Study On Technology Of Astaxanthin Production And Regularization Of Metabolism By Phaffia Rhodozyma

Phaffia rhodozyma is the most potential microbial resource of industrial mass production of natural astaxanthin.However,low-production of astaxanthin restricts its large-scale fermentation production for industrial application.In the present work,the reutilization of the fermentation supernatant was studied and the fed-batch process with ethanol was subsequently established during Phaffia rhodozyma fermentation.Then,the metabolic mechanism of astaxanthin synthesis promoted by ethanol was explored by means of metabolic flux analysis(MFA).Finally,astaxanthin metabolism was regulated by key enzyme inhibitors and ethanol to understand astaxanthin synthesis of Phaffia rhodozyma in detail The main conclusions were as follows:The fermentation supernatant was used to cultivate astaxanthin-producing strain Phaffia rhodozyma JMU-VDL668 and JMU-MVP14.As a result,astaxanthin yield of JMU-VDL668 was increased by 28% with 50% supernatant and the yield was increased by 35% with the complete medium,while the yield of Phaffia rhodozyma JMU-MVP14 reached 57.98 mg/L with the addition of 100% supernatant,as well as a yield of 61.51 mg/L was achieved with the complete medium.Repeated fed-batch in flasks for five batches could basically ensure the production of astaxanthin during the fermentation process.As repeated fed-batch in 7-l bioreactor for two batches,the astaxanthin yield of Phaffia rhodozyma JMU-VDL668 was increased by 38%,while the astaxanthin yield in Phaffia rhodozyma JUM-MVP14 was increased by 63% with a final harvest of 153.88 mg/L.Low concentration of ethanol could improve the ability of astaxanthin synthesis of the two strains,while relatively high concentration of ethanol inhibited the cell growth.After 5g/L ethanol was added in the Phaffia rhodozyma JMU-VDL668 culture,astaxanthin yield was 2.4 times higher than the control group.Meanwhile,a final astaxanthin production of 55.32 mg/L was reached when ethanol concentration was maintained at 2 g/L and glucose was supplemented every 24 h during Phaffia rhodozyma JMU-MVP14 fermentation,which was 38.4% higher than the control.What's more,compare with the cycle-flow supernatant in 7-l bioreactor,it was found that low concentration of sugar combined with ethanol addition was more favorable for cell growth and astaxanthin accumulation,in which astaxanthin yield of Phaffia rhodozyma JMU-VDL668 was increased by 2.7 times.By means of MFA,it was found that ethanol could promote the flux of Phaffia rhodozyma at the metabolic regulation points of pyruvic acid and acetyl coenzyme A,resulting in improvement of the astaxanthin synthesis.As ethanol was consumed by yeast cells,,the fluxes of JMU-VDL668 strain were increased at the metabolic regulation points of acetyl coenzyme A,?-ketoglutarate,and 5-ribulose phosphate.According to the MFA results,amino acids were used to regulate astaxanthin synthesis.regulating the metabolic points of ?-ketoglutarate and 5-ribulose phosphate with ?-ketoglutarate stimulated astaxanthin accumulation in both strains.Similarly,the biomass of Phaffia rhodozyma JMU-VDL668 could reach 8.2 g/L and the astaxanthin yield of Phaffia rhodozyma JMU-MVP14 was up to 67.88 mg/L when 3 g/L of glutamate was added in medium,which was 1.7 times higher than that of control group.Five specific inhibitors of the key enzyme in astaxanthin biosynthesis pathway were selected to regulate fermentation process of Phaffia rhodozyma.It can be found that the addition of nicotine and isoniazid enhanced the intracellular content of ?-cryptoxanthin in Phaffia rhodozyma.Likewise,the intermediate carotenoids in astaxanthin biosynthesis pathway of Phaffia rhodozyma JMU-MVP14 were totally converted to astaxanthin after simvastatin addition,and the proportion of astaxanthin was increased from 82.37% to 98.64%.The combined addition of inhibitor and ethanol in the culture medium was studied and revealed that ethanol showed a remedial effect on the metabolic inhibition induced by simvastatin and N-methyl morpholine,while ethanol combination with nicotine or ?-ionone showed superimposed inhibitory effects.