Biosynthesis of β-carotene in engineered E. coli using the MEP and MVA pathways

Abstract

Backgroundβ-carotene is a carotenoid compound that has been widely used not only in the industrial production of pharmaceuticals but also as nutraceuticals, animal feed additives, functional cosmetics, and food colorants. Currently, more than 90% of commercial β-carotene is produced by chemical synthesis. Due to the growing public concern over food safety, the use of chemically synthesized β-carotene as food additives or functional cosmetic agents has been severely controlled in recent years. This has reignited the enthusiasm for seeking natural β-carotene in large-scale fermentative production by microorganisms.ResultsTo increase β-carotene production by improving the isopentenyl pyrophosphate (IPP) and geranyl diphospate (GPP) concentration in the cell, the optimized MEP (methylerythritol 4-phosphate) pathway containing 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and isopentenyl pyrophosphate isomerase (FNI) from Bacillus subtilis, geranyl diphosphate synthase (GPPS2) from Abies grandis have been co-expressed in an engineered E. coli strain. To further enhance the production of β-carotene, the hybrid MVA (mevalonate) pathway has been introduced into an engineered E. coli strain, co-expressed with the optimized MEP pathway and GPPS2. The final genetically modified strain, YJM49, can accumulate 122.4±6.2 mg/L β-carotene in flask culture, approximately 113-fold and 1.7 times greater than strain YJM39, which carries the native MEP pathway, and YJM45, which harbors the MVA pathway and the native MEP pathway, respectively. Subsequently, the fermentation process was optimized to enhance β-carotene production with a maximum titer of 256.8±10.4 mg/L. Finally, the fed-batch fermentation of β-carotene was evaluated using the optimized culture conditions. After induction for 56 h, the final engineered strain YJM49 accumulated 3.2 g/L β-carotene with a volumetric productivity of 0.37 mg/(L · h · OD600) in aerobic fed-batch fermentation, and the conversion efficiency of glycerol to β-carotene (gram to gram) reached 2.76%.ConclusionsIn this paper, by using metabolic engineering techniques, the more efficient biosynthetic pathway of β-carotene was successfully assembled in E. coli BL21(DE3) with the optimized MEP (methylerythritol 4-phosphate) pathway, the gene for GPPS2 from Abies grandis, the hybrid MVA (mevalonate) pathway and β-carotene synthesis genes from Erwinia herbicola.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-014-0160-x) contains supplementary material, which is available to authorized users.

Metabolic engineering of Escherichia coli using CRISPR–Cas9 meditated genome editing

“…In fed-batch fermentation, our best producer ZF237T produced 2.0 g/L β-carotene, which was similar to the report by Zhao et al (2013) and was still lower than the reports by Nam et al (2013) and Yang and Guo (2014). Besides combinatorially introducing well designed modifications, using our CRISPR-Cas9 based method to fine-tune the expression level of key genes may further increase β-carotene titer (Coussement et al, 2014;Li et al, 2013;Wang et al, 2009).…”

Metabolic engineering of the Stevia rebaudiana ent-kaurene biosynthetic pathway in recombinant Escherichia coli

“…In particular, the MEP pathway has undergone endogenous engineering to increase the supply of isoprenoid precursors in E. coli (Kajiwara et al, 1997). As isoprenoid production could be enhanced by overexpressing key enzymes of MEP pathway such as DXS, IDI, and IspA Yang and Guo, 2014), the three key enzymes from E. coli were overexpressed as one of strategies to improve ent-kaurene production. For the overexpression of the enzymes, four plasmids (pBBR-idi, pBBR-ispA, pUCM-dxs and pBBR-idi-ispA) were constructed and transformed into strain MG1655 expressing the (Table 1).…”

Enhanced β-Carotene Biosynthesis in Recombinant Escherichia Coli Harboring the Bottom Portion of Mevalonate Pathway of Enterococcus Faecium VTCC-B-935 Isolated in Vietnam

“…This observation is in agreement with several previous reports. 6,[11][12][13] To date, β-carotene has not been shown as a toxic agent to E. coli. As expected, over expression of three mva genes significantly up regulates the biosynthesis of β-carotene ( Figure 4B).…”

“…Several studies have proven the feasibility of this strategy. 6,12,13,18 In genetic engineering studies focusing on production of recombinant proteins, it is common that different sources of target genes resulted in different level of expression because of the suitability between genetic code of foreign genes with the genetic reading system/mechanism of specific host. In this study, we test another set of bottom portion of MVA pathway derived from E. faecium isolated in Vietnam.…”

“…To address this issue, addition of the exogenous mevalonate (MVA) pathway was shown as a reasonable strategy. 6,12,18,20,21 The MVA pathway is divided into two portions, the upper (from acetyl-CoA to MVA) and the lower (from MVA to DMAPP and IPP). Natural E. coli harbors MEP pathway that enable biosynthesis of FPP from G3P and pyruvate, as well as IPP isomerase catalyze the two-way conversion of IPP and DMAPP, the precursors of carotenoid (yellow part).…”