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Recombinant Expression of Trichoderma reesei Cel61A in Pichia pastoris: Optimizing Yield and N-terminal Processing.

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  • معلومة اضافية
    • المصدر:
      Publisher: Springer Country of Publication: Switzerland NLM ID: 9423533 Publication Model: Print Cited Medium: Internet ISSN: 1559-0305 (Electronic) Linking ISSN: 10736085 NLM ISO Abbreviation: Mol Biotechnol Subsets: MEDLINE
    • بيانات النشر:
      Publication: [Cham] : Springer
      Original Publication: Totowa, NJ : Humana Press, c1994-
    • الموضوع:
      Fungal Proteins/*biosynthesis ; Mixed Function Oxygenases/*biosynthesis ; Pichia/*metabolism ; Trichoderma/*enzymology; Amino Acid Sequence ; Biomass ; Cellulose/chemistry ; Cellulose/genetics ; DNA, Fungal/genetics ; Fermentation ; Fungal Proteins/genetics ; Hydrolysis ; Mating Factor ; Mixed Function Oxygenases/genetics ; Molecular Sequence Data ; Peptides/genetics ; Peptides/metabolism ; Polysaccharides/chemistry ; Saccharomyces cerevisiae/metabolism ; Sequence Analysis, DNA ; Trichoderma/genetics
    • نبذة مختصرة :
      The auxiliary activity family 9 (AA9, formerly GH61) harbors a recently discovered group of oxidative enzymes that boost cellulose degradation. Indeed, these lytic polysaccharide monooxygenases (LPMOs) are able to disrupt the crystalline structure of cellulose, thereby facilitating the work of hydrolytic enzymes involved in biomass degradation. Since these enzymes require an N-terminal histidine residue for activity, their recombinant production as secreted protein is not straightforward. We here report the expression optimization of Trichoderma reesei Cel61A (TrCel61A) in the host Pichia pastoris. The use of the native TrCel61A secretion signal instead of the alpha-mating factor from Saccharomyces cerevisiae was found to be crucial, not only to obtain high protein yields (>400 mg/L during fermentation) but also to enable the correct processing of the N-terminus. Furthermore, the LPMO activity of the enzyme is demonstrated here for the first time, based on its degradation profile of a cellulosic substrate.
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    • Contributed Indexing:
      Keywords: Auxiliary activity family 9; Cellulose hydrolysis; Lytic polysaccharide monooxygenase; N-terminal processing; Pichia pastoris; Trichoderma reesei Cel61A (TrCel61A)
    • الرقم المعرف:
      0 (DNA, Fungal)
      0 (Fungal Proteins)
      0 (Peptides)
      0 (Polysaccharides)
      61194-02-3 (Mating Factor)
      9004-34-6 (Cellulose)
      EC 1.- (Mixed Function Oxygenases)
    • الموضوع:
      Date Created: 20150820 Date Completed: 20160809 Latest Revision: 20181113
    • الموضوع:
      20231215
    • الرقم المعرف:
      10.1007/s12033-015-9887-9
    • الرقم المعرف:
      26285758