The Differences Of Lipids And Arachidonic Acid Accumulation And Transcriptomic Analysis Of Three Coccoid Green Microalgae

Microalgae can synthesize and accumulate different types of bioactive substances through photosynthesis,which have become an important resource for nutritional products,cosmetics and medicines.Three coccoid green microalgae originally named as Paretochloris incisa SAG 2468,Lobosphaera incisa CAUP H4301 and Myrmecia bisecta SAG 2043,belong to Trebouxiphyceae,Chlorophyta.The three species are similar in morphology and can accumulate high amounts of lipids and arachidonic acid?AA?under certain culture conditions,especially under stress conditions.However,whose taxonomic position is unclear so far.Here,we identified the three strains combined with morphological,ultrstructural observation and phylogenetic analyses.Then the strains were evaluated and optimized by exploring the effects of different nutrient factors?nitrogen sources and nitrogen concentrations,initial concentrations of nitrogen,phosphorus and sulphur in the mBG-11 medium,CO₂ concentration,medium tape?and the photo-bioreactor light path on growth,lipids and AA accumulation through multi-factor combination management.Finally,from the perspective of transcriptomics,the molecular characteristics of the differences in the lipids and AA accumulation of three microalgae under N-free conditions were analyzed.Results showed that the three strains were belonged to the genus of Lobosphaera,and Parietochloris incisa SAG 2468 was revised to Lobosphaera incisa SAG 2468.L.incisa SAG 2468 and Lobosphaera incisa CAUP H4301 were identified to different geographical strains of Lobosphaera,and Myrmecia bisecta SAG 2043 was amended to Lobosphaera bisecta SAG 2043.Nutrient management had significant effects on the biomass,lipids and AA accumulation of three strains.The effects of nitrogen sources and nitrogen levels on the accumulation of biomass,lipids and AA of the three strains were studied.The results revealed that NaNO₃ and urea were superior to ammonium to enhance the growth,and low nitrogen concentration?3 mmol/L?significantly promoted the accumulation of lipids and AA.Experiments with different combination cultivation mode of nitrogen,phosphorus and sulphur concentrations revealed that SAG 2043possessed the potential to accumulate higher biomass,lipids and AA contents compared to the other two strains.The increases of phosphorus and sulphur concentrations were beneficial for the growth.Compared to phosphorus and sulphur stress,nitrogen stress has the most significant effect on the promotion of lipids and AA,and is superior to multiple nutrient stress.The highest lipids and AA productivity could be up to 177.21and 44.46 mg L^-1 d^-1 in the combination cultivation mode of nitrogen deficiency-phosphorus and sulfur repletion.1%CO₂ and mEndo medium were most favorable for growth and AA accumulation of SAG 2043 and the maximum lipids and AA productivity were 170.39 and 41.75 mg L^-1 d^-1,respectively,in low nitrogen concentration?3 mmol/L?and urea used as nitrogen source.The highest biomass concentration of 10.58 g L^-1 was attained in the smaller light path?3 cm?photo-bioreactor,which was increased significantly compared to the wider ones?4.5 cm and 6 cm?,especially in high initial nitrogen concentration?18mmol/L?with mEndo medium,which biomass was nearly 2 times of normal mBG-11medium.Meanwhile,the remarkable lipids and AA productivity of 245.66 and 73.41mg L^-1 d^-1 were obtained following rapid accumulation of biomass,which were 2 and2.66 times of mBG-11 medium,respectively.RNA-Seq technology was performed to analyze the differential expression pattern of lipid metabolism pathways under N-sufficient and N-free conditions in three closely coccoid green microalgae.A comprehensive model for the lipid metabolic pathways of three algae was reconstructed for the first time,including the de novo synthesis of fatty acids,the synthesis of polyunsaturated fatty acids?PUFAs?,chloroplast lipids?mainly MGDG?and triacylglycerols?TAG?.The three strains shared similar AA synthesis pathways??6?.It was indicated that the upstream pathway like C4-like CCM mechanism and PDHC pathway were actively up-regulated,which may played a role in supplementing carbon flow and reducing power.Consequently,these processes resulted in pushing more Acetyl-CoA precursor into the synthesis of fatty acids.Up-regulation of the fatty acid de novo pathway and down-regulation of the MGDG synthesis pathway would enrich more fatty acyl groups for constitution of glycerolipids.In addition,the TAG synthesis pathway were up-regulated.On the whole,multi-level regulation ensured the conversion efficiency from carbon to the synthesis of lipids,which significantly promoted the accumulation of TAG in these three coccoid green microalgae.Relatively speaking,the copy numbers of responsing N-free genes and the transcriptional expression abundance and up-regulation tendency were even more significant in the above process of SAG 2043.It was found that the key enzyme,desaturase and elongase,in polyunsaturated?PUFAs?fatty acid synthesis pathway were significantly up-regulated.Meanwhile,the related genes acted as roll of"pull"and"export"in the fatty acid de novo pathway were significantly up-regulated,paving the way for the supply of substance foundation and greatly promoting the accumulation of AA.Compared to the other two coccoid green microalgae,the copy numbers of up-regulated genes,transcriptional expression abundance and up-regulation tendency in the AA accumulation were relatively more significant in L.incisa SAG 2468.In conlusion,L.bisecta SAG 2043 has strong ability of photosynthetic efficiency and stress-resistance,and possesses the advantages of fast growth,easy culturing,easy settling.It can gain competitive contents and productivities of lipids and AA.It may be a potential candidate for large-scale production of AA.