Reconstitution Of Biosynthesis Pathway Of Arachidonic Acid And Eicosapentaenoic Acid, And Modification Of Soybean Seed-specific Promoter

Polyunsaturated fatty acids (PUFAs), especially very long chain polyunsaturatedfatty acids (VLC-PUFAs) with20carbons or more in length, are of great importancefor the normal development and metabolism of all organisms, and are essential formaintaining human health. Currently, the most available sources of PUFAs are marinefishes and marine algae. However, owing to long-term over-fishing andenvironmental pollution of the marine ecosystems as well as expensive cost to obtainPUFAs from marine algae, it is urgent for scientists to find an alternative source ofPUFAs for a sustainable source for VLC-PUFAs.With the rapid development of transgenic technologies, producing PUFAs bytransgenic oilseed crops has been demonstrated to be a promising alternative sourcefor PUFAs. Soybean is one of the most important oilseed crops as well as linoleicacid (LA) and α-linolenic acid (ALA) which are substrates of the synthesis of PUFAsin soybean oil account for up to55%and13%of total fatty acids, respectively. So,soybean has been an important host producing PUFAs by metabolic engineering. Thetwo pathways, conventional Δ6pathway and alternative Δ8pathway, for PUFAsbiosynthesis in transgenic plants have been described so far and genes encodingelongases and desaturases involved have been identified. Thanks to the bottlenecks ofcomplex substrate conversion in Δ6pathway, in present study, Δ8pathways ofarachidonic acid (ARA) and Eicosapentaenoic acid (EPA) biosynthsis werereconstituted in Saccharomyces cerevisiae and soybean to obtain marker-freetransgenic soybean producing ARA and EPA and soybean seed-specific promoterBCSP952was modified to improve the level of ARA and EPA biosynthsis in thetransgenic soybean.First, three genes presented in Δ8pathway, Δ9elongase gene (IgASE2), Δ8desaturase gene (efd2) and Δ5desaturase gene (ptd5) were cloned from Isochrysisgalbana H29, Euglena gracilis and Phaeodactylum tricornutum, repectively, andcharacterized by their heterologous expression in S. cerevisiae INVSc1. The IgASE2gene was1653bp in length, contained a786bp open reading frame (ORF) encoding a protein of261amino acids that shared87%identity with the reported Δ9elongase,IgASE1, and possessed a44bp5’-untranslated region and a823bp3’-untranslatedregion. IgASE2expressed in S. cerevisiae, elongated LA to eicosadienoic acid (EDA)and ALA to eicosatrienoic acid (EtrA) with57.6%(LA to EDA) and56.1%(ALA toEtrA) conversion ratio, respectively, confirming that IgASE2gene was a novelC18-Δ9-specific PUFAs elongase gene. The ORF of efd2was1266bp and encodeda protein of431amino acids that shared96%identity with the reported Δ8desaturase,EFD1. EFD2expressed in S. cerevisiae converted EDA to dihomo-γ-linolenic acid(DGLA) and EtrA to eicosatetraenoic acid (ETA) with substrate conversion ratio31.2%and46.3%, respectively, confirming that efd2was a Δ8-specific PUFAsdesaturase gene. Ptd5has an ORF of1410bp that encodes469amino acids. PTD5expressed in S. cerevisiae specifically catalyzed DGLA to ARA and ETA to EPA withsubstrate conversion ratio28.7%and37.2%, respectively, confirming that ptd5wasaΔ5-specific PUFAs desaturase gene.Then, using the cloned IgASE2, efd2and ptd5genes, we constructed thealternative Δ8pathway of ARA and EPA biosynthsis in S. cerevisiae and soybean andobtained engineered strain of S. cerevisiae producing ARA and EPA and free-markertransgenic soybean producing ARA and EPA. Based on the Δ8(ω6-Δ8, ω3-Δ8)pathway of ARA and EPA biosynthesis, the co-expression vector pYAE5of S.cerevisiae INVSc1was constructed by cloning the expression cassettes of IgASE2,efd2and ptd5into pYES2and transformed into INVSc1to obtain engineered strainYAE985producing ARA and EPA. When YAE985was cultivated in the inductionmedium with exogenous substrates LA and ALA, YAE985converted LA to ARA andALA to EPA with substrate conversion ratio10.1%and16.9%, respectively, as wellas ARA and EPA in YAE985accounted for up to1.6%and2.5%of total fatty acids,respectively. These results showed that the Δ8(ω6-Δ8, ω3-Δ8) pathway of ARA andEPA biosynthesis was successfully constructed in S. cerevisiae INVSc1.Genetic stability of YAE985was identified by determining the transcription ofIgASE2, efd2and ptd5in YAE985using real-time PCR (RT-PCR). IgASE2, efd2andptd5remained1:1:1at the transcription level when YAE985was continuouslyinoculated and cultured for20generations, confirming that the genes were not lost. The results verified that the engineered strain YAE985was stable.The expression cassettes of IgASE2, efd2and ptd5containing BCSP952wereconstructed by ligating them to the downstream of BCSP952, respectively. They werecloned into the vector pBX to construct the marker-free expression vector pX9AE5inthe transgenic soybean. pX9AE5was transformed into soybean by Agrobacteriumtumefaciens-mediated transformation (ATMT) and the transgenic soybean harboringpX9AE5was identified by kanamycin resistence and PCR. In order to delete theselectable marker gene in the transgenic soybean, the transgenic soybean was inducedwith β-estradiol and the marker-free transgenic soybean was constructed. In the seedsof the marker-free transgenic soybean, ARA and EPA were about6.8%and3.6%ofof total fatty acids. These results showed that the Δ8(ω6-Δ8, ω3-Δ8) pathway ofARA and EPA biosynthesis was successfully constructed in the marker-freetransgenic soybean.Finally, in order to improve the expression level of ARA and EPA in transgenicsoybean, we undertook the function analysis of BCSP952and further modified theBCSP952to improve its strength according to the function analysis of BCSP952. Inorder to understand the regulatory mechanism of BCSP952, a series of5′-deletedpromoters BCSP666, BCSP471, BCSP285and BCSP156were constructed and fusedto the β-glucuronidase (GUS) gene in pBI121as well as transformed into Arabidopsisthaliana via ATMT. The GUS activities of the promoters were detected in differenttissues. The GUS activities of BCSP666, BCSP471, BCSP285and BCSP156were96.5%,69.4%,15.5%and10.1%of BCSP952GUS activity, and the other promotersonly expressed in seed except BCSP156. The results showed that:(a) The type andamount of the seed-specific promoter elements directly impacted on the strength ofthe promoter.(b) Within the-594region, the more the seed-specific promoterelements, the stronger the promoter. However, beyond this region, the strength of thepromoter was limitedly improved if the elements were increased.In order to improve the strength of BCSP952, BCSP952-aa, BCSP952-as andBCSP952-ss were constructed by inserting ABRE and Sph elements into the-140siteof BCSP952and fused to the GUS gene in pBI121as well as transformed into A.thaliana via ATMT. The histovhemical analysis and fluorometric analysis showed that: GUS genes controlled by the modified BCSP952were only expressed in seed of A.thaliana, confirming that they were all seed-specific promoters, and GUS activities ofBCSP952-as, BCSP952-aa and BCSP952-ss were180%,112%and88%of BCSP952GUS activity, repectively.The southern hybridization and real-time PCR (RT-PCR) on the4lines oftransgenic A. thaliana containing different kinds of promoter in which the GUSactivities were the highest were conducted to further identified the strength of themodified BCSP952. The southern hybridization showed that the GUS genespresented in transgenic A. thaliana were single copy except a line containingBCSP952-ss. RT-PCR showed that at the transcription level, GUS gene controlled byBCSP952-as was expressed at highest level, followed by BCSP952-aa, whereas GUSgene controlled by BCSP952-ss was expressed at the lowest level, which wereconsistent with the GUS activities. The results indicated that the strength of BCSP952was improved by modification, and BCSP952-as inserted a ABRE and a Sph elementwas the strongest, followed by BCSP952-aa inserted two ABREs, whereas the thestrength of BCSP952-ss inserted two Sph elements decreased.