Design of fusion proteins for biocatalysis.

Abstract

The use of enzymes to convert substrates into valuable products has been an integral part of biocatalysis. However, some reactions are energy-demanding that requires the use of NAD(P)H to proceed. This NAD(P)H can be costly impeding the progress of enzyme usage at a bigger scale. The rise of sophisticated cloning methods has allowed the possibility of constructing multi-enzyme complexes such as coupling NAD(P)H-requiring enzymes with NADH-regeneration systems such as formate dehydrogenases. This allows a more-efficient way to recycle co-factors or co-substrates with cheaper sacrificial substrate such as formate for formate dehydrogenases or glucose for glucose dehydrogenases. However, the design of fusion proteins requires careful attention especially on the peptide linker that will be used to connect two protein domains. The length and the property of the linker and even the orientation of the genes encoding for the proteins in the open reading frame can significantly affect the outcome of the fusion protein. In this chapter, we present a step-by-step procedure for the design of a fusion protein construct via Gibson assembly and how to design linker libraries from one construct using site-directed mutagenesis.