Metabolic characteristics of an isocitrate dehydrogenase defective derivative of <i>escherichia coli</i> BL21(DE3)

Aoshima, Miho; Ishii, Masaharu; Yamagishi, Akihiko; Oshima, Tairo; Igarashi, Yasuo

doi:10.1002/bit.10832

Cited by 21 publications

(11 citation statements)

References 21 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In mutant strains with icd inactivated (HL2671k and HL26715k), there was also an accumulation of TCA cycle C 6 intermediates such as citrate and isocitrate (data not shown). Others have shown similar effects when icd was inactivated in E. coli (Aoshima et al, 2003). This accumulation is less, though, in HL26715k than in HL2671k due to the glyoxylate cycle.…”

Section: Effect Of Mutations On Aerobic Metabolite Production and Celmentioning

confidence: 73%

Genetic reconstruction of the aerobic central metabolism inEscherichia coli for the absolute aerobic production of succinate

Lin

Bennett

San

2004

Biotechnol. Bioeng.

101

View full text Add to dashboard Cite

Most reported efforts to enhance production of the industrially valuable specialty chemical succinate have been done under anaerobic conditions, where E. coli undergoes mixed-acid fermentation. These efforts have often been hampered by the limitations of NADH availability, poor cell growth, and slow production. An aerobic succinate production system was strategically designed that allows E. coli to produce and accumulate succinate efficiently and substantially as a product under absolute aerobic conditions. Mutations in the tricarboxylic acid cycle (sdhAB, icd, iclR) and acetate pathways (poxB, ackA-pta) of E. coli were created to construct the glyoxylate cycle for aerobic succinate production. Experiments in flask studies showed that 14.28 mM of succinate could be produced aerobically with a yield of 0.344 mole/mole using 55 mM glucose. In aerobic batch reactor studies, succinate production rate was faster, reaching 0.5 mole/mole in 24 h with a concentration of 22.12 mM; further cultivation showed that succinate production reached 43 mM with a yield of 0.7. There was also substantial pyruvate and TCA cycle C(6) intermediate accumulation in the mutant. The results suggest that more metabolic engineering improvements can be made to this system to make aerobic succinate production more efficient. Nevertheless, this aerobic succinate production system provides the first platform for enhancing succinate production aerobically in E. coli based on the creation of a new aerobic central metabolic network.

show abstract

Section: Effect Of Mutations On Aerobic Metabolite Production and Celmentioning

confidence: 73%

Genetic reconstruction of the aerobic central metabolism inEscherichia coli for the absolute aerobic production of succinate

Lin

Bennett

San

2004

Biotechnol. Bioeng.

101

View full text Add to dashboard Cite

show abstract

“…Isocitrate lyase (Icl): 50 mM morpholipepropanesulfonic acid (MOPS; pH, 7.3), 1 mM EDTA, 5 mM MgCl 2 , 4 mM phenylhydrazine HCl (FH) and 12.5 mM L-isocitrate [80]. Isocitrate dehydrogenase (Icdh): 50 mM phosphate buffer (pH 7.5), 5 mM MgCl 2 , 2 mM NADP + , 2.5 mM D,L isocitrate [81]. Tryptophanase (TnaA): 1000 mM potassium phosphate (pH, 8.3), 0.81 mM pyridoxal 5-phosphate, 50 mM L-tryptophan (pH 10.8), trichloroacetic acid 6.1 N, toluene, p-dimethylaminobenzaldehyde solution 5%(w/v), hydrochloric acid-alcohol 895 mM [82].…”

Section: Methodsmentioning

confidence: 99%

New insights into Escherichia coli metabolism: carbon scavenging, acetate metabolism and carbon recycling responses during growth on glycerol

et al. 2012

View full text Add to dashboard Cite

BackgroundGlycerol has enhanced its biotechnological importance since it is a byproduct of biodiesel synthesis. A study of Escherichia coli physiology during growth on glycerol was performed combining transcriptional-proteomic analysis as well as kinetic and stoichiometric evaluations in the strain JM101 and certain derivatives with important inactivated genes.ResultsTranscriptional and proteomic analysis of metabolic central genes of strain JM101 growing on glycerol, revealed important changes not only in the synthesis of MglB, LamB and MalE proteins, but also in the overexpression of carbon scavenging genes: lamB, malE, mglB, mglC, galP and glk and some members of the RpoS regulon (pfkA, pfkB, fbaA, fbaB, pgi, poxB, acs, actP and acnA). Inactivation of rpoS had an important effect on stoichiometric parameters and growth adaptation on glycerol. The observed overexpression of poxB, pta, acs genes, glyoxylate shunt genes (aceA, aceB, glcB and glcC) and actP, suggested a possible carbon flux deviation into the PoxB, Acs and glyoxylate shunt. In this scenario acetate synthesized from pyruvate with PoxB was apparently reutilized via Acs and the glyoxylate shunt enzymes. In agreement, no acetate was detected when growing on glycerol, this strain was also capable of glycerol and acetate coutilization when growing in mineral media and derivatives carrying inactivated poxB or pckA genes, accumulated acetate. Tryptophanase A (TnaA) was synthesized at high levels and indole was produced by this enzyme, in strain JM101 growing on glycerol. Additionally, in the isogenic derivative with the inactivated tnaA gene, no indole was detected and acetate and lactate were accumulated. A high efficiency aromatic compounds production capability was detected in JM101 carrying pJLBaroGfbrtktA, when growing on glycerol, as compared to glucose.ConclusionsThe overexpression of several carbon scavenging, acetate metabolism genes and the absence of acetate accumulation occurred in JM101 cultures growing on glycerol. To explain these results it is proposed that in addition to the glycolytic metabolism, a gluconeogenic carbon recycling process that involves acetate is occurring simultaneously in this strain when growing on glycerol. Carbon flux from glycerol can be efficiently redirected in JM101 strain into the aromatic pathway using appropriate tools.

show abstract

“…Even in A. niger the enhancement of anaplerotic reactions replenishing TCA cycle intermediates predisposes the cells to form high amounts of citric acid [41]. Enhancement of biosynthetic reactions due to shortage of TCA cycle intermediates was also observed in citric acid accumulating E. coli K and B strains in the form of increased glyoxylate pathway [42], [43]. However in our study, similar increase in flux through glyoxylate shunt was not apparent as evident from very low and unaltered ICL activity ( Table 3 ).…”

Section: Discussionmentioning

confidence: 99%

Artificial Citrate Operon Confers Mineral Phosphate Solubilization Ability to Diverse Fluorescent Pseudomonads

et al. 2014

View full text Add to dashboard Cite

Citric acid is a strong acid with good cation chelating ability and can be very efficient in solubilizing mineral phosphates. Only a few phosphate solubilizing bacteria and fungi are known to secrete citric acids. In this work, we incorporated artificial citrate operon containing NADH insensitive citrate synthase (gltA1) and citrate transporter (citC) genes into the genome of six-plant growth promoting P. fluorescens strains viz., PfO-1, Pf5, CHAO1, P109, ATCC13525 and Fp315 using MiniTn7 transposon gene delivery system. Comprehensive biochemical characterization of the genomic integrants and their comparison with plasmid transformants of the same operon in M9 minimal medium reveals the highest amount of ∼7.6±0.41 mM citric and 29.95±2.8 mM gluconic acid secretion along with ∼43.2±3.24 mM intracellular citrate without affecting the growth of these P. fluorescens strains. All genomic integrants showed enhanced citric and gluconic acid secretion on Tris-Cl rock phosphate (TRP) buffered medium, which was sufficient to release 200–1000 µM Pi in TRP medium. This study demonstrates that MPS ability could be achieved in natural fluorescent pseudomonads by incorporation of artificial citrate operon not only as plasmid but also by genomic integration.

show abstract

Metabolic characteristics of an isocitrate dehydrogenase defective derivative of escherichia coli BL21(DE3)

Cited by 21 publications

References 21 publications

Genetic reconstruction of the aerobic central metabolism inEscherichia coli for the absolute aerobic production of succinate

Genetic reconstruction of the aerobic central metabolism inEscherichia coli for the absolute aerobic production of succinate

New insights into Escherichia coli metabolism: carbon scavenging, acetate metabolism and carbon recycling responses during growth on glycerol

Artificial Citrate Operon Confers Mineral Phosphate Solubilization Ability to Diverse Fluorescent Pseudomonads

Contact Info

Product

Resources

About