title: Production of itaconic acid using metabolically engineered Escherichia coli.
abstract: 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 Δicd 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 Δicd 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 Δicd strain. Simultaneous overexpression of acnB with the expression of cad in the Δicd 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.
authors.affiliation: Department of Biobased Materials Science, Kyoto Institute of Technology.
authors.forename: Shusuke
authors.initials: S
authors.lastname: Okamoto
authors.forename: Taejun
authors.initials: T
authors.lastname: Chin
authors.forename: Ken
authors.initials: K
authors.lastname: Hiratsuka
authors.forename: Yuji
authors.initials: Y
authors.lastname: Aso
authors.forename: Yasutomo
authors.initials: Y
authors.lastname: Tanaka
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Takahashi
authors.forename: Hitomi
authors.initials: H
authors.lastname: Ohara
mesh_terms: D002262:Carboxy-Lyases / Q000235:genetics / Q000378:metabolism; D003470:Culture Media / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D017353:Gene Deletion; D015870:Gene Expression; D060847:Metabolic Engineering; D013386:Succinates / Q000378:metabolism*; D013696:Temperature; D013997:Time Factors
chemical_list: D003470:Culture Media; D013386:Succinates; D002262:Carboxy-Lyases; C018742:aconitate decarboxylase; C005229:itaconic acid