Homologous Expression Of ω9Fatty Acid Desaturase From Oleaginous Fungus Mortierella Alpina And Its Effect

Mortierella alpina is one of the most important industrial production strains forpolyunsaturated fatty acids (PUFAs), in which the fatty acid metabolism pathway has beenextensively studied. The annotation of genome reveals that there are three distinct genesencoding Δ9fatty acid desaturase. Unlike Δ9-I and Δ9-II, ω9desaturase gene (FADS9-III)displayed negligible transcriptionally activity under culture conditions, which indicated thatω9fatty acid desaturase wasn’t involved in the Δ9desaturation during the cultivation period.Thus, the intrinsic effect and its substrate specificity remain to be determined. Its significanceof existence is worth studying.In this study, the ω9fatty acid desaturase gene (FADS9-III) from M. alpina ATCC32222was cloned and homologously expressed. The effect in vivo of ω9fatty acid desaturase and itssubstrate specificity in the unsaturated fatty acid synthesis were determined. The temperatureeffect on the ω9desaturation was also preliminarily investigated. The activities of ω9fattyacid desaturase with long-chain saturated fatty acid substrates were studied through specificfatty acids supplementation.Firstly, the expression vector pBIG2-ura5s-FADS9-III was successfully constructed onthe basis of pBIG2-ura5s and pET28a-ITs. Recombination strain of A. tumefaciens C58C1was obtained after electro-transformation.Secondly, the gene FADS9-III was successfully intergrated into the genome of uracilauxotrophic M. alpina by A.tumefaciens-mediated transformation (ATMT). The optimalATMT condition was200μM AS, OD600=0.8of the bacterial cell suspension concentration,co-cultivation at23°C and co-cultivation duration for32h.Thirdly, stable transformants were obtained through mitotic stability tests and PCRverification. Growth rhythms of the recombination strain was also described. RT-qPCR andfatty acid analysis revealed that the transcriptional level of FADS9-III increased and ω9fattyacid desaturase showed certain activities, which indicated the homologous expression of ω9fatty acid desaturase.Fourthly, the effect on fatty acid profile by homologous expression of ω9fatty aciddesaturase was studied. Fatty acid analysis revealed that ω9fatty acid desaturase hadmoderate activities with both palmitic acid (C16:0) and stearic acid (C18:0) as substrates.Palmitoleic acid (C16:1) and oleic acid (C18:1) contents increased3.25-and1.95-fold in ω9over-expressing clones compared to the control strain, reached to0.39%and38.33%of totalfatty acids, respectively. Also, the expression of ω9fatty acid desaturase had little impact onthe contents of other fatty acid components. Thus, the extent of fatty acid unsaturation in cellswas improved. The temperature effect on the ω9desaturation was investigated by shaking flask culture at12°C for216h, which indicated that low temperature condition may enhanceω9fatty acid desaturase activity. The C16:1content (1.34%) increased3.44-fold and theconversion rate of C16:0was raised by9.24-fold. The C18:1content also rose to42.48%oftotal fatty acids. A small amount of C24:1(ω-9)(nervonic acid) was detected in ω9geneover-expressing strains with its content of0.50%of total fatty acids.Lastly, substrate specificities of ω9fatty acid desaturase with long-chain saturated fattyacids were examined. Both the recombination strain and prototrophic strain were able toabsorb exogenous substrates (C20:0、C22:0、C24:0). In contrast with the control strain, thecontent of C20:1didn’t change and no C22:1was detected in the over-expressing strain.However, the yield of C24:1(ω-9) could improve by5-fold with its content reached2.50%oftotal fatty acids, which indicated that ω9fatty acid desaturase may display specific activitieswith C24:0or longer-chain saturated fatty acid substrates.