Identification and characterization of a novel enzyme related to the synthesis of PUFAs derived from Thraustochytrium aureum ATCC 34304

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Identification and characterization of a novel enzyme related to the synthesis of PUFAs derived from Thraustochytrium aureum ATCC 34304
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  1 Identification and characterization of a novel enzymerelated to the synthesis of PUFAs derived from Thraustochytrium aureum ATCC 34304 By Dong-Hoon Kang A THESISSubmitted to the faculty of INHA UNIVERSITYIn partial fulfillment of the requirementsFor the degree of MASTERS OF BIOLOGYCAL ENGINEERINGDepartment of Biological EngineeringFebruary 2010  2 Contents Figure………………………………………………………………………………..Table………………………………………………………………………………….1. Abstract……………………………………………………………………………2. Introduction………………………………………………………………………3. Materials and Method…………………………………………………………… 3.1. Materials3.2. Strains and growth conditions3.3. PCR-based cloning of the novel elongase gene3.4. Expression of  TaNE  in  P. pastoris GS1153.5. Fatty acid analysis3.6. Sequencing and phylogenetic analysis 4. Results……………………………………………………………………………. 4.1. Heterologous expression of the T. aureum novel enzyme ( TaNE  ) ORF in  P. pastoris 4.2. Effects of temperature on the synthesis of PUFAs 5. Discussion…………………………………………………………………………6. References…………………………………………………………………………  3 Figure Fig. 1. Schematic diagram of the PUFA biosynthetic pathway. Fig. 2. Schematic diagrams of constructs used for the functional analysis of the TaNE  gene in  P. pastoris. pPTaNE  was integrated into the  AOX  locus of the  P. pastoris chromosome viahomologous gene replacement. The  pPTaNE  transformants were selected in minimal mediumwithout histidine. 5’PAOX1- 5’ AOX promoter, TaN  ORF- T. aureum Novel gene, TT – TerminalTerminator, His – Histidine synthesizing gene, 3’AOX’ – AOX homologous sequence of 3’direction, AmpR – ampicillin resistant gene, pBR322 –   E. coli srcin for replication. Fig. 3. GC analysis of FAMEs from the yeast strain expressing TaNE  with endogenous substrates(a, b) and with exogenous substrates (c-g). Fig. 4. Comparison of the amino acid sequence of the T. aureum novel gene with other known∆5-desaturase sequences (see the gene abbreviations and GenBank accession number in Table 3).The cytochrome b5-like domain and the three histidine-rich domains are boxed. Fig. 5. Comparison of the amino acid sequence of the T. aureum novel gene with other knownElo-like enzyme sequences (see the gene abbreviations and GenBank accession number in Table4). The histidine and tyrosine boxes are boxed. Fig. 6. A) Phylogenetic tree displaying the relatedness of  TaNE  gene with various ∆5-desaturasegenes (see the gene abbreviations and GenBank accession number in Table 3), B) with variousElo-like enzyme genes (see the gene abbreviations and GenBank accession number in Table 4). Fig. 7. Prediction of transmembrane regions of the TaNE   protein coding sequence (see the geneabbreviations and GenBank accession number in Table 3 for ∆5-desaturases and Table 4 for Elo-like enzymes).  4 Table Table 1. Fatty acid profiles of yeast cells containing empty vector and Tad5, TaElo and TaNE  grown inthe presence of endogenous substrates. Table 2. The conversion rate of each function at 18 ℃ . Table 3. The sequence similarity of the TaNE  with other known  ∆5- desaturases. Table 4. The sequence similarity of  TaNE  with other known Elo-type enzymes.   Table 5. Comparisons of substrate conversion rates according to exogenous substrates at A) 30 ℃ , B)20 ℃ , and C) 10 ℃ .  5 Abstract In Thraustochytrids, Thraustochytrium aureum ATCC 34304 was able to produce highlevels of several polyunsaturated fatty acids. In the present study, a novel gene encoding proteinwas cloned from the DHA rich microbe, T. aureum ATCC 34304. The functional analysis of anovel gene was demonstrated by its heterologous expression in  Pichia pastoris . The gene wasable to synthesize C20 and C22 PUFAs, as well as to mediate different elongations (∆9, ∆6, ∆5)and one ∆5 desaturation activities. The conversion rates of the ∆9 elongation (n-3) and ∆5desaturation products were found to be higher in response to the novel enzyme than the controls( TaElo and Tad5 , respectively). The other ∆9 elongation (n-6) and ∆5 elongation products wereslightly lower than those of the control ( TaElo ). The full length of the 1374 bp gene contained458 amino acids that showed very limited homology with desaturases and elongases fromvarious organisms. In addition, the rates of synthesis of PUFAs were evaluated at temperaturesranging from 10 to 30 ºC. The elongation products were found to decrease dramatically and thedesaturation products were found to increase dramatically at 10 ºC. TaNE  was confirmed to be amultifunctional enzyme with higher activity towards ∆6 elongations than ∆9, ∆5 elongations and∆5 desaturation. Key words:  polyunsaturated fatty acids, Thraustochytium aureum , desaturase, elongase,  Pichia pastoris , heterologous gene expression
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