Itaconic Acid Production Research Articles

The interplay between transport and metabolism in fungal itaconic acid production

Besides enzymatic conversions, many eukaryotic metabolic pathways also involve transport proteins that shuttle molecules between subcellular compartments, or into the extracellular space. Fungal itaconate production involves two such transport steps, involving an itaconate transport protein (Itp), and a mitochondrial tricarboxylate transporter (Mtt). The filamentous ascomycete Aspergillus terreus and the unicellular basidiomycete Ustilago maydis both produce itaconate, but do so via very different molecular pathways, and under very different cultivation conditions. In contrast, the transport proteins of these two strains are assumed to have a similar function. This study aims to investigate the roles of both the extracellular and mitochondrial transporters from these two organisms by expressing them in the corresponding U. maydis knockouts and monitoring the extracellular product concentrations. Both transporters from A. terreus complemented their corresponding U. maydis knockouts in mediating itaconate production. Surprisingly, complementation with At_MfsA from A. terreus led to a partial switch from itaconate to (S)-2-hydroxyparaconate secretion. Apparently, the export protein from A. terreus has a higher affinity for (S)-2-hydroxyparaconate than for itaconate, even though this species is classically regarded as an itaconate producer. Complementation with At_MttA increased itaconate production by 2.3-fold compared to complementation with Um_Mtt1, indicating that the mitochondrial carrier from A. terreus supports a higher metabolic flux of itaconic acid precursors than its U. maydis counterpart. The biochemical implications of these differences are discussed in the context of the biotechnological application in U. maydis and A. terreus for the production of itaconate and (S)-2-hydroxyparaconate.

Enhanced itaconic acid production in Yarrowia lipolytica via heterologous expression of a mitochondrial transporter MTT.

Itaconic acid, a promising platform chemical, has been applied in many fields of industrial production. As a potential candidate for itaconic acid production, Yarrowia lipolytica possesses several innate abilities such as the tolerance of low-pH and high-shear stress, fast growth rate, cultivation flexibility, and easy for genetic manipulation. Here, Y. lipolytica Po1f which was tested to show high tolerance to itaconic acid could accumulate itaconic acid (0.363g/L) by expressing the Aspergillus terreus cis-aconitic acid decarboxylase (CAD). Then, we tried to improve the supply and transport of the immediate precursor cis-aconitic acid by overexpressing a series of genes; these results indicate that overexpression of mitochondrial cis-aconitate transporter MTT is beneficial to the itaconic acid biosynthesis in Y. lipolytica. Further culture optimization enabled 22.03g/L of itaconic acid to be produced in bioreactors, about 60-fold improvement over the initial titer, which is the highest itaconic acid production achieved at low pH by yeast reported worldwide, to data. This study demonstrates the great potential of Y. lipolytica as an industrial platform for itaconic acid production.

Techno-economic analysis of downstream processes in itaconic acid production from fermentation broth

Itaconic acid is a biomolecule with potential application in various products, substituting feedstocks within the petrochemical industry. Simulations of different downstream trains were done to compare the method currently used in industry for recovery of itaconic acid with a) adsorption, b) reactive extraction and c) electrodialysis, to evaluate the most advantageous process considering production costs. In these simulations, adsorption is the method with greater potential to substitute the crystallization as the main downstream method, followed by reactive extraction, which showed values close to those of crystallization. The only non-competitive method was electrodialysis. The processing costs of itaconic acid were estimated at 1.13 US$ kg−1 for the upstream and fermentation, and extra 0.89, 0.63, 0.88, 1.50 US$ kg−1 for crystallization, adsorption, extraction, and electrodialysis-based processes, respectively.

Consolidated bioprocessing of lignocellulosic biomass to itaconic acid by metabolically engineering Neurospora crassa.

Neurospora crassa is a filamentous fungus potent in secreting cellulase and degrading lignocellulosic materials. Here, heterologous cis-aconitic acid decarboxylase was expressed in N. crassa to synthesize itaconic acid which is a high potential platform chemical with applications as an alternative for petroleum-based products. The present study demonstrated that itaconic acid can be produced directly from cellulose and other lignocellulosic materials by the engineered strain, with the highest production of 20.414 ± 0.674mg/L. The multivariate data analysis methods were used in the parameter analysis of the conversion process. It was found by the hot map analysis that itaconic acid production can promote the secretion of cellulase in N. crassa. Principal component analysis suggested that itaconic acid production was closely related to the concentration of the glucose degraded from lignocelluloses, indicating that the secretion of cellulase is key to the direct conversion of cellulose to itaconic acid in the engineered N. crassa. This work demonstrates that N. crassa could be considered as a new platform in the application of cellulose conversion to itaconic acid.

Itaconic Acid Production by Novel Aspergillus Niveus in Solid State Fermentation Using Agrowastes

Itaconic acid (IA) is an organic compound. This dicarboxylicacid is a white solid that is soluble in water, ethanol, and acetone. Historically, itaconic acid was obtained by the distillation of citricacid, but currently it is produced by fermentation. Itaconic acid is essentially utilized as a co-monomer in the generation of polymers, for example, tar, plastic, elastic, paints, surfactant with applications in the paper and structural covering industry.. The fermentative production of Itaconic acid was probably achieved by the filamentous fungus Aspergillus terrus, Aspergillus itaconicus and mainly by Aspergillus niger. In the present work, an attempt was made to produce IA by Aspergillus niveus employing Solid State Fermentation (SSF) from various agro wastes like rice bran, rice husk,tamarind seed,wheat stuff and sugarcane bagasse as carbon substrates, which was pretreated in order to soften it.10 g of each substrate was taken in a 500 ml conical flasks separately and supplemented with 40 mL nutrient solution containing glucose, at pH 3.5. One milliliter inoculum containing 1×107 spores was added and moisture was maintained at 70%. After incubation at 32°C for 14 days, the acid production was estimated by spectrophotometric method and by HPLC analysis. Interestingly, the yield of itaconic acid was promising with all the above substrates, where tamarind seed, sugarcane bagasse and rice bran supported higher yields.

High oxygen tension increases itaconic acid accumulation, glucose consumption, and the expression and activity of alternative oxidase in Aspergillus terreus

Itaconic acid is a five-carbon dicarboxylic acid with an unsaturated alkene bond, frequently used as a building block for the industrial production of a variety of synthetic polymers. It is also one of the major products of fungal "overflow metabolism" which can be produced in submerged fermentations of the filamentous fungus Aspergillus terreus. At the present, molar yields of itaconate are lower than those obtained in citric acid production in Aspergillus niger. Here, we have studied the possibility that the yield may be limited by the oxygen supply during fermentation and hence tested the effect of the dissolved oxygen concentration on the itaconic acid formation rate and yield in lab-scale bioreactors. The data show that a dissolved oxygen concentration of 2% saturation was sufficient for maximal biomass formation. Raising it to 30% saturation had no effect on biomass formation or the growth rate, but the itaconate yield augmented substantially from 0.53 to 0.85mol itaconate/mol glucose. Furthermore, the volumetric and specific rates of itaconic acid formation ameliorated by as much as 150% concurrent with faster glucose consumption, shortening the fermentation time by 48h. Further increasing the dissolved oxygen concentration over 30% saturation had no effect. Moreover, we show that this increase in itaconic acid production coincides with an increase in alternative respiration, circumventing the formation of surplus ATP by the cytochrome electron transport chain, as well as with increased levels of alternative oxidase transcript. We conclude that high(er) itaconic acid accumulation requires a dissolved oxygen concentration that is much higher than that needed for maximal biomass formation, and postulate that the induction of alternative respiration allows the necessary NADH reoxidation ratio without surplus ATP production to increase the glucose consumption and the flux through overflow metabolism.

Whole-cell Immobilization of Engineered Escherichia coli JY001 with Barium-alginate for Itaconic Acid Production

Itaconic acid is an important organic acid and a major component of various polymers. It is used in resins, superabsorbent polymers, and substitutes for petrochemicalbased monomers such as acrylic and methacrylic acids. Itaconic acid is primarily produced by the fungus Aspergillus terreus, which yields a high titer with albeit long fermentation period and by-products. In our previous study, Escherichia coli JY001 was reported to produce itaconic acid using citric acid in whole-cell reaction resulting in higher itaconic acid productivity with less by-products formation. The present study aimed to increase whole-cell enzyme stability and reusability, via immobilization of E. coli JY001 using barium-alginate beads. We optimized the cations, temperature, pH, alginate, BaCl2 concentration, cell density per bead, and CTAB content to improve transfer rate of substrates and products. Under the optimized conditions, immobilized whole cells were stable for four repeated cycles of itaconic acid production. The present results would strengthen the basis for a continuous itaconic acid production.

Itaconic acid mediates crosstalk between macrophage metabolism and peritoneal tumors.

Control of cellular metabolism is critical for efficient cell function, although little is known about the interplay between cell subset–specific metabolites in situ, especially in the tumor setting. Here, we determined how a macrophage-specific (Mϕ-specific) metabolite, itaconic acid, can regulate tumor progression in the peritoneum. We show that peritoneal tumors (B16 melanoma or ID8 ovarian carcinoma) elicited a fatty acid oxidation–mediated increase in oxidative phosphorylation (OXPHOS) and glycolysis in peritoneal tissue–resident macrophages (pResMϕ). Unbiased metabolomics identified itaconic acid, the product of immune-responsive gene 1–mediated (Irg1-mediated) catabolism of mitochondrial cis-aconitate, among the most highly upregulated metabolites in pResMϕ of tumor-bearing mice. Administration of lentivirally encoded Irg1 shRNA significantly reduced peritoneal tumors. This resulted in reductions in OXPHOS and OXPHOS-driven production of ROS in pResMϕ and ROS-mediated MAPK activation in tumor cells. Our findings demonstrate that tumors profoundly alter pResMϕ metabolism, leading to the production of itaconic acid, which potentiates tumor growth. Monocytes isolated from ovarian carcinoma patients’ ascites fluid expressed significantly elevated levels of IRG1. Therefore, IRG1 in pResMϕ represents a potential therapeutic target for peritoneal tumors.

Factors Affecting Production of Itaconic Acid from Mixed Sugars by Aspergillus terreus.

Itaconic acid (IA; a building block platform chemical) is currently produced industrially from glucose by fermentation with Aspergillus terreus. In order to expand the use of IA, its production cost must be lowered. Lignocellulosic biomass has the potential to serve as low-cost source of sugars for IA production. It was found that the fungus cannot produce IA from dilute acid pretreated and enzymatically saccharified wheat straw hydrolysate even at 100-fold dilution. The effects of typical compounds (acetic acid, furfural, HMF and Mn2+, enzymes, CaSO4), culture conditions (initial pH, temperature, aeration), and medium components (KH2PO4, NH4NO3, CaCl2·2H2O, FeCl3·6H2O) on growth and IA production by A. terreus NRRL 1972 using mixed sugar substrate containing glucose, xylose, and arabinose (4:3:1, 80gL-1) mimicking the wheat straw hydrolysate were investigated. Acetic acid, furfural, Mn2+, and enzymes were strong inhibitors to both growth and IA production from mixed sugars. Optimum culture conditions (pH 3.1, 33°C, 200rpm) and medium components (0.8g KH2PO4, 3g NH4NO3, 2.0g CaCl2·2H2O, 0.83-3.33mg FeCl3·6H2O per L) as well as tolerable levels of inhibitors (0.4g acetic acid, < 0.1g furfural, 100mg HMF, < 5.0ppb Mn2+, 24mg CaSO4 per L) for mixed sugar utilization were established. The results will be highly useful for developing a bioprocess technology for IA production from lignocellulosic feedstocks.

Synthesis of itaconic acid from agricultural waste using novel Aspergillus niveus

Filamentous fungi from the genus Aspergillus are of high importance for the production of organic acids. Itaconic acid (IA) is considered as an important component for the production of synthetic fibers, resin, plastics, rubber, paints, coatings, adhesives, thickeners and binders. Aspergillus niveus MG183809 was isolated from the soil sample (wastewater unit) which was collected from Avadi, Chennai, India. In the present study, itaconic acid was successfully produced by isolated A. niveus by submerged batch fermentation. In the fermentation process, various low-cost substrates like corn starch, wheat flour and sweet potato were used for itaconic acid production. Further, the factor influencing parameters such as substrate concentration and incubation period were optimized. Maximum yield of itaconic acid (15.65 ± 1.75 g/L) was achieved by using A. niveus from corn starch at a concentration of 120 g/L after 168 hr (pH 3.0). And also extraction of itaconic acid from the fermentation was performed with 91.96 ± 1.57 degree of extraction.

Synthetic auxotrophs for stable and tunable maintenance of plasmid copy number

Although plasmid-based expression systems have advantages in multi-copy expression of genes, heterogeneity of plasmid copy number (PCN) in individual cells is inevitable even with the addition of antibiotics. Here, we developed a synthetic auxotrophic system for stable and tunable maintenance of the PCN in Escherichia coli without addition of antibiotics. This auxotroph expresses infA, one of the essential genes encoding a translation initiation factor, on a plasmid instead of on the chromosome. With this system, the gene expression was stably maintained for 40 generations with minimized cell-to-cell variation under antibiotic-free conditions. Moreover, varying the expression level of infA enabled us to rationally tune the PCN by more than 5.6-fold. This antibiotic-free PCN control system significantly improved the production of itaconic acid and lycopene compared to the conventional system based on antibiotics (2-fold). Collectively, the developed strategy could be a platform for the production of value-added products in antibiotic-free cultivation.

Itaconate itinerary changed: Now it’s a potential metabolite to switch-off inflammation!

Industry interest of itaconate Itaconic acid is well known as a precursor for polymer synthesis and has been involved in industrial processes for decades. Itaconate was first identified in 1836 as a molecular product of citric acid distillation. In 1931, it was reported that itaconate could be synthesized in vivo by an Aspergillus species (subsequently named Aspergillus itaconcus) isolated from dried salted plums. This has spurred the development of emerging metabolic engineering strategies and process optimization technologies to enhance itaconic acid production. Today, itaconic acid has a huge world market and feeds into the preparation of industrial polymers and bioactive compounds in the personal care, agriculture, pharmaceutical, and medicine sectors. Research into more efficient methods of biomanufacturing itaconate is extremely active, because it represents a sustainable alternative to traditional crude oil- based polymers.

Iterative algorithm-guided design of massive strain libraries, applied to itaconic acid production in yeast

Metabolic engineering requires multiple rounds of strain construction to evaluate alternative pathways and enzyme concentrations. Optimizing multigene pathways stepwise or by randomly selecting enzymes and expression levels is inefficient. Here, we apply methods from design of experiments (DOE) to guide the construction of strain libraries from which the maximum information can be extracted without sampling every possible combination. We use Saccharomyces cerevisiae as a host for a novel six-gene pathway to itaconic acid, selected by comparing alternative shunt pathways that bypass the mitochondrial TCA cycle. The pathway is distinctive for the use of acetylating acetaldehyde dehydrogenase to increase cytosolic acetyl-CoA pools, a bacterial enzyme to synthesize citrate in the cytosol, and an itaconic acid exporter. Precise control over the expression of each gene is enabled by a set of promoter-terminator pairs that span a 174-fold range. Two large combinatorial libraries (160 variants, 2.4 Mb and 32 variants, 0.6 Mb) are designed where the expression levels are selected by statistical methods (I-optimal response surface methodology, full factorial, or Plackett-Burman) with the intent of extracting different types of guiding information after the screen. This is applied to the design of a third library (24 variants, 0.5 Mb) intended to alleviate a bottleneck in cis-aconitate decarboxylase (CAD) expression. The top strain produces 815 mg/l itaconic acid, a 4-fold improvement over the initial strain achieved by iteratively balancing pathway expression. Including a methylated product in the total, the strain produces 1.3 g/l combined itaconic acids. Further, a regression analysis of the libraries reveals the optimal expression level of CAD as well as pairwise interdependencies between genes that result in increased titer and purity of itaconic acid. This work demonstrates adapting algorithmic design strategies to guide automated yeast strain construction and learn information after each iteration.

Manufacturing Multienzymatic Complex Reactors In Vivo by Self-Assembly To Improve the Biosynthesis of Itaconic Acid in Escherichia coli.

The self-assembly of multienzyme into bioreactors is of extensive interest to spatially regulate valuable reactions. Despite the important progresses achieved, methods to precisely manufacture multienzymatic complex reactors (MECRs) are still poorly proposed both in vivo and in vitro, particularly for more than three biocatalytically relevant enzymes. Here, we developed a sequential self-assembly system to form multitude MECRs involving three enzymes in the itaconic acid (IA) pathway with two pairs of protein-peptide interactions. The MECRs were identified as nanoscale particle-like structures when self-assembled in vitro and produced higher IA production than the unassembled and linearly assembled systems when applied in vivo coupling with CRISPR-Cas9 based metabolic engineering. This work provides novel insights into the construction of multifarious multienzyme complex into bioreactors by the self-assembly strategy for multistep cascades to sequentially control metabolic fluxes inside cells.

Process design and economic analysis of a biorefinery co-producing itaconic acid and electricity from sugarcane bagasse and trash lignocelluloses

Itaconic acid has economic potential as a commodity biochemical for the sugar industry, but its production is limited due to high production costs. Using cheaper and alternative lignocellulosic feedstocks together with achieving higher product titres have been identified as potential strategies for viable IA production. Consequently the use of sugarcane bagasse and trash for the production of itaconic acid (IA) and electricity have been investigated for an integrated biorefinery, where the production facility is annexed to an existing sugar mill and new combined heat and power (CHP) plant. Three IA biorefinery scenarios were designed and simulated in Aspen Plus®. Subsequent economic analyses indicated that cheaper feedstocks reduced the IA production cost from 1565.5 US$/t for glucose to 616.5 US$/t, but coal supplementation was required to sufficiently lower the production cost to 604.3 US$/t for a competitive IA selling price of 1740 US$/t, compared to the market price of 1800 US$/t.

Biotechnological production of itaconic acid-things you have to know.

Itaconic acid is one of the basic chemicals for the polymer industry, which can be produced on the basis of renewable raw materials. Since the middle of the twentieth century, itaconic acid has been produced industrially using the filamentous fungus Aspergillus terreus. But the demand for the organic acid is low due to the high production costs compared to alternative petrochemical manufactured raw materials. The high production costs are based on a low final titer, low productivities, and the usage of pure sugars, purified molasses, or starch hydrolysates, since the fungus reacts very sensitively to impurities in a culture medium. This review provides a comprehensive overview of the most recent developments, including a spectrum of studied microorganisms and their capabilities for the production of itaconic acid. The technological achievements in the biotechnological production of itaconic acid are presented. Particular attention is paid to current achievements in terms of suitable alternative substrates and their applicability in fermentation processes. Also, the pathway of itaconic acid and especially the influences on the fermentation process, which must be known in order to achieve a high final titer of itaconic acid, a yield close to the theoretical yield, and high productivity.

Metabolic responses of Aspergillus terreus under low dissolved oxygen and pH levels

The metabolic responses of Aspergillus terreus NRRL1960 to stress conditions (low dissolved oxygen and pH with limited nitrogen and phosphate) in the two-phase fermentation were investigated in this study. The fermentation kinetics suggested that itaconate production was suppressed under low dissolved oxygen (DO) concentrations. A slight change in pH caused a significant change in itaconate production. The transcriptomic data revealed that under low DO concentration, the glycolytic pathway was uncoupled from the oxidative phosphorylation, resulting in the activation of substrate-level phosphorylation as an alternative route for ATP regeneration. The downregulation of pdh genes, the genes encoding ATP synthase and succinate dehydrogenase, confirmed the observation of the uncoupling of the oxidative TCA cycle from glycolysis. It was found that the upregulation of pyc resulted in a large pool of oxaloacetate in the cytosol. This induced the conversion of oxaloacetate to malate. The upregulation of the gene encoding fumarate hydratase with the subsequent formation of fumarate was found to be responsible for the regeneration of NADPH and ATP under the condition of a low dissolved oxygen level. The large pool of oxaloacetate drove itaconic acid production also via the oxidative TCA cycle. Nevertheless, the downregulation of ATP synthase genes resulted in the deficiency of the proton-pumping H+ ATPase and the subsequent stress due to the failure to maintain the physiological pH. This resulted in itaconate production at a low titer. The fermentation kinetics and the transcriptomic data provided in this study can be used for further process optimization and control to improve itaconate production performance.

World market and biotechnological production of itaconic acid.

The itaconic acid (IA) world market is expected to exceed 216 million of dollars by 2020 as a result of an increasing demand for bio-based chemicals. The potential of this organic acid produced by fermentation mainly with filamentous fungi relies on the vast industrial applications of polymers derived from it. The applications may be as a superabsorbent polymer for personal care or agriculture, unsaturated polyester resin for the transportation industry, poly(methyl methacrylate) for electronic devices, among many others. However, the existence of other substitutes and the high production cost limit the current IA market. IA manufacturing is done mainly in China and other Asia-Pacific countries. Higher economic feasibility and production worldwide may be achieved with the use of low-cost feedstock of local origin and with the development of applications targeted to specific local markets. Moreover, research on the biological pathway for IA synthesis and the effect of medium composition are important for amplifying the knowledge about the production of that biochemical with great market potential.

Itaconic acid production in microorganisms.

Itaconic acid, 2-methylidenebutanedioic acid, is a precursor of polymers, chemicals, and fuels. Many fungi can synthesize itaconic acid; Aspergillus terreus and Ustilago maydis produce up to 85 and 53gl-1, respectively. Other organisms, including Aspergillus niger and yeasts, have been engineered to produce itaconic acid. However, the titer of itaconic acid is low compared with the analogous major fermentation product, citric acid, for which the yield is >200gl-1. Here, we review two types of pathway for itaconic acid biosynthesis as well as recent advances by metabolic engineering strategies and process optimization to enhance itaconic acid productivity in native producers and heterologous hosts. We also propose further improvements to overcome existing problems.

Production of itaconic acid from acetate by engineering acid-tolerant Escherichia coli W.

Utilization of abundant and cheap carbon sources can effectively reduce the production cost and enhance the economic feasibility. Acetate is a promising carbon source to achieve cost-effective microbial processes. In this study, we engineered an Escherichia coli strain to produce itaconic acid from acetate. As acetate is known to inhibit cell growth, we initially screened for a strain with a high tolerance to 10 g/L of acetate in the medium, and the W strain was selected as the host. Subsequently, the WC strain was obtained by overexpression of cad (encoding cis-aconitate decarboxylase) using a synthetic promoter and 5' UTR. However, the WC strain produced only 0.13 g/L itaconic acid because of low acetate uptake. To improve the production, the acetate assimilating pathway and glyoxylate shunt pathway were amplified by overexpression of pathway genes as well as its deregulation. The resulting strain, WCIAG4 produced 3.57 g/L itaconic acid (16.1% of theoretical maximum yield) after 88 hr of fermentation with rapid acetate assimilation. These efforts support that acetate can be a potential feedstock for biochemical production with engineered E. coli.