scholarly journals Expression of the Escherichia coli pntAB genes encoding a membrane-bound transhydrogenase in Corynebacterium glutamicum improves l-lysine formation

2007 ◽  
Vol 75 (1) ◽  
pp. 47-53 ◽  
Author(s):  
Armin Kabus ◽  
Tobias Georgi ◽  
Volker F. Wendisch ◽  
Michael Bott
Keyword(s):  
Escherichia Coli ◽  
Corynebacterium Glutamicum ◽  
Genes Encoding ◽  
Membrane Bound

scholarly journals Transcriptional control and essential roles of the Escherichia coli ccm gene products in formate-dependent nitrite reduction and cytochrome c synthesis

1998 ◽  
Vol 334 (2) ◽  
pp. 355-365 ◽  
Author(s):  
Sutipa TANAPONGPIPAT ◽  
Eleanor REID ◽  
Jeffrey A. COLE ◽  
Helen CROOKE
Keyword(s):  
Escherichia Coli ◽  
Cytochrome C ◽  
Transcriptional Control ◽  
Regulatory System ◽  
Nitrite Reduction ◽  
Anaerobic Growth ◽  
Current View ◽  
Fnr Protein ◽  
Genes Encoding ◽  
Membrane Bound

The eight ccm genes located at minute 47 on the Escherichia coli chromosome, in the order ccmABCDEFGH, encode homologues of proteins which are essential for cytochrome c assembly in other bacteria. The ccm genes are immediately downstream from the napFDAGHBC genes encoding a periplasmic nitrate reductase. CcmH was previously shown to be essential for cytochrome c assembly. Deletion analysis and a two-plasmid strategy have now been used to demonstrate that CcmA, B, D, E, F and G are also essential for cytochrome c assembly, and hence for cytochrome-c-dependent nitrite reduction. The ccm genes are transcribed from a ccmA promoter located within the adjacent gene, napC, which is the structural gene for a 24 kDa membrane-bound c-type cytochrome, NapC. Transcription from this ccmA promoter is induced approximately 5-fold during anaerobic growth, independently of a functional Fnr protein: it is also not regulated by the ArcB–ArcA two-component regulatory system. The ccmA promoter is an example of the ‘extended -10 sequence ’ group of promoters with a TGX motif immediately upstream of the -10 sequence. Mutagenesis of the TG motif to TC, CT or CC resulted in loss of about 50% of the promoter activity. A weak second promoter is suggested to permit transcription of the downstream ccmEFGH genes in the absence of transcription readthrough from the upstream napF and ccmA promoters. The results are consistent with, but do not prove, the current view that CcmA, B, C and D are part of an essential haem transport mechanism, that CcmE, F and H are required for covalent haem attachment to cysteine-histidine motifs in cytochrome c apoproteins in the periplasm, and that CcmG is required for the reduction of cysteine residues on apocytochromes c in preparation for haem ligation.

Cloning of the histidine biosynthetic genes from Corynebacterium glutamicum: Organization and analysis of the hisG and hisE genes

2000 ◽  
Vol 46 (9) ◽  
pp. 848-855 ◽  
Author(s):  
Joon-Hye Kwon ◽  
Jae-Yeon Chun ◽  
Heung-Shick Lee ◽  
Choong-Ill Cheon ◽  
Eun-Sook Song ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Corynebacterium Glutamicum ◽  
Biosynthetic Pathway ◽  
Amino Acid Sequences ◽  
Sequencing Analysis ◽  
Gene Products ◽  
Gene Library ◽  
Biosynthetic Genes ◽  
Coding Regions ◽  
Genes Encoding

The physically linked hisG and hisE genes, encoding for ATP-phosphoribosyltransferase and phosphoribosyl-ATP-pyrophosphohydrolase were isolated from the Corynebacterium glutamicum gene library by complementation of Escherichia coli histidine auxotrophs. They are two of the nine genes that participate in the histidine biosynthetic pathway. Molecular genetics and sequencing analysis of the cloned 9-kb insert DNA showed that it carries the hisG and hisE genes. In combining this result with our previous report, we propose that all histidine biosynthetic genes are separated on the genome by three unlinked loci. The coding regions of the hisG and hisE genes are 279 and 87 amino acids in length with a predicted size of about 30 and 10 kDa, respectively. Computer analysis revealed that the amino acid sequences of the hisG and hisE gene products were similar to those of other bacteria.

scholarly journals The Glycolytic Flux in Escherichia coli Is Controlled by the Demand for ATP

2002 ◽  
Vol 184 (14) ◽  
pp. 3909-3916 ◽  
Author(s):  
Brian J. Koebmann ◽  
Hans V. Westerhoff ◽  
Jacky L. Snoep ◽  
Dan Nilsson ◽  
Peter R. Jensen
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Atp Hydrolysis ◽  
Molecular Genetic ◽  
Glycolytic Flux ◽  
E Coli ◽  
Strong Stimulation ◽  
Genes Encoding ◽  
Membrane Bound ◽  
Efficiency And Productivity

ABSTRACT The nature of the control of glycolytic flux is one of the central, as-yet-uncharacterized issues in cellular metabolism. We developed a molecular genetic tool that specifically induces ATP hydrolysis in living cells without interfering with other aspects of metabolism. Genes encoding the F1 part of the membrane-bound (F1F0) H+-ATP synthase were expressed in steadily growing Escherichia coli cells, which lowered the intracellular [ATP]/[ADP] ratio. This resulted in a strong stimulation of the specific glycolytic flux concomitant with a smaller decrease in the growth rate of the cells. By optimizing additional ATP hydrolysis, we increased the flux through glycolysis to 1.7 times that of the wild-type flux. The results demonstrate why attempts in the past to increase the glycolytic flux through overexpression of glycolytic enzymes have been unsuccessful: the majority of flux control (>75%) resides not inside but outside the pathway, i.e., with the enzymes that hydrolyze ATP. These data further allowed us to answer the question of whether catabolic or anabolic reactions control the growth of E. coli. We show that the majority of the control of growth rate resides in the anabolic reactions, i.e., the cells are mostly “carbon” limited. Ways to increase the efficiency and productivity of industrial fermentation processes are discussed.

Expression of the Escherichia coli pntA andpntB Genes, Encoding Nicotinamide Nucleotide Transhydrogenase, in Saccharomyces cerevisiae and Its Effect on Product Formation during Anaerobic Glucose Fermentation

1999 ◽  
Vol 65 (6) ◽  
pp. 2333-2340 ◽  
Author(s):  
Mikael Anderlund ◽  
Torben L. Nissen ◽  
Jens Nielsen ◽  
John Villadsen ◽  
Jan Rydström ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Anaerobic Fermentation ◽  
Product Formation ◽  
Physiological Effect ◽  
Glucose Fermentation ◽  
Genes Encoding ◽  
Membrane Bound ◽  
Glycerol Formation ◽  
High Level

ABSTRACT We studied the physiological effect of the interconversion between the NAD(H) and NADP(H) coenzyme systems in recombinantSaccharomyces cerevisiae expressing the membrane-bound transhydrogenase from Escherichia coli. Our objective was to determine if the membrane-bound transhydrogenase could work in reoxidation of NADH to NAD+ in S. cerevisiaeand thereby reduce glycerol formation during anaerobic fermentation. Membranes isolated from the recombinant strains exhibited reduction of 3-acetylpyridine-NAD+ by NADPH and by NADH in the presence of NADP+, which demonstrated that an active enzyme was present. Unlike the situation in E. coli, however, most of the transhydrogenase activity was not present in the yeast plasma membrane; rather, the enzyme appeared to remain localized in the membrane of the endoplasmic reticulum. During anaerobic glucose fermentation we observed an increase in the formation of 2-oxoglutarate, glycerol, and acetic acid in a strain expressing a high level of transhydrogenase, which indicated that increased NADPH consumption and NADH production occurred. The intracellular concentrations of NADH, NAD+, NADPH, and NADP+were measured in cells expressing transhydrogenase. The reduction of the NADPH pool indicated that the transhydrogenase transferred reducing equivalents from NADPH to NAD+.

scholarly journals Improving cell-free glycoprotein synthesis by characterizing and enriching native membrane vesicles

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jasmine M. Hershewe ◽  
Katherine F. Warfel ◽  
Shaelyn M. Iyer ◽  
Justin A. Peruzzi ◽  
Claretta J. Sullivan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Synthetic Biology ◽  
Membrane Protein ◽  
Membrane Vesicles ◽  
Processing Methods ◽  
Glycoprotein Synthesis ◽  
On Demand ◽  
Free Gene ◽  
Membrane Bound

AbstractCell-free gene expression (CFE) systems from crude cellular extracts have attracted much attention for biomanufacturing and synthetic biology. However, activating membrane-dependent functionality of cell-derived vesicles in bacterial CFE systems has been limited. Here, we address this limitation by characterizing native membrane vesicles in Escherichia coli-based CFE extracts and describing methods to enrich vesicles with heterologous, membrane-bound machinery. As a model, we focus on bacterial glycoengineering. We first use multiple, orthogonal techniques to characterize vesicles and show how extract processing methods can be used to increase concentrations of membrane vesicles in CFE systems. Then, we show that extracts enriched in vesicle number also display enhanced concentrations of heterologous membrane protein cargo. Finally, we apply our methods to enrich membrane-bound oligosaccharyltransferases and lipid-linked oligosaccharides for improving cell-free N-linked and O-linked glycoprotein synthesis. We anticipate that these methods will facilitate on-demand glycoprotein production and enable new CFE systems with membrane-associated activities.

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