• Title/Summary/Keyword: Catabolite repression

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Rewiring carbon catabolite repression for microbial cell factory

  • Vinuselvi, Parisutham;Kim, Min-Kyung;Lee, Sung-Kuk;Ghim, Cheol-Min
    • BMB Reports
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    • v.45 no.2
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    • pp.59-70
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    • 2012
  • Carbon catabolite repression (CCR) is a key regulatory system found in most microorganisms that ensures preferential utilization of energy-efficient carbon sources. CCR helps microorganisms obtain a proper balance between their metabolic capacity and the maximum sugar uptake capability. It also constrains the deregulated utilization of a preferred cognate substrate, enabling microorganisms to survive and dominate in natural environments. On the other side of the same coin lies the tenacious bottleneck in microbial production of bioproducts that employs a combination of carbon sources in varied proportion, such as lignocellulose-derived sugar mixtures. Preferential sugar uptake combined with the transcriptional and/or enzymatic exclusion of less preferred sugars turns out one of the major barriers in increasing the yield and productivity of fermentation process. Accumulation of the unused substrate also complicates the downstream processes used to extract the desired product. To overcome this difficulty and to develop tailor-made strains for specific metabolic engineering goals, quantitative and systemic understanding of the molecular interaction map behind CCR is a prerequisite. Here we comparatively review the universal and strain-specific features of CCR circuitry and discuss the recent efforts in developing synthetic cell factories devoid of CCR particularly for lignocellulose-based biorefinery.

Characterization of the $\alpha$-Galactosidase Gene from Leuconostoc mesenteroides SY1

  • KIM JONG HWAN;PARK JAE-YONG;JEONG SEON-JU;CHUN JIYEON;LEE JONG HOON;CHUNGZ DAE KYUN;KIM JEONG HWAN
    • Journal of Microbiology and Biotechnology
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    • v.15 no.4
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    • pp.800-808
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    • 2005
  • Leuconostoc mesenteroides SY1, an isolate from kimchi, was able to ferment $\alpha$-galactosides, such as melibiose and raffinose. $\alpha$-Galactosidase ($\alpha$-Gal) activity was higher in cells grown on melibiose and raffinose than cells grown on galactose, sucrose, and fructose. $\alpha$-Gal activity was not detected in cells grown on glucose, indicating the operation of carbon catabolite repression (CCR). A 6 kb DNA fragment was PCR amplified using a primer set based on the nucleotide sequence of a putative $\alpha$-galactosidase gene (aga) from L. mesenteroides ATCC 8293. Nucleotide sequencing of the 6 kb fragment confirmed the presence of aga and other genes involved in the galactosides utilization, and the gene order was galR (transcriptional regulator)-aga-gaIK (galactokinase)-gaIT (galactose-1-phosphate uridylyltransferase). Northern blotting experiment showed that aga, gaIK, and gaIT constituted the same operon, that the transcription was induced by galactosides, such as melibiose and raffinose, whereas gaIR was independently transcribed as a monocistronic gene, and that the level of transcription was fairly constant. The aga was overexpressed in E. coli BL21 (DE3) using pET26b(+) vector, and $\alpha$-Gal was accumulated in E. coli as an inclusion body.

Control of Catabolite Repression by Limit Feed of Cellobiose in Cellulomomas sp. (Cellulomonas sp.에 있어서 셀로비오스의 미량공급에 의한 생성물 저해의 조절)

  • Choi, Woo-Young
    • Korean Journal of Agricultural Science
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    • v.3 no.2
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    • pp.235-243
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    • 1976
  • As an investigation on the catabolite repression system in cellulase production by Cellulomons sp. CS1-1, the organism was tested on the avicel overlay plates containing glucose or cellobiose at a range of concentration and was grown in continuous culture vessel, supplied by cellobiose medium, aiming the enhanced production of extracellular CM-cellulase at low dilution rates. Product inhibition of cellulase action by cellobiose was also tested. The results obtained are: i) no inhibition of CM-cellulase was observed up to 10 mM(3.4mg/ml) cellobiose in the reaction mixture, however 30% inhibition was observed at 20mM and 55% at 50mM, ii) the tests of catabolite repression on the solid media were successful, and avicel degradation was markedly repressed by glucose or cellobiose, iii) at low concentrations of cellobiose, dilution rate 0.05 and $1.0hour^{-1}$, no significant increase was observed in the production of either intra or extracellular CM-cellulase.

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Enhanced Production of Antifungal Substance(PAFS) Bioxynthesized by Pseudomonas aeruginosa and Examination of Its Physiological Characteristics in Fermentation (Pseudomonas aeruginosa에 의핸 생합성되는 향진균성물질(PAFS)의 생산성 증가 및 생산균주의 배양생리학적 특성 연구)

  • 박선옥;송성기;윤권상;정연호;이상종;정용섭;전계택
    • Microbiology and Biotechnology Letters
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    • v.28 no.6
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    • pp.341-348
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    • 2000
  • Selection of high producer strain, optimization of production medium and cultivation in bioreactor system were carried out in order to produce an antifungal substance, PAFS in large amounts which sources and 41 kinds of nitrogen sources, a synthetic medium consisting of fructose(70 g/1) and ammonium sulfate (10g/l) and a complex medium including galactose(30g/l), fructose(20g/l) and cottonseed flour(35g/l) were determined as opti-mized media for PAFS production. In bioreactor studies examining physiological characteristics of the pro- ducer microorganism with the complex medium, typical pattern of diauxic growth was observed as demonstrated by the result that fructose was not used before almost exhaustion on readily utilizable carbon source, galactose. When galactose was supplemented additionally during the fermentation period. PAFS pro-ductivity did no increases any more, indicating that large portion of the added galactose was used for cell growth instead of biosynthesis of the secondary metabolite. It was deduced that PAFS production could be enhananced by employing fed-batch operation in order to overcome the apparent phenomenon of catabolite repression and /or inhibition.

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Isolation of Bacillus alcalophilus AX2000 Producing Alkaling Xylanase and Its Enzyme Production (알칼리성 Xylanase를 생산하는 Bacillus alcalojnhilus AX2000의 분리와 효소 생산)

  • 박영서;김태영
    • Microbiology and Biotechnology Letters
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    • v.31 no.2
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    • pp.157-164
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    • 2003
  • An alkali-tolerant bacterium producing the xylanase was isolated from soil and identified as Bacillus alcaiophilus. This strain, named B. alcalophilus AX2000, was able to grow and produce xylanase optimally at pH 10.5 and $37^{\circ}C$. The maximum xylanase production was obtained when 0.5%(w/v) birchwood xylan and 0.5%(w/v) polypeptone and yeast extract were used as carbon source and nitrogen source, respectively. The biosynthesis of xylanase was under the catabolite repression by glucose in the culture medium, and inhibited in the presence of high concentration of xylose. The maximum activity of xylanase was observed at pH 10.0 and $50^{\circ}C$ and the enzyme activity remained was over 80% at $60^{\circ}C$ and from pH 5.0 to 11.0.

Nocardia orientalis 변이주에 의한 고농도 혼합당을 이용한 반코마이신 생산

  • 김창호;고영환;고중환
    • 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.

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Biosynthetic Regulation of Invertase from Thermophilic and Alkalophilic Bacillus sp. TA-11 (고온성이며 호알칼리성인 Bacillus sp. TA-11이 생성하는 Invertase의 생합성 조절)

  • Kim, Jae-Ho;Kim, Na-Mi;Kim, Dong-Woo
    • The Korean Journal of Food And Nutrition
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    • v.15 no.2
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    • pp.126-130
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    • 2002
  • Regulation of invertase biosynthesis was studied in thermophilic and alkalophilic Bacillus sp. TA-11. Biosynthesis of the invertase was effectively induced in the presence of 10 mM sucrose for 180 min. Glucose repressed the invertase induction by sucrose and as late as addition time of glucose, the invertase formation was increased, indicating that glucose repression was occurred by inducer exclusion. Catabolite repression was reduced a little by the addition of cAMP for 180 min of induction.

Effect of Temperature and Carbon Source on the Expression of $\beta$-Galactosidase Gene of Lactococcus lactis ssp. lactis ATCC 7962

  • Kim, Tea-Youn;Lee, Jung-Min;Chang, Hae-Choon;Chung, Dae-Kyun;Lee, Jong-Hoon;Kim, Jeong-Hwan;Lee, Hyong-Joo
    • Journal of Microbiology and Biotechnology
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    • v.9 no.2
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    • pp.201-205
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    • 1999
  • The effects of growth temperature and a carbon source on the expression of $\beta$-galactosidase gene of Lactococcus lactis ssp. lactis ATCC 7962 (L. lactis 7962) were investigated. At $25^{\circ}C$, L. lactis 7962 had a higher $\beta$-galactosidase activity than cells grown at $30^{\circ}C$ or $37^{\circ}C$, although cells grew most quickly at $37^{\circ}C$ The highest $\beta$-galactosidase activity was observed in cells grown in M17 with lactose (l %) followed by cells grown in a galactose (1 %) medium. L. lactis 7962 exhibited the minimum $\beta$-galactosidase activity in glucose media, indicating catabolite repression. When the cellular levels of $\beta$-galactosidase mRNA were examined using slot blot hybridization, no significant differences were observed between cells grown at $25^{\circ}C$ and cells at $30^{\circ}C$ or $37^{\circ}C$ in the same media. This suggests that the quantity of $\beta$-galactosidase mRNA may not be the reason for the higher $\beta$-galactosidase activities of L. lactis 7962 at $25^{\circ}C$ The level of ccpA (Catabolite Control Protein) transcript remained almost constant during the exponential growth phase irrespective of a carbon sourse.

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Impact of Lactic Acid and Hydrogen Ion on the Simultaneous Fermentation of Glucose and Xylose by the Carbon Catabolite Derepressed Lactobacillus brevis ATCC 14869

  • Jeong, Kyung Hun;Israr, Beenish;Shoemaker, Sharon P.;Mills, David A.;Kim, Jaehan
    • Journal of Microbiology and Biotechnology
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    • v.26 no.7
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    • pp.1182-1189
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    • 2016
  • Lactobacillus brevis ATCC 14869 exhibited a carbon catabolite derepressed phenotype that has ability to consume fermentable sugars simultaneously with glucose. To evaluate this unusual phenotype under harsh conditions during fermentation, the effects of lactic acid and hydrogen ion concentrations on L. brevis ATCC 14869 were examined. Kinetic equations describing the relationship between specific cell growth rate and lactic acid or hydrogen ion concentration were deduced empirically. The change of substrate utilization and product formation according to lactic acid and hydrogen ion concentration in the media were quantitatively described. Although the simultaneous utilization has been observed regardless of hydrogen ion or lactic acid concentration, the preference of substrates and the formation of two-carbon products were changed significantly. In particular, acetic acid present in the medium as sodium acetate was consumed by L. brevis ATCC 14869 under extreme pH of both acid and alkaline conditions.

Fermentation Studies on Pseudomonas aeruginosa Producing Antifungal Secondary Metabolite, PAFS. (항진균물질을 생합성하는 Pseudomonas aeruginosa의 배양생리적 특성 연구)

  • 송성기;윤권상;정용섭;전계택
    • Microbiology and Biotechnology Letters
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    • v.32 no.1
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    • pp.52-59
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    • 2004
  • When both fructose and galactose were added to a production medium as carbon sources, the productivity of PAFS (Psedomonas Antifungal Substance) biosynthesized by Pseudomonas aeruginosa was observed to be reduced significantly due to the well-known phenomenon of catabolite repression. In order to overcome this phenomenon by use of fermentation bioprocess, fed-batch cultivation method was examined. In addition, a high producer mutant strain, AP-20 obtained by a rational screening method was tested for its productivity of PAFS in both batch and fed-batch fermentation processes. Notably fed-batch operation showed approximately 4 fold higher PAFS productivity than traditional batch operation process. It was appeared that galactose was utilized principally for the cell growth of Pseudomonas aeruginosa whereas large portion of fructose was used for the biosynthesis of PAFS. Furthermore it was observed that composition and feeding rate of production media should be optimized even in the fed-batch fermentation bioprocess. As an example, very slow feeding of carbon sources gave rather negative effect on the production of PAFS due to significant limitation of carbon and energy sources available for the producer microorganism.