Heterologous expression of Mus musculus immunoresponsive gene 1 (irg1) in Escherichia coli results in itaconate production

Abstract

Itaconic acid, a C5-dicarboxylic acid, is a potential biobased building block for the polymer industry. It is obtained from the citric acid cycle by decarboxylation of cis-aconitic acid. This reaction is catalyzed by CadA in the native itaconic acid producer Aspergillus terreus. Recently, another enzyme encoded by the mammalian immunoresponsive gene 1 (irg1), was found to decarboxylate cis-aconitate to itaconate in vitro. We show that heterologous expression of irg1 enabled itaconate production in Escherichia coli with production titres up to 560 mg/L.

Keywords: Escherichia coli; cis-aconitate decarboxylase; codon harmonization; codon optimization; immunoresponsive gene 1; itaconic acid; metabolic engineering.

FIGURE 1

Itaconate production pathway in Escherichia coli. The bold arrows indicate the introduced pathway consisting of genes encoding citrate synthase (gltA) and aconitase (acnA) from Corynebacterium glutamicum and cis-aconitate decarboxylase (cadA) from Aspergillus terreus or immunoresponsive gene 1 (irg1) from Mus musculus. The dotted lines indicate that phosphate acetyltransferase (pta) and lactate dehydrogenase (ldhA) were deleted.

FIGURE 2

Conversion of cis-aconitate to itaconate (μmol itaconate/min/mg cells) by whole cells of E. coli BW25113 (DE3) Δpta–ΔldhA containing pKV-GA, pKV-GAC^opt, pKV-GAC^har, pKV-GAI^opt, and pKV-GAI^har. The average values of duplicate measurements are given.

FIGURE 3

Itaconate production in bioreactor cultures containing E. coli BW25113 (DE3) Δpta–ΔldhA with pKV-GA, pKV-GAC^opt, or pKV-GAC^har(A) or pKV-GA, pKV-GAI^opt, or pKV-GAI^har (B). The average values and SD of duplicate cultures are given.