Breeding Of Lycopene-Overproducting Strain Blakeslea Trispora And Optimization Of Its Fermentation Process

Lycopene, a natural functional pigment with strong ability of clearing free radicals, is widely used as food additives, animal feed supplements, pharmaceutical and cosmetics, and also has a certain therapeutic effect on some certain cancers and chronic diseases. Thus, the market demand for lycopene is increasing. However, synthetic lycopene is restrictive to use in above-mentioned areas due to its safety issues; besides, there is little natural lycopene because of its high cost, which caused high market price of lycopene. Currently, Blakeslea trispora, which has huge market potential, has achieved industrial production of lycopene. In this study, spores of B. trispora NRRL 2896 (-) were treated with N+ ion implatation and a strain with high-yield of lycopene was isolated, then the fermentation process was optimized and its high yield mechanism was studied preliminarily, which may advance the industrial production process of lycopene produced by B. trispora. The specific contents and results are as follows:(1) Spores of B. trispora (-) were treated with N+ion implantation, and further selected from the screening plates supplemented with fermentation broth extract and lovastatin, then isolated on solid fermentation medium, after which the selected mutants were tested with B. trispora NRRL 2895 (+) on the wort plates. According to the test results, the selected mutants fermented in shake flasks with B. trispora (+), finally a strain named B. trispora (-) 4c with higher yield of lycopene and dry biomass was obtained, which lycopene yield and biomass reached 31.66 mg-g-1 and 46.80 g-L-1 respectively, and increased by 45% and 5.20% respectively, compared with the original strain.(2) Compared with the original strain, enzyme activity of superoxide dismutase(SOD) and catalase(CAT) in mycelium of the mutant strain increased by 52% and 35% respectivelly; and compared with the original strain, the intracellular and extracellular antioxidase activity also increased when the mutant cultured with B. trispora (+), which all promoted the accumulation of lycopene, and ultimately improved the yield of lycopene.(3) Compared with the original strain, the transcription level of key genes hmgR, carB and carRA in mutant strain increased by 3.20 times,3.76 times and 3.45 times, respectivelly. The transcription level of key genes hmgR, carB and carRA also increased when the mutant cultured with B. trispora (+). For hmgR, a maximum 3.66-fold increase in mRNA steady state level was observed 24 h after B. trispora (-) 4c spores inoculation in mated cultures; however, a maximum 3.50-fold increase for carB and a maximum 3.20-fold increase for carRA occured 48 h after spores inoculation in mated cultures.(4) The optimization of fermentation medium:to determine the optimal carbon source and nitrogen source and their optimal levels, single factor test and response surface methodology were utilized to optimize fermentation medium used for the B. trispora (-) 4c strain. The optimized fermentation medium composition are as follows:corn flour 53.06 g-L-1, soybean flour 27.76 g-L-1, cottonseed oil 45.81 mL-L-1, KH2PO41.50 g-L-1, Mg2SO4·7H2O 0.50 g-L-1. Finally, the lycopene production reached 39.85 mg-g-1, and dry biomass reached 41.60 g-L-(5) Based on the optimum medium composition, two aspects of fermentation conditions, inoculation interval time and time of adding inhibitors of lycopene cyclase were optimized. The optimal inoculation interval time is 20 h and the optimal time for adding inhibitors of lycopene cyclase is 32 h. Fermentation time decreased to 96 h, and the yield of lycopene and dry biomass reached 43.61 mg-g-1,41.20 g-L-1 respectively; the volumetric production of lycopene reached 1.79 g-L-1.