title: Production and characterization of in planta transiently produced polygalacturanase from Aspergillus niger and its fusions with hydrophobin or ELP tags.
abstract: BACKGROUND
  Pectinases play an important role in plant cell wall deconstruction and have potential in diverse industries such as food, wine, animal feed, textile, paper, fuel, and others. The demand for such enzymes is increasing exponentially, as are the efforts to improve their production and to implement their use in several industrial processes. The goal of this study was to examine the potential of producing polygalacturonase I from Aspergillus niger in plants and to investigate the effects of subcellular compartmentalization and protein fusions on its accumulation and activity.
  
  
  RESULTS
  Polygalacturonase I from Aspergillus niger (AnPGI) was transiently produced in Nicotiana benthamiana by targeting it to five different cellular compartments: apoplast, endoplasmic reticulum (ER), vacuole, chloroplast and cytosol. Accumulation levels of 2.5%, 3.0%, and 1.9% of total soluble protein (TSP) were observed in the apoplast, ER, and vacuole, respectively, and specific activity was significantly higher in vacuole-targeted AnPGI compared to the same enzyme targeted to the ER or apoplast. No accumulation was found for AnPGI when targeted to the chloroplast or cytosol. Analysis of AnPGI fused with elastin-like polypeptide (ELP) revealed a significant increase in the protein accumulation level, especially when targeted to the vacuole where the protein doubles its accumulation to 3.6% of TSP, while the hydrophobin (HFBI) fusion impaired AnPGI accumulation and both tags impaired activity, albeit to different extents. The recombinant protein showed activity against polygalacturonic acid with optimum conditions at pH 5.0 and temperature from 30 to 50°C, depending on its fusion. In vivo analysis of reducing sugar content revealed a higher release of reducing sugars in plant tissue expressing recombinant AnPGI compared to wild type N. benthamiana leaves.
  
  
  CONCLUSIONS
  Our results demonstrate that subcellular compartmentalization of enzymes has an impact on both the target protein accumulation and its activity, especially in the case of proteins that undergo post-translational modifications, and should be taken into consideration when protein production strategies are designed. Using plants to produce heterologous enzymes for the degradation of a key component of the plant cell wall could reduce the cost of biomass pretreatment for the production of cellulosic biofuels.
authors: Eridan Orlando
authors: EO
authors: Pereira
authors: Igor
authors: I
authors: Kolotilin
authors: Andrew Jonathan
authors: AJ
authors: Conley
authors: Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON N5V 4T3, Canada. Rima.Menassa@agr.gc.ca.
authors: Rima
authors: R
authors: Menassa
mesh_terms: D001234:Aspergillus niger / Q000201:enzymology*; D002736:Chloroplasts / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D011096:Polygalacturonase / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D013696:Temperature; D014026:Nicotiana / Q000378:metabolism*; D014617:Vacuoles / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D011096:Polygalacturonase