18.05.2021
+++ News Ticker Science #83 +++ Biocatalysts +++
Unspecific peroxygenases: New method for producing new enzymes in yeast.
Unspecific peroxygenases (UPOs) can hydroxylate a wide range of substrates and are therefore of enormous interest as biocatalysts for shortening industrial synthesis pathways. The enzyme class occurs naturally in higher fungi, which are increasingly proving to be a promising source of various UPOs. Since more and more higher fungi have been sequenced in the recent past, more than 4000 potential peroxygenase genes have already been identified. However, heterologous expression of the coveted enzymes - for example in yeasts - turns out to be extremely difficult: the proteins are highly glycosylated and bear a haem-thiolate active site. Scientists at IPB, together with partners at MLU, have now developed a modular Golden Gate-based secretion system that enables the expression of active UPOs in yeast, including their one-step purification and activity testing.
In this work, 17 different signal peptide sequences were combined with the genes of eight UPOs and seven C-terminal protein tags for subsequent purification and split-GFP detection. Two of the UPO gene sequences used had only been derived from genome and secretome data and had not yet been characterized as peroxygenases before. After testing the UPOs heterologously expressed in S. cerevisiae for activity and substrate specificity, the Halle scientists transferred the complete secretion system to Pichia pastoris, a biotechnologically used expression yeast, resulting in excellent amounts of UPO expression.
UPOs were first discovered at the beginning of the millennium. They catalyze a variety of hydroxylation reactions very efficiently and require only hydrogen peroxide as a cosubstrate. However, their use as biocatalysts in selected synthetic and biotechnological processes is limited by their suboptimal regio- and enantioselectivities towards certain substrates. In addition, currently a limited choice of UPOs is available, which hinders the search for a suitable catalyst for the desired reaction. With their study, the Halle chemists are laying the foundation for better screening and exploitation of the enormous potential of previously unused or even unknown UPOs from the fungal kingdom.
