scholarly journals Identification of Enzymes and Quantification of Metabolic Fluxes in the Wild Type and in a Recombinant Aspergillus oryzae Strain

1999 ◽  
Vol 65 (1) ◽  
pp. 11-19 ◽  
Author(s):  
Henrik Pedersen ◽  
Morten Carlsen ◽  
Jens Nielsen
Keyword(s):  
Growth Rate ◽  
Pentose Phosphate Pathway ◽  
Isocitrate Dehydrogenase ◽  
Type Strain ◽  
Wild Type Strain ◽  
Specific Growth ◽  
Pentose Phosphate ◽  
Central Metabolism ◽  
Wild Type ◽  
Metabolic Fluxes

ABSTRACT Two α-amylase-producing strains of Aspergillus oryzae, a wild-type strain and a recombinant containing additional copies of the α-amylase gene, were characterized with respect to enzyme activities, localization of enzymes to the mitochondria or cytosol, macromolecular composition, and metabolic fluxes through the central metabolism during glucose-limited chemostat cultivations. Citrate synthase and isocitrate dehydrogenase (NAD) activities were found only in the mitochondria, glucose-6-phosphate dehydrogenase and glutamate dehydrogenase (NADP) activities were found only in the cytosol, and isocitrate dehydrogenase (NADP), glutamate oxaloacetate transaminase, malate dehydrogenase, and glutamate dehydrogenase (NAD) activities were found in both the mitochondria and the cytosol. The measured biomass components and ash could account for 95% (wt/wt) of the biomass. The protein and RNA contents increased linearly with increasing specific growth rate, but the carbohydrate and chitin contents decreased. A metabolic model consisting of 69 fluxes and 59 intracellular metabolites was used to calculate the metabolic fluxes through the central metabolism at several specific growth rates, with ammonia or nitrate as the nitrogen source. The flux through the pentose phosphate pathway increased with increasing specific growth rate. The fluxes through the pentose phosphate pathway were 15 to 26% higher for the recombinant strain than for the wild-type strain.

CreA influences the metabolic fluxes of Aspergillus nidulans during growth on glucose and xylose

Microbiology ◽  
2005 ◽  
Vol 151 (7) ◽  
pp. 2209-2221 ◽  
Author(s):  
Helga David ◽  
Astrid Mørkeberg Krogh ◽  
Christophe Roca ◽  
Mats Åkesson ◽  
Jens Nielsen
Keyword(s):  
Growth Rate ◽  
Specific Growth Rate ◽  
Mutant Strain ◽  
Pentose Phosphate Pathway ◽  
Reference Strain ◽  
Specific Growth ◽  
Pentose Phosphate ◽  
Central Metabolism ◽  
Metabolic Fluxes

The physiological phenotype of Aspergillus nidulans was investigated for different genetic and environmental conditions of glucose repression through the quantification of in vivo fluxes in the central carbon metabolism using 13C-metabolic-flux analysis. The particular focus was the role of the carbon repressor CreA, which is the major regulatory protein mediating carbon repression in many fungal species, in the primary metabolism of A. nidulans. Batch cultivations were performed with a reference strain and a deletion mutant strain (creAΔ4) using [1-13C]glucose as carbon source. The mutant strain was also grown on a mixture of [1-13C]glucose and unlabelled xylose. Fractional enrichment data were measured by gas chromatography-mass spectrometry. A model describing the central metabolism of A. nidulans was used in combination with fractional enrichment data, and measurements of extracellular rates and biomass composition for the estimation of the in vivo metabolic fluxes. The creA-mutant strain showed a lower maximum specific growth rate than the reference strain when grown on glucose (0·11 and 0·25 h−1, respectively), whereas the specific growth rate of the mutant strain grown on the glucose/xylose mixture was identical to that on glucose (0·11 h−1). Different patterns and increased levels of extracellular polyols were observed both upon deletion of the creA gene and upon addition of xylose to the growth medium of the mutant strain. Concerning metabolic fluxes, the major change observed in the flux distribution of A. nidulans upon deletion of the creA gene was a 20 % decrease in the flux through the oxidative part of the pentose-phosphate pathway. Addition of xylose to the growth medium of the mutant resulted in an increase of about 40 % in the activity of the oxidative part of the pentose-phosphate pathway, as well as decreases in the fluxes through the Embden–Meyerhof–Parnas pathway and the tricarboxylic acid cycle (in the range of 20–30 %). The derepression of key pathways leads to alterations in the demands for cofactors, thereby imposing changes in the central metabolism due to the coupling of the many different reactions via the redox and energy metabolism of the cells.

scholarly journals Physiological Role of β-Phosphoglucomutase inLactococcus lactis

2001 ◽  
Vol 67 (10) ◽  
pp. 4546-4553 ◽  
Author(s):  
Fredrik Levander ◽  
Ulrika Andersson ◽  
Peter Rådström
Keyword(s):  
Growth Rate ◽  
Carbon Source ◽  
Specific Growth Rate ◽  
Type Strain ◽  
Wild Type Strain ◽  
Specific Growth ◽  
Maximum Specific Growth Rate ◽  
Wild Type ◽  
Cell Extracts

ABSTRACT A β-phosphoglucomutase (β-PGM) mutant of Lactococcus lactis subsp. lactis ATCC 19435 was constructed using a minimal integration vector and double-crossover recombination. The mutant and the wild-type strain were grown under controlled conditions with different sugars to elucidate the role of β-PGM in carbohydrate catabolism and anabolism. The mutation did not significantly affect growth, product formation, or cell composition when glucose or lactose was used as the carbon source. With maltose or trehalose as the carbon source the wild-type strain had a maximum specific growth rate of 0.5 h−1, while the deletion of β-PGM resulted in a maximum specific growth rate of 0.05 h−1 on maltose and no growth at all on trehalose. Growth of the mutant strain on maltose resulted in smaller amounts of lactate but more formate, acetate, and ethanol, and approximately 1/10 of the maltose was found as β-glucose 1-phosphate in the medium. Furthermore, the β-PGM mutant cells grown on maltose were considerably larger and accumulated polysaccharides which consisted of α-1,4-bound glucose units. When the cells were grown at a low dilution rate in a glucose and maltose mixture, the wild-type strain exhibited a higher carbohydrate content than when grown at higher growth rates, but still this content was lower than that in the β-PGM mutant. In addition, significant differences in the initial metabolism of maltose and trehalose were found, and cell extracts did not digest free trehalose but only trehalose 6-phosphate, which yielded β-glucose 1-phosphate and glucose 6-phosphate. This demonstrates the presence of a novel enzymatic pathway for trehalose different from that of maltose metabolism in L. lactis.

scholarly journals Adaptation of central metabolite pools to variations in growth rate and cultivation conditions in Saccharomyces cerevisiae

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Kanhaiya Kumar ◽  
Vishwesh Venkatraman ◽  
Per Bruheim
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Growth Rate ◽  
Amino Acid ◽  
Growth Rates ◽  
Pentose Phosphate Pathway ◽  
Pentose Phosphate ◽  
Relative Reduction ◽  
Central Metabolism ◽  
Cultivation Conditions ◽  
Biological Studies

Abstract Background Saccharomyces cerevisiae is a well-known popular model system for basic biological studies and serves as a host organism for the heterologous production of commercially interesting small molecules and proteins. The central metabolism is at the core to provide building blocks and energy to support growth and survival in normal situations as well as during exogenous stresses and forced heterologous protein production. Here, we present a comprehensive study of intracellular central metabolite pool profiling when growing S. cerevisiae on different carbon sources in batch cultivations and at different growth rates in nutrient-limited glucose chemostats. The latest versions of absolute quantitative mass spectrometry-based metabolite profiling methodology were applied to cover glycolytic and pentose phosphate pathway metabolites, tricarboxylic acid cycle (TCA), complete amino acid, and deoxy-/nucleoside phosphate pools. Results Glutamate, glutamine, alanine, and citrate were the four most abundant metabolites for most conditions tested. The amino acid is the dominant metabolite class even though a marked relative reduction compared to the other metabolite classes was observed for nitrogen and phosphate limited chemostats. Interestingly, glycolytic and pentose phosphate pathway (PPP) metabolites display the largest variation among the cultivation conditions while the nucleoside phosphate pools are more stable and vary within a closer concentration window. The overall trends for glucose and nitrogen-limited chemostats were increased metabolite pools with the increasing growth rate. Next, comparing the chosen chemostat reference growth rate (0.12 h−1, approximate one-fourth of maximal unlimited growth rate) illuminates an interesting pattern: almost all pools are lower in nitrogen and phosphate limited conditions compared to glucose limitation, except for the TCA metabolites citrate, isocitrate and α-ketoglutarate. Conclusions This study provides new knowledge-how the central metabolism is adapting to various cultivations conditions and growth rates which is essential for expanding our understanding of cellular metabolism and the development of improved phenotypes in metabolic engineering.

scholarly journals Glutathione Reductase from Lactobacillus sanfranciscensis DSM20451T: Contribution to Oxygen Tolerance and Thiol Exchange Reactions in Wheat Sourdoughs

2007 ◽  
Vol 73 (14) ◽  
pp. 4469-4476 ◽  
Author(s):  
André Jänsch ◽  
Maher Korakli ◽  
Rudi F. Vogel ◽  
Michael G. Gänzle
Keyword(s):  
Growth Rate ◽  
Glutathione Reductase ◽  
Mutant Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Wild Type ◽  
Exchange Reactions ◽  
Aerobic Growth ◽  
Oxygen Tolerance ◽  
Lactobacillus Sanfranciscensis

ABSTRACT The effect of the glutathione reductase (GshR) activity of Lactobacillus sanfranciscensis DSM20451T on the thiol levels in fermented sourdoughs was determined, and the oxygen tolerance of the strain was also determined. The gshR gene coding for a putative GshR was sequenced and inactivated by single-crossover integration to yield strain L. sanfranciscensis DSM20451TΔgshR. The gene disruption was verified by sequencing the truncated gshR and surrounding regions on the chromosome. The gshR activity of L. sanfranciscensis DSM20451TΔgshR was strongly reduced compared to that of the wild-type strain, demonstrating that gshR indeed encodes an active GshR enzyme. The thiol levels in wheat doughs fermented with L. sanfranciscensis DSM20451 increased from 9 μM to 10.5 μM sulfhydryl/g of dough during a 24-h sourdough fermentation, but in sourdoughs fermented with L. sanfranciscensis DSM20451TΔgshR and in chemically acidified doughs, the thiol levels decreased to 6.5 to 6.8 μM sulfhydryl/g of dough. Remarkably, the GshR-negative strains Lactobacillus pontis LTH2587 and Lactobacillus reuteri BR11 exerted effects on thiol levels in dough comparable to those of L. sanfranciscensis. In addition to the effect on thiol levels in sourdough, the loss of GshR activity in L. sanfranciscensis DSM20451TΔgshR resulted in a loss of oxygen tolerance. The gshR mutant strain exhibited a strongly decreased aerobic growth rate on modified MRS medium compared to either the growth rate under anaerobic conditions or that of the wild-type strain, and aerobic growth was restored by the addition of cysteine. Moreover, the gshR mutant strain was more sensitive to the superoxide-generating agent paraquat.

scholarly journals Cloning and Inactivation of a Branched-Chain-Amino-Acid Aminotransferase Gene from Staphylococcus carnosus and Characterization of the Enzyme

2002 ◽  
Vol 68 (8) ◽  
pp. 4007-4014 ◽  
Author(s):  
Søren M. Madsen ◽  
Hans Christian Beck ◽  
Peter Ravn ◽  
Astrid Vrang ◽  
Anne Maria Hansen ◽  
...  
Keyword(s):  
Growth Rate ◽  
Amino Acid ◽  
Mutant Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Biomass Yield ◽  
Degradation Products ◽  
Wild Type ◽  
Staphylococcus Carnosus ◽  
Branched Chain

ABSTRACT Staphylococcus carnosus and Staphylococcus xylosus are widely used as aroma producers in the manufacture of dried fermented sausages. Catabolism of branched-chain amino acids (BCAAs) by these strains contributes to aroma formation by production of methyl-branched aldehydes and carboxy acids. The first step in the catabolism is most likely a transamination reaction catalyzed by BCAA aminotransferases (IlvE proteins). In this study, we cloned the ilvE gene from S. carnosus by using degenerate oligonucleotides and PCR. We found that the deduced amino acid sequence was 80% identical to that of the corresponding enzyme in Staphylococcus aureus and that the ilvE gene was constitutively expressed as a monocistronic transcript. To study the influence of ilvE on BCAA catabolism, we constructed an ilvE deletion mutant by gene replacement. The IlvE protein from S. carnosus was shown mainly to catalyze the transamination of isoleucine, valine, leucine, and, to some extent, methionine using pyridoxal 5′-phosphate as a coenzyme. The ilvE mutant degraded less than 5% of the BCAAs, while the wild-type strain degraded 75 to 95%. Furthermore, the mutant strain produced approximately 100-fold less of the methyl-branched carboxy acids, 2-methylpropanoic acid, 2-methylbutanoic acid, and 3-methylbutanoic acid, which derived from the BCAA catabolism, clearly emphasizing the role of IlvE in aroma formation. In contrast to previous reports, we found that IlvE was the only enzyme that catalyzed the deamination of BCAAs in S. carnosus. The ilvE mutant strain showed remarkably lower growth rate and biomass yield compared to those of the wild-type strain when grown in rich medium. Normal growth rate and biomass yield were restored by addition of the three BCAA-derived α-keto acids, showing that degradation products of BCAAs were essential for optimal cell growth.

scholarly journals Determining the Optimal Thymidine Concentration for Growing Thy−Escherichia coli Strains

1998 ◽  
Vol 180 (11) ◽  
pp. 2992-2994 ◽  
Author(s):  
Felipe Molina ◽  
Alfonso Jiménez-Sánchez ◽  
Elena C. Guzmán
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Growth Medium ◽  
Type Strain ◽  
Wild Type Strain ◽  
Wild Type ◽  
Strain Mg1655 ◽  
E Coli ◽  
Replication Time ◽  
Detectable Difference

ABSTRACT Changes of thymidine concentration in the growth medium affect the chromosome replication time of Thy− strains without at the same time causing a detectable difference in the growth rate (R. H. Pritchard and A. Zaritsky, Nature 226:126–131, 1970). Consequently, the optimal thymidine concentration cannot be determined by ascertaining which concentration produces the highest growth rate. Here we present a method for determining the optimal thymidine concentration of any Thy− Escherichia coli strain. Using this method, we found that the E. coli “wild-type” strain MG1655 has a partial Thy− phenotype.

scholarly journals 4-Chlorobenzoate Uptake in Comamonas sp. Strain DJ-12 Is Mediated by a Tripartite ATP-Independent Periplasmic Transporter

2006 ◽  
Vol 188 (24) ◽  
pp. 8407-8412 ◽  
Author(s):  
Jong-Chan Chae ◽  
Gerben J. Zylstra
Keyword(s):  
Growth Rate ◽  
Gene Cluster ◽  
Type Strain ◽  
Wild Type Strain ◽  
Proton Motive Force ◽  
Significant Inhibition ◽  
Motive Force ◽  
Wild Type ◽  
Knockout Mutant

ABSTRACT The fcb gene cluster involved in the hydrolytic dehalogenation of 4-chlorobenzoate is organized in the order fcbB-fcbA-fcbT1-fcbT2-fcbT3-fcbC in Comamonas sp. strain DJ-12. The genes are operonic and inducible with 4-chloro-, 4-iodo-, and 4-bromobenzoate. The fcbT1, fcbT2, and fcbT3 genes encode a transporter in the secondary TRAP (tripartite ATP-independent periplasmic) family. An fcbT1T2T3 knockout mutant shows a much slower growth rate on 4-chlorobenzoate compared to the wild type. 4-Chlorobenzoate is transported into the wild-type strain five times faster than into the fcbT1T2T3 knockout mutant. Transport of 4-chlorobenzoate shows significant inhibition by 4-bromo-, 4-iodo-, and 4-fluorobenzoate and mild inhibition by 3-chlorobenzoate, 2-chlorobenzoate, 4-hydroxybenzoate, 3-hydroxybenzoate, and benzoate. Uptake of 4-chlorobenzoate is significantly inhibited by ionophores which collapse the proton motive force.

scholarly journals The Gene yggE Functions in Restoring Physiological Defects of Escherichia coli Cultivated under Oxidative Stress Conditions

2005 ◽  
Vol 71 (5) ◽  
pp. 2762-2765 ◽  
Author(s):  
SunYoung Kim ◽  
Motomu Nishioka ◽  
Shuhei Hayashi ◽  
Hiroyuki Honda ◽  
Takeshi Kobayashi ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Escherichia Coli ◽  
Type Strain ◽  
Wild Type Strain ◽  
Specific Growth ◽  
Maximum Specific Growth Rate ◽  
Oxygen Species ◽  
Wild Type ◽  
Amino Acid Requirement ◽  
E Coli

ABSTRACT DNA microarray analysis showed that yfiD, yggB, and yggE genes were up-regulated when superoxide dismutase (SOD)-deficient Escherichia coli IM303 (I4) was cultivated under the oxidative stress generated by photoexcited TiO2, and pYFD, pYGB, and pYGE were constructed by inserting the respective genes into a pUC 19 vector. The content of reactive oxygen species (ROS) in IM303 (I4) cells carrying pYGE was reduced to 31% of ROS content in the control cells with pUC 19. In the culture of wild-type strain, E. coli MM294, in the medium with paraquat (10 μmol/l), maximum specific growth rate of the cells with pYGE was about five times higher than that of the control cells, with a decreased ROS content in the former cells. The introduction of pYGE also suppressed the occurrence of the cells with altered amino acid requirement in the culture of MM294 cells with paraquat.

scholarly journals Threshold and adaptation in Phycomyces. Their interrelation and regulation by light.

1984 ◽  
Vol 84 (1) ◽  
pp. 119-132 ◽  
Author(s):  
P Galland ◽  
V E Russo
Keyword(s):  
Growth Rate ◽  
Dark Adaptation ◽  
Type Strain ◽  
Wild Type Strain ◽  
Light Sensitivity ◽  
Growth Conditions ◽  
Continuous Illumination ◽  
Growth Responses ◽  
Wild Type

The absolute light sensitivity of Phycomyces sporangiophores was determined by analyzing the intensity dependence of the phototropic bending rate and of the light growth and dark growth responses to step changes of the intensity. We found that the different methods give approximately the same results for the wild-type strain, as well as for several behavioral mutants with defects in the genes madA, madB, and madC. A crucial factor in the determination of thresholds is the light intensity at which the strains grow during the 4 d after inoculation and prior to the experiment. When the wild-type strain grows in the dark, its threshold for the bending rate is 10(-9) W X m-2, compared with 2 X 10(-7) W X m-2 when it is grown under continuous illumination. Further, the maximal bending rate is twice as high in dark-grown strains. This phenomenon is further complicated by the fact that the diameter and growth rate of the sporangiophores also depend on the illumination conditions prior to the experiment: light-grown sporangiophores have an increased diameter and an increased growth rate compared with dark-grown ones. Some of the behavioral mutants, however, are indifferent to this form of light control. Another factor that is controlled by the growth conditions is adaptation: the kinetics of dark adaptation are slower in light-grown sporangiophores than in dark-grown ones. We found empirically a positive correlation between the slower dark adaptation constant and the threshold of the bending rate, which shows that the two underlying phenomena are functionally related.

scholarly journals Comparative study on growth and morphological characteristics of a wild type strain Rhizobium spp. (RCA-220) and a genetically engineered E. coli BL21

2014 ◽  
Vol 20 ◽  
pp. 75-82
Author(s):  
MM Rahman ◽  
M Salah Uddin ◽  
S Zaman ◽  
MA Saleh ◽  
AE Ekram ◽  
...  
Keyword(s):  
Growth Rate ◽  
Carbon Source ◽  
Salt Concentration ◽  
Type Strain ◽  
Wild Type Strain ◽  
Genetically Modified ◽  
Growth Characteristics ◽  
Wild Type ◽  
E Coli ◽  
Rhizobium Spp

Context: Comparison between a wild type strain Rhizobium spp. (RCA-220) and a genetically modified strain E. coli BL21 in context of growth features. Objective: To observe the comparative growth characteristics of a genetically modified E. coli BL21 and an isolated wild type strain Rhizobium spp. (RCA-220). Materials and Methods: Different kinds of investigations were accomplished in both Luria-Bertani (LB) liquid and semi-solid media to observe the growth and maintenance of these strains. For the isolation of Rhizobium spp. selective Yeast Extract Manitol Agar (YEMA) was used. Colony morphology, pH, temperature, carbon source, salt concentration and light were taken under consideration and optimized for growth characteristics. Results: For the strain E. coli BL21, the maximum growth rate was 1.9 at incubation time 72 h, pH 7.2, temperature 37°C (optimized) while for Rhizobium spp. the growth rate was significantly higher (OD 2) at pH 6.8, temperature 28°C (optimized). Among all used carbon sources, strains grown in the medium supplemented with peptone showed rapid and good performance. So, peptone was proved as the best carbon source for both strains. The maximum growths of these strains were observed at 0g/100ml NaCl salt concentration. RCA-220 strain was comparatively more tolerable to salt than E. coli BL21 strain. In this work, E. coli BL21 showed rapid and good performance in presence of light while Rhizobium spp. showed better performance in absence of light. Statistical analysis showed that the growth rate of Rhizobium spp. was significantly higher than E. coli BL21. Conclusion: From the experimental results, it can be concluded that naturally obtained microbial strains were stable and could tolerate any stress condition where the modified strains lose their growth capability and the overall growth performances were reduced or slowed down than the wild type strain. DOI: http://dx.doi.org/10.3329/jbs.v20i0.17718 J. bio-sci.  20:  75-82, 2012

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