Biosynthetic mechanism of very long chain polyunsaturated fatty acids in Thraustochytrium sp. 26185
2016; Elsevier BV; Volume: 57; Issue: 10 Linguagem: Inglês
10.1194/jlr.m070136
ISSN1539-7262
AutoresDauenpen Meesapyodsuk, Xiao Qiu,
Tópico(s)Algal biology and biofuel production
ResumoThraustochytrium, a unicellular marine protist, has been used as a commercial source of very long chain PUFAs (VLCPUFAs) such as DHA (22:6n-3). Our recent work indicates coexistence of a Δ4-desaturation-dependent pathway (aerobic) and a polyketide synthase-like PUFA synthase pathway (anaerobic) to synthesize the fatty acids in Thraustochytrium sp. 26185. Heterologous expression of the Thraustochytrium PUFA synthase along with a phosphopantetheinyl transferase in Escherichia coli showed the anaerobic pathway was highly active in the biosynthesis of VLCPUFAs. The amount of Δ4 desaturated VLCPUFAs produced reached about 18% of the total fatty acids in the transformant cells at day 6 in a time course of the induced expression. In Thraustochytrium, the expression level of the PUFA synthase gene was much higher than that of the Δ4 desaturase gene, and also highly correlated with the production of VLCPUFAs. On the other hand, Δ9 and Δ12 desaturations in the aerobic pathway were either ineffective or absent in the species, as evidenced by the genomic survey, heterologous expression of candidate genes, and in vivo feeding experiments. These results indicate that the anaerobic pathway is solely responsible for the biosynthesis for VLCPUFAs in Thraustochytrium. Thraustochytrium, a unicellular marine protist, has been used as a commercial source of very long chain PUFAs (VLCPUFAs) such as DHA (22:6n-3). Our recent work indicates coexistence of a Δ4-desaturation-dependent pathway (aerobic) and a polyketide synthase-like PUFA synthase pathway (anaerobic) to synthesize the fatty acids in Thraustochytrium sp. 26185. Heterologous expression of the Thraustochytrium PUFA synthase along with a phosphopantetheinyl transferase in Escherichia coli showed the anaerobic pathway was highly active in the biosynthesis of VLCPUFAs. The amount of Δ4 desaturated VLCPUFAs produced reached about 18% of the total fatty acids in the transformant cells at day 6 in a time course of the induced expression. In Thraustochytrium, the expression level of the PUFA synthase gene was much higher than that of the Δ4 desaturase gene, and also highly correlated with the production of VLCPUFAs. On the other hand, Δ9 and Δ12 desaturations in the aerobic pathway were either ineffective or absent in the species, as evidenced by the genomic survey, heterologous expression of candidate genes, and in vivo feeding experiments. These results indicate that the anaerobic pathway is solely responsible for the biosynthesis for VLCPUFAs in Thraustochytrium. Very long chain PUFAs (VLCPUFAs), such as arachidonic acid (ARA, 20:4n-6) and DHA (22:6n-3), are essential components of cell membranes and precursors for biologically active signaling molecules in mammals. VLCPUFAs and their derived signaling molecules, such as eicosanoids and docosanoids, regulate the neurotransmission process in the brain, thereby affecting mood, cognition, and other neurological behaviors (1Bazinet R.P. Laye S. Polyunsaturated fatty acids and their metabolites in brain function and disease.Nat. Rev. Neurosci. 2014; 15: 771-785Crossref PubMed Scopus (818) Google Scholar). In addition, VLCPUFAs and their derivatives can also regulate other physiological processes, such as blood circulation, metabolic pathways, and inflammatory status in mammals (2Guichardant M. Calzada C. Bernoud-Hubac N. Lagarde M. Vericel E. Omega-3 polyunsaturated fatty acids and oxygenated metabolism in atherothrombosis.Biochim. Biophys. Acta. 2015; 1851: 485-495Crossref PubMed Scopus (32) Google Scholar, 3Wang W. Zhu J. Lyu F. Panigrahy D. Ferrara K.W. Hammock B. Zhang G. omega-3 polyunsaturated fatty acids-derived lipid metabolites on angiogenesis, inflammation and cancer.Prostaglandins Other Lipid Mediat. 2014; 113–115: 13-20Crossref PubMed Scopus (99) Google Scholar). Imbalances of different types of VLCPUFAs and their derivatives in the body have been shown to implicate various pathogeneses in humans, such as neurological disorders, cardiovascular diseases, metabolic syndrome, and inflammatory conditions (4Robinson L.E. Mazurak V.C. N-3 polyunsaturated fatty acids: relationship to inflammation in healthy adults and adults exhibiting features of metabolic syndrome.Lipids. 2013; 48: 319-332Crossref PubMed Scopus (57) Google Scholar, 5Janssen C.I. Kiliaan A.J. Long-chain polyunsaturated fatty acids (LCPUFA) from genesis to senescence: the influence of LCPUFA on neural development, aging, and neurodegeneration.Prog. Lipid Res. 2014; 53: 1-17Crossref PubMed Scopus (336) Google Scholar). Appropriate dietary supplementation of these fatty acids is thus encouraged to provide protection against chronic diseases and improve performance of the brain, eyes, and immune system. There are two distinct pathways in nature for the biosynthesis of VLCPUFAs (6Qiu X. Biosynthesis of docosahexaenoic acid (DHA, 22:6-4,7,10,13,16,19): two distinct pathways.Prostaglandins Leukot. Essent. Fatty Acids. 2003; 68: 181-186Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 7Khozin-Goldberg I. Iskandarov U. Cohen Z. LC-PUFA from photosynthetic microalgae: occurrence, biosynthesis, and prospects in biotechnology.Appl. Microbiol. Biotechnol. 2011; 91: 905-915Crossref PubMed Scopus (160) Google Scholar). The aerobic pathway follows an alternating desaturation and elongation process and occurs mainly in animals and eukaryotic microorganisms (8Sprecher H. Luthria D.L. Mohammed B.S. Baykousheva S.P. Reevaluation of the pathways for the biosynthesis of polyunsaturated fatty acids.J. Lipid Res. 1995; 36: 2471-2477Abstract Full Text PDF PubMed Google Scholar, 9Harwood J.L. Guschina I.A. The versatility of algae and their lipid metabolism.Biochimie. 2009; 91: 679-684Crossref PubMed Scopus (231) Google Scholar, 10Meesapyodsuk D. Qiu X. The front-end desaturase: structure, function, evolution and biotechnological use.Lipids. 2012; 47: 227-237Crossref PubMed Scopus (108) Google Scholar, 11Graham I.A. Larson T. Napier J.A. Rational metabolic engineering of transgenic plants for biosynthesis of omega-3 polyunsaturates.Curr. Opin. Biotechnol. 2007; 18: 142-147Crossref PubMed Scopus (82) Google Scholar). The anaerobic pathway utilizes a polyketide synthase (PKS)-like PUFA synthase and takes place only in microorganisms (12Metz J.G. Roessler P. Facciotti D. Levering C. Dittrich F. Lassner M. Valentine R. Lardizabal K. Domergue F. Yamada A. et al.Production of polyunsaturated fatty acids by polyketide synthases in both prokaryotes and eukaryotes.Science. 2001; 293: 290-293Crossref PubMed Scopus (549) Google Scholar, 13Okuyama H. Orikasa Y. Nishida T. Watanabe K. Morita N. Bacterial genes responsible for the biosynthesis of eicosapentaenoic and docosahexaenoic acids and their heterologous expression.Appl. Environ. Microbiol. 2007; 73: 665-670Crossref PubMed Scopus (108) Google Scholar, 14Morita N. Tanaka M. Okuyama H. Biosynthesis of fatty acids in the docosahexaenoic acid-producing bacterium Moritella marina strain MP-1.Biochem. Soc. Trans. 2000; 28: 943-945Crossref PubMed Scopus (34) Google Scholar). In the aerobic pathway, synthesis of VLCPUFAs, such as DHA in mammals, starts from α-linolenic acid (ALA, 18:3n-3) and goes through a retro-conversion process via a controlled β-oxidation step in the peroxisome for two carbon chain shortening (15Voss A. Reinhart M. Sankarappa S. Sprecher H. The metabolism of 7,10,13,16,19-docosapentaenoic acid to 4,7,10,13,16,19-docosahexaenoic acid in rat liver is independent of a 4-desaturase.J. Biol. Chem. 1991; 266: 19995-20000Abstract Full Text PDF PubMed Google Scholar, 16Sprecher H. Chen Q. Yin F.Q. Regulation of the biosynthesis of 22:5n-6 and 22:6n-3: a complex intracellular process.Lipids. 1999; 34: S153-S156Crossref PubMed Google Scholar), while the synthesis of DHA in eukaryotic microorganisms starts from stearic acid (SA, 18:0) and ends with the final Δ4 desaturation step (17Qiu X. Hong H. MacKenzie S.L. Identification of a Delta 4 fatty acid desaturase from Thraustochytrium sp. involved in the biosynthesis of docosahexanoic acid by heterologous expression in Saccharomyces cerevisiae and Brassica juncea.J. Biol. Chem. 2001; 276: 31561-31566Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). In the anaerobic pathway, the VLCPUFA synthesis catalyzed by a PUFA synthase (12Metz J.G. Roessler P. Facciotti D. Levering C. Dittrich F. Lassner M. Valentine R. Lardizabal K. Domergue F. Yamada A. et al.Production of polyunsaturated fatty acids by polyketide synthases in both prokaryotes and eukaryotes.Science. 2001; 293: 290-293Crossref PubMed Scopus (549) Google Scholar) differs from the aerobic pathway in that it does not require oxygen-dependent desaturation steps to introduce double bonds. Instead, double bonds are introduced during the process of fatty acid extension, as seen in the biosynthesis of unsaturated fatty acids in Escherichia coli (18Cronan J.E. Thomas J. Bacterial fatty acid synthesis and its relationships with polyketide synthetic pathways.Methods Enzymol. 2009; 459: 395-433Crossref PubMed Scopus (188) Google Scholar). It is now known that de novo biosynthesis of VLCPUFAs occurs only in certain types of oceanic microorganisms, while animals and plants lack the PUFA synthase system and possess just part of the entire aerobic biosynthetic pathway, and thus are unable to completely synthesize these fatty acids. For those marine microbes that can de novo synthesize VLCPUFAs, the biosynthetic process goes through either an aerobic pathway employing desaturases and elongases to introduce double bonds and extend carbon chains of preexisting fatty acids for producing the final products or an anaerobic pathway employing a PUFA synthase to carry out all reactions required for conversion of initial acetyl-CoA to VLCPUFAs. Thraustochytrium sp. 26185 is a unicellular marine protist that can produce more than 50% of its total fatty acids as VLCPUFAs in membrane and storage lipids. Our previous research indicates that there is an aerobic pathway in this species for the biosynthesis of VLCPUFAs and DHA can be synthesized through the final step of Δ4 desaturation (17Qiu X. Hong H. MacKenzie S.L. Identification of a Delta 4 fatty acid desaturase from Thraustochytrium sp. involved in the biosynthesis of docosahexanoic acid by heterologous expression in Saccharomyces cerevisiae and Brassica juncea.J. Biol. Chem. 2001; 276: 31561-31566Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). However, our recent genome sequencing revealed existence of an alternative pathway employing a PUFA synthase for the biosynthesis of VLCPUFAs in this species, as seen in Schizochytriun sp., a close relative of the Thraustochytrium sp. 26185 (12Metz J.G. Roessler P. Facciotti D. Levering C. Dittrich F. Lassner M. Valentine R. Lardizabal K. Domergue F. Yamada A. et al.Production of polyunsaturated fatty acids by polyketide synthases in both prokaryotes and eukaryotes.Science. 2001; 293: 290-293Crossref PubMed Scopus (549) Google Scholar, 19Hauvermale A. Kuner J. Rosenzweig B. Guerra D. Diltz S. Metz J.G. Fatty acid production in Schizochytrium sp.: Involvement of a polyunsaturated fatty acid synthase and a type I fatty acid synthase.Lipids. 2006; 41: 739-747Crossref PubMed Scopus (122) Google Scholar, 20Metz J.G. Kuner J. Rosenzweig B. Lippmeier J.C. Roessler P. Zirkle R. Biochemical characterization of polyunsaturated fatty acid synthesis in Schizochytrium: release of the products as free fatty acids.Plant Physiol. Biochem. 2009; 47: 472-478Crossref PubMed Scopus (63) Google Scholar). The goal of this research was, thus, to interrogate the function and importance of the two pathways for the biosynthesis of VLCPUFAs in the species. Thraustochytrium sp. 26185 was purchased from the American Type Culture Collection and cultured on BY+ medium (pH 6.0–6.5) consisting of 0.1% (w/v) yeast extract, 0.1% (w/v) peptone, 0.5% (w/v) D-glucose, and 4% (w/v) artificial sea salts (Sigma) at 25°C. The E. coli strain, BL21(star)DE3, was obtained from Novagen. By sequence analysis of the Thraustochytrium genome database, three putative genes encoding the PUFA synthase were identified (intronless genes). In order to clone the full-length genes, the genomic DNA isolated from Thraustochytrium was used as a template for PCR amplification using Q5, proofreading DNA polymerase (BioLabs). All specific primers were designed according to the sequence information obtained from the Thraustochytrium genome sequence, as listed in supplemental Table S1. The first open reading frame (ORF) of 8,439 bp encodes the PUFA synthase subunit-A, the second ORF of 6,150 bp codes for subunit-B, and the third ORF of 4,494 bp encodes subunit-C. All ORF sequences had both start and stop codons. To facilitate cloning and capacity of fidelity for DNA polymerase to synthesize a large DNA fragment, ORF-A was divided into three pieces according to internal restriction sites, PstI and AscI. Part I contained 3,254 bp including a start site with EcoRI restriction site and extended to a unique PstI site; part II contained 3,194 bp including PstI and AscI restriction sites; and part III contained 1,991 bp including AscI and a stop codon with HindIII restriction site. The ORF-B was divided into two pieces by restriction enzyme, BglII, first fragment (3,437 bp) contained a start site with EcoRI restriction site and extended to a unique BglII, and second fragment (2,713 bp) started from BglII until stop codon with HindIII restriction site. The ORF-C was directly amplified with primers from start until stop codons with EcoRI and HindIII, respectively, at both ends. All PCR fragments were cloned by TA cloning into pGEMT (Promega). All plasmids were sequenced to verify the amplification fidelity. In order to get the full-length of ORF-A, part I, part II, and part III were combined by restriction cloning procedure. First, the PstI fragment was digested from part II-plasmid and cloned into part I-plasmid digested with PstI, producing plasmid-ORF-A-I+II. Second, the AscI and HindIII fragment was digested from part III-plasmid and cloned into plasmid-ORF-A-I+II digested with AscI and HindIII, producing plasmid-ORF-A-FL. The same method was applied to clone the full-length of ORF-B where part I and II were ligated at BglII restriction site, producing plasmid-ORF-B-FL. Previous studies showed that E. coli endogenous phosphopantetheinyl transferase (PPTase) enzymes were unable to activate the apo-acyl carrier protein (ACP) into holo-ACP domains of PUFA synthases (19Hauvermale A. Kuner J. Rosenzweig B. Guerra D. Diltz S. Metz J.G. Fatty acid production in Schizochytrium sp.: Involvement of a polyunsaturated fatty acid synthase and a type I fatty acid synthase.Lipids. 2006; 41: 739-747Crossref PubMed Scopus (122) Google Scholar). A PPTase gene (HetI) from Nostoc sp. PCC7120 was chemically synthesized by Integrated DNA Technologies (USA) and subcloned into pGEM-T vector. The confirmed plasmids were digested by NdeI and KpnI and cloned into the second cloning site of ORF-A/pCOLADuet-1 digested with the same restriction enzymes. To identify genes encoding PPTase from Thraustochytrium and S. limacinum, HetI protein sequence from Nostoc PCC 7120 was used as a query to search Thraustochytrium sp. 26185 and S. limacinum genome databases. Homologous genes were identified from the two species. The full-length PPTase gene from Thraustochytrium was amplified by PCR using genomic DNA as template. The ORF was 867 bp encoding 288 amino acids with a molecular mass of 31.6 kDa. The full-length PPTase gene from Schizochytrium was 816 bp encoding a polypeptide of 271 amino acids and was chemically synthesized at Integrated DNA Technologies. A single colony of Thraustochytrium cells was grown in 10 ml of BY+ medium at 25°C overnight with 240 rpm. The cells were then diluted with the same medium and condition starting OD600 at 0.1. After two double times (8 h), cells were quickly harvested by centrifugation at 5,000 rpm and divided into two parts. The first part was used for total fatty acid analysis and the second part for the total RNA using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Genomic DNA contamination was eliminated by on-column DNase I digestion with RNase-Free DNase (Qiagen). The first-strand cDNA was synthesized from 1 μg of the total RNA by qScript cDNA Supermix (Quanta Biosciences). Three genes, actin, GAPDH, and HSP90, were employed as internal references. Primers were designed using Primer3Plus online software (http://www.primer3plus.com/cgi-bin/dev/primer3plus.cgi). The real-time quantitative PCR was performed using SYBRgreen SuperMix (Quanta Biosciences) according to manufacturer's recommendation. Reactions were carried out on a Bio-Rad CFX real-time PCR system (Bio-Rad, Mississauga, Ontario, Canada) with the following cycling reaction: 50°C for 2 min, 95°C for 2 min, 95°C for 15 s, 65°C for 30 s. Three biological replicates with three technical replicates were performed with each sample. Data obtained from the iCycler software (Bio-Rad) was used to analyze the expression of TcPUFAs and TcD4 compared with three internal standards. Their relative expression ratios were calculated using the comparative Ct method. E. coli transformant cells containing pORF-C/ORF-B/ORF-A and either pHetI or pScPPTase or pTcPPTase were grown in LB or 765 medium supplemented with 10% glycerol, kanamycin (50 μg/ml), and spectinomycin (50 μg/ml) at 37°C. The overnight culture was inoculated to 50 vol of new medium containing the same supplementation. The bacteria were grown at 37°C to OD600 = 0.5–1.0. Induction was achieved by addition of 0.5 mM isopropyl-β-D-thiogalactopyranoside. The cells continued to grow at 20°C with 220 rpm and then were collected for lipid and total fatty acid analysis at the time course to check the expression level by centrifugation (3,000 rpm, 15 min). Total fatty acids in E. coli were converted to fatty acid methyl esters (FAMEs) by adding 3 N methanolic HCl (Sigma-Aldrich) at 80°C for 2 h. After the transmethylation process, the sample was cooled down at room temperature before adding 1 ml of 0.9% NaCl and 2 ml of hexane. The sample was then mixed by vortex and centrifuged at 2,400 rpm for 5 min for phase separation. The hexane phase containing FAMEs was removed and dried under N2. After drying, the sample was resuspended in hexane and used for GC analysis. Two microliter samples of total FAME derivatives were analyzed on an Agilent 6890N gas chromatograph equipped with a DB-23 column (30 m × 0.25 mm) with 0.25 μm film thickness (J&W Scientific). The column temperature was maintained at 160°C for 1 min, and then raised to 240°C at a rate of 4°C/min. For mass spectrometry analysis, the mass selective detector was run under standard electron impact conditions (70 eV), scanning an effective m/z range of 40–700 at 2.26 scans/s. To further study the biosynthesis and assembly of VLCPUFAs in Thraustochytrium sp. 26185, we recently sequenced the whole genome of this species. From the genome sequence, we identified nearly all members in the aerobic pathway for the biosynthesis of DHA, except for acyl-CoA Δ9 desaturase, including five putative front-end desaturases (three Δ6 desaturases, one Δ5 desaturase, and one Δ4 desaturase) and three putative methyl-end desaturases for oxygenic desaturation, as well as at least three ELO type condensing enzymes for fatty acid elongations. In addition, a large type I FAS representing a fusion of fungus FASα and FASβ subunits for the synthesis of SA (18:0) and three large subunits of a type I PKS-like PUFA synthase, highly homologous to those of Schizochytrium for the biosynthesis of VLCPUFAs (19Hauvermale A. Kuner J. Rosenzweig B. Guerra D. Diltz S. Metz J.G. Fatty acid production in Schizochytrium sp.: Involvement of a polyunsaturated fatty acid synthase and a type I fatty acid synthase.Lipids. 2006; 41: 739-747Crossref PubMed Scopus (122) Google Scholar), were also identified. These data indicate that both aerobic and anaerobic pathways might coexist in the species (Fig. 1A, B). To interrogate whether both pathways were functional for the VLCPUFA biosynthesis, we attempted to clone all putative genes in the two pathways that were not biochemically characterized previously, and then expressed them in yeast or E. coli for the functional analysis. In the aerobic pathway, several genes encoding front-end desaturases and elongases had been previously cloned and functionally characterized from the species. For instance, one Δ5 desaturase and one Δ4 desaturase could introduce a double bond into eicosatetraenoic acid (20:4n-3) and docosapentaenoic acid (DPA, 22:5n-3), giving EPA (20:5n-3) and DHA, respectively (17Qiu X. Hong H. MacKenzie S.L. Identification of a Delta 4 fatty acid desaturase from Thraustochytrium sp. involved in the biosynthesis of docosahexanoic acid by heterologous expression in Saccharomyces cerevisiae and Brassica juncea.J. Biol. Chem. 2001; 276: 31561-31566Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). One functional elongase capable of elongating both 18C and 20C PUFAs was characterized and used for stepwise engineering of DHA in an oilseed crop (21Wu G. Truksa M. Datla N. Vrinten P. Bauer J. Zank T. Cirpus P. Heinz E. Qiu X. Stepwise engineering to produce high yields of very long-chain polyunsaturated fatty acids in plants.Nat. Biotechnol. 2005; 23: 1013-1017Crossref PubMed Scopus (262) Google Scholar). In addition to these genes, however, three putative front-end and three putative methyl-end desaturase genes in the aerobic pathway identified from the genome were previously uncharacterized. To functionally analyze these genes, pairs of specific primers targeting outside the individual ORFs were designed and used for reverse transcriptase-PCR amplifications with the total RNA as the template. The amplified coding regions of these putative desaturase genes were individually cloned into a yeast vector and expressed in yeast. The results showed that one of three putative Δ6 desaturase genes indeed encoded an effective front-end desaturase introducing a Δ6 double bond into linoleic acid (LA, 18:2-9,12) and ALA (18:3-9,12,15), giving γ-linolenic acid (GLA, 18:3-6,9,12) and stearidonic acid (18:4-6,9,12,15), respectively (Fig. 2A). The rest did not possess any fatty acid desaturation activity. One of three putative methyl-end desaturase genes, when expressed in yeast, showed little Δ12 desaturase activity on oleic acid (18:1-9) to give product LA (18:2-9,12) at the level of less than 0.1% of the total fatty acids, as compared with the positive control (the yeast transformant with a Δ12 desaturase from fungus Claviceps purpurea) where a substantial amount of two Δ12 desaturated products (16:2-9,12 and 18:2-9,12) on two substrates (16:1-9 and 18:1-9) was observed (22Meesapyodsuk D. Reed D.W. Covello P.S. Qiu X. Primary structure, regioselectivity, and evolution of the membrane-bound fatty acid desaturases of Claviceps purpurea.J. Biol. Chem. 2007; 282: 20191-20199Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar) (Fig. 2B). Another putative methyl-end desaturase gene conferred ω3 desaturation activity converting 20C ω6 fatty acids, such as ARA (20:4-5,8,11,14) and dihomo-GLA (20:3-8,11,14), into their corresponding ω3 fatty acids, such as EPA and eicosatetraenoic acid (20:4-8,11,14,17) (Fig. 2C, supplemental Fig. S1). However, the third one did not show any desaturase activity when expressed in yeast. Collectively, these data indicate that all catalytic steps, except for two ineffective ones (Δ9 and Δ12) from SA (18:0) to LA, in the aerobic pathway might be functional in this species. To confirm that the aerobic pathway might be incomplete by missing the two critical desaturation steps in the species, we fed Thraustochytrium with several key fatty acid intermediates in the aerobic pathway to test the desaturation and elongation activities in vivo. The results showed that Thraustochytrium could effectively elongate both an 18C Δ6 desaturated fatty acid, such as GLA (18:3-6,9,12), and a 20C Δ5 desaturated fatty acid, such as ARA (20:4-5,8,11,14), with particularly highly efficient elongation on 18C PUFAs. The elongation efficiency from 18:3-6,9,12 to 20:3-8,11,14 reached more than 50% (Table 1). The front-end and ω3 desaturation activities were also observed in the species on the fed substrates. However, no Δ9 desaturation on fed 18:0 and no Δ12 desaturation on fed 18:1-9 were observed. In Thraustochytrium, a very small amount of 18:0 (∼1% of the total fatty acids) was produced. When fed with a large amount of 18:0, it still did not produce any 18:1-9 from the fed substrate (Fig. 3A). Thraustochytrium did not produce any 18:1-9. When fed with this fatty acid, it did not produce any detectable 18:2-9,12 from the fed substrate (Fig. 3B, Table 1). These data unequivocally confirmed that the aerobic pathway was incomplete in Thraustochytium sp. 26185 where two critical desaturation activities (Δ9 and Δ12) were either ineffective or missing from the pathway.TABLE 1In vivo desaturation and elongation activities on fatty acids fed to Thraustochytrium sp. 26185Fatty Acid SubstrateDesaturated or Elongated ProductConversion Efficiency18:018:1-90.0 ± 0.0018:1-918:2-9,12 (Δ12 desaturation)0.0 ± 0.0018:2-9,1218:3-9,12,15 (Δ15 desaturation)2.7 ± 0.1118:3-6,9,12 (Δ6 desaturation)5.7 ± 0.1018:3-6,9,1220:3-8,11,14 (Δ6 elongation)56.8 ± 2.4918:4-6,9,12,15 (Δ15 desaturation)0.0 ± 0.0020:3-8,11,1420:4-5,8,11,14 (Δ5 desaturation)16.4 ± 0.2220:4-8,11,14,17 (Δ17 desaturation)29.6 ± 2.9520:4-5,8,11,1422:4-7,10,13,16 (Δ5 elongation)34.2 ± 2.2920:5-5,8,11,14,17 (Δ17 desaturation)27.0 ± 3.79Conversion efficiency was calculated by [products/(substrate plus products)] × 100. Values are mean ± SD of four replicates. Open table in a new tab Conversion efficiency was calculated by [products/(substrate plus products)] × 100. Values are mean ± SD of four replicates. Next, we looked into the anaerobic pathway for the VLCPUFA biosynthesis in Thraustochytium. From the genome sequence, three large ORFs (∼8.5 kb, 6.2 kb, and 4.5 kb) encoding three subunits (ORF-A, 2,813 amino acids; ORF-B, 2,049 amino acids; and ORF-C, 1,497 amino acids) of the mega-enzyme that was highly homologous to a PUFA synthase of Schizochytrium (12Metz J.G. Roessler P. Facciotti D. Levering C. Dittrich F. Lassner M. Valentine R. Lardizabal K. Domergue F. Yamada A. et al.Production of polyunsaturated fatty acids by polyketide synthases in both prokaryotes and eukaryotes.Science. 2001; 293: 290-293Crossref PubMed Scopus (549) Google Scholar, 19Hauvermale A. Kuner J. Rosenzweig B. Guerra D. Diltz S. Metz J.G. Fatty acid production in Schizochytrium sp.: Involvement of a polyunsaturated fatty acid synthase and a type I fatty acid synthase.Lipids. 2006; 41: 739-747Crossref PubMed Scopus (122) Google Scholar) were identified. Most catalytic domains in the three subunits were predicted based on the presence of characteristic active sites (23Keatinge-Clay A.T. The structures of type I polyketide synthases.Nat. Prod. Rep. 2012; 29: 1050-1073Crossref PubMed Scopus (215) Google Scholar). Subunit I comprised one β-ketoacyl-ACP synthase (KS1) domain, one malonyl-CoA:ACP acyltransferase domain, eight ACP domains, one ketoacyl reductase domain, and one dehydratase (DH1) domain. Subunit II comprised two KS domains (KS2 and KS3), one acyltransferase domain, and one enoyl-ACP reductase (ER1) domain. Subunit III consisted of two DH domains (DH2 and DH3) and one ER domain (ER2) (Fig. 4). Furthermore, a putative gene encoding PPTase required for attaching a phosphopantetheine prosthetic group to the ACP domains (19Hauvermale A. Kuner J. Rosenzweig B. Guerra D. Diltz S. Metz J.G. Fatty acid production in Schizochytrium sp.: Involvement of a polyunsaturated fatty acid synthase and a type I fatty acid synthase.Lipids. 2006; 41: 739-747Crossref PubMed Scopus (122) Google Scholar) was also identified from the genome. To functionally characterize the putative PUFA synthase, the three ORFs were first individually cloned into three E. coli vectors, each behind a T7 promoter (pCOLADuet-1 for ORF-A, pCDFDuet-1 for ORF-B, and pETDuet-1 for ORF-C). In addition, a PPTase (HetI) from Nostoc PCC7120 (24Black T.A. Wolk C.P. Analysis of a Het- mutation in Anabaena sp. strain PCC 7120 implicates a secondary metabolite in the regulation of heterocyst spacing.J. Bacteriol. 1994; 176: 2282-2292Crossref PubMed Scopus (139) Google Scholar) was also included in pCOLADuet-1 beside ORF-A for the expression analysis (Fig. 5A). As the ORFs were large, cloning them into the expression vectors was challenging. To facilitate the cloning process, ORF-A was divided into three pieces, and ORF-B was divided into two pieces according to the internal restriction sites for sequential cloning. The final constructs were built by assembling these pieces together according to their order using a restriction-cloning procedure. To reconstitute the anaerobic pathway heterologously, all three recombinant plasmids were simultaneously transformed into the E. coli strain BL21(star)DE3. The functional analysis showed that the transformant produced two new fatty acids, DHA and DPA (22:5n-6), compared with the strain transformed with empty vectors. Although the amount of the two Δ4 desaturated VLCPUFAs produced was at only about 1–2% of the total fatty acids in the transformant (Fig. 6A), the result confirmed that the PUFA synthase from Thraustochytrium was functional for the biosynthesis of VLCPUFAs. To improve production of VLCPUFAs in E. coli, we further attempted to coordinate expressions of three ORFs of the PUFA synthase in an operon by constructing one recombinant plasmid comprising the three ORFs behind a single promoter with a ribosome binding site motif
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