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      Biochemical Characterization of a Type II Diacylglycerol Acyltransferase from Claviceps purpurea

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      Date
      2009
      Author
      Mavraganis, Ioannis
      Type
      Thesis
      Degree Level
      Masters
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      Abstract
      Claviceps purpurea, a fungal pathogen, of ergot diseases in agriculturally important cereal crops, produces high levels of glycerides containing ricinoleic acid (12-hydroxyoctadec-cis-9-enoic acid) in its sclerotia. A fatty acid hydroxylase (CpFAH) involved in the biosynthesis of ricinoleic acid was recently identified from C. purpurea. This research describes the biochemical characterization of a type II diacylglycerol acyltransferase (CpDGAT2) involved in the assembly of this fatty acid into triglycerides from C. purpurea. Expression of CpDGAT2 in a quadruple mutant Saccharomyses cerevisiae H1246, in which all four triacylglycerol (TG) biosynthesis genes (DGA1, LOR1, ACAT1 and ACAT2) were disrupted, restored the ability of the mutant to synthesize TGs in vivo. In vitro enzymatic assays of microsomal preparations of the transformants indicated that CpDGAT2 preferentially use ricinoleic acid over linoleic acid, oleic acid and linolenic acids as acyl donor, and 1,2-dioleoyl-sn-glycerol over 1,2-dipalmitoyl-sn-glycerol as acyl acceptor. CpDGAT2 did not show any activities for the formation of wax esters and estolides when 1-hexadecanol and triricinolein were used as acyl acceptors. Co-expression of CpFAH and CpDGAT2 in yeast resulted in increased accumulation of ricinoleic acids compared to expression of CpFAH along with the yeast native DGAT2 (ScDGA1) or expression of CpFAH alone. Northern blot analysis indicated that CpFAH is solely expressed in sclerotium cells and no transcripts of this gene were detected in mycelium and conidium cells. CpDGAT2 is more widely expressed in cell types examined except for conidiospores where the expression is low. The highest expression of CpDGAT2 was detected in 20 day-old sclerotium cells where the highest levels of ricinoleate glycerides are accumulated. Collectively, these data indicate CpDGAT2 and CpFAH are two key enzymes coordinating the biosynthesis and bioassembly of ricinoleic acid in C. purpurea.
      Degree
      Master of Science (M.Sc.)
      Department
      Applied Microbiology and Food Science
      Program
      Applied Microbiology and Food Science
      Supervisor
      Qiu, Xiao
      Committee
      Tyler, Robert T.; Stone, Scott J.; Vujanovic, Vladimir; Smith, Mark
      Copyright Date
      2009
      URI
      http://hdl.handle.net/10388/etd-05192009-110707
      Subject
      DGAT2 specificity
      DGAT2
      Claviceps
      ricinoleic acid
      Hydroxylase
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