Significantly enhanced production of isoprene by ordered coexpression of genes dxs, dxr, and idi in Escherichia coli

Abstract

We constructed a biosynthetic pathway of isoprene production in Escherichia coli by introducing isoprene synthase (ispS) from Populus alba. 1-deoxy-D-xylulose 5-phosphate synthase (dxs), 1-deoxy-D-xylulose 5-phosphate reductoisomerase (dxr) and isopentenyl diphosphate (IPP) isomerase (idi) were overexpressed to enhance the isoprene production. The isoprene production was improved 0.65, 0.16, and 1.22 fold over the recombinant BL21 (pET-30a-ispS), respectively, and idi was found to be a key regulating point for isoprene production. In order to optimize the production of isoprene in E. coli, we attempted to construct polycistronic operons based on pET-30a with genes dxs, dxr, and idi in various orders. The highest isoprene production yield of 2.727 mg g(-1) h(-1) (per dry weight) was achieved by E. coli transformed with pET-30a-dxs/dxr/idi. Interestingly, the gene order was found to be consistent with that of the metabolic pathway. This indicates that order of genes is a significant concern in metabolic engineering and a sequential expression pattern can be optimized according to the biosynthetic pathway for efficient product synthesis.

Enhanced limonene production by optimizing the expression of limonene biosynthesis and MEP pathway genes in E. coli

“…Our result showed that those E. coli strains expressing ls-gpps or ls-Ecgpps produced larger amounts of limonene than the strains expressing gpps-ls or Ecgpps-ls. This result disputes the current notion that the order of the limiting genes in polycistronic operons should correspond to that of terpenoid metabolic pathway in the natural and fine regulation mechanism of microorganisms [19]. In addition, the expression strength of vectors used for limonene biosynthesis was also indicated to be a very important determinant for the final limonene yield.…”

“…Although overexpression of rate-limiting enzymes in the MEP pathway has been used to produce many terpenoids including isoprene, taxol precursor and levopimaradiene [14][15][16][17][18][19], this method has not been used in the most common engineered strain Escherichia coli to produce limonene. In a previous study, an engineered Escherichia coli, harboring a heterologous GPPS gene from Abies grandis and a LS gene from Mentha spicata, was reported to produce limonene in a titer of merely 5 mg/L (24 h) [4].…”

Characterization of promoters in Escherichia coli and application for xylitol synthesis

“…Escherichia coli is the most widely used platform organism not only for the production of a wide range of important high-value compounds, such as taxol precursor [1], isoprene [2,3], terpenoids [4], 5-Aminolevulinic acid [5], coenzyme Q 10 [6] and plant-specific phenylpropanoids [7], but also for the solution of global energy-related issues such as biofuel production and biomass conversion [8,9]. In these studies, the construction of synthetic pathway frequently involves the expression of multiple genes.…”

Combination of Entner-Doudoroff Pathway with MEP Increases Isoprene Production in Engineered Escherichia coli

“…34°C), its collection from plants is difficult [7]. Hence, much attention and effort have been directed toward isoprene production through metabolically engineered microorganisms [8], [9].…”

“…Although these two pathways begin with different precursors, both culminate with the production of two universal 5-carbon isoprenoid precursors, isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) [10], [11]. The DMAPP can be converted to isoprene by isoprene synthases (IspS), which are cloned from plants and heterologously expressed in microbial hosts [8], [9].…”