Pyruvic acid production by a lipoic acid auxotroph of Escherichia coliW1485

A lipoic acid auxotroph of Escherichia coli K-12, strain W1485lip2 (ATCC25645), produced pyruvic acid aerobically from glucose under the lipoic acid-deficient conditions, while the prototrophic parent strain, W1485 (ATCC12435), produced 2-oxoglutaric acid aas the main product. The mechanism of the pyruvic acid production by strain W1485lip2 was found to be the impaired oxidative decarboxylation of pyruvic acid caused by the decrease in the activity of pyruvate dehydrogenase complex under the conditions of lipoic acid deficiency. Under the optimum culture conditions using the pH-controlled jar fermentor, 25.5 g/l pyruvic acid was obtained from 50 g/l glucose after the culture for 32–40 h at pH6.0. The relationship between the pyruvic acid productivity and the pyruvate dehydrogenase complex activity in jar-fermentor culture was discussed.     

Pyruvic Acid Production by an F1-ATPase-defective Mutant of Escherichia coli W1485lip2

An F₁-ATPase-defective mutant, TBLA-1, was constructed by the transduction of a defective gene for the a subunit of F₁-ATPase, atpA401, into Escherichia coli W1485lip2, a lipoic acid-requiring pyruvic acid producer. The pyruvic acid production of the strain TBLA-1 was found to be improved markedly compared with that of strain W1485lip2. In cultures using a jar fermentor, the strain W1485lip2 consumed 50 g/liter of glucose and produced 25 g/liter of pyruvic acid after culture for 32 h, while strain TBLA-1 consumed the same amount of glucose, and produced more than 30 g/liter of pyruvic acid in a 24-h culture. A revertant, No. 63–1, derived from the strain TBLA-1, had a normal level of F₁-ATPase activity, and showed a similar pattern of pyruvic acid production to that of strain W1485lip2.     

Enzymatic production of tryptophan using a lipoic acid and thiamine double auxotroph of Enterobacter aerogenes having both pyruvic acid productivity and high tryptophanase activity

Washed cells of a newly-developed double auxotroph for lipoic acid and thiamine, Enterobacter aerogenes LT-94, produced about 30 g/l of pyruvic acid from 5% glucose, and subsequently converted the acid to tryptophan in a yield of 16.7 g/l by the reverse reaction of tryptophanase, which had been induced under derepressed conditions. These yields were higher than those of the parent strain, L-12, showing only lipoic acid auxotrophy.     

Ubiquinone deficiency in an auxotroph of Escherichia coli requiring 4-hydroxybenzoic acid

1. Escherichia coli 156:53D2 synthesized ubiquinone only when the growth medium was supplemented with 4-hydroxybenzoate acid. 2. Little or no vitamin K(2) was formed by the mutant under the growth conditions employed, in contrast with wild-type strains. 3. In the mutant ubiquinone deficiency was correlated with low respiration and with low particulate NADH-oxidase and NADH-cytochrome b(1)-reductase activity. 4. Preincubation of ubiquinone-deficient particles with ubiquinone-30 largely restored the NADH-oxidase and NADH-cytochrome b(1)-reductase activities. 5. Various NADH-dye-linked reductases which may be associated with NADH dehydrogenase were not affected by the absence of ubiquinone. 6. The succinate-oxidase complex was less affected than the particulate NADH oxidase by ubiquinone deficiency. 7. A pathway for electrons in the NADH-oxidase complex of the auxotroph of E. coli is proposed and its relationship to the pathway in the wild-type strain is discussed.     

Optimization of octanoic acid and sulfur donor concentrations for lipoic acid production by Pseudomonas reptilivora

Using the optimal concentrations of octanoic acid (0.75 mM) and ethyl mercaptan (2 mM), as the most effective sulfur donor, Pseudomonas reptilivora produced 74 mug lipoic acid per dry cell weight at pH 7.5 and 30 degrees C in a fermenter over 9 h. The dry cell weight was 13.9 g l(-1).     

Endogenous production of lipoic acid is essential for mouse development

alpha-Lipoic acid (LA) is a cofactor for mitochondrial alpha-ketoacid dehydrogenase complexes and is one of the most potent, natural antioxidants. Reduction of oxidative stress by LA supplementation has been demonstrated in patients with diabetic neuropathy and in animal models. To determine how normal development or pathological conditions are affected by genetic alterations in the ability of mammalian cells to synthesize LA and whether dietary LA can circumvent its endogenous absence, we have generated mice deficient in lipoic acid synthase (Lias). Mice heterozygous for disruption of the Lias gene develop normally, and their plasma levels of thiobarbituric acid-reactive substances do not differ from those of wild-type mice. However, the heterozygotes have significantly reduced erythrocyte glutathione levels, indicating that their endogenous antioxidant capacity is lower than those of wild-type mice. Homozygous embryos lacking Lias appear healthy at the blastocyst stage, but their development is retarded globally by 7.5 days postcoitum (dpc), and all the null embryos die before 9.5 dpc. Supplementing the diet of heterozygous mothers with LA (1.65 g/kg of body weight) during pregnancy fails to prevent the prenatal deaths of homozygous embryos. Thus, endogenous LA synthesis is essential for developmental survival and cannot be replaced by LA in maternal tissues and blood.     

Medium optimization for L-phenylalanine production by a tryptophan auxotroph of Corynebacterium glutamicum

Summary By screening 15,000 mutants, tyrosine auxotrophs T6, T7, and tryptophan auxotrophs P6, P8, were obtained. After primary production test, mutant P6 was chosen for further investigation. Fractional factorial design(FFD) and steepest ascent method(SAM) were used to optimize the medium component Mutant P6 had 17.1 g/l L-phenylalanine production when 0.44 g/l tryptophan was added. When Corynebacterium glutamicum P6 was cultivated in the optimum medium, L-phenylaianine production increased 22% as compared with the parent strain CCRC 18335, and the interference of tryptophan during the purification process was removed.     

Ribosomal distribution in a polyamine auxotroph of Escherichia coli.

The distribution of ribosomal particles has been studied in a polyamine-deficient mutant of Escherichia coli by sucrose gradient centrifugation analysis. Lysates from starved cells contained less 70S monomers and 30S subunits but more 50S particles than those prepared from bacteria supplemented with putrescine. The addition of the polyamine to putrescine-depleted cells induced a rapid change of the ribosomal profile. A similar effect could be obtained in vitro by equilibrium dialysis against a polyamine-containing solution. The ribosomal pattern obtained from starved bacteria was specific for polyamine deficiency. We conclude that the changes in ribosomal profiles upon restoration of putrescine levels in previously starved cells denote a shift of the equilibrium between 30S-50S couples and ribosomal subunits.     

Unusual growth characteristics of a methionine-cyano-B12 auxotroph of Escherichia coli

Escherichia coli K-12, strain AB1172, substrain 2276, a methionine(-)-cyano-B(12) (-) auxotroph grew poorly when placed in a medium containing optimal levels of cyano-B(12) and suboptimal amounts of l-methionine. A marked depression of N(5,10)-methylene-H(4)-folate reductase was observed in the inhibited cells, although no significant alterations in other enzymes involved in the metabolism of N(5,10)-methylene-H(4)-folate were observed. Guanosine was effective in preventing the growth inhibition.     

On the role of lipoic acid as a cofactor for oxidative phosphorylation in Escherichia coli.

Using an auxotrophic mutant of $\text{Escherichia coli}$ and the technique of quenching of atebrin fluorescence by membrane particles it has been shown that lipoic acid is not required for either respiration or ATP-driven proton tranlocation.     

Amino acid transport in a polyaromatic amino acid auxotroph of Saccharomyces cerevisiae

The initiation of growth of a polyaromatic auxotrophic mutant of Saccharomyces cerevisiae was inhibited by several amino acids, whereas growth of the parent prototroph was unaffected. A comparative investigation of amino acid transport in the two strains employing (14)C-labeled amino acids revealed that the transport of amino acids in S. cerevisiae was mediated by a general transport system responsible for the uptake of all neutral as well as basic amino acids. Both auxotrophic and prototrophic strains exhibited stereospecificity for l-amino acids and a K(m) ranging from 1.5 x 10(-5) to 5.0 x 10(-5) M. Optimal transport activity occurred at pH 5.7. Cycloheximide had no effect on amino acid uptake, indicating that protein synthesis was not a direct requirement for amino acid transport. Regulation of amino acid transport was subject to the concentration of amino acids in the free amino acid pool. Amino acid inhibition of the uptake of the aromatic amino acids by the aromatic auxotroph did not correlate directly with the effect of amino acids on the initiation of growth of the auxotroph but provides a partial explanation of this effect.     

Stimulation of prostaglandin biosynthesis by lipoic acid.

Enzyme preparations from sheep seminal vesicles display an enhanced ability to synthesize prostaglandins, particularly prostaglandin F from polyunsaturated fatty acids if alpha-lipoic acid is present in the incubation mixture prior to the addition of fatty acid. The stimulation by lipoate is reversible, time dependent, and involves modifications of V and Km for oxygenase activity. Product studies, structure vs. activity studies, and purification data indicate that lipoate exerts it effect by a mechanism distinct from a glutathione-like metabolism of the endoperoxide linkage in prostaglandin G and prostaglandin H. In addition, product studies suggest that lipoate is not a cofactor for the endoperoxide isomerase component of prostaglandin synthetase. Purification of the endoperoxide synthesizing activity by ion-exchange chromatography and isoelectric focusing yields preparations which are more responsive to lipoate than microsomal preparations.     

Phase transitions of phospholipid bilayers from an unsaturated fatty acid auxotroph of Escherichia coli.

Total phospholipids were extracted from cells of temperature sensitive unsaturated fatty acid auxotrophs of Escherichia coli (K-12 UFAts) grown at 28degrees C (PL28), and at 42degrees C in the presence of 2% KCl as an osmotic stabilizer (PL42 (KCl)). From the analysis of fatty acids, it was shown that the content of unsaturated fatty acids of PL42 (KCl) is only 9% of the total fatty acids, while that of PL28 is 54%. The thermal phase transitions of the bilayers prepared from the phospholipid fractions were studied by proton magnetic resonance. The line widths of the methylene signals and the sums of the methylene and methyl signal intensities were plotted against reciprocal values of absolute temperature 1/T or temperature itself. From the plots phase transitions were detected at about 19degrees C for PL28 and at 43degrees C for PL42 (KCl). In spite of its complex composition of fatty acids a highly cooperative transition was observed in the case of PL42 (KCl). It was also suggested that the phospholipids bilayers in the biomembranes of this strain at the growth temperature (42 degrees C) are in the state where the gel and liquid crystalline phases coexist.