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Cyanophycinase CphE from P. alcaligenes produced in different compartments of N. benthamiana degrades high amounts of cyanophycin in plant extracts

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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.

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Acknowledgements

We thank Prof. Dr. Steinbüchel (Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität, Münster, Germany) and Dr. Krehenbrink (Cysal GmbH, Münster, Germany) for providing the CphE encoding plasmid. Likewise, we thank Dr. Gleba and Dr. Giritch (Nomad BioScience; Halle/Saale, Germany) for the MagnICON expression vectors and for their constructive discussions. In addition, we would like to express the warmest of thanks to Alex Rajewski for thoroughly proof-reading the manuscript and the helpful suggestions and comments.

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  1. 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 & Inge Broer

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  1. Henrik Nausch
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Nausch, H., Broer, I. Cyanophycinase CphE from P. alcaligenes produced in different compartments of N. benthamiana degrades high amounts of cyanophycin in plant extracts. Appl Microbiol Biotechnol 101, 2397–2413 (2017). https://doi.org/10.1007/s00253-016-8020-8

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