The DeoR-Type Regulator SugR Represses Expression of ptsG in Corynebacterium glutamicum

ABSTRACT Corynebacterium glutamicum grows on a variety of carbohydrates and organic acids. Uptake of the preferred carbon source glucose via the phosphoenolpyruvate-dependent phosphotransferase system (PTS) is reduced during coutilization of glucose with acetate, sucrose, or fructose compared to growth on glucose as the sole carbon source. Here we show that the DeoR-type regulator SugR (NCgl1856) represses expression of ptsG, which encodes the glucose-specific PTS enzyme II. Overexpression of sugR resulted in reduced ptsG mRNA levels, decreased glucose utilization, and perturbed growth on media containing glucose. In mutants lacking sugR, expression of the ptsG′-′cat fusion was increased two- to sevenfold during growth on gluconeogenic carbon sources but remained similar during growth on glucose or other sugars. As shown by DNA microarray analysis, SugR also regulates expression of other genes, including ptsS and the putative NCgl1859-fruK-ptsF operon. Purified SugR bound to DNA regions upstream of ptsG, ptsS, and NCgl1859, and a 75-bp ptsG promoter fragment was sufficient for SugR binding. Fructose-6-phosphate interfered with binding of SugR to the ptsG promoter DNA. Thus, while during growth on gluconeogenic carbon sources SugR represses ptsG, ptsG expression is derepressed during growth on glucose or under other conditions characterized by high fructose-6-phosphate concentrations, representing one mechanism which allows C. glutamicum to adapt glucose uptake to carbon source availability.

Download Full-text

Mannose utilization in Escherichia coli requires cyclic AMP but not an exogenous inducer

Canadian Journal of Microbiology ◽

10.1139/m80-251 ◽

1980 ◽

Vol 26 (12) ◽

pp. 1508-1511 ◽

Cited By ~ 8

Author(s):

Ann D. E. Fraser ◽

Hiroshi Yamazaki

Keyword(s):

Escherichia Coli ◽

Carbon Source ◽

Cyclic Amp ◽

Adenylyl Cyclase ◽

Sole Carbon Source ◽

Receptor Protein ◽

Deficient Mutant ◽

Phosphotransferase System ◽

Cyclic Amp Receptor Protein ◽

Cyclic Monophosphate

It has not been clarified whether the utilization of mannose by Escherichia coli requires adenosine 3′,5′-cyclic monophosphate (cyclic AMP). Using an adenylyl cyclase deficient mutant (CA8306B) and a cyclic AMP receptor protein (CRP) deficient mutant (5333B) we have shown that the utilization of mannose is dependent on the cyclic AMP–CRP complex. 2-Deoxyglucose (DG) is a nonmetabolizable glucose analog specific for the phosphotransferase system (PTS) which transports mannose (termed here PTSM). Growth of CA8306B on glycerol is unaffected by addition of the analog, whereas growth of the strain on glycerol plus cyclic AMP ceases im mediately upon addition of DG. These results suggest that the formation of PTSM is dependent on cyclic AMP. In addition, CA8306B grown on glycerol plus cyclic AMP can immediately utilize mannose when transferred to a medium containing mannose as a sole carbon source, whereas the same strain grown on glycerol without cyclic AMP cannot utilize mannose when so transferred. These results suggest that the formation of PTSM does not require an exogenous inducer.

Download Full-text

Metabolic Fluxes in Corynebacterium glutamicum during Lysine Production with Sucrose as Carbon Source

Applied and Environmental Microbiology ◽

10.1128/aem.70.12.7277-7287.2004 ◽

2004 ◽

Vol 70 (12) ◽

pp. 7277-7287 ◽

Cited By ~ 71

Author(s):

Christoph Wittmann ◽

Patrick Kiefer ◽

Oskar Zelder

Keyword(s):

Carbon Source ◽

Corynebacterium Glutamicum ◽

Metabolic Flux ◽

Tca Cycle ◽

Pentose Phosphate ◽

Flux Analysis ◽

Phosphotransferase System ◽

Lysine Production ◽

Metabolic Fluxes ◽

Fructose 1,6 Bisphosphate

ABSTRACT Metabolic fluxes in the central metabolism were determined for lysine-producing Corynebacterium glutamicum ATCC 21526 with sucrose as a carbon source, providing an insight into molasses-based industrial production processes with this organism. For this purpose, 13C metabolic flux analysis with parallel studies on [1-13CFru]sucrose, [1-13CGlc]sucrose, and [13C6 Fru]sucrose was carried out. C. glutamicum directed 27.4% of sucrose toward extracellular lysine. The strain exhibited a relatively high flux of 55.7% (normalized to an uptake flux of hexose units of 100%) through the pentose phosphate pathway (PPP). The glucose monomer of sucrose was completely channeled into the PPP. After transient efflux, the fructose residue was mainly taken up by the fructose-specific phosphotransferase system (PTS) and entered glycolysis at the level of fructose-1,6-bisphosphate. Glucose-6-phosphate isomerase operated in the gluconeogenetic direction from fructose-6-phosphate to glucose-6-phosphate and supplied additional carbon (7.2%) from the fructose part of the substrate toward the PPP. This involved supply of fructose-6-phosphate from the fructose part of sucrose either by PTSMan or by fructose-1,6-bisphosphatase. C. glutamicum further exhibited a high tricarboxylic acid (TCA) cycle flux of 78.2%. Isocitrate dehydrogenase therefore significantly contributed to the total NADPH supply of 190%. The demands for lysine (110%) and anabolism (32%) were lower than the supply, resulting in an apparent NADPH excess. The high TCA cycle flux and the significant secretion of dihydroxyacetone and glycerol display interesting targets to be approached by genetic engineers for optimization of the strain investigated.

Download Full-text

Comparative metabolomics of Phialemonium curvatum as an omnipotent fungus cultivated on crude palm oil versus glucose

Microbial Cell Factories ◽

10.1186/s12934-020-01434-w ◽

2020 ◽

Vol 19 (1) ◽

Author(s):

Arief Izzairy Zamani ◽

Susann Barig ◽

Sarah Ibrahim ◽

Hirzun Mohd. Yusof ◽

Julia Ibrahim ◽

...

Keyword(s):

Carbon Source ◽

Organic Acids ◽

Vegetable Oil ◽

Carbon Metabolism ◽

Sole Carbon Source ◽

Carbon Sources ◽

Tca Cycle ◽

Central Carbon Metabolism ◽

Targeted Metabolomics ◽

Central Carbon

Abstract Background Sugars and triglycerides are common carbon sources for microorganisms. Nonetheless, a systematic comparative interpretation of metabolic changes upon vegetable oil or glucose as sole carbon source is still lacking. Selected fungi that can grow in acidic mineral salt media (MSM) with vegetable oil had been identified recently. Hence, this study aimed to investigate the overall metabolite changes of an omnipotent fungus and to reveal changes at central carbon metabolism corresponding to both carbon sources. Results Targeted and non-targeted metabolomics for both polar and semi-polar metabolites of Phialemonium curvatum AWO2 (DSM 23903) cultivated in MSM with palm oil (MSM-P) or glucose (MSM-G) as carbon sources were obtained. Targeted metabolomics on central carbon metabolism of tricarboxylic acid (TCA) cycle and glyoxylate cycle were analysed using LC–MS/MS-TripleQ and GC–MS, while untargeted metabolite profiling was performed using LC–MS/MS-QTOF followed by multivariate analysis. Targeted metabolomics analysis showed that glyoxylate pathway and TCA cycle were recruited at central carbon metabolism for triglyceride and glucose catabolism, respectively. Significant differences in organic acids concentration of about 4- to 8-fold were observed for citric acid, succinic acid, malic acid, and oxaloacetic acid. Correlation of organic acids concentration and key enzymes involved in the central carbon metabolism was further determined by enzymatic assays. On the other hand, the untargeted profiling revealed seven metabolites undergoing significant changes between MSM-P and MSM-G cultures. Conclusions Overall, this study has provided insights on the understanding on the effect of triglycerides and sugar as carbon source in fungi global metabolic pathway, which might become important for future optimization of carbon flux engineering in fungi to improve organic acids production when vegetable oil is applied as the sole carbon source.

Download Full-text

Characterization of a Corynebacterium glutamicum Lactate Utilization Operon Induced during Temperature-Triggered Glutamate Production

Applied and Environmental Microbiology ◽

10.1128/aem.71.10.5920-5928.2005 ◽

2005 ◽

Vol 71 (10) ◽

pp. 5920-5928 ◽

Cited By ~ 132

Author(s):

Corinna Stansen ◽

Davin Uy ◽

Stephane Delaunay ◽

Lothar Eggeling ◽

Jean-Louis Goergen ◽

...

Keyword(s):

Lactate Dehydrogenase ◽

Carbon Source ◽

Corynebacterium Glutamicum ◽

Specific Activity ◽

Temperature Shift ◽

Pcr Analysis ◽

Mrna Levels ◽

Reverse Transcription Pcr ◽

Glutamate Production ◽

Lactate Utilization

ABSTRACT Gene expression changes of glutamate-producing Corynebacterium glutamicum were identified in transcriptome comparisons by DNA microarray analysis. During glutamate production induced by a temperature shift, C. glutamicum strain 2262 showed significantly higher mRNA levels of the NCgl2816 and NCgl2817 genes than its non-glutamate-producing derivative 2262NP. Reverse transcription-PCR analysis showed that the two genes together constitute an operon. NCgl2816 putatively codes for a lactate permease, while NCgl2817 was demonstrated to encode quinone-dependent l-lactate dehydrogenase, which was named LldD. C. glutamicum LldD displayed Michaelis-Menten kinetics for the substrate l-lactate with a Km of about 0.51 mM. The specific activity of LldD was about 10-fold higher during growth on l-lactate or on an l-lactate-glucose mixture than during growth on glucose, d-lactate, or pyruvate, while the specific activity of quinone-dependent d-lactate dehydrogenase differed little with the carbon source. RNA levels of NCgl2816 and lldD were about 18-fold higher during growth on l-lactate than on pyruvate. Disruption of the NCgl2816-lldD operon resulted in loss of the ability to utilize l-lactate as the sole carbon source. Expression of lldD restored l-lactate utilization, indicating that the function of the permease gene NCgl2816 is dispensable, while LldD is essential, for growth of C. glutamicum on l-lactate.

Download Full-text

Identification and analysis of a Saccharomyces cerevisiae copper homeostasis gene encoding a homeodomain protein.

Molecular and Cellular Biology ◽

10.1128/mcb.14.12.7792 ◽

1994 ◽

Vol 14 (12) ◽

pp. 7792-7804 ◽

Cited By ~ 23

Author(s):

S A Knight ◽

K T Tamai ◽

D J Kosman ◽

D J Thiele

Keyword(s):

Saccharomyces Cerevisiae ◽

Carbon Source ◽

Sole Carbon Source ◽

Copper Resistance ◽

Yeast Cells ◽

Homeodomain Protein ◽

Mrna Levels ◽

Reading Frame ◽

Cup1 Gene ◽

Intracellular Copper

Yeast metallothionein, encoded by the CUP1 gene, and its copper-dependent transcriptional activator ACE1 play a key role in mediating copper resistance in Saccharomyces cerevisiae. Using an ethyl methanesulfonate mutant of a yeast strain in which CUP1 and ACE1 were deleted, we isolated a gene, designated CUP9, which permits yeast cells to grow at high concentrations of environmental copper, most notably when lactate is the sole carbon source. Disruption of CUP9, which is located on chromosome XVI, caused a loss of copper resistance in strains which possessed CUP1 and ACE1, as well as in the cup1 ace1 deletion strain. Measurement of intracellular copper levels of the wild-type and cup9-1 mutant demonstrated that total intracellular copper concentrations were unaffected by CUP9. CUP9 mRNA levels were, however, down regulated by copper when yeast cells were grown with glucose but not with lactate or glycerol-ethanol as the sole carbon source. This down regulation was independent of the copper metalloregulatory transcription factor ACE1. The DNA sequence of CUP9 predicts an open reading frame of 306 amino acids in which a 55-amino-acid sequence showed 47% identity with the homeobox domain of the human proto-oncogene PBX1, suggesting that CUP9 is a DNA-binding protein which regulates the expression of important copper homeostatic genes.

Download Full-text

Host engineering for improved valerolactam production in Pseudomonas putida

10.1101/660795 ◽

2019 ◽

Cited By ~ 2

Author(s):

Mitchell G. Thompson ◽

Luis E. Valencia ◽

Jacquelyn M. Blake-Hedges ◽

Pablo Cruz-Morales ◽

Alexandria E. Velasquez ◽

...

Keyword(s):

Metabolic Engineering ◽

Carbon Source ◽

Pseudomonas Putida ◽

Aromatic Compounds ◽

Sole Carbon Source ◽

Carbon Sources ◽

Shotgun Proteomics ◽

Rich Media ◽

Amide Hydrolase

ABSTRACTPseudomonas putida is a promising bacterial chassis for metabolic engineering given its ability to metabolize a wide array of carbon sources, especially aromatic compounds derived from lignin. However, this omnivorous metabolism can also be a hindrance when it can naturally metabolize products produced from engineered pathways. Herein we show that P. putida is able to use valerolactam as a sole carbon source, as well as degrade caprolactam. Lactams represent important nylon precursors, and are produced in quantities exceeding one million tons per year[1]. To better understand this metabolism we use a combination of Random Barcode Transposon Sequencing (RB-TnSeq) and shotgun proteomics to identify the oplBA locus as the likely responsible amide hydrolase that initiates valerolactam catabolism. Deletion of the oplBA genes prevented P. putida from growing on valerolactam, prevented the degradation of valerolactam in rich media, and dramatically reduced caprolactam degradation under the same conditions. Deletion of oplBA, as well as pathways that compete for precursors L-lysine or 5-aminovalerate, increased the titer of valerolactam from undetectable after 48 hours of production to ~90 mg/L. This work may serve as a template to rapidly eliminate undesirable metabolism in non-model hosts in future metabolic engineering efforts.

Download Full-text

Transcriptional control of the Saccharomyces cerevisiae PGK gene by RAP1

Molecular and Cellular Biology ◽

10.1128/mcb.9.12.5516-5524.1989 ◽

1989 ◽

Vol 9 (12) ◽

pp. 5516-5524

Author(s):

A Chambers ◽

J S Tsang ◽

C Stanway ◽

A J Kingsman ◽

S M Kingsman

Keyword(s):

Carbon Source ◽

Binding Sites ◽

Transcriptional Control ◽

Carbon Sources ◽

Phosphoglycerate Kinase ◽

Yeast Cells ◽

Regulation Of Transcription ◽

Mrna Levels ◽

Upstream Activation Sequence ◽

Activation Sequence

The promoter of the yeast glycolytic gene encoding phosphoglycerate kinase (PGK) contains an upstream activation sequence between bases -538 and -402 upstream of the initiating ATG. The upstream activation sequence contains multiple functional elements, including an essential region called the activator core (AC) sequence and three copies of the pentamer 5'-CTTCC-3'. The AC sequence shows strong homology to the consensus binding sites for the yeast proteins RAP1 (GRF1) and TUF. We have demonstrated that the yeast protein which interacts with the AC sequence is the DNA-binding protein RAP1. Expression of the PGK gene is found to be regulated according to the carbon source in the growth medium. PGK mRNA levels are high in yeast cells grown in glucose medium but low in yeast cells grown in media containing carbon sources such as pyruvate and acetate. This carbon source regulation of transcription was found to be mediated, in part, via regulation of RAP1 binding to the AC sequence. The promoters of many other yeast glycolytic genes also contain consensus RAP1-binding sites and copies of the CTTCC pentamer. This suggests that RAP1 may be involved in transcriptional control of many other glycolytic genes in addition to the PGK gene.

Download Full-text

Saccharomyces cerevisiae glucose signalling regulator Mth1p regulates the organellar Na+/H+ exchanger Nhx1p

Biochemical Journal ◽

10.1042/bj20100796 ◽

2010 ◽

Vol 432 (2) ◽

pp. 343-352 ◽

Cited By ~ 2

Author(s):

Keiji Mitsui ◽

Masafumi Matsushita ◽

Hiroshi Kanazawa

Keyword(s):

Saccharomyces Cerevisiae ◽

Carbon Source ◽

Sole Carbon Source ◽

Glucose Sensor ◽

Ph Regulation ◽

Gene Knockout ◽

Carbon Sources ◽

Acidic Ph ◽

In Cells

Organelle-localized NHEs (Na+/H+ exchangers) are found in cells from yeast to humans and contribute to organellar pH regulation by exporting H+ from the lumen to the cytosol coupled to an H+ gradient established by vacuolar H+-ATPase. The mechanisms underlying the regulation of organellar NHEs are largely unknown. In the present study, a yeast two-hybrid assay identified Mth1p as a new binding protein for Nhx1p, an organellar NHE in Saccharomyces cerevisiae. It was shown by an in vitro pull-down assay that Mth1p bound to the hydrophilic C-terminal half of Nhx1p, especially to the central portion of this region. Mth1p is known to bind to the cytoplasmic domain of the glucose sensor Snf3p/Rgt2p and also functions as a negative transcriptional regulator. Mth1p was expressed in cells grown in a medium containing galactose, but was lost (possibly degraded) when cells were grown in medium containing glucose as the sole carbon source. Deletion of the MTH1 gene increased cell growth compared with the wild-type when cells were grown in a medium containing galactose and with hygromycin or at an acidic pH. This resistance to hygromycin or acidic conditions was not observed for cells grown with glucose as the sole carbon source. Gene knockout of NHX1 increased the sensitivity to hygromycin and acidic pH. The increased resistance to hygromycin was reproduced by truncation of the Mth1p-binding region in Nhx1p. These results implicate Mth1p as a novel regulator of Nhx1p that responds to specific extracellular carbon sources.

Download Full-text

Transcriptional control of the Saccharomyces cerevisiae PGK gene by RAP1.

Molecular and Cellular Biology ◽

10.1128/mcb.9.12.5516 ◽

1989 ◽

Vol 9 (12) ◽

pp. 5516-5524 ◽

Cited By ~ 70

Author(s):

A Chambers ◽

J S Tsang ◽

C Stanway ◽

A J Kingsman ◽

S M Kingsman

Keyword(s):

Carbon Source ◽

Binding Sites ◽

Transcriptional Control ◽

Carbon Sources ◽

Phosphoglycerate Kinase ◽

Yeast Cells ◽

Regulation Of Transcription ◽

Mrna Levels ◽

Upstream Activation Sequence ◽

Activation Sequence

Download Full-text

Identification of a second β-glucoside phosphoenolpyruvate : carbohydrate phosphotransferase system in Corynebacterium glutamicum R

Microbiology ◽

10.1099/mic.0.029496-0 ◽

2009 ◽

Vol 155 (11) ◽

pp. 3652-3660 ◽

Cited By ~ 13

Author(s):

Yuya Tanaka ◽

Haruhiko Teramoto ◽

Masayuki Inui ◽

Hideaki Yukawa

Keyword(s):

Corynebacterium Glutamicum ◽

Catabolite Repression ◽

Gene Disruption ◽

Single Gene ◽

Carbon Sources ◽

Phosphotransferase System ◽

Carbohydrate Transport ◽

Encoding Genes ◽

Redundant Role

The phosphoenolpyruvate : carbohydrate phosphotransferase system (PTS) catalyses carbohydrate transport by coupling it to phosphorylation. Previously, we reported a Corynebacterium glutamicum R β-glucoside PTS encoded by bglF. Here we report that C. glutamicum R contains an additional β-glucoside PTS gene, bglF2, organized in a cluster with a putative phospho-β-glucosidase gene, bglA2, and a putative antiterminator, bglG2. While single gene disruption strains of either bglF or bglF2 were able to utilize salicin or arbutin as sole carbon sources, a double disruption strain exhibited defects in utilization of both carbon sources. Expression of both bglF and bglF2 was induced in the presence of either salicin or arbutin, although disruption of bglG2 affected only bglF2 expression. Moreover, in the presence of either salicin or arbutin, glucose completely repressed the expression of bglF but only slightly repressed that of bglF2. We conclude that BglF and BglF2 have a redundant role in β-glucoside transport even though the catabolite repression control of their encoding genes is different. We also show that expression of both bglF and bglF2 requires the general PTS.

Download Full-text