The Role of Metal Ions in Fungal Organic Acid Accumulation

Karaffa, Levente; Fekete, Erzsébet; Kubicek, Christian P.

doi:10.3390/microorganisms9061267

Cited by 20 publications

(14 citation statements)

References 83 publications

(93 reference statements)

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“…Therefore, the effects of the mRDM medium on 3-HP production were further optimized by alternation of individual components. Maintenance of manganese at a very low concentration (~ 10 ppb) is required to support high-yield production of citric acid in A. niger [ 31 , 45 ] as well as itaconic acid in A. pseudoterreus [ 46 ]. In this study, however, we found that 100-fold higher concentrations of manganese in the culture medium support 3-HP production highlighting the difference in metabolism between production of 3-HP and the organic acids typically produced by these organisms.…”

Section: Discussionmentioning

confidence: 99%

Metabolic engineering to improve production of 3-hydroxypropionic acid from corn-stover hydrolysate in Aspergillus species

Dai

Pomraning

Deng

et al. 2023

Biotechnol Biofuels

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Background Fuels and chemicals derived from non-fossil sources are needed to lessen human impacts on the environment while providing a healthy and growing economy. 3-hydroxypropionic acid (3-HP) is an important chemical building block that can be used for many products. Biosynthesis of 3-HP is possible; however, low production is typically observed in those natural systems. Biosynthetic pathways have been designed to produce 3-HP from a variety of feedstocks in different microorganisms. Results In this study, the 3-HP β-alanine pathway consisting of aspartate decarboxylase, β-alanine-pyruvate aminotransferase, and 3-hydroxypropionate dehydrogenase from selected microorganisms were codon optimized for Aspergillus species and placed under the control of constitutive promoters. The pathway was introduced into Aspergillus pseudoterreus and subsequently into Aspergillus niger, and 3-HP production was assessed in both hosts. A. niger produced higher initial 3-HP yields and fewer co-product contaminants and was selected as a suitable host for further engineering. Proteomic and metabolomic analysis of both Aspergillus species during 3-HP production identified genetic targets for improvement of flux toward 3-HP including pyruvate carboxylase, aspartate aminotransferase, malonate semialdehyde dehydrogenase, succinate semialdehyde dehydrogenase, oxaloacetate hydrolase, and a 3-HP transporter. Overexpression of pyruvate carboxylase improved yield in shake-flasks from 0.09 to 0.12 C-mol 3-HP C-mol−1 glucose in the base strain expressing 12 copies of the β-alanine pathway. Deletion or overexpression of individual target genes in the pyruvate carboxylase overexpression strain improved yield to 0.22 C-mol 3-HP C-mol−1 glucose after deletion of the major malonate semialdehyde dehydrogenase. Further incorporation of additional β-alanine pathway genes and optimization of culture conditions (sugars, temperature, nitrogen, phosphate, trace elements) for 3-HP production from deacetylated and mechanically refined corn stover hydrolysate improved yield to 0.48 C-mol 3-HP C-mol−1 sugars and resulted in a final titer of 36.0 g/L 3-HP. Conclusions The results of this study establish A. niger as a host for 3-HP production from a lignocellulosic feedstock in acidic conditions and demonstrates that 3-HP titer and yield can be improved by a broad metabolic engineering strategy involving identification and modification of genes participated in the synthesis of 3-HP and its precursors, degradation of intermediates, and transport of 3-HP across the plasma membrane.

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Section: Discussionmentioning

confidence: 99%

Metabolic engineering to improve production of 3-hydroxypropionic acid from corn-stover hydrolysate in Aspergillus species

Dai

Pomraning

Deng

et al. 2023

Biotechnol Biofuels

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“…However, while iron—the most abundant metal in stainless steel—was supplemented here to the growth medium in 0.1 mg L −1 concentration, other citric acid production formulas advocate using up to 1.3 mg L −1 Fe 2+ ions ( Shu and Johnson, 1948b ). Secondly, it is plausible—though not yet confirmed experimentally—that the inhibitory effect of iron in concentrations higher than above on the citric acid yield is actually related to the manganese effect, as even analytical grade iron salts may contain up to 0.5% (w/w) Mn 2+ as impurities ( Karaffa et al, 2021 ). Such Fe/Mn interactions obviously do not exist in our experimental setup.…”

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…Specific molar citric acid yields (Y p/s ) are the ratio between the moles of citric acid produced and the moles of D-glucose consumed after the complete depletion of the D-glucose. A high-yield citric acid fermentation (shake-flask or bioreactor alike) was defined as a culture reaching a Y p/s of >0.8 (Karaffa et al, 2021). Three aspects of mycelial morphology were monitored by microscopy: 1) swollen, yeast-like hyphae; 2) filamentous hyphae; and 3) pellet-like mycelial clumps (aggregates) (Bartoshevich et al, 1990;Paul and Thomasn, 1998;Sándor et al, 2021).…”

Section: Methodsmentioning

confidence: 99%

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Bioreactor as the root cause of the “manganese effect” during Aspergillus niger citric acid fermentations

Fekete

Bíró²,

Márton³

et al. 2022

Front. Bioeng. Biotechnol.

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High-yield citric acid production by the filamentous Ascomycete fungus Aspergillus niger requires a combination of extreme nutritional conditions, of which maintaining a low manganese (II) ion concentration (<5 μg L−1) is a key feature. Technical-scale production of citric acid predominantly uses stainless-steel tank fermenters, but glass bioreactors used for strain improvement and manufacturing process development also contain stainless steel components, in which manganese is an essential alloying element. We show here that during citric acid fermentations manganese (II) ions were leaching from the bioreactor into the growth media, resulting in altered fungal physiology and morphology, and significant reduction of citric acid yields. The leaching of manganese (II) ions was dependent on the fermentation time, the acidity of the culture broth and the sterilization protocol applied. Manganese (II) ion leaching was partially mitigated by electrochemical polishing of stainless steel components of the bioreactor. High concentrations of manganese (II) ions during early cultivation led to a reduction in citric acid yield. However, the effect of manganese (II) ions on the reduction of citric acid yield diminished towards the second half of the fermentation. Since maintaining low concentrations of manganese (II) ions is costly, the results of this study can potentially be used to modify protocols to reduce the cost of citric acid production.

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“…Based on the work on A. oryzae [3,26], three factors were likely responsible for the observed response. These were insoluble solids, metal ions, and pH [13,24,27].…”

Section: Introductionmentioning

confidence: 99%

The Effect of pH, Metal Ions, and Insoluble Solids on the Production of Fumarate and Malate by Rhizopus delemar in the Presence of CaCO3

et al. 2022

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Calcium carbonate has been extensively used as a neutralising agent in acid-forming microbial processes. The effect of increasing calcium carbonate concentrations on Rhizopus delemar has not been previously investigated. In this study, an evaluation of fumaric acid (FA) and malic acid (MA) production was conducted at three CaCO3 concentrations in shake flask cultivations. Increased CaCO3 concentrations resulted in the co-production of FA and MA in the first 55 h of the fermentation (regime 1), and the subsequent depletion of FA thereafter (regime 2). Three factors were highlighted as likely causes of this response: insoluble solids, metal ion concentrations, and pH. Further shake flask cultivations as well as a continuous fermentation with immobilised R. delemar were used to explore the effect of the three factors on regime 1 and 2. Insoluble solids were found to have no effect on the response in either regime 1 or 2. Increasing the aqueous calcium ion concentrations to 10g/L resulted in a three-fold increase in MA titres (regime 1). Moreover, an increase in pH above 7 was associated with a drop in FA concentrations in regime 2. Further tests established that this was due to the hydration of FA to MA, influenced by high pH conditions ( 7 or higher), nitrogen starvation, and glucose depletion. Anaerobic conditions were also found to significantly improve the hydration process. This study presents the first investigation in which the production of FA followed by in situ hydration of FA to MA with R. delemar has been achieved.

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The Role of Metal Ions in Fungal Organic Acid Accumulation

Cited by 20 publications

References 83 publications

Metabolic engineering to improve production of 3-hydroxypropionic acid from corn-stover hydrolysate in Aspergillus species

Metabolic engineering to improve production of 3-hydroxypropionic acid from corn-stover hydrolysate in Aspergillus species

Bioreactor as the root cause of the “manganese effect” during Aspergillus niger citric acid fermentations

The Effect of pH, Metal Ions, and Insoluble Solids on the Production of Fumarate and Malate by Rhizopus delemar in the Presence of CaCO3

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