Heterologous Expression Of Δ12 Fatty Acid Desaturase In Geotrichum Candidum And Its Application In Mold-ripened Cheese

Mold-ripened cheese contains rich nutrients and its lipid content is up to 30.0%,but it lacks unsaturated fatty acids（UFAs）.Geotrichum candidum is the dominant strain in mold-ripened cheeses,but it has low ability to produce high level of essential fatty acids.Linoleic acid（LA）andα-linoleic acid（ALA）,as essential fatty acids,are the precursor fatty acids for de novo biosynthesis ofω3/ω6 polyunsaturated fatty acids,whileΔ12 fatty acid desaturase（FADS12）plays an important role in the biosynthesis of LA and ALA.Mucor circinelloides,an oleaginous filamentous fungus producing commercialγ-linolenic acid,is capable of accumulating 28.3%oleic acid（OA）and in which the conversion rate of FADS12 reaches 67.0%,the highest OA conversion rate in wild-type fungi.Furthermore,M.circinelloides has a clear genetic background and a long history of gene manipulation,which offers a solid basis for the heterologous expression of the FADS12 gene from M.circinelloides in G.candidum to improve LA production.Therefore,through the expression of exogenous gene in G.candidum to enhance the essential fatty acid biosynthesis,and apply it to make mold-ripened cheese for increasing UFA levels,which is of great significance and application value.In this study,the catalytic activity and function of FADS12s from G.candidum and M.circinelloides were analyzed,and the FADS12 gene was heterologously expressed in G.candidum to obtain high-yielding LA recombinant strain.Moreover,the recombinant strain was used to make mold-ripened cheese and increased the level of UFAs in the cheese.The main results are as follows:1.Study on catalytic activity and function for FADS12:The heterologous expression vectors for Gc FADS12 and Mc FADS12 were constructed under the skeleton of plasmid p YES2/NT C and expressed in Saccharomyces cerevisiae.By adding exogenous substrate（OA or LA）,the activity and function of two FADS12s were determined.The results showed that Gc FADS12 was a bifunctional enzyme desaturating both OA and LA at different concentrations,but showed substrate preference for OA and its conversion rate was 25.0±0.4%,while the Mc FADS12 can only catalyze OA,and its catalytic activity was up to 67.8±1.0%.2.Heterologous expression of Mc FADS12 gene in G.candidum:First,a plasmid harboring the Mc FADS12 gene was constructed under the skeleton of plasmid pc DNA3.1（+）and overexpressed in G.candidum using electroporation method;after cerulenin-triphenyltetrazolium chloride screening and characterization of transformants,Gc-Mc FADS12 recombinant strains were obtained.Subsequently,the fatty acid profile of G.candidum transformants was determined.The results showed that Mc FADS12 gene was successfully expressed in G.candidum,and its relative transcript level was 3.5 times higher than that of the control strain and the level of LA was reached 31.1±1.4%,which resulted in a 2.8-fold increase compared to LA level in wild-type G.candidum.3.Essential fatty acid production in Gc-Mc FADS12 recombinant strains:The effects of different carbon/nitrogen sources,temperature,p H and culture time on biomasss,LA,and ALA production in the recombinant Gc-Mc FADS12 were investigated.In addition,the free form fatty acid and triglyceride form fatty acid were supplemented to investigate EFA production.The results showed that the LA production grown on maltose and urea were up to（124.9±4.3）mg/L and（123.8±10.8）mg/L,respectively,which were significantly higher than that grown on other carbon/nitrogen sources.The ALA yield reached（20.0±1.3）mg/L and（23.8±1.9）mg/L when grown in maltose and urea.The LA and ALA production reached（143.4±8.5）mg/L and（27.6±2.2）mg/L in recombinant Gc-Mc FADS12 grown at 30°C,respectively.However,the LA and ALA production grown on other culture conditions were no more than carbon/nitrogen source.To raise the level of LA,free form fatty acid was used as exogenous substrate,and 0.250%OA increased the LA production up to（154.2±5.7）mg/L.Considering the cost of LA production in G.candidum and the stability of free form substrate,rapeseed oil was used as source of OA.When recombinant Gc-Mc FADS12 was grown on 0.45 g/L rapeseed oil,the highest LA yield was up to（188.9±5.8）mg/L.4.Quality characteristics and fatty acid profile of mold-ripened cheese fermented with recombinant Gc-Mc FADS12:The recombinant Gc-Mc FADS12 was cultivated under high LA-producing conditions and used for cheese manufacturing.The sensory properties,physico-chemical（moisture,p H,ash,protein,and fat）and technological parameters（color,texture profile,taste properties and fatty acid profile）were determined during ripening.The results showed that mold-ripened cheese fermented with recombinant Gc-Mc FADS12 had high sensory acceptance in middle ripening;the p H,moisture,protein,and fat content of the cheese were gradually decreased,while the ash content was constant increased with the extension of ripening time.In the color,the L^*color parameter first decreased and then tended to be stable,and the a^*and b^*color parameters were gradually increased.During the ripening period,the hardness and chewiness of cheese increased steadily,the viscosity of cheese fluctuated significantly,and the changes of cohesion and springiness of cheese were relatively slow.As the increase of ripening time,the saltiness of cheese was gradually increased,while the sweetness and sourness was gradually decreased,and the umami first increased and then decreased.On the 30^th day of ripening,the level of LA reached（7.4±0.4）g/100 g and UFAs accounted for 39.1±2.6%of the total fatty acids,after supplementing okara and fermented,the level of LA was improved to（12.6±0.4）g/100 g and the ratio of UFAs to saturated fatty acids was up to 1.3:1.