Metabolic Process Analysis And Metabolic Regulation Fermentation Of Astaxanthin Producing By Phaffia Rhodozyma

Astaxanthin,a kind of carotenoid,has been extensively used in food,feed,cosmetic and pharmaceutical industry because of its property of antioxidation,antitumor and enhancing immunity.Phaffia rhodozyma is an important natural resource for astaxanthin production,which has some advantages such as shorter period of fermentation,easy to culture in a high density,and so on.However,it still can't meet the increasing industrial deamand as astaxanthin productivity is low in wild strains of Phaffia rhodozyma.In order to improve the production of astaxanthin further,five strains of Phaffia rhodozyma preserved in our laboratory were used to studied according to following aspects.Above all,the fermentation characteristics of the five Phaffia strains were studied,and results were shown as following.Firstly,it was found that JMU-MVP14 was an astaxanthin over-producing strain,with highest production of total carotenoids of 40.06 mg/L,ten times of the production compared with the other four strains,and JMU-17 W was a strain of the highest biomass,which reached 10.83 g/L.In addition,total carotenoids were analyzed by HPLC after fermentation for 120 h,and it was lieing a significant difference in the composition and content of total carotenoids.Moreover,metabolic flux analysis was done to the five strains of Phaffia rhodozyma,and the results were shown as follows: improving the flux of EMP together with an appropriate controlling on the flux of HMP was in favour of the synthesis of astaxanthin.Appropriate enhancement of TCA cycle could offer energy for the synthesis of astaxanthin.But,an excess of carbon source going to TCA cycle would cause energy wastage,resulting a low production of astaxanthin.Energy could also be lost with the synthesis of ethanol.Controlling the synthesis of ethanol would reduce the loss of energy and was good for the synthesis of other metabolite.The process pyruvic acid to generate acetyl-CoA offered precursor for the synthesis of astaxanthin.Increasing the flux of this approach was in favour of the synthesis of astaxanthin.Later,the strains of JMU-VDL668 and JMU-MVP14 were used as the starting strains to conduct metabolic control breeding,and 14 mutant strains were obtained as a result.After studied the fermentation characteristics of the 8 mutant strains,the metabolic flux analysis of the JMU-15 V,JMU-VP471,JMU-15 M and JMU-15MP4 strains were investigated.It was shown that the flux of EMP,HMP and TCA cycle increased in the strain of JMU-15 V,but the ethanol synthesis pathway was basicly blocked.At the meantime,the flux of EMP,TCA cycle and ethanol synthesis pathway in the strain of JMU-VP471 increased by various extents,but only,the flux of EMP was inhibited.Besides,the flux of the HMP and EMP decreased in both JMU-15 M and JUM-15MP4 strains,while the flux of TCA cycle and the acetyl-CoA synthetic way increased,which can provide plenty of energy and precursor chemicals for the synthesis of the astaxanthin.Finally,to further improve the production of astaxanthin in Phaffia rhodozyma,we carried out a single and combination of metabolic regulation of regulating fermentation research for original strain JMU-VDL668 and JMU-MVP and its corresponding mutants.After the regulated fermentation of combination,the optimal combination for each strain was selected,and made the fermentated metabolic flux analysis in the course of the regulation period during at 20-24 h.It was shown that the EMP flux of the majority strains have increased when the sodium gluconate,which is the accelerator of EMP,was added to the medium for combinational regulation fermentation,except the EMP flux of JMU-MVP14 strain.The HMP flux of JMU-MVP14 strain has decreased when the sodium phosphate,which is the inhibitor of HMP,was added to the medium.However,the HMP flux of JMU-15MP4,JMU-VDL668 and JMU-VP471 strains have significantly increased.Therefore,the effect of combinational regulation is not the simple superimposition of several metabolic regulating agents,the function of each kind of metabolic regulating agent will change for different combinational regulations or strains.