Enabling brighter living by enzyme engineering: From structure inspired trial and error to structure guided design

Enzymes evolve in nature to enable life. They can provide a competitive edge to organisms to survive under changing environmental conditions. In industry, we apply enzymes under conditions and for reactions that can be quite different from those for which the enzyme evolved in nature. For this reason, enzyme properties are often not fit for the intended industrial applications. Enzyme engineering is therefore an important tool to overcome these limitations and unlocks the potential of enzymes for many applications. This presentation will provide an overview of different enzyme applications developed by us and enabled by Enzyme Engineering. Examples are selected to show how enzyme engineering methods have evolved over time since our first commercial production of non-animal derived chymosin in 1988 [1]. One of the early enzyme engineering work to enable “Green Routes” for beta-lactam antibiotics was the directed evolution of a glutarylacylase into an adipylacylase. In a first mutagenesis round sites contributing to the adipyl activity where explored followed by saturation mutagenesis of these residues. Only in hindsight, the identified mutations could be rationalized based on the enzyme structure [2]. Another interesting example of enzyme engineering was the development of a thermostable phytases. The developed `consensus approach\u27 showed that sequence information of homologous, mesophilic enzymes contains sufficient information to allow rapid design of a thermostabilized, fully functional phytase [3]. More recently, we apply computational methods to create “smart libraries” with more reliable predictions of beneficial mutations. Together with the University of California in San Francisco, we designed a new computational tool for the Rosetta computational design software package, which out-performs previous design tools [4]. Such methods are especially important for enzyme engineering problems in which the throughput of the applied screening methods is limited, for example, due to the complexity of matrices we apply for food enzyme developments. [1] J.R. Shuster et al. (1990) Kluyveromyces as a host for heterologous gene expression: Expression and secretion of prochymosin. Bio/Technology 8 (2), 135-139 [2] C.F. Sio, A.M. Riemens, J.-M. van der Laan, R.M.D. Verhaert, W.J. Quax (2002) Directed evolution of a glutaryl acylase into an adipyl acylase. Eur.J.Biochem.269, 4495–4504. [3] M. Lehmann, L. Pasamontes, S.F. Lassen, M. Wyss (2000) The consensus concept for thermostability engineering of proteins Biochimica et Biophysica Acta 1543, 408-415. [4] N. Ollikainen, R.M. de Jong, T. Kortemme (2015) Coupling Protein Side-Chain and Backbone Flexibility Improves the Re-design of Protein-Ligand Specificity. PLOS Computational Biology 11(9)

Peroxicretion: a novel secretion pathway in the eukaryotic cell

Background: Enzyme production in microbial cells has been limited to secreted enzymes or intracellular enzymes followed by expensive down stream processing. Extracellular enzymes consists mainly of hydrolases while intracellular enzymes exhibit a much broader diversity. If these intracellular enzymes could be secreted by the cell the potential of industrial applications of enzymes would be enlarged. Therefore a novel secretion pathway for intracellular proteins was developed, using peroxisomes as secretion vesicles. Results: Peroxisomes were decorated with a Golgi derived v-SNARE using a peroxisomal membrane protein as an anchor. This allowed the peroxisomes to fuse with the plasma membrane. Intracellular proteins were transported into the peroxisomes by adding a peroxisomal import signal (SKL tag). The proteins which were imported in the peroxisomes, were released into the extracellular space through this artificial secretion pathway which was designated peroxicretion. This concept was supported by electron microscopy studies. Conclusion: Our results demonstrate that it is possible to reroute the intracellular trafficking of vesicles by changing the localisation of SNARE molecules, this approach can be used in in vivo biological studies to clarify the different control mechanisms regulating intracellular membrane trafficking. In addition we demonstrate peroxicretion of a diverse set of intracellular proteins. Therefore, we anticipate that the concept of peroxicretion may revolutionize the production of intracellular proteins from fungi and other microbial cells, as well as from mammalian cells.

On the enzymatic cycle of p-hydroxybenzoate hydroxylase

Het enzym para-hydroxy benzoaat hydroxylase (PHBH) uit de bacterie Pseudomonas fluorescence katalyseert de volgende reactie: (zie samenvatting) Het enzym bestaat uit een keten opgebouwd uit 392 aminozuren met als prosthetische groep flavine adenine dinucleotide (FAD), dat niet covalent, doch wel op zeer specifieke wijze aan het eiwit gebonden is. De eigenschappen van de flavine worden in belangrijke mate door de interacties met het eiwit bepaald. In PHBH activeert de flavine een zuurstof molecule waarvan één atoom op één zeer specifieke plaats wordt ingebouwd in het substraat, terwijl het andere atoom een watermolecuul vormt. Hiervoor zijn vier electronen nodig: twee worden geleverd door het coenzym NADPH, de andere twee door het substraat. De beschreven reactie is een eerste stap in de afbraak van de zeer stabiele aromatische ring. ... Zie: Samenvatting