Metabolic engineering of itaconate production in Escherichia coli

Abstract

Interest in sustainable development has led to efforts to replace petrochemical-based monomers with biomass-based ones. Itaconic acid, a C5-dicarboxylic acid, is a potential monomer for the chemical industry with many prospective applications. cis-aconitate decarboxylase (CadA) is the key enzyme of itaconate production, converting the citric acid cycle intermediate cis-aconitate into itaconate. Heterologous expression of cadA from Aspergillus terreus in Escherichia coli resulted in low CadA activities and production of trace amounts of itaconate on Luria-Bertani (LB) medium (<10 mg/L). CadA was primarily present as inclusion bodies, explaining the low activity. The activity was significantly improved by using lower cultivation temperatures and mineral medium, and this resulted in enhanced itaconate titres (240 mg/L). The itaconate titre was further increased by introducing citrate synthase and aconitase from Corynebacterium glutamicum and by deleting the genes encoding phosphate acetyltransferase and lactate dehydrogenase. These deletions in E. coli's central metabolism resulted in the accumulation of pyruvate, which is a precursor for itaconate biosynthesis. As a result, itaconate production in aerobic bioreactor cultures was increased up to 690 mg/L. The maximum yield obtained was 0.09 mol itaconate/mol glucose. Strategies for a further improvement of itaconate production are discussed.

Challenges in the production of itaconic acid by metabolically engineeredEscherichia coli

“…The engineered E. coli produced 4.34 g/L of IA when grown for 105 h in LB medium supplemented with glucose, under pH-stat conditions. 4 Similarly to what observed by Vuoristo et al, we did not detect Cad activity in the engineered E. coli at 37 C. 5 In our study, cad was cloned into the IPTG-inducible pETHis vector 15 containing the P T7 promoter, and protein expression was carried out in E. coli BW25113 (DE3) pLysS cells at 20 C or at 37 C. Cells were induced with IPTG for 18 h in 2 mL of LB medium. The presence of the pLysS plasmid considerably improved the expression levels of Cad; other expression systems bearing the inducible promoters P LAC and P BAD were unable to yield acceptable quantities of Cad.…”

“…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.…”

“…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. 5 Cells thus modified produced up to 0.69 g/L of IA when grown in mineral medium supplemented with glucose, in an aerobic bioreactor.…”

Itaconic acid production from glycerol using Escherichia coli harboring a random synonymous codon‐substituted 5′‐coding region variant of the cadA gene

“…To circumvent disadvantages arising from physiological characteristics of A. terreus, including spore formation, filamentous growth and sensitivity to shear stress, several groups have recently sought to create a non-natural host that produces IA based on wellknown industrial microorganisms, such as Escherichia coli and Saccharomyces cerevisiae (Blazeck et al, 2014;Vuoristo et al, 2015). These hosts have many advantages as a production host, including well-established molecular tools for genetic modification and relatively easy operating conditions for fermentation.…”

P gas , a Low-pH-Induced Promoter, as a Tool for Dynamic Control of Gene Expression for Metabolic Engineering of Aspergillus niger

“…Aspergillus terrous was the natural producer of itaconate, with a production level of 80 g/liter. After the gene encoding the key enzyme AtCAD was identified in 2008 (22), many studies were performed to utilize this gene to produce itaconate in various hosts, including Escherichia coli (33), Corynebacterium glutamicum (34), Saccharomyces cerevisiae (35), Yarrowia lipolytica (36), and A. niger. Because A. niger produced large amounts of citrate (180 g/liter), the precursor of itaconate, there was great interest in transforming the natural citrate producer into an itaconate producer; however, constitutive expression of the CAD gene alone led to only small amounts of itaconate production.…”