Directed evolution of Escherichia coli LacZ gene to create diversity in glycosidic bonds hydrolysis

Abstract

Starting with LacZ of Escherichia coli, coding for β-galactosidase, the aim of the thesis project is to apply in vitro directed evolution techniques to help create other glycosidic bond hydrolysis activities. This was done using the main β-galactosidase backbone with limited amino acid sequence change. Any altered glycosyl hydrolase activity would lead to changed substrate specificity. Moreover, genetic changes leading to improved beta-galactosidase activity was also investigated. Error-prone PCR was applied to the LacZ gene (β-galactosidase) to achieve the desired aims. The technique used to introduce random mutagenesis was based on modifications of method developed by Xu et al., 1999.Optimization was performed with DNA polymerase selection, PCR conditions and various Mn and dITP concentrations to obtain best amplified PCR product for random mutagenesis library construction. Plasmid pTZ1 containing the entire coding sequence of LacZ was used a whole plasmid random Mutagenesis library construction strategy. The complete pTZ1 plasmid sequence had to be done in order to help establish a framework for primer design and establish a complete restriction map of the plasmid including the lacZ gene. The sequence analysis of the plasmid revealed that it has 5,502bp. Screening of random mutagenesis libraries was based on the colour development resulting from the glycosidic hydrolysis of chromogenic substrate to identify any glycosidic activity towards particular glycosyl hydrolase on LB plates or M9 plates. We have screened random mutagenesis libraries for any possible activity for β-glucosidase, β-xylosidase or for an improved β-galactosidase activity. Colonies that showed colour development on substrate even after retransformation of plasmid DNA for β-xylosidase activity were selected and its mutated plasmid DNA was sequenced. Two of the variants in which one has mutation at K552E position and another at N959Y were isolated, from two different clear blue colonies on β-xylosidase substrate. However, to the issue of change in substrate specificity (colour development on plates) was not clear. The direct evolution method applied here is seems simpler and promising in creating random mutagenesis libraries in order to select variants with useful novel properties.