Identification, Characterization And Expression Of The Key Enzyme Genes In Biosynthesis Of Polyunsaturated Fatty Acids In Pavlova Viridis

Polyunsaturated fatty acids(PUFAs)are unbranched fatty acids containing more than one double bond.They are essential components of human health including EPA, DHA and so on.And EPA/DHA are associated with various physiological and pathophysiological processes,thereby affecting human health.Clinical studies show that DHA is essential for the growth and development of the brain in infants and for maintenance of normal brain function in adults.Cell growth and division,platelet aggregation,inflammatory responses,hemorrhage,vasoconstriction and vasodilation and immune functions are associated with EPA/DHA.Studies have shown their role in prevention and treatment of coronary heart disease,hypertension,type 2 diabetes, arthritis,cancer and other inflammatory and autoimmune disorders.At present,fish oil is the major source of EPA/DHA.However,the demand of EPA/DHA is constantly increasing while the fish sources producing them are depleting.Marine micralgae,the primary producers of PUFAs,are being explored as an alternative source for yielding these essential elements of health.Desaturases and elongase systems are critical enzymes involved in biosynthesis of PUFAs and occur in most living cells.Desaturases are iron-containing enzymes that introduce a double bond in a specific position counted from the carbonyl end of the fatty acids,aerobically.The elongase systems are responsible for the addition of two carbon units to the carboxyl end of a fatty acid chain.And the genes of desaturases and elongase systems have important biotechnological appeal from genetic engineering point of view.The identification and prospecting of these genes becomes a novel method for enhanced PUFA production.Pavlova viridis,a marine microalga,is rich in PUFAs,which represents an attractive production system for PUFAs such as EPA and DHA.In this dissertation,P. viridis has been used as a useful source for cloning fatty acid desaturase and elongases genes.The results and significance of this study is summarized as follows:1.Optimization of P.viridis growth conditions.P.viridis contains plenty of polyunsaturated fatty acids(PUFA).In this dissertation,we've studied the impacts of environmental factors on growth of P. viridis,and investigated those factors' effects on content of EPA/DHA in total fatty acids through gas chromatograph(GC)analysis.Through mensurating the curve of growth under different environmental factors,it was indicated that the growth of P. viridis under light was visible better than which in dark,the consistency of cell reached 2.49×10~6 cells/mL after 12 days under light.However,the cell consistency was only 1.60×10~6 cells/mL after 12 days in dark.The results also indicated that high light density was helpful with accumulation of total fatty acids,especially,could increased the content of EPA/DHA.Meanwhile,the growth of P.viridis under 25℃was visible better than which under 18℃and 16℃.However,the contents of EPA/DHA declined while the growth temperature rised,which showed that lower growth temperature was helpful to the accumulation of EPA/DHA.We've invesgated the fatty acid composition of P.viridis through gas chromatograph mass spectra(GC/MS)analysis.The results showed there were various kind of fatty acids in P.viridis,making it an ideal candidate for studying the PUFA synthetic pathway.2.Improvation of RNA extraction and construction of P.viridis cDNA library and EST analysis.High quality RNA is critical in molecular manipulation such as the cDNA synthesis,cDNA library construction and RACE.However,P.viridis is rich in polysacchrides and polyphenols,which could interfere with the extraction of total RNA from cells.In this study,an improved CTAB method was constructed based on the traditional methods of RNA extract by Trizol agent and acid-guanidine-phenol-chloroform method.The method could effectively eliminate the interferences of polysacchrides and polyphenols.Upon the method mentioned above,total RNA was extracted from P.virids cells. Poly~+A RNA was then isolated and purified and the mRNA template was reversely transcriptionized to a single strand cDNA with modified Oligo(dT)primer using SMART(Switching Mechanism At 5' end of RNA Transcript)technology. Consequently,the synthesized ds cDNA was ligated with vectorλTriplex2.The recombined vectors were packaged in vitro.The titer of the unamplified constructed cDNA library was 6×10~6 pfu/mL and the recombination rate was 98%.Meanwhile, the length of the inserted DNA fragment was between 500 bp and 15 kb.All of these suggested that the library could be suitable for screening low abundant mRNA for cDNA clones.The results also indicated that the improved CTAB method was appropriate for RNA extraction of microalage.The expressed sequence tags(ESTs)was a rapid method which can be used to obtain the new genes from cDNA library.The clones containing cDNA longer than 500 bp were selected for sequencing.200 ESTs were obtained by sequencing from the 5' end of the cDNA clones.Then these ESTs were compared with sequences in the GenBank data of NCBI using Blast.Partial sequence of some functional genes was identified,including atp I and atpH genes for subunit of ATPase complex,rRNA gene, carboxypeptidase A,cytochrome b,chloroplast photosystemⅡ12 kD extrinsic protein and so on.The results provided the basis for further study of P.viridis gene expression.3.Identification,characterization and overexpression of a new C20-elongase gene elkj.A novel elongase gene was isolated for the first time from P.viridis via reverse transcriptase-polymerase chain reaction(RT-PCR)using the primers designed from conserved motifs and 5'/3' RACE,named as elkj(GenBank accession No.486525). Sequence analysis indicated that elkj gene was with high identity with a functionally characterized C20-elongase of P.lutheri.And the elkj gene was 945 bp in length, encoding a protein of 314 amino acids with an estimated molecular weight of 34 kDa. The predicted protein ELKj contained seven transmembrane domains with its C-terminal in the cytoplasm and located in the endoplasmic reticulum.The heterologous expression in E.coli demonstrated that elkj encoded a C20-elongase that mediated the elongation of EPA into docosapentaenoic acid(DPA, 22:5n-3).Therefore,the identification and characterization of the elkj gene also confirmed the two-step conversion existed in marine microalga.When the elkj gene was overexpressed heterologously,the recombinant strains grew much slowly compared with the host strain,indicating that the ELKJ protein as a membrane protein brought toxic effects to E.coli.Thus,the ELKJ protein was fused with green fluorescent protein(GFP),to monitor the overexpression,solubilization and purification of the ELKJ-GFP protein,instead of laborious and time-consuming process.4.Identification of two novel desaturase genes:the△4-desaturase gene pkjDes4 and the△5-desaturase gene pkjDes5,respectively.The molecular biology of marine microalga hasn't been developed until recent years and little was known about their genetic manipulation.The efforts of selecting P. viridis cDNA library by probe bloting were made to clone the desaturase genes involved in the biosynthesis of PUFA but with no progress.Thus,two partial cDNA sequences with identity to△4-and△5-desaturase genes were obtained via RT-PCR using primers designed according to the conserved histidine motifs of previously reported desaturase genes.And then the entire sequences of the two desaturase genes were isolated using SMART RACE together with SEFA PCR(self-formed adaptor PCR).Sequence analysis revealed that the△4-desaturase gene pkjDes4 was 1440 bp in length,encoding a protein with 449 amino acids(GenBank accession No.486526) and the△5-desaturase gene pkjDes5 1278 bp in length,with 425 amino acids (GenBank accession No.486527). 5.Co-expression of elkj with pkjDes4 in Pichia pastoris.In marine microalgae,the conversion of EPA to DHA involved a C20-elongase together with a△4-desaturase,which was considered as the efficient two-step conversion.In this study,the C20-elongase gene elkj was ligated with the expression vector pPIC3.5K and integrated into P.pastoris.Functional analysis through GC chromatograph indicated that the recombinant strain had the ability of elongating EPA into DPA.Then,the△4-desaturase pkjDEs4 was also ligated into the pPIC3.5K vector, which could be used to reconstructing the DHA synthesis pathway in P.pastoris.6.Construction of a novel membrane protein expression system in Lactococcus lactis.Eukaryotic membrane proteins are often difficult to produce in large quantities, which is a significant obstacle for further structural and biochemical investigation.L. lactis has several properties that are ideal for enhanced expression of eukaryotic membrane proteins.In this study,a novel lactococcal vector was constructed,by inserting a green fluorescent protein sequence into the vector pNZ8148,which is a commonly used vector in the NICE system(NIsin Controlled gene Expression system).The resulting vector pKj-gfp was applied to the overexpression of an elongase-green fluorescent protein fusion in L.lactis.During expression,the fusion was monitored through in-gel fluorescence and no degradation was observed. Furthermore,the targeted protein was exclusively directed into the cytoplasmic membrane,and accounted for approximately 15%of the total membrane protein. These results indicated that the lactococcal system can be used for the overproduction of eukaryotic membrane protein.The identification of different elonagase and desaturase genes involved in the EPA/DHA production has important biotechnological applications.These genes can be used in the production of PUFA-rich transgenic plant oils for therapeutic and prophylactic use.Also,advances in understanding gene regulation in PUFA biosynthesis will also impact the single-cell oil industry,such that growth conditions of the microalgae can be manipulated to enhance the production of EPA/DHA.This in turn will impact the marine fish-farming industry which depends on microalgae for enhancing the levels of PUFAs in fish.All the above will eventually afford the public an economical source of balanced n-3/n-6 PUFA-enriched oils that will greatly impact general health and nutrition in the near future.