title: Cyanophycinase CphE from P. alcaligenes produced in different compartments of N. benthamiana degrades high amounts of cyanophycin in plant extracts.
abstract: One of the major constraints in pig and poultry farming is the supply of protein-rich forage, containing sufficient amounts of key amino acids such as arginine (Ufaz and Galili 2008). Since these are underrepresented in plant proteins, the usage of plants as feed is limited. The heterologous production of the cyanobacterial storage polymer cyanophycin granule polypeptide (CGP) in plastids increases the amount of arginine substantially (Huhns et al. 2008; Huhns et al. 2009; Nausch et al. 2016a). CGP degradation releases arginine-aspartate dipeptides. CGP is stable in plants because its degradation is exclusively restricted to bacterial cyanophycinases (CGPases; Law et al. 2009). Since animals are also unable to digest CGP, CGPases need to be co-delivered with CGP-containing plant feed in order to release the dipeptides in the gastrointestinal tract of animals during digestion. Therefore, an extracellular CGPase, CphE from Pseudomonas alcaligenes DIP-1, was targeted to the cytosol, ER, and apoplasm of Nicotiana benthamiana. Translocation to the chloroplast was not successful. Although CphE accumulated in high amounts in the cytosol, only moderate levels were present in the ER, while the enzyme was nearly undetectable in the apoplasm. This correlates with the higher instability of post-translationally modified CphE in crude plant extracts. In addition, the production in the ER led to an increased number and size of necroses compared with cytosolic expression and might therefore interfere with the endogenous metabolism in the ER. Due to the high and robust enzyme activity, even moderate CphE concentrations were sufficient to degrade CGP in plant extracts.
authors.affiliation: Faculty of Agricultural and Environmental Sciences, Department of Agrobiotechnology and Risk Assessment for Bio- und Gene Technology, University of Rostock, Justus-von-Liebig Weg 8, 18059, Rostock, VM, Germany. henrik.nausch@uni-rostock.de.
authors.forename: Henrik
authors.initials: H
authors.lastname: Nausch
authors.affiliation: Faculty of Agricultural and Environmental Sciences, Department of Agrobiotechnology and Risk Assessment for Bio- und Gene Technology, University of Rostock, Justus-von-Liebig Weg 8, 18059, Rostock, VM, Germany.
authors.forename: Inge
authors.initials: I
authors.lastname: Broer
mesh_terms: D000595:Amino Acid Sequence; D000821:Animal Feed; D001120:Arginine / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D003600:Cytosol / Q000378:metabolism; D004044:Dietary Proteins / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D006868:Hydrolysis; D008958:Models, Molecular; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism*; D059828:Plant Cells / Q000378:metabolism; D010936:Plant Extracts / Q000737:chemistry*; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D044202:Pseudomonas alcaligenes / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*; D019076:Transgenes
chemical_list: D001426:Bacterial Proteins; D004044:Dietary Proteins; D010936:Plant Extracts; D011994:Recombinant Proteins; C024250:cyanophycin; D001120:Arginine; D010447:Peptide Hydrolases; C120585:cyanophycinase
keywords: Cyanophycinase CphE; Enzyme activity; Nicotiana benthamiana; Post-translational modifications; Protein stability