Transketolase enzyme discovery and engineering

Transketolases (TKs) are important C-C bond forming enzymes that in vivo transfer a two carbon ketol unit to the acceptors d-ribose-5-phosphate or d-erythrose-4-phosphate. There is significant interest in biocatalytic applications where frequently the donor β-hydroxypyruvic acid is used. In recent years there has been interest in the discovery of new TKs with unique or robust properties that are an excellent starting point for mutagenesis, or that are able to accept new acceptors or donors. Similarly, TK mutagenesis has led to TKs with alternative substrate profiles. In this chapter, firstly an overview of the substrates accepted by TKs is briefly summarized. Then, metagenomic strategies for the discovery of unique TKs and how this approach has developed with an early example, and a more recent study on the discovery of 'split'-TKs, are described with methods. Finally, enzyme evolution methods and approaches to develop a wide range of TKs with modified substrate acceptance and improved stabilities are detailed.