| Long chain omega(ω)-3 polyunsaturated fatty acids(PUFAs),such as α-Linolenic acid(ALA),Stearidonic acid(SDA),Eicosatetraenoic acid(ETA),Eicosapentaenoic acid(EPA)and Docosahexaenoic acid(DHA),play an important role as structural components of the phospholipids in cellular membranes with high commercial values.Mucor circinelloides was the first oleaginous microorganism to be used in industrial production of γ-Linolenic acid(GLA),and the high-lipid-producer strain M.circinelloides WJ11 is able to accumulate lipids content up to 36% of its cell dry weight(CDW)and 13% of GLA content of total fatty acids.Due to lacks C20 desaturase and elongase,it cannot synthesize the C20 very-long-chain PUFAs.However,the genetically engineering strain WJ11 has been reported to produce dihomo-γ-linolenic acid(DGLA,20:3,n-6)by our research team.Therefore,in this thesis we elucidated the biosynthetic pathways of very-long-chain PUFAs,and integrated some key desaturase genes to construct a suitablae cell factory for ETA and EPA production by expression of Δ17 and Δ5 desaturase genes,respectively.The heterologous expression of Δ17 and Δ5 desaturase genes in the recombinant strains was studied,and their effects on the growth,lipid accumulation and fatty acid composition of M.circinelloides were analyzed.The main findings of the study are as follows:(1)The Δ17 fatty acid desaturase gene was cloned from Pythium aphanidermatum and Phytophthora parasitica,respectively,and the recombinant strains Mc-Pa D17 and Mc-Pp D17 were successfully constructed,and construct a suitablae cell factory for ETA production.The biomass of the recombinant strains Mc-Pa D17 and Mc-Pp D17 after 96 h of fermentation was up to 25.98 g/L and 23.34 g/L,respectively,which were increased by 40.58% and 26.29%,respectively,compared with the control strain,and the total fatty acid content also increased by23.52% and 32.38%,respectively,compared with the control,and a new long-chain PUFA-ETA was detected in the recombinant strain Mc-Pp D17,up to 108.74 mg/L.(2)The Δ5 fatty acid desaturase gene was cloned from Thraustochytrium,Mortierella alpina and Phaeodactylum tricornutum,and the recombinant strains Mc-TSD5,Mc-MAD5 and Mc-PTD5 were successfully constructed,and construct a suitablae cell factory for EPA production.After 72 h fermentation,the biomass of recombinant strain Mc-MAD5,reached a maximum of 20.60 g/L,which was 5.21% higher than that of the control strain,lipid content was also increased by 22.07%,and the production of ETA and EPA was also detected with a content of 31.26 and 77.72 mg/L.After 96 h of fermentation of recombinant strain Mc-TSD5,its biomass reached at maximum level,which increased by 18.69% and lipid content by 16.55% compared to the control,while ETA and EPA were also detected at 22.38 and 54.45 mg/L,respectively.After 96 h of fermentation of the recombinant strain Mc-PTD5,its biomass reached a maximum of 11.03% and lipid content increased by 7.10% compared to the control,while the yields of ETA and EPA were also detected 24.05 and 63.24 mg/L,respectively.(3)The recombinant strains Mc-MAD5-MAD5 and Mc-Pp D17-Pp D17 were constructed by double overexpression of double copy of Δ17 and Δ5 desaturases,which are better expressed,by linking the genes together through T2 A,after 96 h of fermentation the biomass of the recombinant strain Mc-MAD5-MAD5 reached 21.54 g/L,which was 16.56% higher than that of the control strain,while lipid content reached 28.04%,which was 14.82% higher than the control,and increased 4.86% compared with the single copy overexpression strain,and the yield of ETA and EPA reached 10.62 and 18.72 mg/L.In the recombinant strain Mc-Pp D17-Pp D17,the biomass was increased by 29.76% after 96 h of fermentation,compared with the control,and there is no significant changes were observed on its lipid content compared with the control,while the yield and proportion of ETA decreased compared with the single copy overexpression strain,and the yield of ETA was only 59.87 mg/L.(4)The strain with the highest ETA yield from the above experiments was fermented under different fermentation conditions,and the effect of different fermentation conditions on the growth and lipid composition of M.circinelloides was investigated.When the fermentation temperature was reduced,the total fatty acid content of Mc-Pp D17 decreased significantly,but the proportion of ETA in the total fatty acid increased to 3.71% when the fermentation temperature was 24℃.At 10% dissolved oxygen levels for fermentation,the growth of the recombinant strain was inhibited,and its biomass was significantly reduced compared with the fermentation without controlled dissolved oxygen,and subsequently lipid yield was also significantly reduced.The percentage of ETA in the total fatty acids increased slightly,but its yield was only 48.22 mg/L.When GLA was added for fermentation,the percentage of ETA in the total fatty acids increased slightly,but its biomass did not change significantly,and the lipid content decreased significantly,and the yield of ETA was only 72.95 mg/L.In summary,this study has successfully constructed a recombinant strain of M.circinelloides for synthesizing ETA and EPA fatty acids by overexpression exogenous genes into M.circinelloides through the genetic engineering strategy,which laid the foundation for further construction of ETA and EPA industrial production cell factories. |
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