• KSBB Journal
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• v.15 no.2
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• pp.181-187
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• 2000

The production of heterologous protein using GAL promoter in conventional S. cerevisiae has several problems to s이ve for c commercialization. In this research, S. cerevisiae mutant(reg1-501, gaI1), which cannot use galactose and has alleviated g glucose repression level, is used as host for optimizing induction of GAL promoter. In this experiment, the effects of specific g growth rate on specific recombinant protein expression rate were tested in both cases and optimum fed batch fermentation m method was obtained in both cases. Through these experiments, optimum condition of recombinant protein production by G GAL promoter using S. cerevisiae mutant (reg1-501, gal1) were found.

• Microbiology and Biotechnology Letters
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• v.24 no.5
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• pp.604-612
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• 1996

To improve the fermentation characteristics of the haploid starch-fermenting recombinant yeast strain K114/YIpMS$\Delta$R(LEU2/URA3) secreting both $\alpha$-amylase and glucoamylase was rare-mated with polyploid industrial yeast Saccharomyces sp. K35. The K35 strain had good fermentation-characteristics such as ethanol-tolerance, high temperature and sugar-tolerance, and high fermentation rate. Among the resulting 66 hybrids, the best strain RH51 was selected. The RH51 exhibited amylolytic activity of K114/YIpMS$\Delta$R(LEU2/URA3) as well as ethanol and sugar tolerance of K35. The optimum temperature of hybrid RH51 for starch fermentation was 34$\circ$C which was same as that of K35 but different from that (30$\circ$C) of K114/YIpMS$\Delta$R(LEU2/URA3). The optimum pH was 5.0. The optimum size of inoculum was 2% as the pellet (w/v) of yeast cells. The hybrid strain RH51 produced 7.0% ethanol (w/v) from 20% (w/v) soluble starch while K35 formed almost no ethanol, 0.3% (w/v). RH51 strain produced 7.5% (w/v) ethanol after 8 days in a 2.5 l fermenter containing 800 ml of 20% (w/v) soluble starch. The residual starch content in the fermentation medium was 1.68% (w/v), and therefore almost all the starch was fermented completely.

• Journal of Microbiology and Biotechnology
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• v.22 no.9
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• pp.1258-1263
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• 2012

2,3-Butanediol (2,3-BD) is a major metabolite produced by Klebsiella pneumoniae KCTC2242, which is a important chemical with wide applications. Three genes important for 2,3-BD biosynthesis acetolactate decarboxylase (budA), acetolactate synthase (budB), and alcohol dehydrogenase (budC) were identified in K. pneumoniae genomic DNA. With the goal of enhancing 2,3-BD production, these genes were cloned into pUC18K expression vectors containing the lacZ promoter and the kanamycin resistance gene to generate plasmids pSB1-7. The plasmids were then introduced into K. pneumoniae using electroporation. All strains were incubated in flask experiments and 2,3-BD production was increased by 60% in recombinant bacteria harboring pSB04 (budA and budB genes), compared with the parental strain K. pneumoniae KCTC2242. The maximum 2,3-BD production level achieved through fed-batch fermentation with K. pneumoniae SGJSB04 was 101.53 g/l over 40 h with a productivity of 2.54 g/l.h. These results suggest that overexpression of 2,3-BD synthesis-related genes can enhance 2,3-BD production in K. pneumoniae by fermentation.

• KSBB Journal
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• v.20 no.4
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• pp.299-304
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• 2005

A two-dimensional (2D) spectrofluorometer was used to monitor various fermentation processes with recombinant E. coli for the production of 5-aminolevulinic acid (ALA). The whole fluorescence spectral data obtained during a process were analyed using artificial neural networks, i.e. self-organizing map (SOM) and feedforward backpropagation neural network (BPNN).Based on the classified fluorescence spectra a supervised BPNN algorithm was used to predict some of the process parameters. It was also shown that the BPNN models could elucidate some sections of the process performance, e.g. forecasting the process performance.

• Mycobiology
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• v.47 no.4
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• pp.512-520
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• 2019

Statistical experimental methods were used to optimize the medium for mass production of a novel laccase3 (Lac3) by recombinant Saccharomyces cerevisiae TYEGLAC3-1. The basic medium was composed of glucose, casamino acids, yeast nitrogen base without amino acids (YNB w/o AA), tryptophan, and adenine. A one-factor-at-a-time approach followed by the fractional factorial design identified galactose, glutamic acid, and ammonium sulfate, as significant carbon, nitrogen, and mineral sources, respectively. The steepest ascent method and response surface methodology (RSM) determined that the optimal medium was (g/L): galactose, 19.16; glutamic acid, 5.0; and YNB w/o AA, 10.46. In this medium, the Lac3 activity (277.04 mU/mL) was 13.5 times higher than that of the basic medium (20.50 mU/mL). The effect of temperature, pH, agitation (rpm), and aeration (vvm) was further examined in a batch fermenter. The best Lac3 activity was 1176.04 mU/mL at 25 ℃, pH 3.5, 100 rpm, and 1 vvm in batch culture.

• Journal of Microbiology and Biotechnology
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• v.20 no.11
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• pp.1534-1538
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• 2010

Fed-batch cultures of Hansenula polymorpha were studied to develop an efficient biosystem to produce recombinant human serum albumin (HSA). To comply with this purpose, we used a high-purity oxygen-supplying strategy to increase the viable cell density in a bioreactor and enhance the production of target protein. A mutant strain, H. polymorpha GOT7, was utilized in this study as a host strain in both 5-l and 30-l scale fermentors. To supply high-purity oxygen into a bioreactor, nearly 100% high-purity oxygen from a commercial bomb or higher than 93% oxygen available in situ from a pressure swing adsorption (PSA) oxygen generator was employed. Under the optimal fermentation of H. polymorpha with highpurity oxygen, the final cell densities and produced HSA concentrations were 24.6 g/l and 5.1 g/l in the 5-l fermentor, and 24.8 g/l and 4.5 g/l in the 30-l fermentor, respectively. These were about 2-10 times higher than those obtained in air-based fed-batch fermentations. The discrepancies between the 5-l and 30-l fermentors with air supply were presumably due to the higher contribution of surface aeration over submerged aeration in the 5-l fermentor. This study, therefore, proved the positive effect of high-purity oxygen in enhancing viable cell density as well as target recombinant protein production in microbial fermentations.

• Journal of Microbiology and Biotechnology
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• v.20 no.3
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• pp.550-557
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• 2010

Escherichia coli (E. coli) heat-labile enterotoxin B subunit (LTB) was regarded as one of the most powerful mucosal immunoadjuvants eliciting strong immunoresponse to coadministered antigens. In the research, the high-level secretory expression of functional LTB was achieved in P. pastoris through high-density fermentation in a 5-1 fermentor. Meanwhile, the protein was expressed in E. coli by the way of inclusion body, although the gene was cloned from E. coli. Some positive yeast and E. coli transformants were obtained respectively by a series of screenings and identifications. Fusion proteins LTB-6$\times$His could be secreted into the supernatant of the medium after the recombinant P. pastoris was induced by 0.5% (v/v) methanol at $30^{\circ}C$, whereas E. coli transformants expressed target protein in inclusion body after being induced by 1 mM IPTG at $37^{\circ}C$. The expression level increased dramatically to 250-300 mg/l supernatant of fermentation in the former and 80-100 mg/l in the latter. The LTB-6$\times$His were purified to 95% purity by affinity chromatography and characterized by SDS-PAGE and Western blot. Adjuvant activity of target protein was analyzed by binding ability with GMI gangliosides. The MW of LTB-6$\times$His expressed in P. pastoris was greater than that in E. coli, which was equal to the expected 11 kDa, possibly resulted from glycosylation by P. pastoris that would enhance the immunogenicity of co-administered antigens. These data demonstrated that P. pastoris producing heterologous LTB has significant advantages in higher expression level and in adjuvant activity compared with the homologous E. coli system.

• Biotechnology and Bioprocess Engineering:BBE
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• v.6 no.2
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• pp.144-149
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• 2001

For the production and purification of a single chain human insulin precursor, four types of fusion peptides $\beta$-galactosidase (LacZ), maltose binding protein (MBP), glutathione-S-transferase (GST), and (His)(sub)6-tagged sequence (HTS) were investigated. Recombinant E. coli harboring hybrid genes was cultivated at 37$\^{C}$ for 1h, and gene induction occurred when 0.2mM of isopropyl-D-thiogalactoside (IPTG) was added to the culture broth, except for E. coli BL21 (DE3) pLysS harboring a pET-BA cultivation with 1.0mM IPTG, followed by a longer than 4h batch fermentation respectively. DEAE-Sphacel and Sephadex G-200 gel filtration chromatography, amylose affinity chromatography, glutathione-sepharose 4B affinity chromatography, and a nickel chelating affinity chromatography system as a kind of immobilized metal ion affinity chromatography (IMAC) were all employed for the purification of a single chain human insulin precursor. The recovery yields of the HTS-fused, GST-fused, MBP-fused, and LacZ-fused single chain human insulin precursors resulted in 47%, 20%, 20%, and 18% as the total protein amounts respectively. These results show that a higher recovery yield of the finally purified recombinant peptides was achieved when affinity column chromatography was employed and when the fused peptide had a smaller molecular weight. In addition the pET expression system gave the highest productivity of a fused insulin precursor due to a two-step regulation of the gene expression, and the HTS-fused system provided the highest recovery of a fused insulin precursor based on a simple and specific separation using the IMAC technique.

• Journal of Applied Biological Chemistry
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• v.64 no.3
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• pp.309-316
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• 2021

Kaempferol 3-O-galactoside (Trifolin), a member of the flavonol group, has been reported to have anticancer effects against promyelocytic leukemia, histocytic lymphoma, skin melanoma and lung cancer. Trifolin has been extracted and used from several plants, but the extraction process is complicated and the final yield is low. Biotransformation is an alternative tool to produce high value-added chemicals from inexpensive compounds. To synthesis trifolin from naringenin, three genes (PeFLS and OsUGE-PhUGT) were introduced into Escherichia coli, respectively. In order to synthesis trifolin from naringenin, a co-culture fermentation system was established by optimizing the cell concentration, biotransformation temperature and medium, isopropyl-β-D-thiogalactoside (IPTG) concentration, substrate supply concentration, and recombinant protein induction time. The established optimal conditions for trifolin production were a 3:1 ratio of BL-UGTE to BL-FLS, induction of recombinant protein at 25 ℃ for 4 h after addition of 2.0 mM IPTG, biotransformation at 30 ℃, and supply of 300 μM naringenin. Through the optimized co-culture fermentation system, trifolin was biosynthesized up to 67.3 mg/L.

• Proceedings of the Korean Institute of Information and Commucation Sciences Conference
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• 2019.05a
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• pp.533-536
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• 2019

Recombinant baculoviruses are widely used to express heterologous genes in cultured insect cells. Recombinant baculoviruses can serve as gene-transfer vectors for expression of recombinant proteins in a wide range of mammalian cell types. Baculovirus system has significant benefits in view of safety, large-scale, and high level of gene expression. In this study, baculoviral vectors which were reconstructed from pOPINEneo-3C-GFP vector, were recombined with cytomegalovirus (CMV) promoter, green fluorescent protein (GFP), and p53 with NcoI and XhoI. These recombinant vectors were infected with various cells and cell lines. The baculovirus vector thus developed was analyzed by comparing the metastasis and expression of the recombinant genes with conventional vectors. These results suggest that the baculovirus vector has higher efficiency in metastasis and expression than the control vector. This work was supported by a grant from Mid-Career Researcher Program(NRF-2016R1A2B4016552) through the National Research Foundation of Korea(NRF) funded by the Ministry of Science, ICT & Future Planning(MSIP).