• Microbiology and Biotechnology Letters
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• v.29 no.2
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• pp.72-77
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• 2001

Expression of f3 ~agarase Gene and Catabolite Repression in Escherichia coli by the Promoter of Alginate Lyase Gene Isolated from Marine Pseudomonas sp. Jin, Cheal~Ho, J~Hyeon Park, Jeong-Hyun Han, YoonM Hyeok Chae, Jong~Hee Lee, Jung-Kee Lee!, and In-800 Kong*. Faculty of Food Science and Biotechnology, Pukyong National UniversitYt Pusan 608-737, Korea, llnBioNet Co. 1690-3 Taejon 306-230, Korea - Promoter is a key factor for expression of the recombinant protein. There are many promoters for overexpression of protein in various organisms. The aly promoter of Pseudomonas sp. W7 isolated from marine environment was known to be a constitutive expression promoter of the alginate lyase gene, and it's promoter activity is repressed by glucose in Escherichia coli. To investigate the catabolite repression of the aly promoter ~md association between the promoter mutants, f3 agarase gene, which was also cloned from Pseudomonas sp. W7 was connected to the aly promoter with the sequence the coding 46 N-terminal amino acids ofthe alginate lyase gene. The constructed plasmid was introduced into E. coli and the agarase activity was measured. Fourty six amino acids of the alginate lyase gene was serially deleted using peR to the direction of 5' upstream region and subcloned. The agarase was overexpressed by the aly promoter and the production of agarase was repressed by the addition of glucose into culture media. Fourty six amino acids of alginate lyase did not affect the production of agarase at all. The deletion of a putative stem-loop structure in the aly promoter induced the decrease of f3 -agarase productivity.

• Journal of Microbiology and Biotechnology
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• v.8 no.1
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• pp.14-20
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• 1998

Syntheses of the B. stearothermophilus xylanolytic enzymes such as xylanases, ${\beta}$-xylosidases, ${\alpha}$-arabinofurano-sidases, and esterases, were observed to be regulated by the carbon source present in the culture media. Xylan induced synthesis of ${\beta}$-xylosidase at the highest level while xylose gave about 30% of the ${\beta}$-xylosidase activity induced by xylan. The lowest syntheses of the xylanolytic enzymes above mentioned were detected in the basal medium containing glucose as a sole carbon source. When a mixture of xylan and glucose was used as a carbon source, we could observe glucose repression of xylanase (about 70-fold) and ${\beta}$-xylosidase (about 40-fold) syntheses. Whereas, the level of the glucose repression of the expression of the xylA gene encoding the major ${\beta}$-xylosidase of B. stearothermophilus was assessed to be about l0-fold when the relative amounts of the xylA transcript were determined. From the sequence of the xylA gene, we could find two CRE-like sequences (CRE-l: nucleotides ＋124 to ＋136 and CRE-2:＋247 to ＋259) within the reading frame of the xylA gene, either or both of which were suspected to be involved in catabolite repression of the xylA gene.

• Microbiology and Biotechnology Letters
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• v.24 no.4
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• pp.404-407
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• 1996

The effects of carbon sources on vancomycin production were investigated using Nocardia orientalis CSVC 3300. Among carbon sources tested, glucose, maltose and fructose were effective for the production of vancomycin. Glucose was favored for growth, but decrease the production of vancomycin at the concentration above 7.5%. In comparison, maltose did not decrease the production of vancomycin up to the concentration of 20%. When the mixture of glucose and maltose was used in the ratio 1:3 to 1:4, the highest production of vancomycin was achieved. When glucose concentration was set at 3.0%, catabolite repression could not be observed up to total sugar concentration of 16.0%. Fermentation was carried out using commercial hydrolyzed starch composed of glucose, maltose, maltotriose and maltotetraose, The initial glucose concentration was set at 3.0% and subsequent oligosaccharide consumption was monitored by checking their supernatant with HPLC. During initial cultivation for 38 hour, glucose was the sole carbon source leading to rapid growth. After cell growth stopped, the maltose and glucose concentrations increased due to degradation of maltotriose and maltotetraose, but glucose level was maintained at around 3.0%. After 70 hour fermentation, maltose slowly converted to glucose, and vancomycin production continued during the period.

• Korean Journal of Microbiology
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• v.30 no.2
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• pp.78-82
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• 1992

The dikaryon of Trimorphonzycc,.~ papilioncicc.ous, one of basidiornycetous yeast needed thiamine as a growth factor and required relatively simple nutrient components. This organism grem best at 25$^{\circ}$C. anci showed broad pH range (pH 4.0-7.0). It was groM,n in liquid minimal media with various carbon sources and they could be classilied into 3 groups as follows. Glucose. fructose. mannose, sucrose and xylose (A gi.oup) supportecl good growth (>OD 0.8), and showed poor laccase activity (less than 1.5 u'mg protein). Galactose and gluconate (B group) showed moderate growth (01) 0.3-0.6). and hail moderatc crlzyrne activity (4-6u). Arabinosc. lactose. maltose ant1 pyruvate (C 5roup) showed poor growth (OD 0.1-0.2). and showed high enzyme activity (higher than 8 u). Kibosc, acetate. citrate. lactate and oxaloacetate showed no growth. When the yeast was grown in a medium which had two carbon sources (glucose and arabinose). laccase was regul;~tecl by the cutahoiitc repression.

• 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.

• Journal of Microbiology and Biotechnology
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• v.16 no.4
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• pp.543-549
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• 2006

DNA microarray was used to study the transcription profiling of Escherichia coli adapting to acetate as a sole carbon source. Bacteria grown in glucose minimal media were used as a reference. The dynamic expression levels of 3,497 genes were monitored at seven time points during this adaptation. Among the central metabolic genes, the glycolytic and glucose phosphotransferase genes were repressed as the bacteria entered stationary phase, whereas the glyoxylate pathway, TCA cycle, and gluconeogenic genes were induced. Distinct induction or repression patterns were recognized among different pathway genes. For example, the repression of glycolytic genes and the induction of gluconeogenic ones started immediately after glucose was depleted. On the other hand, the regulation of the pentose phosphate pathway genes and glyoxylate genes gradually responded to the glucose depletion or was more related to growth in acetate. When the whole genome was considered, many of the CRP, FadR, and Cra regulons were immediately responsive to the glucose depletion, whereas the $\sigma^s$, Lrp, and IHF regulons were gradually responsive to the glucose depletion. The expression profiling also provided differential regulations between isoenzymes; for example, malic enzymes A (sfcA) and B (maeB). The expression profiles of three genes were confirmed with RT-PCR.

• KSBB Journal
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• v.14 no.4
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• pp.476-481
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• 1999

S. cerevisiae mutant(reg1-501, gal1), which cannot use galactose and has alleviated glucose repression level, is used as host for optimizing induction of GAL promoter. The optimum concentration of galactose as inducer for recombinant protein production and the galactose consumption rate have been tested with S. cerevisiae mutant and compared with conventional S. cerevisiae. The extent of glucose repression were investigated for both strain and the degradation pattern of produced foreign protein have been compared in both cases. The effect of pH on foreign protein degradation pattern were studied for both strains. The secetion efficiency of both strains were carried out. Through these experiments, optimum condition of recombinant protein production by GAL promoter using S. cerevisiae mutant (reg1-501, gal1) were found.

• Journal of Microbiology and Biotechnology
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• v.27 no.3
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• pp.514-523
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• 2017

Carbon catabolite repression is a crucial regulation mechanism in microorganisms, but its characteristic in Rhizopus is still unclear. We extracted a carbon regulation gene, cre, that encoded a carbon catabolite repressor protein (CRE) from Rhizopus stolonifer TP-02, and studied the regulation of CRE by real-time qPCR. CRE responded to glucose in a certain range, where it could significantly regulate part of the cellulase genes (eg, bg, and cbh2) without cbh1. In the comparison of the response of cre and four cellulase genes to carboxymethylcellulose sodium and a simple carbon source (lactose), the effect of CRE was only related to the concentration of reducing sugars. By regulating the reducing sugars to range from 0.4% to 0.6%, a glucose-containing medium with lactose as the inducer could effectively induce cellulases without the repression of CRE. This regulation method could potentially reduce the cost of enzymes produced in industries and provide a possible solution to achieve the largescale synthesis of cellulases.

• Microbiology and Biotechnology Letters
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• v.14 no.4
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• pp.293-298
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• 1986

Sisomicin, which is one of aminoglycoside antibiotics, was produced by Micromonospora inyoensis. The effects of carbon sources on sisomicin production were studied in batch cultures. Starch, dextrin and maltose were good carbon sources for the production of sisomicin. However, when glucose was used, the antibiotic productivity decreased significantly due to a carbon catabolite regulation. The carbon catabolite regulation depends mostly on carbon catabolite repression, but not on carbon catabolite inhibition. On the other hand, the growth-production curves of batch cultures show that sisomicin is produced most actively during the idiophase.

• Journal of Microbiology and Biotechnology
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• v.11 no.1
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• pp.131-137
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• 2001

Previous work has identified that only the catabolite responsive element A (creA; previously called cre-2) out of two potential cre sequences (cre-1: nucleotide +160 to +173 and cre-2: +173 to +186), recognized within the coding region of the xylanaseA gene (xynA) of Bacillus stearothermophilus No.236, was actually, was actually involved in the carbon catabolite repression(CCR) of xynA expression in B. subtilis. However, the level of CCR of xynA expression in the original B.stearothermophilus No.236 strain (70-fold repression). Therefore, to search for an additional cre element in the promoter region, the upstream region of the xynA gene was subcloned by chromosome walking, and as a result, another potential cre element (nucleotide -124∼-137; designated creB) was recognized in this region. The cre-like sequence revealed a high homology to the cre consensus sequence. The xylanase activity of B. subtilis MW15 bearing pWPBR14 (containing creA and creB) cultured in a medium containing xylose as the sole carbon source was about 7.7 times higher than that observed for the same culture containing glucose. B. subtilis MW15 bearing pWPBR23 (containing only creA) produced an activity about 2.4 times higher. This pattern of CCR was confirmed using derivatives of xynA::aprA fusion plasmids. Furthermore, a measurement of the amounts of the xynA transcript showed a similar pattern as that for the production of xylanase. In addition, the synthesis of xylanase in B. subtilis QB7115 [a catabolite control protein A (ccpA) mutant strain] carrying pWPBR14 was almost completely relieved from glucose repression. Together, these results lead to a conclusion that the CCR of the expression of the xynA gene is mediated by CcpA binding at creA and creB sites in B. subtilis.