Search

The aim of this research was to test the role of the glyoxylate cycle enzyme malate synthase (MLS) in lipid utilization, gluconeogenesis, and seedling growth in Arabidopsis. We hypothesized that in the absence of MLS, succinate produced by isocitrate lyase (ICL) could still feed into the tricarboxylic acid cycle, whereas glyoxylate could be converted to sugars using enzymes of the photorespiratory pathway. To test this hypothesis we isolated knock-out mls mutants and studied their growth and metabolism in comparison to wild type and icl mutant seedlings. In contrast to icl seedlings, which grow slowly and are unable to convert lipid into sugars (Eastmond, P. J., Germain, V., Lange, P. R., Bryce, J. H., Smith, S. M. & Graham, I. A. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 5669–5674), mls seedlings grow faster, use their lipid more rapidly, and are better able to establish as plantlets. Transcriptome and metabolome analyses show that icl seedlings exhibit many features characteristic of carbohydrate starvation, whereas mls seedlings differ relatively little from wild type. In the light mls seedlings generate more sugars than icl seedlings, and when fed with [14C]acetate, 14C-labeling of sugars is three times greater than in icl seedlings and more than half that in wild type seedlings. The mls seedlings also accumulate more glycine and serine than icl or wild type seedlings, consistent with a diversion of glyoxylate into these intermediates of the photorespiratory pathway. We conclude that, in contrast to bacteria and fungi in which MLS is essential for gluconeogenesis from acetate or fatty acids, MLS is partially dispensable for lipid utilization and gluconeogenesis in Arabidopsis seedlings.

In the amino-acid-producing microorganism Corynebacterium glutamicum, the specific activities of the acetate-activating enzymes acetate kinase and phosphotransacetylase and those of the glyoxylate cycle enzymes isocitrate lyase and malate synthase were found to be high when the cells were grown on acetate (0.8, 2.9, 2.1, and 1.8 U/mg protein, respectively). When the cells were grown on glucose or on other carbon sources such as lactate, succinate, or glutamate, the specific activities were two- to fourfold (acetate kinase and phosphotransacetylase) and 45- to 100-fold (isocitrate lyase and malate synthase) lower, indicating that the synthesis of the four enzymes is regulated by acetate in the growth medium. A comparative Northern (RNA) analysis of the C. glutamicum isocitrate lyase and malate synthase genes (aceA and aceB) and transcriptional cat fusion experiments revealed that aceA and aceB are transcribed as 1.6- and 2.7-kb monocistronic messages, respectively, and that the regulation of isocitrate lyase and malate synthase synthesis is exerted at the level of transcription from the respective promoters. Surprisingly, C. glutamicum mutants defective in either acetate kinase or phosphotransacetylase showed low specific activities of the other three enzymes (phosphotransacetylase, isocitrate lyase, and malate synthase or acetate kinase, isocitrate lyase, and malate synthase, respectively) irrespective of the presence or absence of acetate in the medium. This result and a correlation of a high intracellular acetyl coenzyme A concentration with high specific activities of isocitrate lyase, malate synthase, acetate kinase, and phosphotransacetylase suggest that acetyl coenzyme A or a derivative thereof may be a physiological trigger for the genetic regulation of enzymes involved in acetate metabolism of C. glutamicum.

Background Tuberculosis (TB) remains a major world health problem. Around 2 billions of people are infected by Mycobacterium tuberculosis, the causal agent of this disease. This fact accounts for a third of the total world population and it is expected that 9 million people will become infected each year. Only approximately 10% of the infected people will develop disease. However, health care workers (HCW) are continually exposed to the bacilli at endemic sites presenting increased chance of becoming sick. The objective of this work was to identify LTBI (latent tuberculosis infection) among all asymptomatic HCW of a Brazilian Central Hospital, in a three year follow up, and evaluate the humoral response among HCW with previous and recent LTBI to recombinant HspX and GlcB from M. tuberculosis. Methods Four hundred and thirty seven HCW were screened and classified into three different groups according to tuberculin skin test (TST) status: uninfected, previous LTBI and recent LTBI. ELISA test were performed to determine the humoral immune response to HspX and GlcB. Results The levels of IgG and IgM against the HspX and GlcB antigens were the same among HCW with recent and previous LTBI, as well as among non infected HCW. However, the IgM levels to HspX was significantly higher among HCW with recent LTBI (OD = 1.52 ± 0.40) than among the uninfected (OD = 1.09 ± 0.50) or subjects with previous LTBI (OD = 0.96 ± 0.51) (p < 0.001). Conclusion IgG and IgM humoral responses to GlcB antigens were similar amongst all studied groups; nevertheless IgM levels against HspX were higher among the recent LTBI/HCW.

The photosynthetic non-sulfur purple bacterium Rhodobacter capsulatus E1F1 can grow on acetate or dl-malate photoheterotrophically under anerobic conditions or chemoheterotrophically in the dark in the presence of dioxygen. Bacterial cells grown under both anaerobic and aerobic conditions exhibited high amounts of the tricarboxylic acid cycle enzymes especially in dark-aerobic cultures. A high activity of isocitrate lyase was found in cells of R. capsulatus E1F1 and, to a lesser extent, in those of R. capsulatus IP2, Rhodobacter sphaeroides and Rhodospirillum rubrum grown photoheterotrophically on acetate under anaerobic conditions. The second enzyme of the glyoxylate shunt, malate synthase, appears to be constitutive. Itaconate, a powerful inhibitor of isocitrate lyase, severely inhibited growth of R. capsulatus, R. rubrum and R. sphaeroides on acetate, thus corroborating a physiological role of the enzyme in acetate metabolism by Rhodospirillaceae. * Abbreviations: Abbreviations ISL : isocitrate lyase MS : malate synthase DTE : dithioerythrytol pHMB : p -hydroxymercuribenzoate DTNB : 5,5′-dithiobis(2-nitrobenzoic acid)

Mutant strains of Escherichia coli K-12 were shown to be impaired in their ability to assimilate glycolate- 2 - 14 C . One strain (Glc-103) has lost the ability to oxidize glycolate; another strain (Glc-102) was relatively impermeable to the compound. A third strain (Glc-104) had undergone a similar loss in permeability, and, in addition, was deranged in the synthesis of either glyoxylate reductase or malate synthase G.

The following two articles present a way of determining the chirality of doubly labelled methyl groups, and reveal that the condensation reaction catalysed by malate synthase takes place with inversion of configuration.

Given the success in engineering synthetic phenotypes in microbes and mammalian cells, constructing non-native pathways in mammals has become increasingly attractive for understanding and identifying potential targets for treating metabolic disorders. Here we introduced the glyoxylate shunt into mouse liver to investigate mammalian fatty acid metabolism. Mice expressing the shunt showed resistance to diet-induced obesity on a high fat diet despite similar food consumption. This was accompanied by a decrease in total fat mass, circulating leptin levels, plasma triglyceride concentration, and a signaling metabolite in liver, malonyl-CoA, that inhibits fatty acid degradation. Contrary to plants and bacteria, in which the glyoxylate shunt prevents the complete oxidation of fatty acids, this pathway when introduced in mice increases fatty acid oxidation such that resistance to diet-induced obesity develops. This work suggests that using non-native pathways in higher organisms to explore and modulate metabolism may be a useful approach.

Background: Glycolic acid is a C2 hydroxy acid that is a widely used chemical compound. It can be polymerised to produce biodegradable polymers with excellent gas barrier properties. Currently, glycolic acid is produced in a chemical process using fossil resources and toxic chemicals. Biotechnological production of glycolic acid using renewable resources is a desirable alternative.Results: The yeasts Saccharomyces cerevisiae and Kluyveromyces lactis are suitable organisms for glycolic acid production since they are acid tolerant and can grow in the presence of up to 50 g l-1 glycolic acid. We engineered S. cerevisiae and K. lactis for glycolic acid production using the reactions of the glyoxylate cycle to produce glyoxylic acid and then reducing it to glycolic acid. The expression of a high affinity glyoxylate reductase alone already led to glycolic acid production. The production was further improved by deleting genes encoding malate synthase and the cytosolic form of isocitrate dehydrogenase. The engineered S. cerevisiae strain produced up to about 1 g l-1 of glycolic acid in a medium containing d-xylose and ethanol. Similar modifications in K. lactis resulted in a much higher glycolic acid titer. In a bioreactor cultivation with d-xylose and ethanol up to 15 g l-1 of glycolic acid was obtained.Conclusions: This is the first demonstration of engineering yeast to produce glycolic acid. Prior to this work glycolic acid production through the glyoxylate cycle has only been reported in bacteria. The benefit of a yeast host is the possibility for glycolic acid production also at low pH, which was demonstrated in flask cultivations. Production of glycolic acid was first shown in S. cerevisiae. To test whether a Crabtree negative yeast would be better suited for glycolic acid production we engineered K. lactis in the same way and demonstrated it to be a better host for glycolic acid production.

Background The genome of the important industrial host Bacillus subtilis does not encode the glyoxylate shunt, which is necessary to utilize overflow metabolites, like acetate or acetoin, as carbon source. In this study, the operon encoding the isocitrate lyase (aceB) and malate synthase (aceA) from Bacillus licheniformis was transferred into the chromosome of B. subtilis. The resulting strain was examined in respect to growth characteristics and qualities as an expression host. Results Our results show that the modified B. subtilis strain is able to grow on the C2 compound acetate. A combined transcript, protein and metabolite analysis indicated a functional expression of the native glyoxylate shunt of B. lichenifomis in B. subtilis. This metabolically engineered strain revealed better growth behavior and an improved activity of an acetoin-controlled expression system. Conclusions The glyoxylate shunt of B. licheniformis can be functionally transferred to B. subtilis. This novel strain offers improved properties for industrial applications, such as growth on additional carbon sources and a greater robustness towards excess glucose feeding.