Developing a synthetic yeast for the expression of heterologous genes using SCRaMbLE

Abstract

Synthetic genomics is a new and fast emerging multi-disciplinary field of research, representing the largest scale of work underway in synthetic biology. The Saccharomyces cerevisiae version 2 (Sc2.0) project is currently the leading example of synthetic genomics research, and is an attempt to perform the first redesign, synthesis and assembly of a complete synthetic genome for an eukaryotic organism. Major changes are being made to the redesigned DNA sequence of the new yeast genome and these include a variety of different deletions, sequence recodings at every gene and also insertions of DNA motifs. The most significant insertion is the placement of a recombinase recognition site called loxPsym throughout the genome, placed downstream of all non-essential genes. These recombinase sites act as recombination hotspots for Cre recombinase, to bring about recombination-mediated genomic rearrangements of the synthetic chromosomes. Together the Cre recombinase and loxPsym inserts make up the inducible Synthetic Chromosome Rearrangement and Modification by LoxPsym Evolution (SCRaMbLE) system. This thesis describes the design, synthesis, hierarchical assembly and in vivo integration of synthetic DNA for the construction of synthetic chromosome XI for the Sc2.0 project. With the first 90 kb of the synthetic chromosome complete, the SCRaMbLE toolkit was then examined. It was hypothesised that along with causing gene deletions, inversions and duplications, this Cre-lox system could also be implemented to insert heterologous DNA into the synthetic chromosomes. This thesis shows that with the correct formatting of heterologous DNA, SCRaMbLE can be further developed to generate a new synthetic biology method called ‘SCRaMbLE-in’ suitable for the insertion of heterologous genes into synthetic chromosomes as they are rearranging to produce diverse synthetic yeast strains with novel functions. Having successfully developed and investigated SCRaMbLE-in, this method was then used for the simultaneous introduction of multiple genes that can confer a selective benefit to yeast. By providing three heterologous genes encoding enzymes that together reconstitute the oxidoreductase xylose-utilisation pathway, a synthetic yeast strain capable of growth on the lignocellulosic sugar xylose was produced by SCRaMbLE-in. This work thus demonstrates a new approach to constructing strains for metabolic engineering projects, where incorporation of heterologous genes and rapid evolution of the yeast genome can be done simultaneously in one pot.