Mutation Breeding Of High Arachidonic Acid Production Strain And Optimization Of Its Fermentation Conditions

Arachidonic acid(ARA) belongs to the ω-6 group of polyunsaturated fatty acids(PUFAs), which plays an important role in maintaining physiological activities of human body.Mortierella alpina can accumulate substantial quantities PUFAs and have been identified as optimal strains for ARA production. To obtain strains with higher ARA yields, M.alpina was mutated combined with targeted screening strategy. After optimizing fermentation conditions,the ARA production and productivity of this mutated strain were further improved. The main contents of this paper were as follows:Firstly, the basic characteristics and fermentation conditions of the original strain were studied.The dynamic change of key parameters included biomass, lipid and ARA and fermentation period were grasped through the determination of fermentation curve. The influences of different initial glucose concentration on fermentation results were also studied and the initial glucose concentration of 50 g/L was optimal for ARA fermentation.Furthermore, the mycelium morphology in seed culture was controlled through p H level and spores inoculation concentration, which ensure the stability of the fermentation results.Secondly, the original strain was treated with two rounds of mutation combining compound screening strategy. The first round of mutation-screening process was atmospheric and room temperature plasma(ARTP) coupled with FAS inhibitor, cerulenin. The isolated mutant with high ARA production obtained by ARTP mutagenesis was mutated by DES and isolated with TTC, an indicator of desaturase. Based on the efficient screening strategy targeting the key enzymes of ARA biosynthesis, the mutant D20 was isolated. The average biomass, lipid and ARA production were 26.58 g/L, 11.20 g/L and 5.12 g/L, respectively, a40.61% increase of ARA production over the original strain. The mutant D20 has optimal genetic stability and the relative ARA percentage of total fatty acids was enhanced from38.99 % to 45.46% compared with the original strain under identical conditions.The fermentation condition of the mutant was optimized, includingthe nitrogen source and fed-batch culture conditions.When the culture was fed 10 g/L glucose once a day from the second day to the fifth day, the final ARA yield was the highest(6.54 g/L).The maximum glucose uptake rate and biomass were obtained using soy flour as the sole nitrogen source.However, yeast extract as the sole nitrogen source gave the highest nitrogen uptake rate, lipid and ARA production. A mixture of yeast extract and soy flour(1:2, w/w) gave the highest ARA yield(6.82 g/L). A certain amount of Mg SO4、KH2PO4 and Ca Cl2 enhance ARA yield and the addition of Na2SO4 had little effect on ARA production. Experiments also found that the kind and concentration of minerals have no obvious effects on cell morphology.Finally, the effects of p H and various fed-batch modes on ARA fermentation were investigated in a 5 L fermentor. In the whole fermentation process, the optimal p H for both cell growth and ARA production were quite different. Following a kinetic analysis of the whole fermentation process, a two-stage p H control strategy was developed in which the p H was maintained at 5.5 for the first 48 h and then shifted to 6.5 till the end of fermentation.Using this strategy, a maximum ARA production of 8.12 g/L was achieved, which was higher than that of the best results achieved under aconstant p H condition(7.43 g/L of ARA at p H6.5). On the basis of p H control, the effects of fed-batch cultures on ARA productivity were further investigated. A two-stage variable speed glucose feeding strategy was suited for the growth characteristics of M.alpina. A maximum ARA productivity of 1.40 g/L/d was obtained,which was an increase of 38.61% over the productivity obtained via batch fermentation with the two-stage p H control strategy.