Research On Fermentation Process Of Arachidonic Acid Production

Arachidonic acid(AA;5,8,11,14-cis-eicosatetraenoic acid) belongs to omega-6 class polyunsaturated fatty acids(PUFAs).As a precursor of prostaglandins,thromboxane,prostacyclin,and leucotrienes,it owns various physiological functions and has been found wide application in medicine, pharmacology,cosmetics,food industry,agriculture and other fields.Production of AA by cultivation of Mortierella fungi,which are thought to be the most prominent AA source,has been widely reported and industrialized.However,several key problems such as the hardness to control the AA percentage in lipids,high content of saturated and monounsaturated fatty acids and low conversion ratio of glucose to AA are not solved,which lead to high market price of AA.This maybe caused by the complicated fatty aicds biosynthesis mechanism and traditional methods' lack of metabolic network analysis on AA fermentation.In the present work,metabolic engineering was introduced to analyze the mechanism of AA synthesis in Mortierella alpina ME-1,and several strategies were applied to enhance AA production.1.Optimization of media components for biomass and arachidonic acid production by Mortierella alpina ME-1 using response surface methodologyThe media for biomass and arachidonic acid production by Mortierella alpina ME-1 were optimized in shake flask cultures using response surface methodology (RSM).Two imperial polynomial models were developed,and the maximum biomass yield of 36.86 g/L appeared at glucose,yeast extract(YE),KH₂PO₄ and NaNO₃ of 90.16,12.50,3.80 and 3.54 g/L,respectively,while a maximum AA yield of 9.65 g/L appeared at glucose,YE,KH₂PO₄ and NaNO₃ of 103.16,11.66,3.80 and 3.43 g/L,respectively,in 6.5-day fermentation were predicted.These predicted values were also verified by validation experiments which showed excellent correlation between predicted and measured values.The results of bioreactor fermentation also illustrated that the optimized culture medium enhanced both biomass(34.21±1.01 g/L in 5-day fermentation) and AA(9.86±0.45 g/L in 6-day fermentation) production by Mortierella alpina ME-1 in a large-scale fermentation process.2.Metabolic flux analysis on arachidonic acid FermentationAnalysis of flux distributions in metabolic networks has become an important approach for understanding the fermentation characteristics of the process.A model of metabolic flux analysis of arachidonic acid(AA) synthesis in Mortierella alpina ME-1 was established and carbon flux distributions were estimated in different fermentation phases with different concentrations of N-source.During exponential, decelerating and stationary phase,carbon fluxes to AA were 3.28%,8.80%,6.97%, respectively,with sufficient N-source broth based on the flux of glucose uptake,and those were increased to 3.95%,19.21%and 39.29%,respectively,by regulating the shifts of carbon fluxes via fermentation with limited N-source broth and adding 0.05%NaNO₃ at 96h.Eventually AA yield was increased from 1.3 g/L to 3.5 g/L. These results suggest a way to improve AA fermentation,that is,fermentation with limited N-source broth and adding low concentration N-source during stationary phase.3.Dynamic flux analysis model for arachiconic acid fermentationKinetic models of arachidonic acid fermentation by Mortierella alpina ME-1 with limited N-source and glucose were established based on the experimental data. Then a dynamic flux analysis model based on the kinetic models was proposed according to the theory of metabolic flux analysis.And the optimal parameters were evaluated.Further experiment data verified the accuracy of the models and the results showed that the models appeared to provide a reasonable description for the fermentation,and the flux distributions in different time can be easily obtained.