Heterologous Expression Of Acyl-ACP-Δ9 Desaturase Derived From Cat’s Claw Promotes ω-7 Fatty Acid Biosynthesis In Chlorella And Tobacco Leaves

Omega-7 fatty acid(ω-7 FA)is a kind of unusual monounsaturated fatty acid,which contains only one double bond located at the seventh carbon atom from the methyl end of fatty acid chain(also named n-7 fatty acid.Omega-7 fatty acids mainly include palmitoleic acid(C16:1Δ9),cis-11-octadecenoic acid(C18:1Δ11)and cis-11-octanolenoic acid(C20:1Δ13),which have unique functions and many applications in food and nutrition,medicine and healthcare,biofuels and oleochemical industry.Omega-7 fatty acids Just highly synthesized and accumulated in some wild plant seeds,whereas such valued fatty acids are extremely low(<2%)in common oil crop seeds including soybeans and rapeseed.Due to their low yield,limited geographical conditions and poor agronomic traits,co-7 FA high accumulators such as Macfadyena unguis-cati cannot be grown for commercial oil production like common oilseeds.Recently,metabolic engineering technology was employed to assemble co-7 FA biosynthesis pathways in the seeds of common oil crops in order to commercialize the production of ω-7 FAs,such research has been a hot research field with great prospects.In this paper,the acyl-ACP-Δ9 dehydrogenase gene was transformed into high-biomass tobacco(Nicotiana bentharniana)and the commercially-cultivated Chlorella strain CS-SX08,respectively.This enzyme can catalyze palmitic acid(C16:0)to palmioleic acid(C16:lΔ9).The object here is to develope novel germplasms of ω-7 FAs enriched plant vegetable organs and the engineered strains of Chlorella highly synthesizing ω-7 FAs.Such study provides the scientific basis to develop a new approach for the commercial production of omega-7 fatty acids.The main findings are as follows:(1)The optimized culture conditions and growth curve of Chlorella strain CS-SX08 was established,and then the optimum active period of Chlorella cells was determined.The sensitivity of Chlorella to a variety of antibiotics was systematically tested.Hygromycin resistance gene was used as the selective marker for screening the transgenic Chlorella cell lines.The screening pressure was determined as 80 μg·m L-1 and 200 μg m·L-1 of hygromycin for solid culture medium and liquid medium,respectively(2)The cDNA clone encoding an acyl-ACP-Δ9 dehydrogenase(named MucACP-Δ9D)catalyzing palmitic acid(16:0-ACP)to palmioleic acid(16:1 Δ9-ACP)was previously isolated from the developing seeds of Macfadyena unguis-cati In this paper,the over expression vector of MucACP-Δ9D(MucACP-Δ9D-pCABIA1303)was successfully constructed for genetic transformation of Chlorella and transient expressionin tobacco leaves.The vector contains GFP reporter gene and hygromycin resistance gene used as the screening markers(3)The MucA CP-Δ9D-transient expression tobacco leaves were obtained by Agrobacterium-mediated infiltration.Gas chromatography(GC)analysis on fatty acids in the tobacco leaves showed that the total content of ω-7 FAs was only 0.4%in wild-type and empty-vector controls However,total ω-7 FAs increased up to 14.2%(8.8%of C16:1Δ9 and 5.4%of C18:1Δ11,respectively)in the MucA CP-Δ9D-transient expression leaves with corresponding reduction of palmitic acid to 18.7%from 24%in the control leaves.This indicated that the MucACP-Δ9D expressed in the tobacco leaf tissue has the enzyme activity to catalyze the synthesis of palmitoleic acids.This gene can be used for genetic modification in other heterologous higher plants.In addition,linoleic acid(C18:2)and linolenic acid(C18:3)decreased,respectively,from 22.5%and 27.4%in the control to 19.3%and 22%on average in tobacco leaves expressing MucACP-Δ9D.The contents of saturated stearic acid(C18:0)and monounsaturated oleic acid(C18:1Δ9)were not significantly different between the control and MucACP-Δ9D-expressed tobacco leaves.The change in fatty acid profiles may be a corresponding compensation reaction of the host itself to an increase of monoenoic acid despite the mechanism await for further investigation(4)The MucACP-Δ9D gene was transferred into Chlorella using particle bombardment and the positive transformants were screened using hygromycin resistance gene as a selective marker.Molecular examination on the reporer gene GFP and DNA plus RNA reveals that MucACP-Δ9D has been successfully integrated into Chlorella genome,and expressed efficiently(5)Analysis on the growth curve and protein content in the wild-type and trangenic Chlorella showed that the overexpression of MucA CP-Δ9D did not significantly affect algal growth and protein synthesis(6)GC analysis on fatty acid profiles in algal cells displayed that ω-7 FA content in the genetically modified Chlorella cells was increased from very low levels to 15.1%compared to the wild-type algae Among them,palmioleic acid(C16:1Δ9)increased by 8.4%,and its elongated product C18:1Δ11 enhanced to 6.7%in the transgenic algal cellls.Correspondingly,palmitic acid(C16:0)significantly reduced from 22.8%in the wild-type algae to 18.7%in the transgenic algae.This shows that MucACP-Δ9D can normally function as the enzymeto catalyze palmitic acid to palmioleic acid in single-cell microalgaeIn summary,this study demonstrated that ω-7 FA biosynthesis pathways can be assembled in both single-cell microalgae and vegetative organs of higher plants.Acyl-ACP-Δ9 dehydrogenase derived from higher plants can also perform a normal catalytic function in single-cell Chlorella.Vegetative organs of the model plant tobacco can also be developed as bioreactors to produce omega-7 FAs.