Itaconic Acid Production Research Articles

Itaconic acid production is regulated by LaeA in Aspergillus pseudoterreus

The global regulator LaeA controls secondary metabolism in diverse Aspergillus species. Here we explored its role in regulation of itaconic acid production in Aspergillus pseudoterreus. To understand its role in regulating metabolism, we deleted and overexpressed laeA, and assessed the transcriptome, proteome, and secreted metabolome prior to and during initiation of phosphate limitation induced itaconic acid production. We found that secondary metabolite clusters, including the itaconic acid biosynthetic gene cluster, are regulated by laeA and that laeA is required for high yield production of itaconic acid. Overexpression of LaeA improves itaconic acid yield at the expense of biomass by increasing the expression of key biosynthetic pathway enzymes and attenuating the expression of genes involved in phosphate acquisition and scavenging. Increased yield was observed in optimized conditions as well as conditions containing excess nutrients that may be present in inexpensive sugar containing feedstocks such as excess phosphate or complex nutrient sources. This suggests that global regulators of metabolism may be useful targets for engineering metabolic flux that is robust to environmental heterogeneity.

Synergistic effects on itaconic acid production in engineered Aspergillus niger expressing the two distinct biosynthesis clusters from Aspergillus terreus and Ustilago maydis

BackgroundItaconic acid (IA) is a versatile platform chemical widely used for the synthesis of various polymers and current methods for IA production based on Aspergillus terreus fermentation are limited in terms of process efficiency and productivity. To construct more efficient IA production strains, A. niger was used as a chassis for engineering IA production by assembling the key components of IA biosynthesis pathways from both A. terreus and Ustilago maydis.ResultsRecombinant A. niger S1596 overexpressing the A. terreus IA biosynthesis genes cadA, mttA, mfsA produced IA of 4.32 g/L, while A. niger S2120 overexpressing the U. maydis IA gene cluster adi1, tad1, mtt1, itp1 achieved IA of 3.02 g/L. Integration of the two IA production pathways led to the construction of A. niger S2083 with IA titers of 5.58 g/L. Increasing cadA copy number in strain S2083 created strain S2209 with titers of 7.99 g/L and deleting ictA to block IA degradation in S2209 created strain S2288 with IA titers of 8.70 g/L. Overexpressing acoA to enhance the supply of IA precursor in strain S2288 generated strain S2444 with IA titers of 9.08 g/L in shake flask.ConclusionRecombinant A. niger overexpressing the U. maydis IA biosynthesis pathway was capable of IA accumulation. Combined expression of the two IA biosynthesis pathways from A. terreus and U. maydis in A. niger resulted in much higher IA titers. Furthermore, increasing cadA copy number, deleting ictA to block IA degradation and overexpressing acoA to enhance IA precursor supply all showed beneficial effects on IA accumulation.

Iron Deficiency and Overload Modulate the Inflammatory Responses and Metabolism of Alveolar Macrophages.

Alveolar macrophages (AM) are critical to defense against respiratory pathogens. This study evaluated cellular iron imbalance to immunometabolism in endotoxin-polarized porcine AMs (PAMs). PAMs collected from five 6-week-old pigs were treated with a basal media, iron chelator, or ferric ammonium citrate to maintain iron replete or induce iron deficiency or overload, respectively. After 24 h treatment, PAMs were challenged with saline or lipopolysaccharide (LPS) for 6 h. Cells were analyzed for gene, protein, and untargeted metabolome. Cytokines were determined in culture media. Data were assessed using two-way ANOVA. Treatments successfully induced iron deficiency and overload. The mRNA of DMT1 and ZIP14 was increased up to 300-fold by LPS, but unaffected by iron. Surprisingly, both iron deprivation and overload attenuated LPS-induced inflammation, showing less TNFα production and lower mRNA of pro- and anti-inflammatory cytokines than iron-replete PAMs. Forty-eight metabolites were altered by either or both main effects. LPS enhanced the glycolysis and polyol pathways. Iron deprivation disrupted the TCA cycle. Iron overload increased intracellular cholesterol. Interestingly, iron deprivation augmented, whereas iron overload diminished, LPS-induced itaconic acid production, which has anti-microbial and anti-inflammatory properties. Therefore, iron-deficient PAMs may be more resistant to intracellular pathogens which use PAMs as a conduit for infection.

Itaconic acid production by Aspergillus terreus from glucose up to pilot scale and from corn stover and wheat straw hydrolysates using new manganese tolerant medium

Itaconic acid (IA), is a versatile platform chemical that is produced by Aspergillus terreus using glucose as carbon source. Lignocellulosic biomass can be used as a low cost source of sugars for IA production. However, A. terreus does not produce IA even after 100 fold dilution from enzymatically hydrolyzed wheat straw pretreated with dilute acid. In addition to organic inhibitors, Mn2+ strongly inhibits IA production. Mn2+ was present in the hydrolysate at a level 230 times over the inhibitory limit (50 ppb). Recently, PO43− limitation was shown to decrease the inhibitory effect of Mn2+ on IA production and a medium was formulated in which A. terreus produced IA very efficiently in the presence of Mn2+ up to 100 ppm. Here, the effectiveness of this medium was demonstrated at pilot scale using a stainless steel bioreactor and glucose as the substrate. The productions of IA from corn stover cellulosic hydrolysate in a biorefinery concept and mitigated wheat straw hydrolysate using the Mn2+ tolerant medium were also demonstrated at laboratory scale. The fungal strain produced 74% IA from cellulosic hydrolysate and 98% IA from wheat straw hydrolysate in comparison with respective mock sugar solution. This is the first report on using Mn2+ tolerant medium to achieve IA production by A. terreus on hydrolysates.

Tailoring key enzymes for renewable and high-level itaconic acid production using genetic Escherichia coli via whole-cell bioconversion

Renewable chemical productions through carbon-neutral design are widely concerned in recent years. Among all, itaconic acid (IA) is one of the most important building block chemicals from biorefinery. However, IA fermentation by the eukaryotic Aspergillus terreus is time-consuming and less productive. The whole-cell (WC) bioconversion, proposed as an alternative approach by transforming citrate into IA via two key enzymes of aconitase (ACN, EC 4.2.1.3) and cis-aconitate decarboxylase (CAD, EC 4.1.1.6), is attractive. In this study, we screened the best genes from genes library, studied the kinetics parameters of ACN from Corynebacterium glutamicum (Cg) and CAD from Aspergillus terreus (At), thus achieving the maximum IA production. The catalytic activity of CgAcnA was 39-fold of AtCadA, indicating CAD was the rate-determining step. For metal ions effect, copper and ferric ions inhibited 95% and 59% enzyme activity when both enzymes co-worked together. Finally, the engineered Escherichia coli expressing dual genes and cultured in glycerol-included medium reached the highest IA titer of 67 g/L and productivity of 8.375 g/L/h, which demonstrates as a promising renewable process.

A CRISPRi mediated self-inducible system for dynamic regulation of TCA cycle and improvement of itaconic acid production in Escherichia coli

Itaconic acid (ITA), an effective alternative fossil fuel, derives from the bypass pathway of the tricarboxylic acid (TCA) cycle. Therefore, the imbalance of metabolic flux between TCA cycle and ITA biosynthetic pathway seriously limits the production of ITA. The optimization of flux distribution between biomass and production has the potential to the productivity of ITA. Based on the previously constructed strain Escherichia coli MG1655 Δ1-SAS-3 (ITA titer: 1.87 g/L), a CRISPRi-mediated self-inducible system (CiMS), which contained a responsive module based on the ITA biosensor YpItcR/Pccl and a regulative CRISPRi-mediated interferential module, was developed to regulate the flux of the TCA cycle and to enhance the capacity of the strain to produce ITA. First, a higher ITA-yielding strain, Δ4-Prmd-SAS-3 (ITA titer: 3.20 g/L), derived from Δ1-SAS-3, was constructed by replacing the promoter PJ23100, for the expression of ITA synthesis genes, with Prmd and knocking out the three bypass genes poxB, pflB, and ldhA. Subsequently, the CiMS was used to inhibit the expression of key genes icd, pykA, and sucCD to dynamically balance the metabolic flux between TCA cycle and ITA biosynthetic pathway during the ITA production stage. The constructed strain Δ4-Prmd-SAS-3 under the dynamic regulation of the CiMS, showed a 23% increase in the ITA titer, which reached 3.93 g/L. This study indicated that CiMS was a practical strategy to dynamically and precisely regulated the metabolic flux in microbial cell factories.

Photocontrol of Itaconic Acid Synthesis in Escherichia coli.

Metabolic engineering aims to control cellular metabolic flow and maximize the production of a product of interest. Photocontrol of the activities of proteins is an effective method for accurately regulating metabolic pathways. In this study, we inserted the photosensor light-oxygen-voltage-sensing domain 2 of Avena sativa (AsLOV2) into selected sites of isocitrate dehydrogenase (IDH), the key enzyme in the competitive pathway of itaconic acid (ITA) synthesis, to construct photoswitchable IDH-AsLOV2 (ILOVs). These engineered light-sensitive proteins were used to regulate the metabolic flux of the tricarboxylic acid (TCA) cycle in Escherichia coli to improve ITA production. The engineered fusion proteins ILOV2, ILOV3, ILOV6, and ILOV7 exhibited effective reversibility under the oscillation of darkness and blue light illumination in vitro. The efficacies of the intracellular photoswitches were evaluated, and an optimal photocontrol strategy was established in vivo. The ITA titer was significantly enhanced to 3.30 g/L for strain ITAΔ43, which displayed superior photoswitchable potency for ITA production compared with the strains that completely deleted the icd gene. The photocontrol strategy developed here can be extended for process optimization and titer improvement of other high-value bioengineering chemicals.

Engineering Yarrowia lipolytica to Produce Itaconic Acid From Waste Cooking Oil.

Itaconic acid (IA) is a high-value organic acid with a plethora of industrial applications. In this study, we seek to develop a microbial cell factory that could utilize waste cooking oil (WCO) as raw material for circular and cost-effective production of the abovementioned biochemical. Specifically, we expressed cis-aconitic acid decarboxylase (CAD) gene from Aspergillus terreus in either the cytosol or peroxisome of Yarrowia lipolytica and assayed for production of IA on WCO. To further improve production yield, the 10 genes involved in the production pathway of acetyl-CoA, an intermediate metabolite necessary for the synthesis of cis-aconitic acid, were individually overexpressed and investigated for their impact on IA production. To minimize off-target flux channeling, we had also knocked out genes related to competing pathways in the peroxisome. Impressively, IA titer up to 54.55 g/L was achieved in our engineered Y. lipolytica in a 5 L bioreactor using WCO as the sole carbon source.

Acetate-based production of itaconic acid with Corynebacterium glutamicum using an integrated pH-coupled feeding control

To date, most bio-based products of industrial biotechnology stem from sugar-based carbon sources originating from food and feed competing resources. Exemplary for bioproducts converted from glucose, the potential C5 platform chemical itaconic acid is presently produced by the filamentous fungus Aspergillus terreus. Here, an engineered strain of the industrial platform organism Corynebacterium glutamicum ATCC 13032 was used for acetate-based production of itaconic acid to overcome current production difficulties. For this purpose, C. glutamicum ICDR453C (pEKEx2-malEcadopt) with a mutated icd variant for reduced isocitrate dehydrogenase activity was constructed harbouring pEKEx2-malEcadopt, that includes a cis-aconitate dehydrogenase gene originating from A. terreus. Overall, a peak volumetric productivity of 1.01 gL−1h−1 was achieved resulting in an itaconate titer of 29.2 g/L, by using an integrated pH-coupled acetate feeding control in a fed-batch process without base titration. The results support the high potential of acetate as alternative substrate for bioproduction.

Itaconate inhibits TET DNA dioxygenases to dampen inflammatory responses.

As one of the most induced genes in activated macrophages, immune-responsive gene 1 (IRG1) encodes a mitochondrial metabolic enzyme catalysing the production of itaconic acid (ITA). Although ITA has an anti-inflammatory property, the underlying mechanisms are not fully understood. Here we show that ITA is a potent inhibitor of the TET-family DNA dioxygenases. ITA binds to the same site on TET2 as the co-substrate α-ketoglutarate, inhibiting TET2 catalytic activity. Lipopolysaccharide treatment, which induces Irg1 expression and ITA accumulation, inhibits Tet activity in macrophages. Transcriptome analysis reveals that TET2 is a major target of ITA in suppressing lipopolysaccharide-induced genes, including those regulated by the NF-κB and STAT signalling pathways. In vivo, ITA decreases the levels of 5-hydroxymethylcytosine, reduces lipopolysaccharide-induced acute pulmonary oedema as well as lung and liver injury, and protects mice against lethal endotoxaemia, depending on the catalytic activity of Tet2. Our study thus identifies ITA as an immune modulatory metabolite that selectively inhibits TET enzymes to dampen the inflammatory responses.

Production of Itaconic Acid by Aspergillus terreus from Sorghum Bran Hydrolysates and Optimization for Fermentative Production

Production of Itaconic Acid by <i>Aspergillus terreus</i> from Sorghum Bran Hydrolysates and Optimization for Fermentative Production

Alleviating Glucose Repression and Enhancing Respiratory Capacity to Increase Itaconic Acid Production

Alleviating Glucose Repression and Enhancing Respiratory Capacity to Increase Itaconic Acid Production

Environmental assessment of the production of itaconic acid from wheat straw under a biorefinery approach

This study performs the environmental assessment of itaconic acid (IA) production from wheat straw. The Life Cycle Assessment (LCA) methodology is used to determine the environmental hotspots, considering impact categories such as Global Warming (GW), Fossil Resource Scarcity (FRS), Water Consumption (WC), among others. A sensitivity analysis was performed considering an optimization of the steam explosion process and 100% renewable energy. Results report an impact of about 14.33 kg CO2 eq in GW, 4.15 kg of oil eq in FRS, for each kg of IA produced for the baseline scenario. Moreover, the pretreatment and fermentation stages constitute hotspots of the IA production. In addition, using a renewable energy source in production would reduce the impact by 82% in GW, 71% in PM and 82% in FRS categories. The optimization of the steam explosion process presents a better performance in GW and FRS but also lies in an increase in WC.

Recent Advances in Biotechnological Itaconic Acid Production, and Application for a Sustainable Approach.

Intense research has been conducted to produce environmentally friendly biopolymers obtained from renewable feedstock to substitute fossil-based materials. This is an essential aspect for implementing the circular bioeconomy strategy, expressly declared by the European Commission in 2018 in terms of “repair, reuse, and recycling”. Competent carbon-neutral alternatives are renewable biomass waste for chemical element production, with proficient recyclability properties. Itaconic acid (IA) is a valuable platform chemical integrated into the first 12 building block compounds the achievement of which is feasible from renewable biomass or bio-wastes (agricultural, food by-products, or municipal organic waste) in conformity with the US Department of Energy. IA is primarily obtained through fermentation with Aspergillus terreus, but nowadays several microorganisms are genetically engineered to produce this organic acid in high quantities and on different substrates. Given its trifunctional structure, IA allows the synthesis of various novel biopolymers, such as drug carriers, intelligent food packaging, antimicrobial biopolymers, hydrogels in water treatment and analysis, and superabsorbent polymers binding agents. In addition, IA shows antimicrobial, anti-inflammatory, and antitumor activity. Moreover, this biopolymer retains qualities like environmental effectiveness, biocompatibility, and sustainability. This manuscript aims to address the production of IA from renewable sources to create a sustainable circular economy in the future. Moreover, being an essential monomer in polymer synthesis it possesses a continuous provocation in the biopolymer chemistry domain and technologies, as defined in the present review.

Bayesian Inference for Integrating Yarrowia lipolytica Multiomics Datasets with Metabolic Modeling.

Optimizing the metabolism of microbial cell factories for yields and titers is a critical step for economically viable production of bioproducts and biofuels. In this process, tuning the expression of individual enzymes to obtain the desired pathway flux is a challenging step, in which data from separate multiomics techniques must be integrated with existing biological knowledge to determine where changes should be made. Following a design-build-test-learn strategy, building on recent advances in Bayesian metabolic control analysis, we identify key enzymes in the oleaginous yeast Yarrowia lipolytica that correlate with the production of itaconate by integrating a metabolic model with multiomics measurements. To this extent, we quantify the uncertainty for a variety of key parameters, known as flux control coefficients (FCCs), needed to improve the bioproduction of target metabolites and statistically obtain key correlations between the measured enzymes and boundary flux. Based on the top five significant FCCs and five correlated enzymes, our results show phosphoglycerate mutase, acetyl-CoA synthetase (ACSm), carbonic anhydrase (HCO3E), pyrophosphatase (PPAm), and homoserine dehydrogenase (HSDxi) enzymes in rate-limiting reactions that can lead to increased itaconic acid production.

Recent advances in itaconic acid production from microbial cell factories

Itaconic acid is an unsaturated organic acid with two carboxyls and one methylene group. The presence of these functional groups, along with a conjugated double bond, makes itaconic acid a versatile molecule with a vast number of applications. Itaconic acid can be produced through chemical as well as biological routes. Aspergillus terreus is the most prevalent microbial cell factory for the biological production of itaconic acid, reaching titers of >100 g/L. However, it suffers from low yield and volumetric productivities leading to high manufacturing costs. The wider applications of itaconic acid can be enabled with a low-cost production process, which can be achieved with cheaper feedstocks and robust cell factories accumulating itaconic acid efficiently. The current review summarizes the recent advances in the biological production of itaconic acid with a focus on the metabolic engineering of prokaryotic and eukaryotic systems for the overproduction of itaconic acid. It comprehensively describes various microbial cell factories with an insight into the pathway leading to itaconic acid production in natural producers like A. terreus and U. maydis. It also discusses the metabolic engineering approaches to improve strain performance in terms of high itaconic acid productivity, less by-product generation, and the ability to utilize unconventional cheap substrates. Moreover, the alternative strategies for the development of non-native producers through genome engineering and the hurdles related to itaconic acid production have been elaborated.

Inactivation of Aspergillus species in real water matrices using medium pressure mercury lamps.

The aim of this work is to understand the inactivation efficiency of medium pressure mercury lamps, measured in terms of growth inhibition as well as cell death, damage and response, using three strains from three different Aspergillus species (A. fumigatus, A. niger and, A. terreus) spiked in filtered surface water. A complete characterization of the effect of the treatment on each strain of the fungal species was assessed considering spores' morphology, cell wall integrity and enzymatic activity, the formation of pyrimidine dimers in the DNA and proteome analysis. Results showed that, when subjected to medium pressure mercury lamps, A. niger is the most resistant to inactivation, that both A. fumigatus and A. niger suffer more morphological changes and present a higher number of damaged spores and A. terreus presented more dead spores. DNA damages detected in A. niger were able to be repaired to some extent, under both light and dark conditions. Finally, proteome analysis showed that the UV radiation treatment triggered different types of stress response, including cell wall reorganization and DNA repair in A. fumigatus and A. terreus, and oxidative stress responses like the increase in production of citric acid and itaconic acid in A. niger and A. terreus, respectively.

Utilization of ethanol for itaconic acid biosynthesis by engineered Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, ethanol can serve as both a carbon source and NADH donor for the production of acetyl-CoA derivatives. Here we investigated the metabolic regulation of ethanol utilization for itaconic acid production by S. cerevisiae. To understand the interconnection between the TCA cycle and the glyoxylate pathway, mitochondrial membrane transporter proteins SFC1, YHM2, CTP1, DIC1 and MPC1 were knocked out and results showed that SFC1 functions as an important entrance of the glyoxylate pathway into the TCA cycle, and YHM2 is helpful to IA production but not the primary pathway for citric acid supply. To decrease the accumulation of acetic acid, the major ADP/ATP carrier of the mitochondrial inner membrane, AAC2, was upregulated and determined to accelerate ethanol utilization and itaconic acid production. RNA sequencing results showed that AAC2 overexpression enhanced IA titer by upregulating the ethanol-acetyl-CoA pathway and NADH oxidase in the mitochondrial membrane. RNA-seq analysis also suggested that aconitase ACO1 may be a rate-limiting step of IA production. However, the expression of exogenous aconitase didn't increase IA production but enhanced the rate of ethanol utilization and decreased cell growth.

Microbial itaconic acid production from starchy food waste by newly isolated thermotolerant Aspergillus terreus strain

In the present study, we have explored the potential of newly isolated Aspergillus terreus BD strain, which can accumulate itaconic acid (IA) at higher temperature. The shake flask cultivation of thermotolerant strain with medium optimized using Box-Behnken Design at 45 °C resulted in IA accumulation of 28.9 g/L with yield of 0.27 g/g. The enzymatic saccharification of the synthetic food waste (SFW) consisting of potatoes, rice & noodles were optimized using Taguchi method of orthogonal array to maximize the release of fermentable sugar. The maximum glucose release of 0.60 g/g was achieved with 10% biomass loading, 5% enzyme concentration, pH 5.5 and temperature 60 0C. The sugars obtained from SFW was integrated with IA production and maximum IA titer achieved with SFW hydrolysate during bioreactor cultivation was 41.1 g/L with conversion yield of 0.27 g/g while with pure glucose IA titer and yield were 44.7 g/L and 0.30 g/g, respectively.

Production of itaconic acid from alkali pretreated lignin by dynamic two stage bioconversion

Expanding the portfolio of products that can be made from lignin will be critical to enabling a viable bio-based economy. Here, we engineer Pseudomonas putida for high-yield production of the tricarboxylic acid cycle-derived building block chemical, itaconic acid, from model aromatic compounds and aromatics derived from lignin. We develop a nitrogen starvation-detecting biosensor for dynamic two-stage bioproduction in which itaconic acid is produced during a non-growth associated production phase. Through the use of two distinct itaconic acid production pathways, the tuning of TCA cycle gene expression, deletion of competing pathways, and dynamic regulation, we achieve an overall maximum yield of 56% (mol/mol) and titer of 1.3 g/L from p-coumarate, and 1.4 g/L titer from monomeric aromatic compounds produced from alkali-treated lignin. This work illustrates a proof-of-principle that using dynamic metabolic control to reroute carbon after it enters central metabolism enables production of valuable chemicals from lignin at high yields by relieving the burden of constitutively expressing toxic heterologous pathways.