Construction And Culture Optimization Of The Genetic Engineering Bacterium For The Production Of β-carotene

As a typical representative of carotenoid family,β-carotene is not only an important source of vitamin A in human body, it also has some physiological functions to the health of a person. Recent research suggests thatβ-carotene is associated with reduced risk of certain disease, particularly in cacer. Simultaneouly, it can improve the immune ability of the body. In recent years, the producing technologies, especially fermentation, have been studied.Dunaliella salina is one of the major microorganisms commercially employed to produceβ-carotene, it can accumulate much moreβ-carotene than any other organisms under the conditions of high light, high salinity and nutrient deprivation. Therefor, it is of great economical significance to study the mechanisms ofβ-carotene synthesis and accumulation from the perspective of molecular biology, which may explain the molecular regulation of key genes and enzymes of D. salina in the carotenogenic pathway.Lycopeneβ-cyclase (LycB) is the key enzyme of catalyzing lycopene to formβ-carotene. The reason that D. salina highly accumulateβ-carotene is directly related to the catalytic mechanisms of LycB and the molecular regulation of its gene. In order to address the function of LycB, we cloned the open reading frame(ORF) of LycB by RT-PCR strategy. After confirmed by DNA sequencing, we constructed a recombinant plasmid pACCRT-EIB-LycB by cloning the ORF of LycB into the plasmid pACCRT-EIB which harbours the Erwinia uredovora genes necessary for lycopene formation. Than transformed the pACCRT-EIB-LycB into E.coli DH5α, so a genetically engineered bacteria strain which can accumulateβ-carotene stablely was gained.In order to obtain the maximum yield ofβ-carotene, response surface analysis methodology (RSM) was applied to optimize the composition of fermentation medium. Based on single-factor experiments, four-factors-three-levels Box-behnken central compositon experiments design were applied. The results showed that the best fermentation medium was tryptone 20.43g/L, yeast extract 10g/L, NaCl 5g/L, glycerol 1.97g/L, NH4Cl 1.09g/L, Na2HPO4?7H2O 12.84g/L. Validation experiments were carried out at these optimum conditions and the average yield ofβ-carotene is 2.3896mg/L. The experiment data shows the production ofβ-carotene has increased 39.26% compared with the E.coli strain containing pACCRT16ΔX which contains crtY gene of Erwinia uredovora under the same culture conditions.