| γ-linolenic acid is an important polyunsaturated fatty acid that carries out many important physiological function in genital system, immune system, endocrine system and cardiovascular system. The traditional sources of γ-linolenic acid are the seeds of Onagraceae, Boraginaceae and Saxifragaceae plants. However, γ-linolenic acid extracted from plant seeds is easily influenced by region, climate and seasons, resulting in their quantity and quality fluctuating. It can not meet the demand of the increasing market.Mucor circinelloides belongs to oleaginous fungus which accumulate γ-linolenic acid and stores in its cell. In the early 1980 s, there was a precedent of producing γ-linolenic acid by M. circinelloides. To improve the industrial potential of M. circinelloides, and find out the key factor of γ-linolenic acid synthesis pathway in M. circinelloides, in this study, research work has been carried out as follow.Firstly, Δ12, Δ6-1 and Δ6-2 fatty acid desaturases genes were amplified from M.circinelloides CBS 277.49, which were then cloned into the plasmid p MAT1552 to construct expression vectors p MAT-Delta12, p MAT-Delta6-1 and p MAT-Delta6-2. These vectors contained pyr G gene which code for orotidine-5’- monophosphate(OMPdecase), was integrated into M. circinelloides genome at Car RP gene site. The replacement fragments were sequences upstream and downstream of Car RP gene. Both replacement fragments were 1000 bp.Secondly, after protoolasts preparation and electroporation protocols optimization, Δ12, Δ6-1 and Δ6-2 desaturases were transformed into M. circinelloides with improved methods. The transformants were named Mc12, Mc61 and Mc62, and the integration of target genes were verified by PCR. Flask culture experiments showed that the knock out of Car RP gene due to the homologous integration did not influence the biomass and lipid accumulation.Thirdly, transcription analysis showed that transcription level increased remarkably in the transformants. Fatty acid composition analysis showed that the overexpression of Δ12 fatty acid desaturase led to the decline of oleic acid, increase of linoleic acid by 41%, and slightly decrease of γ-linolenic acid. The results suggested that the concentration of linolenic acid in the wild type fungus is saturated for Δ12 fatty acid desaturase, and the overexpression of Δ12 fatty acid desaturase may regulate the expression of Δ6 fatty acid desaturases. Overexpression of two types of Δ6 fatty acid desaturases remarkably increased the γ-linolenic acid content, by 37% with Δ6-1 desaturase and 18% with Δ6-2 desaturase. It proved that Δ6-1 desaturase played a more important role in γ-linolenic acid production than Δ6-2 desaturase. The γ-linolenic acid content reached up to 43% of total fatty acids, when Mc61 grew in a fermentor by preliminarily optimized condition.Lastly, Δ12 and Δ6 fatty acid desaturases were co-expressed in the fungus, and γ-linolenic acid production in the mutant strain were analysized. Δ12 desaturase gene was cloned to plasmid p LEU4, to construct the expression vector p LEU-Delta12, which contained Leu A gene to cover the leucine auxotroph. Together with p MAT-Delta6-1, both plasmids were transformed into Mu402, a leucine and uracil double auxotroph strain derived from R7 B. The Δ12, Δ6-1 fatty acid desaturases co-expressed strain was named as Mc1261. Fermentation data showed that overexpression of both Δ12 and Δ6-1 fatty acid desaturases in the same strain(Mc1261) declined the biomass slightly, but had no effect on lipid accumulation, when cultivated in flasks. In Mc1261, Δ12 and Δ6-1 desaturases were transcripted by same promoter with same transcription level. Compared with the control strain, linoleic acid and γ-linolenic acid increased remarkably. However, the increase were less than overexpressing each fatty acid desaturase. It was possible that there’s mutual regulation of the expression of Δ12 and Δ6 fatty acid desaturases. |
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