• Journal of Microbiology and Biotechnology
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• v.24 no.1
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• pp.70-79
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• 2014

In an attempt to develop a variety of expression vector systems for Corynebacterium glutamicum, six types of promoters, including $P_{tac}$, $P_{sod}$, $P_{sod}$ with a conserved Shine-Dalgarno (SD) sequence from C. glutamicum, $P_{ilvC}$, $P_{ilvC}$ with a conserved SD-1 ($P_{ilvC-M1}$), and $P_{ilvC}$ with a conserved SD-2 ($P_{ilvC-M2}$), were cloned into a modified shuttle vector, pCXM48. According to analysis of promoter strength by quantitative reverse transcription PCR, $P_{sod}$ and $P_{sod-M}$ were superior to tac and ilvC promoters in terms of transcription activity in C. glutamicum. All of the promoters have promoter activities in Escherichia coli, and $P_{sod-M}$ displayed the highest level of transcriptional activity. The protein expression in constructed vectors was evaluated by measuring the fluorescence of green fluorescent protein (GFP) and SDS-PAGE. C. glutamicum harboring plasmids showed GFP fluorescence with an order of activity of $P_{ilvC}$ > $P_{ilvC-M1}$ > $P_{sod}$ > $P_{ilvC-M2}$ > $P_{sod-M}$, whereas all plasmids except pCSP30 with $P_{sod}$ displayed fluorescence activities in E. coli. Of them, the strongest level of GFP was observed in E. coli with $P_{sod-M}$, and this seems to be due to the introduction of the conserved SD sequence in the translational initiation region. These results demonstrate that the expression vectors work well in both C. glutamicum and E. coli for the expression of target proteins. In addition, the vector systems harboring various promoters with different strengths, conserved SD sequences, and multiple cloning sites will provide a comfortable method for cloning and gene expression, and consequently contribute to the metabolic engineering of C. glutamicum.

• Journal of Microbiology and Biotechnology
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• v.33 no.6
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• pp.724-735
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• 2023

NdgR, a global regulator in soil-dwelling and antibiotic-producing Streptomyces, is known to regulate branched-chain amino acid metabolism by binding to the upstream region of synthetic genes. However, its numerous and complex roles are not yet fully understood. To more fully reveal the function of NdgR, phospholipid fatty acid (PLFA) analysis with gas chromatography-mass spectrometry (GC-MS) was used to assess the effects of an ndgR deletion mutant of Streptomyces coelicolor. The deletion of ndgR was found to decrease the levels of isoleucine- and leucine-related fatty acids but increase those of valine-related fatty acids. Furthermore, the defects in leucine and isoleucine metabolism caused by the deletion impaired the growth of Streptomyces at low temperatures. Supplementation of leucine and isoleucine, however, could complement this defect under cold shock condition. NdgR was thus shown to be involved in the control of branched-chain amino acids and consequently affected the membrane fatty acid composition in Streptomyces. While isoleucine and valine could be synthesized by the same enzymes (IlvB/N, IlvC, IlvD, and IlvE), ndgR deletion did not affect them in the same way. This suggests that NdgR is involved in the upper isoleucine and valine pathways, or that its control over them differs in some respect.

• Korean Journal of Microbiology
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• v.46 no.1
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• pp.104-108
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• 2010

We describe the construction of derivatives of wild type and mutant lrp genes that encode 6XHis-tag Lrps. These derivatives of wild type and mutant Lrp could be useful for in vitro studies including Lrp conformational changes. We show that 6XHis-tag Lrp wild type and 6XHis-tag Lrp R145W bind with similar patterns in vitro to 21 bp duplex DNA containing the consensus sequences of Lrp sites of upstream of the ilvIH operon. In addition, we report here the 6XHis-tag Lrp R145W is useful to investigate the conformational changes of Lrp in solution by using its own intrinsic fluorescence characteristics.

• Journal of Microbiology and Biotechnology
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• v.20 no.11
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• pp.1539-1545
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• 2010

In beer, glutathione works as the main antioxidant compound, which also correlates with the stability of the beer flavor. In addition, high residual sugars in beer contribute to major nonvolatile components, which are reflected in a high caloric content. Therefore, in this study, the Saccharomyces cerevisiae GSH1 gene encoding glutamylcysteine synthetase and the Saccharomycopsis fibuligera ALP1 gene encoding ${\alpha}$-amylase were coexpressed in industrial brewing yeast strain Y31 targeting the ${\alpha}$-acetolactate synthase (AHAS) gene (ILV2) and alcohol dehydrogenase gene (ADH2), resulting in the new recombinant strain TY3. The glutathione content in the fermentation broth of TY3 increased to 43.83 mg/l as compared with 33.34 mg/l in the fermentation broth of Y31. The recombinant strain showed a high ${\alpha}$-amylase activity and utilized more than 46% of the starch as the sole carbon source after 5 days. European Brewery Convention tube fermentation tests comparing the fermentation broths of TY3 and Y31 showed that the flavor stability index for TY3 was 1.3-fold higher, whereas its residual sugar concentration was 76.8% lower. Owing to the interruption of the ILV2 gene and ADH2 gene, the contents of diacetyl and acetaldehyde as off-flavor compounds were reduced by 56.93% and 31.25%, respectively, when compared with the contents in the Y31 fermentation broth. In addition, since no drug-resistant genes were introduced to the new recombinant strain, it should be more suitable for use in the beer industry, owing to its better flavor stability and other beneficial characteristics.

• Korean Journal of Microbiology
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• v.50 no.4
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• pp.275-280
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• 2014

Leucine-responsive Regulatory Protein (Lrp) from Escherichia coli is an 18.8 kDa protein composed of 164 amino acids. Wild type Lrp (Lrp Wt) does not possess any tryptophan amino acid which has strong intrinsic fluorescence, whereas the mutant Lrp R145W contains a single tryptophan at the position 145 in the leucine-responsive domain. To investigate the fluorescence character, the Lrp R145W and Lrp Wt proteins were purified. The fluorescence intensity of Lrp R145W is much higher than that of wild type protein, and the intensity of Lrp R145W was decreased by binding to its specific DNA designed from ilvIH operon and to L-leucine. In addition, the tryptophan fluorescence intensity of Lrp R145W was strongly quenched by addition of acrylamide even in the least amount of concentration as well as by urea. The data obtained from this study may give valuable information on the three dimensional structure of Lrp R145W.

• Journal of the Korean Society of Systems Engineering
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• v.2 no.1
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• pp.37-41
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• 2006

The KSLV-I satellite launch vehicle will be launched in a space center currently under construction. The Space Center which is an advance post base of space development of Korea is located on Oenaro island in Kohung, South Cholla Province. A Ground Complex of the Space Center consists of an AC(Assembly Complex), a LC(Launch Complex), and a MCC(Mission Control Center). Assembly and test facilities are located in the AC in which stage assembly, integrated assembly, check-up, certification test, and pre-launch test are made effectively. A launch pad, fuel supply facilities, a launch control center and associated supporting facilities are located in the LC, and the MCC has control over the space center. These ground complex facilities have diverse forms of an interface with mechanical device, electric device, and etc. These should also provide optimum condition and performance during launch operation processes of the launch vehicle. This paper introduces the result of R&D for the AC of the ground complex performed during system design period.

• Archives of Pharmacal Research
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• v.15 no.1
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• pp.30-34
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• 1992

In order to obtain acetaminophen, a popular analgesic-antipyretic, through microbial p-hydroxylation and N-acetylation of aniline, various fungi and bacteria were secreened. Among them, Streptomyces species were chosen for strain improvement by the use of interspecific protoplast fusion technique. Two interspecific fused strains were developed between S. rimosus (N-cetylation function) and S. aureofaciens (p-hydroxylation function) and also between S. lividans and S. globisporus. For efficient protoplast fusion and cell wall regeneration, various conditions were examined. In a typical experiment of mixed S rimosus ($pro^- \;his^-$) and S. aureofaciens ($ilv^-$) protoplasts with 40% (w/v) polythylene glycol 3350 (PEG) for 3 min gave $8.3\times10^{-7}$ of fusion frequency. Treatment of mixed S. lividans (pant-) and S. globisporus (leu-) protoplasts with 50% (w/v) PEG for 3 min at $30^\circ{C}$ gave $1.2\times10^{-6}$ of frequency. Among the fused strains, up to 40-50% increase in p-hydroxylation power was observed. To investigate the possibility of plasmid involvement in p-hydroxylation power was observed. To investigate the possibility of plasmid involvement in p-hydroxylation of acetanilide, plasmid curing was attempted. We found that cells treated with acriflavine (at the frequency of 100%) and cells regenerated from protoplsts of S. auroefaciens (2% frequency) lost their p-hydroxylation function.

• Bulletin of the Korean Space Science Society
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• 2009.10a
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• pp.49.1-49.1
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• 2009

Ground Complex, which is located at Naro Space Center, consists of Assembly Complex(AC) and Launch Complex(LC) which is necessary for successful launch of KSLV-I(NARO). AC consists of Assembly/Testing Building(ATB), Payload Processing Building(PPB), Kick Motor Building(KMB). The purpose of AC is accepting of KSLV-I components, testing, checkout, assembly(disassembly) of the launch vehicle(LV), readiness for transferring LV to LC, accepting of integrated Launch Vehicle(ILV) in case of launch cancellation and short/long time storage, and so on. Qualification tests(QT) for the total system at AC are carried out to check hardware used for operations with first stage unit mockup, upper stage unit Mockup and integrated mockup(GTV). The qualification tests is carried out according to program and procedures of QT. By course of this process, AC is certificated that all the systems and facilities of AC are guaranteed by the fulfillment of technological operations envisioned in the program of qualification tests during the work with the mock-up.

• Proceedings of the Korean Society of Propulsion Engineers Conference
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• 2010.11a
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• pp.656-657
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• 2010

The ground oxidizer supply system in the launch site of NARO space center had operated 9 times from the start of tests with ILV on May, 2009 to the 2nd flight test of the NARO vehicle. This system operated successfully for twice launches of the NARO vehicle. To judge the successful operation of the ground facility, it should have reproducibility and reliability. In this report, we have analyzed the liquid oxygen consumption of the system to judge of its reproducibility and it can be a reference for using this system for the next generation of KSLV system.

• Microbiology and Biotechnology Letters
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• v.47 no.2
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• pp.242-249
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• 2019

Biosynthesis of indole-3-acetate (IAA) from L-tryptophan via indole-3-pyruvate pathway requires three enzymes including aminotransferase, indole-3-pyruvate decarboxylase, and indole-3-acetate dehydrogenase. To establish a bio-based production of IAA, the aspC, ipdC, and iad1 from Escherichia coli, Enterobacter cloacae, and Ustilago maydis, respectively, were expressed under control of the tac, ilvC, and sod promoters in C. glutamicum. Cells harboring ipdC produced tryptophol, indicating that the ipdC product is functional in this host. Analyses of SDS-PAGE and enzyme activity revealed that genes encoding AspC and Iad1 were efficiently expressed from the sod promoter, and their enzyme activities were 5.8 and 168.5 nmol/min/mg-protein, respectively. The final resulting strain expressing aspC, ipdC, and iad1 produced 2.3 g/l and 7.3 g/l of IAA from 10 g/l L-tryptophan, respectively, in flask cultures and a 5-L bioreactor.