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 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 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 icd strain. Simultaneous overexpression of acnB with the expression of cad in the 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.Key words: aconitase; cis-aconitate decarboxylase; isocitrate dehydrogenase; isocitrate lyase; itaconic acid; metabolic engineering IntroductionOver the past several decades, there has been substantial interest in itaconic acid as a dicarboxyvinyl monomer produced by microbial fermentation of biomass (Kobayashi and Nakamura, 1964;Okabe et al., 2009;Willke and Vorlop, 2001). Owing to its useful properties, itaconic acid is used in the manufacturing of synthetic polymers, such as plastics and resins (Milson and Meers, 1985;Tate, 1981). Graft polymers with an itaconic acid-based main chain and oligolactate side chain have been synthesized and characterized in our laboratory Okada et al., 2012). The polymers consisting of itaconic acid had high biomass content and are therefore thought to contribute to carbon emission reduction.Itaconic acid is industrially produced from sugars, such as glucose, by the fungus Aspergillus terreus via submerged fermentation (Bonnarme et al., 1995). To date, many re searchers have reported increased productivity of a native itaconic acid producer, A. terreus, by using techniques associated with mutation breeding (Yahiro et al., 1995) and fermentation conditions (Okabe et al., 1993;Park et al., 1994;Riscaldati et al., 2000;Träger et al., 1989;Yahiro et al., 1997). Specifically, the spontaneous mutant A. terreus TN-484 is used as a target microorganism for optimizing the fermentation process. In a previous study, a typical yield of 82....

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.