Selection Of High-yielding Gamma-linolenic Acid Mutant Strains Of Mortierella Isabellina Based On Metabolic Control Breeding

Gamma linolenic acid (GLA), which is one kind of essential polyunsaturated fatty acids（PUFA） inthe human body, is a precursor of the prostaglandin and interleukins, which have a wide range ofphysiological functions and significant pharmacological effects. Compared with the method of extractionfrom animals and plants, the production of PUFAs by microorganism fermentation shows its obviousadvantages and potentials, therefore, it is getting more and more attention from people.The selection model of resisting malonic acid method and TTC method was established under the keypoint of metabolic regulation of Mortierella Isabillina. A high-yielding mutant strain MortierellaIsabillina U-0116producing lipid was successfully obtained, after the original strain MortierellaIsabillina AS3.3410was mutated by ultraviolet, and was screened by the primary screening method ofresisting malonic acid and the rescreening method of ultrasonic-assistant extraction taken high lipidcontents as indexes. When cultivated in500-mL shaking flask, the biomass, the content of lipid, the lipidyield and the GLA yield of Mortierella Isabillina I-0116reached19.86g/L,70%,13.92g/L,699.70mg/L, and as against the original strain, increased by18.85%,16.67%,38.58%,26.27%, respectively.The results of N+ions implantation experiment showed that the optimal implantation dose was48×1014ions/cm2when the N+ions implantation energy was30KeV. Mutagenizing MortierellaIsabillina U-0116on this optimal condition, a high-yielding mutant strain Mortierella Isabillina I-0281producing GLA was obtained by the screening method of TTC and the rescreening method of gaschromatograph. The GLA yield of Mortierella Isabillina was899.80mg/L, up to60.27%greater thanthe original strain.Single factor experiments and central composite design were used to optimize the fermentationmedium for producing GLA of Mortierella Isabillina I-0281by500-mL flask. The results showed thatthe optimum condition was as follows: the concentration of glucose was77g/L, the concentration ofyeast extract was0.45g/L, the concentration of potassium nitrate was0.98g/L, the concentration ofMgSO4was0.2g/L, the concentration of potassium dihydrogen phosphate was2g/L, the concentrationof sodium citrate was2g/L, and the initial pH was6.0.The impact of exogenous substances such as citric acid, acetic acid, malic acid, tween-80, betaine onMortierella Isabellina fermentation on the biosynthesis of GLA showed that：the addition of0.02%malic acid in the fourth day was beneficial to the improvement of the biomass, but had no obviousimpact on the lipid content and GLA/TFA，and the GLA yield increased by7.16%. Neither the additionof0.02%acetic acid nor malic acid in the fifth day had obvious effect on biomass, while both of thembenefited the synthesis of lipid, increasing6.26%and10.24%GLA yield, respectively. Appropriatedosage of tween-80contributed to improve biomass, lipid yield and GLA yield. When the addition oftween-80was0.21%, GLA yield reached maximum output, increasing10.91%in comparison with thecontrol. Low concentration of betaine had no obvious impact on the growth of the Mortierella Isabellina,while high concentrations of betaine had obvious inhibitory effect on cell growth and the accumulation oflipid, what was worthy to mention was that GLA/TFA increased instead.