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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 production of A. niger transformants. A ): Itaconic acid HPLC analysis of micro titer plate screening of A. niger CAD transformants. B): Itaconic acid screening results in micro-titer plate for the selected GPD and HBD transformants. The parental strain CAD 10.1 was used as a control. The transformants with the highest production and lowest production surrounded by an oval circle were selected further for Southern blot analysis.
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Figure 1: Itaconic acid production of A. niger transformants. A ): Itaconic acid HPLC analysis of micro titer plate screening of A. niger CAD transformants. B): Itaconic acid screening results in micro-titer plate for the selected GPD and HBD transformants. The parental strain CAD 10.1 was used as a control. The transformants with the highest production and lowest production surrounded by an oval circle were selected further for Southern blot analysis.

Mentions: The itaconic acid producing strain AB 1.13 CAD 10.1 pyrG- (CAD 10.1) was selected from a limited number of cadA transformants [4]. In an attempt to isolate a further improved itaconic acid producing strains, new transformants were generated. More than 90 colonies were screened for their itaconic acid production in a 96 well micro-titer plate together with CAD 10.1 as a reference. As presented in Figure 1A, strain CAD 10.1 is shown to be among the best itaconic acid producing strains. Therefore, this strain was used for further genetic modification.


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 production of A. niger transformants. A ): Itaconic acid HPLC analysis of micro titer plate screening of A. niger CAD transformants. B): Itaconic acid screening results in micro-titer plate for the selected GPD and HBD transformants. The parental strain CAD 10.1 was used as a control. The transformants with the highest production and lowest production surrounded by an oval circle were selected further for Southern blot analysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Itaconic acid production of A. niger transformants. A ): Itaconic acid HPLC analysis of micro titer plate screening of A. niger CAD transformants. B): Itaconic acid screening results in micro-titer plate for the selected GPD and HBD transformants. The parental strain CAD 10.1 was used as a control. The transformants with the highest production and lowest production surrounded by an oval circle were selected further for Southern blot analysis.
Mentions: The itaconic acid producing strain AB 1.13 CAD 10.1 pyrG- (CAD 10.1) was selected from a limited number of cadA transformants [4]. In an attempt to isolate a further improved itaconic acid producing strains, new transformants were generated. More than 90 colonies were screened for their itaconic acid production in a 96 well micro-titer plate together with CAD 10.1 as a reference. As presented in Figure 1A, strain CAD 10.1 is shown to be among the best itaconic acid producing strains. Therefore, this strain was used for further genetic modification.

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