Metabolic engineering of escherichia coli for the production of monoterpenoids

Abstract

Monoterpenes have a great potential in many industrial areas including fragrance, alternative fuels, and also therapeutics. However, plant extraction of monoterpenes is possible only at low concentration. In the study described herein, applying systems metabolic engineering strategies, we developed efficient recombinant Escherichia coli producing γ-terpinene, linalool, limonene, and geraniol. By expressing mevalonate pathway, along with selecting geranyl diphosphate synthases and monoterpene synthases from several heterologous hosts, we constructed each monoterpene biosynthetic pathway in E. coli. However, for terpinene synthase (TPS2) which was highly insoluble protein in our strain, expression was remarkably elevated by using fusion proteins. Finally, to increase the precursor pool, flux engineering was conducted in E. coli’s genome. Native zwf was overexpressed to increase cofactor pool, and genomic integration of Bacillus subtilis derived fni was conducted to enhance DXP pathway. Finally, when introducing geraniol biosynthetic pathway, final strain WRNYFZ produced 12.4 mg/L and 90.6 mg/L of geraniol, by shake-flask cultivation and fed-batch fermentation. This achievement is the first report on geraniol biosynthesis using cofactor pool and DXP pathway engineering in genomic DNA of E. coli, having potential for applying to other terpenoids production.