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 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....

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

“…1) CadA_mCherry were recloned into pSCT5 without the mCherry fusion, and their expression patterns and levels in E. coli XL1-Blue were verified by SDS-PAGE and analyzed by using ImageJ, which is Java-based image processing program, respectively. Whereas wildtype CadA was mainly expressed as insoluble aggregates, in accord with previous reports (Okamoto et al, 2014;Otten et al, 2015), more than 95% of total expressed protein in synonymous codon variants No. 2 (scvCadA_No2) and No.…”

“…In addition, IA is produced in proportion to the expression level of CadA in Aspergillus niger (van et al, 2014). However, the cadA gene is mostly expressed as insoluble aggregates in surrogate hosts (Okamoto et al, 2014). Accordingly, problems associated with functional expression of the cadA gene have to resolve for the development of non-natural hosts for IA production.…”

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.…”

“…4 Overexpression of acn likely suppresses the activity of the glyoxylate shunt, resulting in an increase of acetic acid concentration derived from acetylCoA. To further optimize the production of IA, we inactivated both the acetate kinase gene (ack) and the pyruvate oxidase gene (pox), which are involved in acetic acid production, in E. coli expressing cad.…”