Development Of Siraitia Grosvenorii Cell Suspension Culture System And Effective Biosynthesis Of Mogrosides V

The extraction rate of Mogrosides V from fruits is very low,but the secondary metabolites can be increased by adding elicitors to the plant suspension cells.The suspension culture technology is not limited by natural conditions,which is an effective way to increase the yield of secondary metabolites.However,a very few researches have been conducted on Siraitia grosvenorii cells,especially on the scale-up of bioreactor.Thus,this study covers the research onthe following aspects:(1)Screening of elicitors,finding the optimum concentration and addition time for increasing the yield of Mogrosides V.(2)Screening of carbon,nitrogen and phosphorus sources,limiting factors were found.(3)Establishing the additive mode to expansion cultivation of 5L bioreactor.(4)The physiological and biochemical responses of Siraitia grosvenorii cells under salt stress were studied,and the possible pathway for the synthesis of Mogrosides V was analyzed in conjunction with the concentration changes.The results are as follows:1.The effect of elicitors on the growth of Siraitia grosvenorii cells and the synthesis of Mogrosides VConcentrations of phenylalanine,methyl jasmonate,salicylic acid,betain,chitosan and NaF were measured.Furthermore,the the additional time and selectinon of the appropriate elicitor for orthogonal optimization were also studied.The results of the study indicated that the single-factor experiments showed the induction of MJA,SA,Phe was superior.After orthogonalizing,the following results were obtained:being added on the 6th day and the concentration was MJA 120 ?mol/L+SA 40 ?mol/L+Phe 100 ?mol/L,and the yield reached 0.83 mg/g DCW,which was about 10 times as compared with the blank.Moreover,the two routes of precursor feeding and inducer addition can significantly promote the synthesis of Mogrosides V.2.Siraitia grosvenorii culture process optimization and reactor amplification cultureThrough screening of carbon,nitrogen,phosphorus sources,the main influencing factors and concentration ranges were obtained,and then culture modes were established in two bioreactors.The results showed that carbon(Sucrose),nitrogen(NH4NO3),and phosphorus(NaH2PO4)sources had a significant impact on the growth of Siraitia grosvenorii cells,especially nitrogen source,which was a major influencing factor and selected a suitable range of nitrogen sources:30-150 mg/L.When the concentration of NH4NO3 was 150 mg/L,the cell biomass and specific growth rates reached the maximum value,which were 7.15 g/L and 0.0601 d-1,respectively.However,exceeding 450 mg/L or lowering 30 mg/L,cell growth was inhibited.Comparing the 5L bubbling and stirring bioreactor experiments,it can be seen that on the 21st day,the biomass in stirring bioreactor was 2.8 times that of the initial inoculum,reaching 140 g/L;and the biomass in bubbling type was 2 times that of the initial,reaching only 100 g/L.Therefore,the stirring bioreactor is more suitable for Siraitia grosvenorii cells.The bubble force of the bubbling bioreactor is too small and the cells tend to agglomerate,affecting the mass transfer.3.To study the effects of salt stress on the growth of suspension cells of Siraitia grosvenorii and the synthesis of Mogrosides VThe change of K+/Na+ ratio in the initial culture medium has a great influence on the growth and physiological response of the cells.Results showed that the optimum ratio of K+to Na was 15:1 for biomass formation and Mogrosides V biosynthesis,more or less than 15:1 of K+/Na is not beneficial for the cell growth and Mogrosides V production,which were mainly caused salt osmolarity stress.Furthermore,under the optimized condition of K+/Na+,the biomass of Siraitia grosvenorii cells was the highest,reached 11.67 g/L,and the yield of Mogrosides V was also the highest,which was 0.42 mg/g DCW,which was 2 and 1.4 times that of the control,respectively.The peak of Kcull+(mg/Kg DCW),K+/Na+,S(K,Na)are obtained with the highest utilization efficiency of K and the maximum specific cell growth rate(?,d-1).Based on the results of intermediates like organic acids(malate,formate)and amino acids(alanine and branched chain amino acids)produced by sucrose through EMP,HMP and TCA,it is inferred that the metabiloc flux intensity of TCA would be attenuated when Siraitia grosvenorii cells are cultivated under this osmolarity stress condition.However,the flux through MEP and MVP would be intensified,leading to the precursor acetyl CoA utilization for the production of Mogrosides V.