Metabolic engineering of Escherichia coli for the biosynthesis of alpha-pinene

Background: alpha-Pinene is an important natural product that is widely used in flavorings, fragrances, medicines, fine chemicals and high-density renewable fuels. Currently, alpha-Pinene used in industry is mainly produced either by tapping trees (gum turpentine) or as a byproduct of paper pulping (crude sulfate turpentine, CST). However, the extraction of it from trees is tedious and inefficient and requires substantial expenditure of natural resources. Therefore, it is necessary to seek sustainable technologies for alpha-pinene production. Results: To construct the microbial synthetic pathway of alpha-pinene in E. coli, we co-expressed native geranyl diphosphate synthase (IspA) from E. coli and alpha-pinene synthase (Pt30) from Pinus taeda, and then to increase the geranyl diphosphate (GPP) content in the cells, a suitable geranyl diphosphate synthase (GPPS2) was selected from two different origins. Furthermore, to enhance alpha-pinene production, a novel biosynthetic pathway of alpha-pinene was assembled in E. coli BL21(DE3) with the heterologous hybrid mevalonate (MVA) pathway, GPPS2 and alpha-pinene synthase (Pt30). The final genetic strain, YJM28, harboring the above novel biosynthetic pathway of alpha-pinene, accumulated alpha-pinene up to 5.44 mg/L and 0.97 g/L under flask and fed-batch fermentation conditions, respectively. The conversion efficiency of glucose to alpha-pinene (gram to gram) in the metabolically engineered strain reached 2.61%. Conclusions: In this paper, by using metabolic engineering techniques, the more efficient biosynthetic pathway of alpha-pinene was successfully assembled in E. coli BL21(DE3) with the heterologous hybrid MVA pathway, GPPS2 and alpha-pinene synthase (Pt30). In addition, this is the first report on alpha-pinene fed-batch fermentation, and our results represent improvements over previous reports.