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

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.

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.

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 %.

Figure 3

Itaconic-, citric- and oxalic acid production in fermentation media of A. niger . Organic acid content (itaconic- citric- and oxalic- acid) of A. niger strain AB 1.13 CAD pyr + in Medium 12 with varying copper concentration. (♦—Medium12-Cu with 0.005 mM Cu²⁺; ■—Medium12 with 0.01 mM Cu²⁺ ; ▲—Medium12 + Cu with 0.02 mM Cu²⁺) (EFT: Electronic Fermentation Time).

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⁺).