Development of novel metabolite biosensors
thesisposted on 29.03.2022, 00:41 authored by Alexander Carpenter
Adaptive laboratory evolution (ALE) offers a potentially powerful method of genetic engineering for S. cerevisiae cell factories. One of the major limitations in ALE is the limited genetic diversity generation currently capable with traditional mutagenesis methods. The SCRaMbLE technique, outlined as a part of the Synthetic Yeast Genome Project (Sc 2.0) has the capacity to generate significant genetic diversity for use in ALE. Discussed here is the partial construction of synthetic chromosome XIV as a part of Sc 2.0, with successful integration of approximately 80 kilobases of synthetic DNA. Also presented here is the development of two biosensors for use in S. cerevisiae ALE experiments for the metabolites butanol and methionine, both of which are industrially significant chemicals. The butanol biosensor was based on the BmoRp transcription factor from Thauera butanovorans. Treatment of cells containing this biosensor with 5mM of butanol resulted in a 1.7 fold increase in GFP expression. The methionine biosensor was based on the S. cerevisiae MET17 promoter and has a dynamic range of 8.6 fold with the addition of 5mM methionine. Thorough characterisation of the components that comprise these biosensors suggest promising modifications that could improve their function and ultimately enable their use for ALE.