| 초록 |
Microbial production of itaconate has been mainly carried out by Aspergillus terreus. Recently, its production in Escherichia coli has been studied due to the complicated fermentation process and shortage of genetic engineering tools of A. terreus. One of the major issues on itaconate production in industrial strains is the poor availability of its precursor, cis-aconitate, unlike spatially compartmentalized itaconate synthesis of A. terreus. In this study, directed evolution of protein known to have substrate promiscuity to citrate was conducted in E. coli to enhance the cis-aconitate availability. Initially, the itaconate responsive screening system could be constructed by using the itaconate-responsive transcription factor (ItcR) from Yersinia pseudotuberculosis. Thereafter, the mutant library was rationally designed and enriched. Finally, the developed mutant showed significantly enhanced catalytic efficiency to citrate compared to the wild-type enzyme and consequently, its use enabled 4.26-fold increased itaconate production compared to the parental strain, indicating the successful enhancement of cis-aconitate supply as a hijacker of TCA flux. |
