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Shimbata T.,Nippon Flour Mills Company | Inokuma T.,Nippon Flour Mills Company | Sunohara A.,Nippon Flour Mills Company | Vrinten P.,Bioriginal Food and Science Corporation | And 3 more authors.
Journal of Agricultural and Food Chemistry | Year: 2011

Sweet wheat (SW), which lacks functional granule-bound starch synthase I (GBSSI) and starch synthase IIa (SSIIa), accumulates high levels of free sugars in immature seeds. Here, we examined the effects of the lack of these two enzymes on mature kernel composition.Whole grain flour ofSW had higher levels of sugars, particularly maltose, slightly higher ash and protein content, approximately two to three times higher lipid levels, and about twice as much total dietary fiber as parental or wild-type lines. Considerably higher levels of low-molecular-weight soluble dietary fiber (LMW-SDF), largely consisting of fructan, were also detected in SW. Although there were no differences in total amino acid levels, the free amino acid content of SW was approximately 4-fold higher than that of wild type, and the levels of certain free amino acids such as proline were particularly high. Thus, we were able to clearly demonstrate that the lack of GBSSI and SSIIa caused dramatic changes in mature seed composition in SW. These compositional changes suggest that SW flour may provide health benefits when used as a food ingredient. © 2011 American Chemical Society. Source

Mavraganis I.,University of Saskatchewan | Meesapyodsuk D.,University of Saskatchewan | Vrinten P.,Bioriginal Food and Science Corporation | Smith M.,National Research Council Canada | Qiu X.,University of Saskatchewan
Applied and Environmental Microbiology | Year: 2010

Claviceps purpurea, the fungal pathogen that causes the cereal disease ergot, produces glycerides that contain high levels of ricinoleic acid [(R)-12-hydroxyoctadec-cis-9-enoic acid] in its sclerotia. Recently, a fatty acid hydroxylase (C. purpurea FAH [CpFAH]) involved in the biosynthesis of ricinoleic acid was identified from this fungus (D. Meesapyodsuk and X. Qiu, Plant Physiol. 147:1325-1333, 2008). Here, we describe the cloning and biochemical characterization of a C. purpurea type II diacylglycerol acyltransferase (CpDGAT2) involved in the assembly of ricinoleic acid into triglycerides. The CpDGAT2 gene was cloned by degenerate RT-PCR (reverse transcription-PCR). The expression of this gene restored the in vivo synthesis of triacylglycerol (TAG) in the quadruple mutant strain Saccharomyces cerevisiae H1246, in which all four TAG biosynthesis genes (DGA1, LR01, ARE1, and ARE2) are disrupted. In vitro enzymatic assays using microsomal preparations from the transformed yeast strain indicated that CpDGAT2 prefers ricinoleic acid as an acyl donor over linoleic acid, oleic acid, or linolenic acid, and it prefers 1,2-dioleoyl-sn-glycerol over 1,2-dipalmitoyl-sn-glycerol as an acyl acceptor. The coexpression of CpFAH with CpDGAT2 in yeast resulted in an increased accumulation of ricinoleic acid compared to the coexpression of CpFAH with the native yeast DGAT2 (S. cerevisiae DGA1 [ScDGA1]) or the expression of CpFAH alone. Northern blot analysis indicated that CpFAH is expressed solely in Sclerotium cells, with no transcripts of this gene being detected in mycelium or conidial cells. CpDGAT2 was more widely expressed among the cell types examined, although expression was low in conidiospores. The high expression of CpDGAT2 and CpFAH in Sclerotium cells, where high levels of ricinoleate glycerides accumulate, provided further evidence supporting the roles of CpDGAT2 and CpFAH as key enzymes for the synthesis and assembly of ricinoleic acid in C. purpurea. Copyright © 2010, American Society for Microbiology. All Rights Reserved. Source

Cheng B.,Bioriginal Food and Science Corporation | Cheng B.,Agriculture and Agri Food Canada | Wu G.,Bioriginal Food and Science Corporation | Vrinten P.,Bioriginal Food and Science Corporation | And 3 more authors.
Transgenic Research | Year: 2010

Eicosapentaenoic acid (EPA, 20:5n-3) plays an important role in many aspects of human health. In our efforts towards producing high levels of EPA in plants, we investigated the effects of different host species, genes and promoters on EPA biosynthesis. Zero-erucic acid Brassica carinata appeared to be an outstanding host species for EPA production, with EPA levels in transgenic seed of this line reaching up to 25%. Two novel genes, an 18-carbon ω3 desaturase (CpDesX) from Claviceps purpurea and a 20-carbon ω3 desaturase (Pir-ω3) from Pythium irregulare, proved to be very effective in increasing EPA levels in high-erucic acid B. carinata. The conlinin1 promoter from flax functioned reasonably well in B. carinata, and can serve as an alternative to the napin promoter from B. napus. In summary, the judicious selection of host species and promoters, together with the inclusion of genes that enhance the basic very long chain polyunsaturated fatty acid biosynthetic pathway, can greatly influence the production of EPA in plants. © Springer Science+Business Media B.V. 2009. Source

Vrinten P.L.,Bioriginal Food and Science Corporation | Hoffman T.,Bioriginal Food and Science Corporation | Bauer J.,BASF | Qiu X.,Bioriginal Food and Science Corporation | Qiu X.,University of Saskatchewan
Biochemistry | Year: 2010

We describe a condensing enzyme from Pythium irregulare (PirELO) that shows highest activity on the 18-carbon, δ-6 desaturated fatty acids, stearidonic acid and γ-linolenic acid. However, this enzyme is also capable of elongating a number of other fatty acids including the 20-carbon, δ-5 desaturated fatty acid eicosapentaenoic acid. Surprisingly, a Phytophthora infestans condensing enzyme (PinELO) with very high homology to PirELO did not show activity with 20-carbon fatty acids. A series of chimeric proteins for these two enzymes were constructed to investigate the influence of different regions on substrate and product length. The substitution of a region from near the center of PirELO into PinELO resulted in an enzyme having EPA-elongating activity similar to that of PirELO. Only eight amino acids differed between the two proteins in this region; however, substitution of the same region from PinELO into PirELO produced a protein which was almost inactive. The addition of a small region from near the N-terminus of PinELO was sufficient to restore activity with GLA, indicating that amino acids from these two regions interact to determine protein structure or function. Predicted topology models for PirELO and PinELO placed the two regions described here near the luminal?proximal ends of the first and fourth/fifth transmembrane helixes, at the opposite end of the condensing enzyme from four conserved regions thought to form a catalytic ring. Thus, protein characteristics determined by specific luminal?proximal regions of fatty acid condensing enzymes have a major influence on substrate specificity and final product length. © 2010 American Chemical Society. Source

Vrinten P.,Bioriginal Food and Science Corporation | Mavraganis I.,Bioriginal Food and Science Corporation | Mavraganis I.,National Research Council Canada | Qiu X.,University of Saskatchewan | Senger T.,BASF
Lipids | Year: 2013

Sphaeroforma arctica is a unique, recently discovered marine protist belonging to a group falling close to the yeast/animal border. S. arctica is found in cold environments, and accordingly has a fatty acid composition containing a high proportion of very long chain polyunsaturated fatty acids, including the ω3 polyunsaturated fatty acids eicosapentaenoic acid (EPA) and docosahexanoic acid (DHA). Two elongases and five desaturases, representing the complete set of enzymes necessary for the synthesis of DHA from oleic acid, were isolated from this species and characterized in yeast. One elongase showed high conversion rates on a wide range of 18 and 20 carbon substrates, and was capable of sequential elongation reactions. The second elongase had a strong preference for the 20-carbon fatty acids EPA and arachidonic acid, with over 80 % of EPA converted to docosapentaenoic acid (DPA) in the heterologous yeast host. The isolation of a Δ8-desaturase, along with the detection of eicosadienoic acid in S. arctica cultures indicated that this species uses the alternate Δ8-pathway for the synthesis of long-chain polyunsaturated fatty acids. S. arctica also carried a Δ4-desaturase that proved to be very active in the production of DHA from DPA. Finally, a long chain acyl-CoA synthetase from S. arctica improved DHA uptake in the heterologous yeast host and led to an improvement in desaturation and elongation efficiencies. © 2012 AOCS. Source

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