, green fluorescent protein (GFP) was successfully expressed in A. oryzae transformed by pAsOP-GFP. Additionally, we identified a novel fungal α-amylase (PcAmy) gene from Penicillium sp. and cloned the gene into the vector. After transformation by pAsOPPcAmy, the α-amylase PcAmy from Penicillium sp. was successfully expressed in a heterologous host system for the first time. The α-amylase activity in the A. oryzae transformant was increased by 62.3% compared with the untransformed A. oryzae control. The PcAmy protein produced in the system had an optimum pH of 5.0 and optimum temperature of 30^oC. As a cold-adapted enzyme, PcAmy shows potential value in industrial applications because of its high catalytic activity at low temperature. Furthermore, the expression vector reported in this study provides promising utility for further scientific research and biotechnological applications.

• Molecular & Cellular Toxicology
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• v.5 no.4
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• pp.283-290
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• 2009

Differential gene expression profiling was performed in the hepatic tissue of marine medaka fish (Oryzias javanicus) after exposure to benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon (PAH), by heterologous hybridization using a medaka cDNA microarray. Thirty-eight differentially expressed candidate genes, of which 23 were induced and 15 repressed (P<0.01), were identified and found to be associated with cell cycle, development, endocrine/reproduction, immune, metabolism, nucleic acid/protein binding, signal transduction, or non-categorized. The presumptive physiological changes induced by BaP exposure were identified after considering the biological function of each gene candidate. The results obtained in this study will allow future studies to assess the molecular mechanisms of BaP toxicity and the development of a systems biology approach to the stress biology of organic chemicals.

• Journal of Microbiology and Biotechnology
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• v.23 no.10
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• pp.1413-1421
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• 2013

The marine microalga Isochrysis sphaerica is rich in the very-long-chain polyunsaturated fatty acids, including eicosapentaenoic acid (EPA, $C20:5{\omega}-3$) and docosahexaenoic acid (DHA, $C22:6{\omega}-3$) that are important to human health. Here, we report a functional characterization of a ${\Delta}4$-fatty acid desaturase gene (FAD4) from I. sphaerica. IsFAD4 contains a 1,284 bp open reading frame encoding a 427 amino acid polypeptide. The deduced amino sequence comprises three conserved histidine motifs and a cytochrome b5 domain at its N-terminus. Phylogenetic analysis indicated that IsFad4 formed a unique Isochrysis clade distinct from the counterparts of other eukaryotes. Heterologous expression of IsFAD4 in Pichia pastoris showed that IsFad4 was able to desaturate docosapentaenoic acid (DPA) to form DHA, and the rate of converting DPA to DHA was 79.8%. These results throw light on the potential industrial production of specific polyunsaturated fatty acids through IsFAD4 transgenic yeast or oil crops.

• Journal of Microbiology and Biotechnology
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• v.16 no.6
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• pp.952-960
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• 2006

As a unicellular green alga that possesses many of the metabolic pathways present in higher plants, Chlorelia offers many advantages for expression of heterologous proteins. Since strong and constitutive promoters are necessary for efficient expression in heterologous expression systems, the development of such promoters for use in the Chlorella system was the aim of this study. Proteins encoded by the early genes of algal viruses are expressed before viral replication, probably by the host transcriptional machinery, and the promoters of these genes might be useful for heterologous expression in Chlorella. In this study, putative promoter regions of DNA polymerase, ATP-dependent DNA ligase, and chitinase genes were amplified from eight Korean Chlorella virus isolates by using primer sets designed based on the sequence of the genome of PBCV-1, the prototype of the Phycodnaviridae. These putative promoter regions were found to contain several cis-acting elements for transcription factors, including the TATA, CAAT, NTBBF1, GATA, and CCAAT boxes. The amplified promoter regions were placed into Chlorella transformation vectors containing a green fluorescence protein (GFP) reporter gene and the Sh ble gene for phleomycin resistance. C. vulgaris protoplasts were transformed and then selected with phleomycin. The GFP fluorescence intensities of cells transformed with chitinase, DNA polymerase, and DNA ligase gene promoter-GFP fusion constructs were 101.5, 100.8, and 95.8%, respectively, of that of CaMV 35S-GFP-transformed Chlorella cells. These results demonstrate that these viral promoters are active in transformed Chlorella.

• Microbiology and Biotechnology Letters
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• v.35 no.3
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• pp.184-190
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• 2007

Approximately 2.0 kb of the promoter region of the Pichia pastoris phosphoglycerate kinase gene (PGK1) was reduced to a 266 bp fragment and this minimized portion was used for construction of a new episomal constitutive expression vector in P. pastoris. As an approach to developing a constitutive expression vector in P. pastoris, the GAP promoter region of the Pichia expression vector pGAPZB was replaced with sequentially deleted PGK1 promoter fragments fused to a beta-galactosidase gene. When a lacZ gene was used as a reporter gene, PGK1 promoter strength was lower than that of the constitutive GAP promoter but it was higher than TEF1. We report here the development of the pPGKZ-E vector as a new episomal expression vector for heterologous gene expression by removing non-essential regions of the PGK1 promoter. This broadens the choice of episomal expression vectors for controlled constitutive expression in P. pastoris.

• BMB Reports
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• v.31 no.5
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• pp.503-507
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• 1998

To investigate the effects of the Vitreoscilla hemoglobin (VHb) gene on the production of a heterologous protein, a comparative expression system for VHb and ferritin was constructed. First, the VHb gene was inserted into the downstream and upstream regions of the ferritin gene to construct pHF2 and pHF3, respectively. Next, the two plasmids pACHB1 and pVUTFH10, having the VHb gene and the ferritin gene respectively, were constructed in order to express the two genes in different plasmids by using a coplasmid expression system. It was observed that the cell growth was improved in all strains containing the VHb gene. Furthermore, in our coplasmid expression system, the presence of the VHb gene increased production of the ferritin by 1.8 times, as much as that in a strain not having the VHb gene.

• Journal of Microbiology and Biotechnology
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• v.7 no.4
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• pp.229-236
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• 1997

A gene, $phbC_{2.4.1}$ encoding poly-3-hydroxybutyric acid (PHB) synthase of Rhodobacter sphaeroides 2.4.1 was cloned by employing heterologous expression in Escherichia coli. R. sphaeroides chromosomal DNA partially digested with MboI was cloned in pUC19 followed by mobilization into E. coli harbouring $phbA,B_{AC}$ in pRK415, which code for ${\beta}$-ketothiolase and acetoacetyl CoA reductase of Alcaligenes eutrophus, respectively. Two E. coli clones carrying R. sphaeroides chromosomal fragment of $phbC_{2.4.1}$ in pUC19 were selected from ca. 10,000 colonies. The PHB-producing colonies had an opaque white appearance due to the intracellular accumulation of PHB. The structure of PHB produced by the recombinant E. coli as well as from R. sphaeroides 2.4.1 was confirmed by [$H^{+}$]-nuclear magnetic resonance (NMR) spectroscopy. Restriction analysis of the two pUC19 clones revealed that one insert DNA fragment is contained as a part of the other cloned fragment. An open reading frame of 601 amino acids of $phbC_{2.4.1}$ with approximate M.W. of 66 kDa was found from nucleotide sequence determination of the 2.8-kb SaiI-PstI restriction endonuclease fragment which had been narrowed down to support PHB synthesis through heterologous expression in the E. coli harbouring $phbA,B_{AC}$. The promoter (s) of the $phbC_{2.4.1}$ were localized within a 340-bp DNA region upstream of the $phbC_{2.4.1}$ start codon according to heterologous expression analysis.

• Journal of Microbiology and Biotechnology
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• v.30 no.11
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• pp.1777-1784
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• 2020

To increase the availability of microalgae as producers of valuable compounds, it is necessary to develop novel systems for gene expression regulation. Among the diverse expression systems available in microalgae, none are designed to induce expression by low temperature. In this study, we explored a cold-inducible system using the antifreeze protein (AFP) promoter from a polar diatom, Chaetoceros neogracile. A vector containing the CnAFP promoter (pCnAFP) was generated to regulate nuclear gene expression, and reporter genes (Gaussia luciferase (GLuc) and mVenus fluorescent protein (mVenus)) were successfully expressed in the model microalga, Chlamydomonas reinhardtii. In particular, under the control of pCnAFP, the expression of these genes was increased at low temperature, unlike pAR1, a promoter that is widely used for gene expression in C. reinhardtii. Promoter truncation assays showed that cold inducibility was still present even when pCnAFP was shortened to 600 bp, indicating the presence of a low-temperature response element between -600 and -477 bp. Our results show the availability of new heterologous gene expression systems with cold-inducible promoters and the possibility to find novel low-temperature response factors in microalgae. Through further improvement, this cold-inducible promoter could be used to develop more efficient expression tools.