Production of itaconic acid in Escherichia coli expressing recombinant α-amylase using starch as substrate

Abstract

Several studies on fermentative production of a vinyl monomer itaconic acid from hydrolyzed starch using Aspergillus terreus have been reported. Herein, we report itaconic acid production by Escherichia coli expressing recombinant α-amylase, using soluble starch as its sole carbon source. To express α-amylase in E. coli, we first constructed recombinant plasmids expressing α-amylases by using cell surface display technology derived from two amylolytic bacteria, Bacillus amyloliquefaciens NBRC 15535(T) and Streptococcus bovis NRIC 1535. The recombinant α-amylase from S. bovis (SBA) showed activity at 28°C, which is the optimal temperature for production of itaconic acid, while α-amylase from B. amyloliquefaciens displayed no noticeable activity. E. coli cells expressing SBA produced 0.15 g/L itaconic acid after 69 h cultivation under pH-stat conditions, using 1% starch as the sole carbon source. In fact, E. coli cells expressing SBA had similar growth rates when grown in the presence of 1% glucose or starch, thereby highlighting the expression of an active α-amylase that enabled utilization of starch to produce itaconic acid in E. coli.

Challenges in the production of itaconic acid by metabolically engineeredEscherichia coli

“…1 In particular, itaconic acid (IA), a promising vinyl monomer that is industrially produced by Aspergillus terreus, 2 has recently been produced in Escherichia coli, [3][4][5][6] Aspergillus niger, [7][8][9] and Synechocystis sp, 10 which were engineered to express the cisaconitate decarboxylase gene (cad) 11 from A. terreus. Vinyl monomers such as IA are promising building blocks for synthetic polymers, because of their radical polymerizability.…”

“…Compared to fungi, E. coli seems to be a more suitable producer of itaconic acid because of its high growth rate; furthermore, gene manipulation of E. coli is easier than that of fungi. 6 Additionally, E. coli has the potential to produce dicarboxylic acids, such as succinic acid, malic acid, and itaconic acid. 6 Li et al demonstrated that E. coli cells containing the cad gene produced 0.08 g/L of IA after overnight growth in Luria-Bertani (LB) medium.…”

“…6 Additionally, E. coli has the potential to produce dicarboxylic acids, such as succinic acid, malic acid, and itaconic acid. 6 Li et al demonstrated that E. coli cells containing the cad gene produced 0.08 g/L of IA after overnight growth in Luria-Bertani (LB) medium. 3 More recently, Vuoristo et al introduced the citrate synthase and the aconitase genes (acn) from Corynebacterium glutamicum in E. coli expressing cad and inactivating the phosphate acetyltransferase and the lactate dehydrogenase genes.…”

“…To reach this goal, Okamoto et al expressed the a-amylase gene (amy) from Streptococcus bovis in E. coli expressing cad and inactivated icd. 6 In this report, we constructed a new expression vector, pGV3, which is suitable for cell surface display of heterologous proteins in E. coli. Cell surface display represents a unique expression system to decorate protein on the cell surface.…”

“…The amy gene was cloned into pGV3, resulting pGV3-SBA. 6 The Figure 1. SDS-PAGE analysis of the Cad protein in E. coli.…”