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

Abstract

Aspergillus terreus cadA, encoding cis-aconitate decarboxylase, is an essential gene for itaconic acid (IA) biosynthesis, but it is primarily expressed as insoluble aggregates in most industrial hosts. This has been a hurdle for the development of recombinant strategies for IA production. Here, we created a library of synonymous codon variants (scv) of the cadA gene containing synonymous codons in the first 10 codons (except ATG) and screened it in Escherichia coli. Among positive clones, E. coli scvCadA_No8 showed more than 95% of expressed CadA in the soluble fraction, and in production runs, produced threefold more IA than wild-type E. coli in Luria-Bertani broth supplemented with 0.5% glucose. In M9 minimal media containing 0.85 g/L citrate and 1% glycerol, E. coli scvCadA_No8 produced 985.6 ± 33.4 mg/L IA during a 72-h culture after induction with isopropyl β-D-1-thiogalactopyranoside. In a 2-L fed-batch fermentation consisting of two stages (growth and nitrogen limitation conditions), we obtained 7.2 g/L IA by using E. coli by introducing only the scv_cadA gene and optimizing culture conditions for IA production. These results could be combined with metabolic engineering and generate an E. coli strain as an industrial IA producer. Biotechnol. Bioeng. 2016;113: 1504-1510. © 2015 Wiley Periodicals, Inc.

Production of itaconate by whole-cell bioconversion of citrate mediated by expression of multiple cis-aconitate decarboxylase (cadA) genes in Escherichia coli

“…1). Other studies have indicated the importance of cadA expression for itaconate production1920. E. coli BL21(DE3) containing acn and three cadA genes yielded a much higher conversion (64.0%).…”

“…produced itaconate using an E. coli strain expressing heterologous cadA , achieving a titer of 690 mg/L18. Another E. coli strain with random synonymous codon substitutions produced 7.23 g/L of itaconate19. Model-based metabolic engineering of E. coli increased itaconate production to 32 g/L20.…”

Emerging biotechnologies for production of itaconic acid and its applications as a platform chemical

“…Recently, Yang et al [102] reported that co-expression of aconitase (ACO) and CAD in E. coli (uCA) resulted in the production of 117.2 mg IA L −1 after 48 h fermentation while the IA titer was significantly improved to 222.1 mg L −1 by self-assembly of ACO-PDZ (APd) and CAD-PDZlig (CPI) in E. coli (sPP) under the same conditions. Jeon et al [35] reported IA production from glycerol using E. coli harboring a random synonymous codon-substituted 5ʹ-coding region variants of the cadA gene. They showed that functional expression of the cadA gene was improved through synonymous codon substitution at the first ten codons (excluding ATG).…”

Optimization and scale up of itaconic acid production from potato starch waste in stirred tank bioreactor

“…It may be due to the high shear rate generated by mechanical agitation in the STR that may damage fungal cells. Optimum balance between aeration rate and agitation speed needs to be maintained to avoid mechanical stress on the mycelia caused by excessive impeller speed, or conversely poor oxygen transfer rate at lower speed. Hence, agitation and aeration rates were simultaneously varied in the current investigation to find the optimum balance.…”