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The depleting petroleum reserve, increasingly severe energy crisis, and global climate change are reigniting enthusiasm for seeking sustainable technologies to replace petroleum as a source of fuel and chemicals. In this paper, the efficiency of the MVA pathway on isoprene production has been improved as follows: firstly, in order to increase MVA production, the source of the “upper pathway” which contains HMG-CoA synthase, acetyl-CoA acetyltransferase and HMG-CoA reductase to covert acetyl-CoA into MVA has been changed from Saccharomyces cerevisiae to Enterococcus faecalis; secondly, to further enhance the production of MVA and isoprene, a alanine 110 of the mvaS gene has been mutated to a glycine. The final genetic strain YJM25 containing the optimized MVA pathway and isoprene synthase from Populus alba can accumulate isoprene up to 6.3 g/L after 40 h of fed-batch cultivation.

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.

BackgroundUnder aerobic conditions, acetic acid is the major byproduct produced by E. coli during the fermentation. And acetic acid is detrimental to cell growth as it destroys transmembrane pH gradients. Hence, how to reduce the production of acetic acid and how to utilize it as a feedstock are of intriguing interest. In this study, we provided an evidence to produce β-caryophyllene by the engineered E. coli using acetic acid as the only carbon source.ResultsFirstly, to construct the robust acetate-utilizing strain, acetyl-CoA synthases from three different sources were introduced and screened in the E. coli. Secondly, to establish the engineered strains converting acetic acid to β-caryophyllene, acetyl-CoA synthase (ACS), β-caryophyllene synthase (QHS1) and geranyl diphosphate synthase (GPPS2) were co-expressed in the E. coli cells. Thirdly, to further enhance β-caryophyllene production from acetic acid, the heterologous MVA pathway was introduced into the cells. What’s more, acetoacetyl-CoA synthase (AACS) was also expressed in the cells to increase the precursor acetoacetyl-CoA and accordingly resulted in the increase of β-caryophyllene. The final genetically modified strain, YJM67, could accumulate the production of biomass and β-caryophyllene up to 12.6 and 1.05 g/L during 72 h, respectively, with a specific productivity of 1.15 mg h−1 g−1 dry cells, and the conversion efficiency of acetic acid to β-caryophyllene (gram to gram) reached 2.1 %. The yield of β-caryophyllene on acetic acid of this strain also reached approximately 5.6 % of the theoretical yield.ConclusionsIn the present study, a novel biosynthetic pathway for β-caryophyllene has been investigated by means of conversion of acetic acid to β-caryophyllene using an engineered Escherichia coli. This was the first successful attempt in β-caryophyllene production by E. coli using acetic acid as the only carbon source. Therefore, we have provided a new metabolic engineering tool for β-caryophyllene synthesis.

BackgroundTo deal with the increasingly severe energy crisis and environmental consequences, biofuels and biochemicals generated from renewable resources could serve as a promising alternative for replacing petroleum as a source of fuel and chemicals, among which isoprene (2-methyl-1,3-butadiene) in particular is of great significance in that it is an important platform chemical, which has been used in industrial production of synthetic rubber for tires and coatings or aviation fuel.ResultsWe firstly introduced fatty acid decarboxylase (OleTJE) from Jeotgalicoccus species into E. coli to directly convert MVA(mevalonate) into 3-methy-3-buten-1-ol. And then to transform 3-methy-3-buten-1-ol to isoprene, oleate hydratase (OhyAEM) from Elizabethkingia meningoseptica was overexpressed in E. coli. A novel biosynthetic pathway of isoprene in E. coli was established by co-expressing the heterologous mvaE gene encoding acetyl-CoA acetyltransferase/HMG-CoA reductase and mvaS gene encoding HMG-CoA synthase from Enterococcus faecalis, fatty acid decarboxylase (OleTJE) and oleate hydratase (OhyAEM). Furthermore, to enhance isoprene production, a further optimization of expression level of OleTJE, OhyAEM was carried out by using different promoters and copy numbers of plasmids. Thereafter, the fermentation process was also optimized to improve the production of isoprene. The final engineered strain, YJM33, bearing the innovative biosynthetic pathway of isoprene, was found to produce isoprene up to 2.2 mg/L and 620 mg/L under flask and fed-batch fermentation conditions, respectively.ConclusionsIn this study, by using metabolic engineering techniques, the novel MVA-mediated biosynthetic pathway of isoprene was successfully assembled in E. coli BL21(DE3) with the heterologous MVA upper pathway, OleTJE from Jeotgalicoccus species and OhyAEM from Elizabethkingia meningoseptica. Compared with traditional MVA pathway, the novel pathway is shortened by 3 steps. In addition, this is the first report on the reaction of converting MVA into 3-methy-3-buten-1-ol by fatty acid decarboxylase (OleTJE) from Jeotgalicoccus species. In brief, this study provided an alternative method for isoprene biosynthesis, which is largely different from the well-developed MEP pathway or MVA pathway.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0236-2) contains supplementary material, which is available to authorized users.