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Immune-responsive gene 1 protein links metabolism to immunity by catalyzing itaconic acid production.

ABSTRACT: Immunoresponsive gene 1 (Irg1) is highly expressed in mammalian macrophages during inflammation, but its biological function has not yet been elucidated. Here, we identify Irg1 as the gene coding for an enzyme producing itaconic acid (also known as methylenesuccinic acid) through the decarboxylation of cis-aconitate, a tricarboxylic acid cycle intermediate. Using a gain-and-loss-of-function approach in both mouse and human immune cells, we found Irg1 expression levels correlating with the amounts of itaconic acid, a metabolite previously proposed to have an antimicrobial effect. We purified IRG1 protein and identified its cis-aconitate decarboxylating activity in an enzymatic assay. Itaconic acid is an organic compound that inhibits isocitrate lyase, the key enzyme of the glyoxylate shunt, a pathway essential for bacterial growth under specific conditions. Here we show that itaconic acid inhibits the growth of bacteria expressing isocitrate lyase, such as Salmonella enterica and Mycobacterium tuberculosis. Furthermore, Irg1 gene silencing in macrophages resulted in significantly decreased intracellular itaconic acid levels as well as significantly reduced antimicrobial activity during bacterial infections. Taken together, our results demonstrate that IRG1 links cellular metabolism with immune defense by catalyzing itaconic acid production.

SUBMITTER: Michelucci A

PROVIDER: S-EPMC3651434 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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Immune-responsive gene 1 protein links metabolism to immunity by catalyzing itaconic acid production.

Michelucci Alessandro A Cordes Thekla T Ghelfi Jenny J Pailot Arnaud A Reiling Norbert N Goldmann Oliver O Binz Tina T Wegner André A Tallam Aravind A Rausell Antonio A Buttini Manuel M Linster Carole L CL Medina Eva E Balling Rudi R Hiller Karsten K

Proceedings of the National Academy of Sciences of the United States of America 20130422 19

Immunoresponsive gene 1 (Irg1) is highly expressed in mammalian macrophages during inflammation, but its biological function has not yet been elucidated. Here, we identify Irg1 as the gene coding for an enzyme producing itaconic acid (also known as methylenesuccinic acid) through the decarboxylation of cis-aconitate, a tricarboxylic acid cycle intermediate. Using a gain-and-loss-of-function approach in both mouse and human immune cells, we found Irg1 expression levels correlating with the amount ...[more]

PMID: 23610393

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Project description:BackgroundItaconic acid is a promising platform chemical for a bio-based polymer industry. Today, itaconic acid is biotechnologically produced with Aspergillus terreus at industrial scale from sugars. The production of fuels but also of chemicals from food substrates is a dilemma since future processes should rely on carbon sources which do not compete for food or feed. Therefore, the production of chemicals from alternative substrates such as acetate is desirable to develop novel value chains in the bioeconomy.ResultsIn this study, Corynebacterium glutamicum ATCC 13032 was engineered to efficiently produce itaconic acid from the non-food substrate acetate. Therefore, we rewired the central carbon and nitrogen metabolism by inactivating the transcriptional regulator RamB, reducing the activity of isocitrate dehydrogenase, deletion of the gdh gene encoding glutamate dehydrogenase and overexpression of cis-aconitate decarboxylase (CAD) from A. terreus optimized for expression in C. glutamicum. The final strain C. glutamicum ΔramB Δgdh IDHR453C (pEKEx2-malEcadopt) produced 3.43 ± 0.59 g itaconic acid L-1 with a product yield of 81 ± 9 mmol mol-1 during small-scale cultivations in nitrogen-limited minimal medium containing acetate as sole carbon and energy source. Lowering the cultivation temperature from 30 °C to 25 °C improved CAD activity and further increased the titer and product yield to 5.01 ± 0.67 g L-1 and 116 ± 15 mmol mol-1, respectively. The latter corresponds to 35% of the theoretical maximum and so far represents the highest product yield for acetate-based itaconic acid production. Further, the optimized strain C. glutamicum ΔramB Δgdh IDHR453C (pEKEx2-malEcadopt), produced 3.38 ± 0.28 g itaconic acid L-1 at 25 °C from an acetate-containing aqueous side-stream of fast pyrolysis.ConclusionAs shown in this study, acetate represents a suitable non-food carbon source for itaconic acid production with C. glutamicum. Tailoring the central carbon and nitrogen metabolism enabled the efficient production of itaconic acid from acetate and therefore this study offers useful design principles to genetically engineer C. glutamicum for other products from acetate.

Project description:Itaconic acid (IA), or 2-methylenesuccinic acid, has a broad spectrum of applications in the biopolymer industry owing to the presence of one vinyl bond and two acid groups in the structure. Its polymerization can follow a similar mechanism as acrylic acid but additional functionality can be incorporated into the extra beta acid group. Currently, the bio-based production of IA in industry relies on the fermentation of the filamentous fungus Aspergillus terreus. However, the difficulties associated with the fermentation undertaken by filamentous fungi together with the pathogenic potential of A. terreus pose a serious challenge for industrial-scale production. In recent years, there has been increasing interest in developing alternative production hosts for fermentation processes that are more homogenous in the production of organic acids. Pichia kudriavzevii is a non-conventional yeast with high acid tolerance to organic acids at low pH, which is a highly desirable trait by easing downstream processing. We introduced cis-aconitic acid decarboxylase gene (cad) from A. terreus (designated At_cad) into this yeast and established the initial titer of IA at 135 ± 5 mg/L. Subsequent overexpression of a native mitochondrial tricarboxylate transporter (herein designated Pk_mttA) presumably delivered cis-aconitate efficiently to the cytosol and doubled the IA production. By introducing the newly invented CRISPR-Cas9 system into P. kudriavzevii, we successfully knocked out both copies of the gene encoding isocitrate dehydrogenase (ICD), aiming to increase the availability of cis-aconitate. The resulting P. kudriavzevii strain, devoid of ICD and overexpressing Pk_mttA and At_cad on its genome produced IA at 505 ± 17.7 mg/L in shake flasks, and 1232 ± 64 mg/L in fed-batch fermentation. Because the usage of an acid-tolerant species does not require pH adjustment during fermentation, this work demonstrates the great potential of engineering P. kudriavzevii as an industrial chassis for the production of organic acid.

Project description:Methylorubrum extorquens (formerly Methylobacterium extorquens) AM1 is a methylotrophic bacterium with a versatile lifestyle. Various carbon sources including acetate, succinate and methanol are utilized by M. extorquens AM1 with the latter being a promising inexpensive substrate for use in the biotechnology industry. Itaconic acid (ITA) is a high-value building block widely used in various industries. Given that no wildtype methylotrophic bacteria are able to utilize methanol to produce ITA, we tested the potential of M. extorquens AM1 as an engineered host for this purpose. In this study, we successfully engineered M. extorquens AM1 to express a heterologous codon-optimized gene encoding cis-aconitic acid decarboxylase. The engineered strain produced ITA using acetate, succinate and methanol as the carbon feedstock. The highest ITA titer in batch culture with methanol as the carbon source was 31.6 ± 5.5 mg/L, while the titer and productivity were 5.4 ± 0.2 mg/L and 0.056 ± 0.002 mg/L/h, respectively, in a scaled-up fed-batch bioreactor under 60% dissolved oxygen saturation. We attempted to enhance the carbon flux toward ITA production by impeding poly-β-hydroxybutyrate accumulation, which is used as carbon and energy storage, via mutation of the regulator gene phaR. Unexpectedly, ITA production by the phaR mutant strain was not higher even though poly-β-hydroxybutyrate concentration was lower. Genome-wide transcriptomic analysis revealed that phaR mutation in the ITA-producing strain led to complex rewiring of gene transcription, which might result in a reduced carbon flux toward ITA production. Besides poly-β-hydroxybutyrate metabolism, we found evidence that PhaR might regulate the transcription of many other genes including those encoding other regulatory proteins, methanol dehydrogenases, formate dehydrogenases, malate:quinone oxidoreductase, and those synthesizing pyrroloquinoline quinone and thiamine co-factors. Overall, M. extorquens AM1 was successfully engineered to produce ITA using acetate, succinate and methanol as feedstock, further supporting this bacterium as a feasible host for use in the biotechnology industry. This study showed that PhaR could have a broader regulatory role than previously anticipated, and increased our knowledge of this regulator and its influence on the physiology of M. extorquens AM1.

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Immune-responsive gene 1 protein links metabolism to immunity by catalyzing itaconic acid production.

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Immune-responsive gene 1 protein links metabolism to immunity by catalyzing itaconic acid production.

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