Dataset Information

Development of an itaconic acid production process with Ustilaginaceae on alternative feedstocks.

ABSTRACT:

Background

Currently, Aspergillus terreus is used for the industrial production of itaconic acid. Although, alternative feedstock use in fermentations is crucial for cost-efficient and sustainable itaconic acid production, their utilisation with A. terreus most often requires expensive pretreatment. Ustilaginacea are robust alternatives for itaconic acid production, evading the challenges, including the pretreatment of crude feedstocks regarding reduction of manganese concentration, that A. terreus poses.

Results

In this study, five different Ustilago strains were screened for their growth and production of itaconic acid on defined media. The most promising strains were then used to find a suitable alternative feedstock, based on the local food industry. U. cynodontis ITA Max pH, a highly engineered production strain, was selected to determine the biologically available nitrogen concentration in thick juice and molasses. Based on these findings, thick juice was chosen as feedstock to ensure the necessary nitrogen limitation for itaconic acid production. U. cynodontis ITA Max pH was further characterised regarding osmotolerance and product inhibition and a successful scale-up to a 2 L stirred tank reactor was accomplished. A titer of 106.4 g_{itaconic acid}/L with a theoretical yield of 0.50 g_{itaconic acid}/g_sucrose and a space-time yield of 0.72 g_{itaconic acid}/L/h was reached.

Conclusions

This study demonstrates the utilisation of alternative feedstocks to produce ITA with Ustilaginaceae, without drawbacks in either titer or yield, compared to glucose fermentations.

SUBMITTER: Niehoff PJ

PROVIDER: S-EPMC10476437 | biostudies-literature | 2023 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Development of an itaconic acid production process with Ustilaginaceae on alternative feedstocks.

Niehoff Paul-Joachim PJ Müller Waldemar W Pastoors Johannes J Miebach Katharina K Ernst Philipp P Hemmerich Johannes J Noack Stephan S Wierckx Nick N Büchs Jochen J

BMC biotechnology 20230903 1

<h4>Background</h4>Currently, Aspergillus terreus is used for the industrial production of itaconic acid. Although, alternative feedstock use in fermentations is crucial for cost-efficient and sustainable itaconic acid production, their utilisation with A. terreus most often requires expensive pretreatment. Ustilaginacea are robust alternatives for itaconic acid production, evading the challenges, including the pretreatment of crude feedstocks regarding reduction of manganese concentration, that ...[more]

PMID: 37661280

Similar Datasets

Project description:There is a strong interest in itaconic acid in the medical and pharmaceutical sectors, both as an anti-bacterial compound and as an immunoregulator in mammalian macrophages. Fungal hosts also produce itaconic acid, and in addition they can produce two derivatives 2-hydroxyparaconic and itatartaric acid. Not much is known about these two derivatives, while their structural analogy to itaconate could open up several applications. In this study, we report the production of these two itaconate-derived compounds. By overexpressing the itaconate P450 monooxygenase Cyp3 in a previously engineered itaconate-overproducing Ustilago cynodontis strain, itaconate was converted to its lactone 2-hydroxyparaconate. The second product itatartarate is most likely the result of the subsequent lactone hydrolysis. A major challenge in the production of 2-hydroxyparaconate and itatartarate is their co-production with itaconate, leading to difficulties in their purification. Achieving high derivatives specificity was therefore the paramount objective. Different strategies were evaluated including process parameters such as substrate and pH, as well as strain engineering focusing on Cyp3 expression and product export. 2-hydroxyparaconate and itatartarate were successfully produced from glucose and glycerol, with the latter resulting in a higher derivatives specificity due to an overall slower metabolism on this non-preferred carbon source. The derivatives specificity could be further increased by metabolic engineering approaches including the exchange of the native itaconate transporter Itp1 with the Aspergillus terreus itaconate transporter MfsA. Both 2-hydroxyparaconate and itatartarate were recovered from fermentation supernatants following a pre-existing protocol. 2-hydroxyparaconate was recovered first through a process of evaporation, lactonization, and extraction with ethyl acetate. Subsequently, itatartarate could be obtained in the form of its sodium salt by saponification of the purified 2-hydroxyparaconate. Finally, several analytical methods were used to characterize the resulting products and their structures were confirmed by nuclear magnetic resonance spectroscopy. This work provides a promising foundation for obtaining 2-hydroxyparaconate and itatartarate in high purity and quantity. This will allow to unravel the full spectrum of potential applications of these novel compounds.

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.

Dataset Information

Development of an itaconic acid production process with Ustilaginaceae on alternative feedstocks.

Background

Results

Conclusions

Publications

Development of an itaconic acid production process with Ustilaginaceae on alternative feedstocks.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets