Producing Lycopene By Engineered Escherichia Coli

Lycopene is an important carotenoid, which is a proven antioxidant and prevents oxidation of low density lipoprotein (LDL) and cholesterol, thereby reducing the risk of developing atherosclerosis and coronary heart disease. Recent research suggests that lycopene is associated with reduced risk of macular degenerative disease, serum lipid oxidation, and cancers of the lung, bladder, cervix and skin. In light of these potential applications, lycopene, a very useful phytochemical nutraceutical, holds great commercial value. At the same time, the producing technologies, especially fermentation, have been studied.All carotenoids are produced via a common precursor isopentenyl diphosphate (IPP). The biosynthesis of isopentenyl phosphate (IPP) is through either the classical mevalonate pathway or the more recently discovered non-mevalonate pathway. In E. coli, the biosynthesis of IPP branches from central metabolic pathway intermediates pyruvate and glyceraldehyde 3-phosphate (G3P).In this study, the ispA and idi gene that are required to convert IPP to lycopene are overexpressed in E. coli which contained the plasmid pAC-Lyc bearing the E. herbicola's crtE, crtI and crtB genes cloned in pACYC184 (CmR), a vector with p 15 A origin of replication. The genes are under the control of their native promoters. In addition, experiments that combine both metabolic engineering strategies and process optimization have been carried out to maximize lycopene productivity. Four aspects have been investigated:(1) The extraction of lycopene from engineered Escherichia coli was optimized from cell disruption methods, solvents and other extracting conditions. Results showed that acid-hot method was the best to disrupt cell walls and acetone was suitable for extracting lycopene from this E.coli. And other important factors such as disrupting temperature, disrupting time, the concentration and quantity of acid, quantity of acetone, extracting temperature and time had been studied. The optimal conditions were as that hydrochloric acid concentration 4 mol/L, hydrochloric acid addition 10mL / g(wet cells), temperature for breaking cell walls 35℃ timefor disrupting cells 20min, acetone addition 20mL / g(wet cell), extracting temperature 40 °C and extracting time 2h was obtained.(2) The culturing conditions used were as follows: the medium was modified LB+M9 broth (Trypton 10g/L; Yeast Extract 5 g/L; Glycerol 20 g/L; Na₂HPO₄·7H₂O 12.8 g/L; CaCl₂ 0.022 g/L; KH₂PO₄ 3 g/L; NaCl 0.5 g/L; MgSO₄ 0.48 g/L; NH₄Cl1 g/L; (NH₄)₂SO₄ 10 g/L; Stimulus 2 g/L; pH 7.0.), the engineering bacteia were shake cultured at 33°C to OD₆₀₀ of 0.6, the final concentration of the engineering bacteria was further cultured for 24h. The engineering bacteria could grow to 6.48gDCW/ L and accumulate lycopene to 10.6mg/ gDCW under culturing conditions.(3) The conversion from FPP to GGPP is the first bottleneck, followed sequentially by IPP isomerization and FPP synthesis. Removal of these bottle necks results in an E. coli strain providing sufficient precursors for in vivo synthesis of isoprenoids such as lycopene. We cloned and overexpressed isopentenyl diphosphate (IPP) isomerase (encoded by idi) gene and farnesyl diphosphate (FPP) synthase (encoded by ispA) from Escherichia coli. These genes and the gene isopentenyl diphosphate (IPP) isomerase of E. coli and the gene cluster (crtEIB) of the Erwinia sp. were introduced into E. coli to produce lycopene, an orange pigment and antioxidant. The result showed that the production of lycopene in E. coli DH5α(pACCRT-EIB, pUC19-ispA-idi) increased by 69%.(4) We co-expressed the gene idi, ispA without or with the gene crtEIB in the order of ispA-idi-crtEIB. The result showed that E. coli BL21(DE3)(pET-28a-ispA-idi, pACCRT-EIB) and E. coli BL21(DE3)(pET-28a-ispA-idi-EIB) produced less lycopene and grew very slowly.(5) The lycopene fed-batch fermentation showed that the production of lycopene were 20mg/L and 66mg/L in E. coli DH5a(pACCRT-EIB) and E. coli DH5α(pACCRT-EIB, pUC19-idi), respectively.