Direct production of itaconic acid from liquefied corn starch by genetically engineered Aspergillus terreus

Abstract

Background: Itaconic acid is on the DOE (Department of Energy) top 12 list of biotechnologically produced building block chemicals and is produced commercially by Aspergillus terreus. However, the production cost of itaconic acid is too high to be economically competitive with the petrochemical-based products. Itaconic acid is generally produced from raw corn starch, including three steps: enzymatic hydrolysis of corn starch into a glucose-rich syrup by α-amylase and glucoamylase, fermentation, and recovery of itaconic acid. The whole process is very time-consuming and energy-intensive.

Results: In order to reduce the production cost, saccharification and fermentation were integrated into one step through overexpressing the glucoamylase gene in A. terreus under the control of the native PcitA promoter. The transformant XH61-5 produced higher itaconate titer from liquefied starch than WT. To further increase the titer by enhancing the secretion capacity of overexpressed glucoamylase, a stronger signal peptide was selected based on the major secreted protein ATEG_02176 (an acid phosphatase precursor) by A. terreus under the itaconate production conditions. Under the control of the stronger signal peptide, the transformant XH86-8 showed higher itaconate production level than XH61-5 from liquefied starch. The itaconate titer was further enhanced through a two-step process involving the vegetative and production phase, and the transformant XH86-8 produced comparable itaconate titer from liquefied starch to current one (~80 g/L) from saccharified starch hydrolysates in industry. The effects of the new signal peptide and the two-step process on itaconate production were investigated and discussed.

Conclusions: Itaconic acid could be efficiently produced from liquefied corn starch by overexpressing the glucoamylase gene in A. terreus, which will be helpful for constructing a highly efficient microbial cell factory for itaconate production and for further lowering the production cost of itaconic acid.

Figure 1

Itaconic acid production from liquefied corn starch by the transformants of pXH61 and pXH59. The transformants were screened using liquefied corn starch (corresponding to 140 g/L glucose) as the carbon source on a rotary shaker at 37°C for 72 h. Three independents experiments were performed for each strain. The itaconate titers were determined by HPLC.

Figure 2

Itaconic acid production from liquefied corn starch by the transformants of pXH86. The transformants were screened using liquefied corn starch (corresponding to 140 g/L glucose) as the carbon source on a rotary shaker at 37°C for 72 h. Three independents experiments were performed for each strain. The itaconate titers were determined by HPLC.

Figure 3

Comparison of the effects of signal peptides on itaconate production from liquefied corn starch. WT and the transformants XH61-5 and XH86-8 were compared in the one-step (A) and two-step (B) processes using liquefied corn starch (equivalent to 140 g/L glucose) as the carbon source. Cultures were sampled every 12 h. The itaconate titers were determined by HPLC. Itaconic acid production from saccharified corn starch hydrolysate (SCSH) by WT was used as the reference.

Figure 4

Comparison of the effects of signal peptides on glucoamylase activity during the course of growth. WT and the transformants XH61-5 and XH86-8 were compared in the one-step (A) and two-step (B) processes using liquefied corn starch as the carbon source. Cultures were sampled every 12 h. The activity of expressed secreted glucoamylase was assayed at 40°C and pH4.6 using soluble starch as the substrate. Specific activity was defined as one unit of enzyme activity per ml of crude culture filtrates.