Abstract
Backgroundα-Pinene is an important natural product that is widely used in flavorings, fragrances, medicines, fine chemicals and high-density renewable fuels. Currently, α-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 α-pinene production.ResultsTo construct the microbial synthetic pathway of α-pinene in E. coli, we co-expressed native geranyl diphosphate synthase (IspA) from E. coli and α-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 α-pinene production, a novel biosynthetic pathway of α-pinene was assembled in E. coli BL21(DE3) with the heterologous hybrid mevalonate (MVA) pathway, GPPS2 and α-pinene synthase (Pt30). The final genetic strain, YJM28, harboring the above novel biosynthetic pathway of α-pinene, accumulated α-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 α-pinene (gram to gram) in the metabolically engineered strain reached 2.61%.ConclusionsIn this paper, by using metabolic engineering techniques, the more efficient biosynthetic pathway of α-pinene was successfully assembled in E. coli BL21(DE3) with the heterologous hybrid MVA pathway, GPPS2 and α-pinene synthase (Pt30). In addition, this is the first report on α-pinene fed-batch fermentation, and our results represent improvements over previous reports.
Highlights
Background α-Pinene is a natural and active monoterpene, which is derived from the head-to-tail condensation of dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP) [1]. α-Pinene is an important natural product that is widely used in flavorings, fragrances [2], medicines and fine chemicals [3,4]
In this paper, α-pinene production was significantly enhanced by assembling a biosynthetic pathway of α-pinene using the heterologous MVA pathway and the GPPS2 and Pt30 genes in an engineered E.coli strain
To synthesize α-pinene, α-pinene synthase (Pt30) derived from Pinus taeda was introduced into the E. coli strain
Summary
Background αPinene is a natural and active monoterpene, which is derived from the head-to-tail condensation of dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP) [1]. α-Pinene is an important natural product that is widely used in flavorings, fragrances [2], medicines and fine chemicals [3,4]. Because of its compact structure and reactive olefin functionality, it has some potential as a feedstock for high-density renewable fuels such as jet fuel [5,6]. Monoterpenes such as α-pinene, β-pinene, camphene, and limonene are important components of pine resin and are currently produced on an industrial scale either. By using the native MEP pathway to supply the precursor of DMAPP and IPP, Carter et al introduced a monoterpene biosynthesis pathway into E. coli, which led to about 5 mg/L limonene production [13]
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