Genetic map position of the cistron coding for isocitrate dehydrogenase inEscherichia coli K-12

Apostolakos, Diane; Menter, Paul A.; Rampsch, Brian J.; Reeves, Henry C.; Birge, Edward A.

doi:10.1007/bf01570979

Cited by 15 publications

(4 citation statements)

References 13 publications

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“…However, the growth of HY041(pTrcTAD) was inhibited dramatically (Fig. 3a), that may be caused by insufficient supply of α-ketoglutaric acid, a precursor for glutamate biosynthesis [31]. Meanwhile, the rate of acetate consumption was also reduced in HY041(pTrcTAD), and only 43.1 mM was consumed in 48 h (Fig.…”

Section: Resultsmentioning

confidence: 99%

Metabolic engineering of Escherichia coli carrying the hybrid acetone-biosynthesis pathway for efficient acetone biosynthesis from acetate

et al. 2019

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BackgroundThe shortage of food based feedstocks has been one of the stumbling blocks in industrial biomanufacturing. The acetone bioproduction from the traditional acetone–butanol–ethanol fermentation is limited by the non-specificity of products and competitive utilization of food-based substrates. Using genetically modified Escherichia coli to produce acetone as sole product from the cost-effective non-food based substrates showed great potential to overcome these problems.ResultsA novel acetone biosynthetic pathway were constructed based on genes from Clostridium acetobutylicum (thlA encoding for thiolase, adc encoding for acetoacetate decarboxylase, ctfAB encoding for coenzyme A transferase) and Escherichia coli MG1655 (atoB encoding acetyl-CoA acetyltransferase, atoDA encoding for acetyl-CoA: acetoacetyl-CoA transferase subunit α and β). Among these constructs, one recombinant MG1655 derivative containing the hybrid pathway consisting of thlA, atoDA, and adc, produced the highest level of acetone from acetate. Reducing the gluconeogenesis pathway had little effect on acetone production, while blocking the TCA cycle by knocking out the icdA gene enhanced the yield of acetone significantly. As a result, acetone concentration increased up to 113.18 mM in 24 h by the resting cell culture coupling with gas-stripping methods.ConclusionsAn engineered E. coli strain with optimized hybrid acetone biosynthetic pathway can utilize acetate as substrate efficiently to synthesize acetone without other non-gas byproducts. It provides a potential method for industrial biomanufacturing of acetone by engineered E. coli strains from non-food based substrate.Electronic supplementary materialThe online version of this article (10.1186/s12934-019-1054-8) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Metabolic engineering of Escherichia coli carrying the hybrid acetone-biosynthesis pathway for efficient acetone biosynthesis from acetate

et al. 2019

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“…We are interested in how the bacteroid TCA cycle is regulated and have selected ICD for our initial studies because it is a regulated enzyme at a branch point in the TCA cycle (15), because it is regulated by aerobiosis in other gram-negative bacteria (23,24), and because it is a relatively simple enzyme with a single type of subunit that, in other bacteria, is encoded by a single gene (2). In this study, we report the isolation of the gene that encodes ICD in R meliloti and the symbiotic properties of mutants with defects in this gene.…”

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confidence: 99%

Cloning and mutagenesis of the Rhizobium meliloti isocitrate dehydrogenase gene

McDermott

Kahn

1992

J Bacteriol

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The gene encoding Rhizobium meliloti isocitrate dehydrogenase (ICD) was cloned by complementation of an Escherichia coli icd mutant with an R. meliloti genomic library constructed in pUC18. The complementing DNA was located on a 4.4-kb BamHI fragment. It encoded an ICD that had the same mobility as R. meliloti ICD in nondenaturing polyacrylamide gels. In Western immunoblot analysis, antibodies raised against this protein reacted with R. meliloti ICD but not with E. coli ICD. The complementing DNA fragment was mutated with transposon TnS and then exchanged for the wild-type allele by recombination by a novel method that employed the Bacillus subtilis levansucrase gene. No ICD activity was found in the two R. meliloti icd::Tn5 mutants isolated, and the mutants were also found to be glutamate auxotrophs. The mutants formed nodules, but they were completely ineffective. Faster-growing pseudorevertants were isolated from cultures of both R. melloti icd::Tn5 mutants. In addition to lacking all ICD activity, the pseudorevertants also lacked citrate synthase activity. Nodule formation by these mutants was severely affected, and inoculated plants had only callus structures or small spherical structures.In the symbiosis between legumes and rhizobia, the host plant provides the bacteria with reduced carbon as an energy source. The bacteria use this energy to reduce atmospheric nitrogen to ammonia, which they release to the plant. The qualitative nature of this energy source has been the focus of much research. Sucrose is the major photosynthate transported from the shoot to the nodule (41), but biochemical evidence suggests that neither sucrose nor hexoses obtained from sucrose degradation are important sources of energy for the bacteroids (reviewed in references 7 and 34). In support of this conclusion, mutants of various species of rhizobia with defects in sugar metabolism have been found to be effective in symbiosis (7,34). By contrast, dicarboxylic acids appear to be important carbon sources in the establishment of an effective symbiosis. Succinate and malate are found at high concentrations in nodules (14,45,55), are actively transported across the peribacteroid membrane (20,56), are taken up by bacteroids (11,20,42,50), and are quickly oxidized to CO2 after uptake (47). Dicarboxylic acid transport (dct) mutants of Rhizobium meliloti (5) and R. leguminosarum biovars viciae (3, 12) and trifolii (44) We are interested in how the bacteroid TCA cycle is regulated and have selected ICD for our initial studies because it is a regulated enzyme at a branch point in the TCA cycle (15), because it is regulated by aerobiosis in other gram-negative bacteria (23, 24), and because it is a relatively simple enzyme with a single type of subunit that, in other bacteria, is encoded by a single gene (2). In this study, we report the isolation of the gene that encodes ICD in R meliloti and the symbiotic properties of mutants with defects in this gene. MATERIALS AND METHODSBacterial strains and plasmids. The strains of R. meliloti and Es...

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“…However, MA1935 generated from BL21(DE3) showed glutamate auxotrophy, which has been also reported for other ICDH defective E. coli strains (EB106 (Apostolakos et al, 1982) and DEK2004 (Thorsness and Koshland, 1987)), indicating that 2-oxoglutarate, a starting material for the glutamate biosynthesis, can be supplied only from isocitrate by ICDH. As it has been reported that K-12 wild strain (CS101) cannot grow on glutamate owing to the poor glutamate uptake and accumulation abilities (Halpern and Lupo, 1965), growth of W3110 on glutamate was faint or negligible.…”

Section: Growth Characteristicsmentioning

confidence: 63%

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

Aoshima

Ishii

Yamagishi

et al. 2003

Biotech & Bioengineering

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A 7.8 kb fragment containing isocitrate dehydrogenase (ICDH) gene and its flanking regions was cloned from Escherichia coli BL21(DE3) and sequenced. Unlike the case of the K-12 strain, the e14 element was not found. The nucleotide divergence between these two strains was about 2%. Using the cloned fragment, ICDH defective mutant strain, MA1935, was generated from BL21(DE3). Although MA1935 accumulated citrate, citrate synthase activity was not repressed but was rather high. In addition, isocitrate lyase was not highly induced at the stationary phase. MA1935 was shown to be a good host strain for ICDH gene expression.

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Genetic map position of the cistron coding for isocitrate dehydrogenase inEscherichia coli K-12

Cited by 15 publications

References 13 publications

Metabolic engineering of Escherichia coli carrying the hybrid acetone-biosynthesis pathway for efficient acetone biosynthesis from acetate

Metabolic engineering of Escherichia coli carrying the hybrid acetone-biosynthesis pathway for efficient acetone biosynthesis from acetate

Cloning and mutagenesis of the Rhizobium meliloti isocitrate dehydrogenase gene

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

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