| Lycopene, synonymous with ψ- carotene, is one kind of deoxy-carotenoids. Lycopene has many important bioactivities such as antioxidation, free radical scavenging, anti-cancer, anti-aging and enhancing immune capacity. Furthermore, it is also of great significance for preventing cardiovascular disease, atherosclerosis and some other adult diseases. Currently, lycopene production methods mainly include extraction from natural products, chemical synthesis and fermentation. Among them, genetic engineering has been successfully used to build lycopene exogenous expression systems. Through optimizing culture medium and conditions of engineered bacteria, higher lycopene yields could be achieved. Dunaliella bardwil is one of the most commonly used model organism for carotenoids production. Lycopene could be enriched in the ultimate product by inhibiting its conversion to β- carotene, which is the final synthetic reaction in the metabolic pathway( pentenyl pyrophosphate(IPP)- geranyl geranyl pyrophosphate(GGPP)- phytoene- phytofluene- all-trans-lycopene-β- carotene).In this thesis, fermentation conditions of engineered strain W-05, including different media, temperature, carbon, nitrogen and inorganic salts have been optimized to determine the optimal medium and the optimum concentrations for lycopene production. Then, further response surface optimization and amplification culture in fermenter were conducted to determine the optimum medium. After that, different feeding methods were experimented to further increase lycopene yields. In the study, inhibitory effects of concentration gradients of seven different blocking agents including imidazole, piperidine, triethylamine, pyridine, creatinine, and niacin and piperonyl butoxidethe were respectively explored by blocking the final metabolic step. The inhibitory effect of triethylamine was tested to be evident. Consequently, the effects of its concentrations, adding time, extreme environmental stress and et.al on the yield of lycopene were further studied. Finally, by means of real-time PCR, the effect of triethylamine on transcription levels of key genes in the carotene metabolism pathway of Dunaliella bardawil, including Db Psy, Db Pds, Db Zds and Db Lyc B1, were studied.Experimental results showed that the most suitable W-05 medium formula for the production of lycopene is: 10 peptone, yeast extract powder 5, glycerol 7.83(m L / L), ammonium nitrate 3.30, KH 2 PO 4 1.80, Na Cl 11.62(units g / L). The optimum cultivation temperature was 37 ℃ in the initial fermentation period(0-10 hours), which was 33 ℃ in the product expression period(10-20 hours). In the process of amplication cultivation using a fermentor, OD600 value 58 of final biomass and the pigment yield 19.932 mg / L were achieved by means of exponential fed-batch. In the experiments of using Dunaliella bardawil to procuce lycopene, triethylamine showed significant inhibition in contrast to the other six block agents. Lycopene was generated by the four groups 500, 1000, 1500 and 2000 ppm of triethylamine. The optimum conditions determined by optimization experiments were triethylamine concentration 75ppm; the optimum adding time was when alage just went through the logarithmic phase and started entering latent phase( OD630 = 0.944, algal cells at a concentration of 8.81 × 10 6 g / m L). Under the optimum conditions, the highest lycopene yield of 8.98 mg / L was obtained. In researches of extreme nutritional stress, nitrogen deficiency was found to have significant promoting effects on lycopene accumulation in triethylamine-treated algal samples. Lycopene content in a single cell reached 7.603 × 10-4 mg. Quantitative PCR results showed that different concentrations of triethylamine had varying degrees of inhibitory effects on transcriptional level of lycopene cyclase Lyc B1, and had enhancing effects on Db Psy, Db Pds and Db Zds which respectively regulates phytoene synthase, phytoene TRs dehydrogenase and ζ- carotene dehydrogenase. |
PDF Full Text Request