An
Escherichia coli system was engineered for the heterologous production of itaconic acid via the expression of
cis-aconitate decarboxylase gene (
cad), and then maximal itaconic acid levels produced by engineered
E. coli were evaluated. Expression of
cad in
E. coli grown in Luria-Bertani (LB) medium without glucose in a test tube resulted in 0.07 g/L itaconic acid production after 78 h at 20°C. To increase itaconic acid production,
E. coli recombinants were constructed by inactivating the isocitrate dehydrogenase gene (
icd) and/or the isocitrate lyase gene (
aceA). Expression of
cad and inactivation of
icd resulted in 0.35 g/L itaconic acid production after 78 h, whereas
aceA inactivation had no effect on itaconic acid production. The intracellular itaconate concentration in the Δ
icd strain was higher than that in the
cad-expressing strain without
icd inactivation, which suggests that the extracellular secretion of itaconate in
E. coli is the rate-determining step during itaconic acid production. pH-stat cultivation using the
cad-expressing Δ
icd strain in LB medium with 3% glucose in a jar fermenter resulted in 1.71 g/L itaconic acid production after 97 h at 28°C. To further increase itaconic acid production, the aconitase B gene (
acnB) was overexpressed in the
cad-expressing Δ
icd strain. Simultaneous overexpression of
acnB with the expression of
cad in the Δ
icd strain led to 4.34 g/L itaconic acid production after 105 h. Our findings indicate that
icd inactivation and
acnB overexpression considerably enhance itaconic acid production in
cad-expressing
E. coli.
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