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Enhanced itaconic acid production in Aspergillus niger using genetic modification and medium optimization.

Li A, Pfelzer N, Zuijderwijk R, Punt P - BMC Biotechnol. (2012)

Bottom Line: Based on the results of previous transcriptomics studies and research from other groups, two genes : gpdA encoding the glyceraldehyde -3-dehydrogenase (GPD) and hbd1 encoding a flavohemoglobin domain (HBD) were overexpressed in A. niger.The hbd1 transformants (HBD 2.2/2.5) did not improve itaconic acid titer while the gpdA transformant (GPD 4.3) decreased the itaconic acid production.Medium optimization showed that, copper was positively correlated with improved itaconic acid production.

View Article: PubMed Central - HTML - PubMed

Affiliation: TNO Microbiology and Systems biology, PO Box 360, 3700 AJ Zeist, The Netherlands. an.li@tno.nl

ABSTRACT

Background: Aspergillus niger was selected as a host for producing itaconic acid due to its versatile and tolerant character in various growth environments, and its extremely high capacity of accumulating the precursor of itaconic acid: citric acid. Expressing the CAD gene from Aspergillus terreus opened the metabolic pathway towards itaconic acid in A. niger. In order to increase the production level, we continued by modifying its genome and optimizing cultivation media.

Results: Based on the results of previous transcriptomics studies and research from other groups, two genes : gpdA encoding the glyceraldehyde -3-dehydrogenase (GPD) and hbd1 encoding a flavohemoglobin domain (HBD) were overexpressed in A. niger. Besides, new media were designed based on a reference medium for A. terreus. To analyze large numbers of cultures, we developed an approach for screening both fungal transformants and various media in 96-well micro-titer plates. The hbd1 transformants (HBD 2.2/2.5) did not improve itaconic acid titer while the gpdA transformant (GPD 4.3) decreased the itaconic acid production. Using 20 different media, copper was discovered to have a positive influence on itaconic acid production. Effects observed in the micro-titer plate screening were confirmed in controlled batch fermentation.

Conclusions: The performance of gpdA and hbd1 transformants was found not to be beneficial for itaconic acid production using the tested cultivation conditions. Medium optimization showed that, copper was positively correlated with improved itaconic acid production. Interestingly, the optimal conditions for itaconic acid clearly differ from conditions optimal for citric- and oxalic acid production.

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Biomass growth and glucose consumption of A. niger in fermentations with varying copper concentrations. Biomass determination of mycelium in batch fermentation (♦—Medium12-Cu with 0.005 mM Cu2+; ■—Medium12 with 0.01 mM Cu2+ ; ▴—Medium12 + Cu with 0.02 mM Cu2+). The glucose consumption of three batch fermentations were shown on the secondary Axis (X— Medium12-Cu with 0.005 mM Cu2+; □—Medium12 with 0.01 mM Cu2+ ; ●—Medium12 + Cu with 0.02 mM Cu2+).
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Figure 4: Biomass growth and glucose consumption of A. niger in fermentations with varying copper concentrations. Biomass determination of mycelium in batch fermentation (♦—Medium12-Cu with 0.005 mM Cu2+; ■—Medium12 with 0.01 mM Cu2+ ; ▴—Medium12 + Cu with 0.02 mM Cu2+). The glucose consumption of three batch fermentations were shown on the secondary Axis (X— Medium12-Cu with 0.005 mM Cu2+; □—Medium12 with 0.01 mM Cu2+ ; ●—Medium12 + Cu with 0.02 mM Cu2+).

Mentions: The levels of itaconic acid, citric acid and oxalic acid of A. niger transformant AB 1.13 CAD pyr + 4.1 in medium 12 containing three concentrations of copper were analyzed. As presented in Figure 3A, the production rate of itaconic acid is positively correlated with the copper concentration while this correlation is different for citric acid (Figure 3B) and oxalic acid (Figure 3C). In the low-copper medium (0.005 mM Cu2+), high levels of oxalic acid are accumulated whereas the highest amount of citric acid is produced in the medium-copper medium. In addition to organic acids, we determined biomass and glucose consumption. Glucose consumption of A. niger strain AB 1.13 CAD pyr + 4.1 among all three media is nearly the same (Figure 4). Increase in biomass showed a strong positive correlation with copper levels (Figure 4). The three main organic acids were quantified and the average concentrations (g/L) of duplicate measurements are listed in Table 4. As indicated, a two fold increase of the percentage of itaconic acid is obtained in the high-copper medium. (Table 4).


Enhanced itaconic acid production in Aspergillus niger using genetic modification and medium optimization.

Li A, Pfelzer N, Zuijderwijk R, Punt P - BMC Biotechnol. (2012)

Biomass growth and glucose consumption of A. niger in fermentations with varying copper concentrations. Biomass determination of mycelium in batch fermentation (♦—Medium12-Cu with 0.005 mM Cu2+; ■—Medium12 with 0.01 mM Cu2+ ; ▴—Medium12 + Cu with 0.02 mM Cu2+). The glucose consumption of three batch fermentations were shown on the secondary Axis (X— Medium12-Cu with 0.005 mM Cu2+; □—Medium12 with 0.01 mM Cu2+ ; ●—Medium12 + Cu with 0.02 mM Cu2+).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3472327&req=5

Figure 4: Biomass growth and glucose consumption of A. niger in fermentations with varying copper concentrations. Biomass determination of mycelium in batch fermentation (♦—Medium12-Cu with 0.005 mM Cu2+; ■—Medium12 with 0.01 mM Cu2+ ; ▴—Medium12 + Cu with 0.02 mM Cu2+). The glucose consumption of three batch fermentations were shown on the secondary Axis (X— Medium12-Cu with 0.005 mM Cu2+; □—Medium12 with 0.01 mM Cu2+ ; ●—Medium12 + Cu with 0.02 mM Cu2+).
Mentions: The levels of itaconic acid, citric acid and oxalic acid of A. niger transformant AB 1.13 CAD pyr + 4.1 in medium 12 containing three concentrations of copper were analyzed. As presented in Figure 3A, the production rate of itaconic acid is positively correlated with the copper concentration while this correlation is different for citric acid (Figure 3B) and oxalic acid (Figure 3C). In the low-copper medium (0.005 mM Cu2+), high levels of oxalic acid are accumulated whereas the highest amount of citric acid is produced in the medium-copper medium. In addition to organic acids, we determined biomass and glucose consumption. Glucose consumption of A. niger strain AB 1.13 CAD pyr + 4.1 among all three media is nearly the same (Figure 4). Increase in biomass showed a strong positive correlation with copper levels (Figure 4). The three main organic acids were quantified and the average concentrations (g/L) of duplicate measurements are listed in Table 4. As indicated, a two fold increase of the percentage of itaconic acid is obtained in the high-copper medium. (Table 4).

Bottom Line: Based on the results of previous transcriptomics studies and research from other groups, two genes : gpdA encoding the glyceraldehyde -3-dehydrogenase (GPD) and hbd1 encoding a flavohemoglobin domain (HBD) were overexpressed in A. niger.The hbd1 transformants (HBD 2.2/2.5) did not improve itaconic acid titer while the gpdA transformant (GPD 4.3) decreased the itaconic acid production.Medium optimization showed that, copper was positively correlated with improved itaconic acid production.

View Article: PubMed Central - HTML - PubMed

Affiliation: TNO Microbiology and Systems biology, PO Box 360, 3700 AJ Zeist, The Netherlands. an.li@tno.nl

ABSTRACT

Background: Aspergillus niger was selected as a host for producing itaconic acid due to its versatile and tolerant character in various growth environments, and its extremely high capacity of accumulating the precursor of itaconic acid: citric acid. Expressing the CAD gene from Aspergillus terreus opened the metabolic pathway towards itaconic acid in A. niger. In order to increase the production level, we continued by modifying its genome and optimizing cultivation media.

Results: Based on the results of previous transcriptomics studies and research from other groups, two genes : gpdA encoding the glyceraldehyde -3-dehydrogenase (GPD) and hbd1 encoding a flavohemoglobin domain (HBD) were overexpressed in A. niger. Besides, new media were designed based on a reference medium for A. terreus. To analyze large numbers of cultures, we developed an approach for screening both fungal transformants and various media in 96-well micro-titer plates. The hbd1 transformants (HBD 2.2/2.5) did not improve itaconic acid titer while the gpdA transformant (GPD 4.3) decreased the itaconic acid production. Using 20 different media, copper was discovered to have a positive influence on itaconic acid production. Effects observed in the micro-titer plate screening were confirmed in controlled batch fermentation.

Conclusions: The performance of gpdA and hbd1 transformants was found not to be beneficial for itaconic acid production using the tested cultivation conditions. Medium optimization showed that, copper was positively correlated with improved itaconic acid production. Interestingly, the optimal conditions for itaconic acid clearly differ from conditions optimal for citric- and oxalic acid production.

Show MeSH
Related in: MedlinePlus