An exploration of the expression and engineering of a bacterial tyrosinase to improve its biocatalytic potential

Nova Mentis Ltd. is a University College Dublin (UCD) start-up company with a patented technology to produce the potent antioxidant compound hydroxytyrosol (HT). The key to this technology is the tyrosinase enzyme biocatalyst RV145 (accession number: JX272620.1), which was developed in a past project in UCD. However, the potential of the process to produce HT was limited by poor enzyme expression. This project first aimed to understand the issues surrounding the current tyrosinase expression system and then resolve them. Initial results showed the majority of tyrosinase produced by the E. coli expression host was trapped in insoluble inclusion bodies, which while active had reduced activity relative to the small amount of soluble tyrosinase present. Several strategies to remove inclusion bodies were then employed such as altered growth conditions & media, codon optimisation of the tyrosinase gene sequence, the use of alternate plasmid expression vectors and co-expression of the tyrosinase gene with protein folding chaperones. While some of these methods improved expression of soluble protein, none removed inclusion bodies completely. Alterations of the domain architecture of the tyrosinase were then undertaken to improve expression, through protein truncation and use of alternative signal peptide sequences. However, this also did not prevent inclusion body formation. As such, protein engineering was carried out by both random and site directed mutagenesis to find higher activity variants. However, no improved variant of RV145 was identified. Yet this study did result in the identification of key residues within the enzyme which are important for catalysis. Homology modelling and an in-silico substrate docking study were used to explore the substrate range of the parent enzyme of RV145, the R. pseudosolanacearum GMI1000 tyrosinase. This study resulted in the identification of several novel substrates for this enzyme and also the dentification of residues within the enzyme likely involved in catalysis. At the time of writing a manuscript describing the results of this study is under review by the FEBS journal. Attempts to culture E. coli at 5 L scale in bioreactor vessels revealed a surprising export of RV145 into the culture media resulting in a loss of tyrosinase captured in the cell mass. However, dramatic improvements to the hydroxytyrosol production process were achieved, resulting in a bioprocess which increased the titre of HT produced from 300 mM to 600 mM while halving the concentration of homogenate containing enzyme used.