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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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Itaconic acid and citric acid production of A. niger in different media. Itaconic acid (2A) and Citric acid (2B) level in micro-titer screening cultures using 20 different media. Standard deviations were less than 6 %.
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Figure 2: Itaconic acid and citric acid production of A. niger in different media. Itaconic acid (2A) and Citric acid (2B) level in micro-titer screening cultures using 20 different media. Standard deviations were less than 6 %.

Mentions: After cultivation for about 70 hours, cultures from each medium were analyzed for itaconic acid production using HPLC. As indicated in Figure 2A, in comparison to reference medium 1, all strains showed increased itaconic acid production in Medium 12. In this medium, both pyrG + strains have even higher amount of itaconic acid production than the hbd1 and gpdA strains (Figure 2A). In contrast, medium 11 and 13 yield the lowest itaconic acid production. Moreover, transformants grown in medium 8 and 20 also show improved itaconic acid production compared to the reference, except for transformant GPD 4.3. GPD 4.3 has generally low production level compared to other strains through all 20 media. The itaconic acid inducing components in the best media are phosphate (M8), copper (M12) and potassium (M20). The production pattern of citric acid is quite similar to itaconic acid in medium 8, 11, 12 and 13 (Figure 2B). In contrast, medium 4 provided high citric acid but low itaconic acid production. Medium 7 and 19 showed improved itaconic acid production only to the pyrG + strains. Based on the obtained results, we have retested a subset of the above mentioned media (8, 11, 12, 13, 20) and a newly designed medium (M20+) which combined the potentially itaconic acid inducing attributes (phosphorus 3 g/L, copper 0.0025 g/L and potassium 0.52 g/L) from medium 8, 12 and 20. Results are shown in Table 3. This time, strain FHB 2.5 was tested together with the reference strain pyr + 4.1 in triplicate. Similar results were obtained as in the initial screen. Medium 12 still yields the best itaconic acid production, whereas M20+ did not further improve production. In total, the standard deviations of the micro-titer plate screening results were less than 6 %. No oxalic acid was produced in the micro-titer plate cultivations.


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

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

Itaconic acid and citric acid production of A. niger in different media. Itaconic acid (2A) and Citric acid (2B) level in micro-titer screening cultures using 20 different media. Standard deviations were less than 6 %.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Itaconic acid and citric acid production of A. niger in different media. Itaconic acid (2A) and Citric acid (2B) level in micro-titer screening cultures using 20 different media. Standard deviations were less than 6 %.
Mentions: After cultivation for about 70 hours, cultures from each medium were analyzed for itaconic acid production using HPLC. As indicated in Figure 2A, in comparison to reference medium 1, all strains showed increased itaconic acid production in Medium 12. In this medium, both pyrG + strains have even higher amount of itaconic acid production than the hbd1 and gpdA strains (Figure 2A). In contrast, medium 11 and 13 yield the lowest itaconic acid production. Moreover, transformants grown in medium 8 and 20 also show improved itaconic acid production compared to the reference, except for transformant GPD 4.3. GPD 4.3 has generally low production level compared to other strains through all 20 media. The itaconic acid inducing components in the best media are phosphate (M8), copper (M12) and potassium (M20). The production pattern of citric acid is quite similar to itaconic acid in medium 8, 11, 12 and 13 (Figure 2B). In contrast, medium 4 provided high citric acid but low itaconic acid production. Medium 7 and 19 showed improved itaconic acid production only to the pyrG + strains. Based on the obtained results, we have retested a subset of the above mentioned media (8, 11, 12, 13, 20) and a newly designed medium (M20+) which combined the potentially itaconic acid inducing attributes (phosphorus 3 g/L, copper 0.0025 g/L and potassium 0.52 g/L) from medium 8, 12 and 20. Results are shown in Table 3. This time, strain FHB 2.5 was tested together with the reference strain pyr + 4.1 in triplicate. Similar results were obtained as in the initial screen. Medium 12 still yields the best itaconic acid production, whereas M20+ did not further improve production. In total, the standard deviations of the micro-titer plate screening results were less than 6 %. No oxalic acid was produced in the micro-titer plate cultivations.

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