Exploiting SCRaMbLE to increase fatty acid synthesis in yeast
thesisposted on 2022-03-28, 22:01 authored by Thomas Robert Collier
Fatty acids (FAs) and lipids produced in yeast could provide an alternative source for consumable oils in the future. In this work, four recombinant yeast strains were developed using The SCRaMbLEing technology (Synthetic Chromosome Recombination and Modification by Lox-P Mediated Evolution) developed in the global Yeast 2.0 project aiming to build a completely synthetic Saccharomyces cerevisiae. The genes: ACC1 (acetyl-CoA carboxylase 1), ACS1 (acetyl-CoA synthetase), FAS1 (fatty acid synthase 1) and FAS2 (fatty acid synthase 2) enclosed by loxP sites were overexpressed in a semi-synthetic yeast strain equipped with a synthetic chromosome 14 containing about 260 Lox-P sites to enable SCRaMbLE. The lipid stain Nile red stain for neutral lipids was successfully used in conjunction with flow cytometry to develop a high through put screening protocol for yeast strains with increased FA content. The FA profiles of the SCRaMbLEd strains Syn-sXIV,Syn-sACC1, and Syns-FAS2 were then analysed by gas chromatography mass spectrometry and an increase in C16:0, C16:1, C18:0 and C18:1 FA content was detected in strains expressing ACC1, and FAS2.