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

Merkel, Manuel; Kiefer, Dirk; Schmollack, Marc; Blombach, Bastian; Lilge, Lars; Henkel, Marius; Hausmann, Rudolf

doi:10.1016/j.biortech.2022.126994

Cited by 21 publications

(21 citation statements)

References 43 publications

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“…However, no IA could be detected in the culture medium after 72 h. Otten et al . [ 33 ] showed that application of nitrogen limitation increased IA production of C. glutamicum from glucose, hence, we reduced the nitrogen concentration in the medium from a C:N ratio of 2.75:1 to 40:1 [ 33 , 34 ]. By applying nitrogen-limiting conditions, C. glutamicum ITA1 produced 0.32 ± 0.09 g IA L −1 after 72 h, corresponding to a Y P/S of 8 ± 2 mmol IA per mol acetate (Table 1 ).…”

Section: Resultsmentioning

confidence: 99%

“…Recently, we engineered C. glutamicum for IA production from acetate as sole carbon source by expression of the cis -aconitate decarboxylase from A. terreus and attenuation of isocitrate dehydrogenase (IDH) activity. In fed-batch fermentations with an adjusted C:N ratio, the engineered C. glutamicum IDH R453C (pEKEx2- malE cad opt ) strain produced 29 g IA L −1 with a peak volumetric productivity (Q P ) of 1 g L −1 h −1 and a product yield (Y P/S ) of 0.16 g IA per g of acetate [ 34 ]. To further improve the production of IA from acetate with C. glutamicum , we systematically engineered the acetate metabolism and its regulation, perturbed the nitrogen homeostasis and optimized the culture conditions.…”

Section: Introductionmentioning

confidence: 99%

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Metabolic engineering of Corynebacterium glutamicum for acetate-based itaconic acid production

Schmollack

Werner

Huber

et al. 2022

Biotechnol Biofuels

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Background Itaconic 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. Results In 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. Conclusion As 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.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Metabolic engineering of Corynebacterium glutamicum for acetate-based itaconic acid production

Schmollack

Werner

Huber

et al. 2022

Biotechnol Biofuels

Self Cite

View full text Add to dashboard Cite

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“…This was employed for itaconic acid production with Corynebacterium glutamicum. When the DO-concentration surpassed an upper limit upon the depletion of the carbon source, acetate solution was fed which allowed for the production of 29.2 g/L itaconate within 46 h [ 31 ]. Maintaining a low acetate concentration with a continuous or DO-coupled acetate feed could also be evaluated for malic acid production with A. oryzae .…”

Section: Discussionmentioning

confidence: 99%

Optimization of l-malic acid production from acetate with Aspergillus oryzae DSM 1863 using a pH-coupled feeding strategy

et al. 2022

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Background Malic acid, a dicarboxylic acid mainly used in the food industry, is currently produced from fossil resources. The utilization of low-cost substrates derived from biomass could render microbial processes economic. Such feedstocks, like lignocellulosic hydrolysates or condensates of fast pyrolysis, can contain high concentrations of acetic acid. Acetate is a suitable substrate for l-malic acid production with the filamentous fungus Aspergillus oryzae DSM 1863, but concentrations obtained so far are low. An advantage of this carbon source is that it can be used for pH control and simultaneous substrate supply in the form of acetic acid. In this study, we therefore aimed to enhance l-malate production from acetate with A. oryzae by applying a pH-coupled feeding strategy. Results In 2.5-L bioreactor fermentations, several feeding strategies were evaluated. Using a pH-coupled feed consisting of 10 M acetic acid, the malic acid concentration was increased about 5.3-fold compared to the batch process without pH control, resulting in a maximum titer of 29.53 ± 1.82 g/L after 264 h. However, it was not possible to keep both the pH and the substrate concentration constant during this fermentation. By using 10 M acetic acid set to a pH of 4.5, or with the repeated addition of NaOH, the substrate concentration could be maintained within a constant range, but these strategies did not prove beneficial as lower maximum titers and yields were obtained. Since cessation of malic acid production was observed in later fermentation stages despite carbon availability, a possible product inhibition was evaluated in shake flask cultivations. In these experiments, malate and succinate, which is a major by-product during malic acid production, were added at concentrations of up to 50 g/L, and it was found that A. oryzae is capable of organic acid production even at high product concentrations. Conclusions This study demonstrates that a suitable feeding strategy is necessary for efficient malic acid production from acetate. It illustrates the potential of acetate as carbon source for microbial production of the organic acid and provides useful insights which can serve as basis for further optimization.

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“…Among the products obtained from microbial conversion of acetate listed in the reviews cited above are organic acids (malic, succinic and itaconic acid and 3-HP), long-chain alcohols (isobutanol, isopropanol), hydroxyalkanoates (PHB, PHA), lipids (long-chain triacylglycerols, rhamnolipids) and proteins. Very recently, also recombinant C. glutamicum strains have been reported to produce 3-HP to concentrations of up to 17.1 g L −1 (Chang et al, 2022) and itaconic acid with titers of 29 g L −1 from acetate as sole carbon source (Merkel et al, 2022), the latter using an integrated pH-coupled feeding control.…”

Section: Product Formation From Acetate And/or Ethanol: Potential Org...mentioning

confidence: 99%

The Potential of Sequential Fermentations in Converting C1 Substrates to Higher-Value Products

et al. 2022

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Today production of (bulk) chemicals and fuels almost exclusively relies on petroleum-based sources, which are connected to greenhouse gas release, fueling climate change. This increases the urgence to develop alternative bio-based technologies and processes. Gaseous and liquid C1 compounds are available at low cost and often occur as waste streams. Acetogenic bacteria can directly use C1 compounds like CO, CO2, formate or methanol anaerobically, converting them into acetate and ethanol for higher-value biotechnological products. However, these microorganisms possess strict energetic limitations, which in turn pose limitations to their potential for biotechnological applications. Moreover, efficient genetic tools for strain improvement are often missing. However, focusing on the metabolic abilities acetogens provide, they can prodigiously ease these technological disadvantages. Producing acetate and ethanol from C1 compounds can fuel via bio-based intermediates conversion into more energy-demanding, higher-value products, by deploying aerobic organisms that are able to grow with acetate/ethanol as carbon and energy source. Promising new approaches have become available combining these two fermentation steps in sequential approaches, either as separate fermentations or as integrated two-stage fermentation processes. This review aims at introducing, comparing, and evaluating the published approaches of sequential C1 fermentations, delivering a list of promising organisms for the individual fermentation steps and giving an overview of the existing broad spectrum of products based on acetate and ethanol. Understanding of these pioneering approaches allows collecting ideas for new products and may open avenues toward making full use of the technological potential of these concepts for establishment of a sustainable biotechnology.

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

Cited by 21 publications

References 43 publications

Metabolic engineering of Corynebacterium glutamicum for acetate-based itaconic acid production

Metabolic engineering of Corynebacterium glutamicum for acetate-based itaconic acid production

Optimization of l-malic acid production from acetate with Aspergillus oryzae DSM 1863 using a pH-coupled feeding strategy

The Potential of Sequential Fermentations in Converting C1 Substrates to Higher-Value Products

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