• Journal of Microbiology and Biotechnology
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• 제23권2호
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• pp.149-155
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• 2013

Heterologous expression of a putative $K^+/H^+$ antiporter of Streptomyces coelicolor A3(2) (designated as sha4) in E. coli and Streptomyces hygroscopicus JCM4427 showed enhanced tolerance to $K^+$ stress, acidic-pH shock, and/or geldanamycin production under $K^+$ stress. In a series of $K^+$ extrusion experiments with sha4-carrying E. coli deficient in the $K^+/H^+$ antiporter, a restoration of impaired $K^+$ extrusion activity was observed. Based on this, it was concluded that sha4 was a true $K^+/H^+$ antiporter. In different sets of experiments, the sha4-carrying E. coli showed significantly improved tolerances to $K^+$ stresses and acidic-pH shock, whereas sha4-carrying S. hygroscopicus showed an improvement in $K^+$ stress tolerance only. The sha4-carrying S. hygroscopicus showed much higher geldanamycin productivity than the control under $K^+$ stress condition. In another set of experiments with a production medium, the secretion of geldanamycin was also significantly enhanced by the expression of sha4.

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

• KSBB Journal
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• 제13권3호
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• pp.325-330
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• 1998

The methylotrophic yeast, Pichia pastoris, is known to be a potential host to offer many advantages for production of recombinant proteins. Fermentation parameters were optimized to enhance the heterologous ${\beta}$-galactosidase productivity with P. pastoris. Optimum concentration of methanol, used as inducer, was observed to be 8 g/L and the extent of repression of AOX1 promoter by glycerol was lower than by glucose. The degradation of the gene product ${\beta}$-galactosidase by protease was inhibited as the pH increased from 5 to 8 and the yeast extract(1%) as nitrogen source increased expression level 4 times higher compared to yeast nitrogen base(1%) as nitrogen source increased expression level 4 times higher compared to yeast nitrogen base(1%). Induction method, in which methanol is just added to fermentation medium without centrifugation, was found to be as much effective as the one with centrifugation.

• Biotechnology and Bioprocess Engineering:BBE
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• 제5권4호
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• pp.227-233
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• 2000

Although filamentous fungi are used extensively for protein expression, their use for the production of heterologous glycoproteins is constrained by the types of N-glycan structures produced by filamentous fungi as compared to those naturally found on the glycoproteins. Attempts are underway to engineer the N-glycan synthetic pathways in filamentous fungi in order to produce fungal expression strains which can produce heterologous glycoproteins carrying specific N-glycan structures. To fully realize this goal, a detailed understanding of the genetic components of this pathway in filamentous fungi is required. In this review, we discuss the characterization of the $\alpha$-mannosidase gene family in filamentous fungi and its implications for the elucidation of the N-glycan synthetic pathway.

• Journal of Microbiology and Biotechnology
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• 제25권7호
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• pp.953-962
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• 2015

Escherichia coli is the most preferred microorganism to express heterologous proteins for therapeutic use, as around 30% of the approved therapeutic proteins are currently being produced using it as a host. Owing to its rapid growth, high yield of the product, costeffectiveness, and easy scale-up process, E. coli is an expression host of choice in the biotechnology industry for large-scale production of proteins, particularly non-glycosylated proteins, for therapeutic use. The availability of various E. coli expression vectors and strains, relatively easy protein folding mechanisms, and bioprocess technologies, makes it very attractive for industrial applications. However, the codon usage in E. coli and the absence of post-translational modifications, such as glycosylation, phosphorylation, and proteolytic processing, limit its use for the production of slightly complex recombinant biopharmaceuticals. Several new technological advancements in the E. coli expression system to meet the biotechnology industry requirements have been made, such as novel engineered strains, genetically modifying E. coli to possess capability to glycosylate heterologous proteins and express complex proteins, including full-length glycosylated antibodies. This review summarizes the recent advancements that may further expand the use of the E. coli expression system to produce more complex and also glycosylated proteins for therapeutic use in the future.

• 한국미생물·생명공학회지
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• 제31권1호
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• pp.36-41
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• 2003

Saccharomyces cerevisiae로부터 외래 $\alpha$-amylase의 발현 및 분비를 증진시키기 위하여 여러 실험이 수행되었다. ADC1 promoter와 mouse salivary $\alpha$-amylase cDNA gene의 native signal sequence를 효모의 PRB1 promoter와 invertase leader sequence로 대치한 plasmid vector pCNN(AMY)를 제작하였다. 효모세포에서 생성된 $\alpha$-amylase의 세포외로의 분비율은 mouse o-amylase의 native signal sequence인 경우는 약 89.4%이었으며 invertase leader sequence로 치환된 경우는 96.3%로 분비효율이 증진되었다. 야생주인 K8l/pCNN(AMY)와 호흡결여변이주인 K81/pCNN(AMY)p-의 혐기적 조건하에서의 배양 결과 $\alpha$-amylase 생산량이 K8l/pCNN(AMY)보다 K81/pCNN(AMY)p-가 약 5~8배 정도 증가하였다. $\alpha$-Amylase의 생산에 있어서 배지조성에 따른 K81/pCNN(AMY)의 생산증진의 비교는 배지성분인 yeast extract와 peptone의 구성비율을 비교하였을 때 yeast extract 1%와 peptone 2%, NaCl의 경우 100 mM, 2-mercaptoethanol인 경우에는 0.015%(w/v)을 첨가하였을 때 최대 효소 활성을 나타내었고, 특히 2-mercaptoethanol인 경우에는 대조구에 비해 효소 생산량이 약 3배 정도 증진되었다.

• Journal of Plant Biotechnology
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• 제5권3호
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• pp.149-155
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• 2003

Metabolic engineering for production of isoflavones in nonlegume plants could distribute the health benefits of these phytoestrogens in more widely-consumed grains. Series of investigation to check the ability of the heterologous isoflavone synthase enzyme to interact with the endogenous phenylpropanoid pathway have been conducted. Overall, results provide possibility of production of isoflavonoids in several plant tissue systems including soybean and nonlegumes. In tissue that undergoes naturally enhanced synthesis of anthocyanins, genistein production was enhanced. In a monocot cell system, introduced expression of a transcription factor regulating genes of the anthocyanin pathway was effective in conferring the ability to produce genistein in the presence of the isoflavone synthase gene. However, in this case the intermediate accumulated to high levels indicating an inefficiency in its conversion. Introduction of a third gene, chalcone reductase, provided the ability to synthesize an additional substrate of isoflavone synthase resulting in production of the isoflavone daidzein. These research efforts provide insight into requirements for metabolic engineering for isoflavone production in nonlegume dicot and monocot tissues.

• 한국식물생명공학회:학술대회논문집
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• 한국식물생명공학회 2003년도 식물바이오벤처 페스티발
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• pp.77-84
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• 2003

Metabolic engineering for production of isoflavones in non-legume plants could distribute the health benefits of these phytoe-strogens in more widely-consumed grains. We investigate the ability of the heterologous isoflavone synthase enzyme to interact with the endogenous phenylpropanoid pathway. Overall, results provide possibility of production of isoflavonoids in several plant tissue systems including soybean and non-legumes. In tissue that undergoes naturally enhanced synthesis of anthocyanins, genistein production was enhanced. In a monocot cell system, introduced expression of a transcription factor regulating genes of the antho-cyanin pathway was effective in conferring the ability produce genistein in the presence of the isoflavone synthase gene. However, in this case the intermediate accumulated to high levels indicating an inefficiency in its conversion. Introduction of a third gene, chalcone reductase, provided the ability to synthesize an additional substrate of isoflavone synthase resulting in production of the isoflavone daidzein. These research efforts provide insight into requirements for metabolic engineering for isoflavone production in non-legume dicot and monocot tissues.

• KSBB Journal
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• 제14권4호
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• pp.482-489
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• 1999

As host for the production of eucaryotic heterologous proteins, methylotrophic yeast Pichia pastoris and Hansenula polymorpha are the most highly developed of a small group of alternative yeast species chosen for their perceived advantages. This paper describes the method to enhance the recombinant protein productivity with P. pastoris and H. Plymorpha. In these experiments, the effects of methanol induction timing, induction method, pH, culture temperature and kinds of nitrogen sources on foreign protein production were tested with P. pastoris and compared with H. polymorpha.. In addition, optimum methanol concentration as inducer and the effects of carbon sources on AOX1 or MOX promoter repression and secretion efficiency were also studied in both cases.

• Journal of Microbiology and Biotechnology
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• 제21권11호
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• pp.1143-1146
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• 2011

Metabolic engineering of plant-specific phenylpropanoid biosynthesis has attracted an increasing amount of attention recently, owing to the vast potential of flavonoids as nutraceuticals and pharmaceuticals. Recently, we have developed a recombinant Streptomyces venezuelae as a heterologous host for the production of flavonoids. In this study, we successfully improved flavonoid production by expressing two sets of genes predicted to be involved in malonate assimilation. The introduction of matB and matC encoding for malonyl-CoA synthetase and the putative dicarboxylate carrier protein, respectively, from Streptomyces coelicolor into the recombinant S. venezuelae strains expressing flavanone and flavone biosynthetic genes resulted in enhanced production of both flavonoids.