Optimizing tyrosine production in yeast via in vivo continuous evolution
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Tyrosine is an aromatic amino acid with a wide range of uses. Tyrosine is a precursor for numerous biological compounds ranging from neurotransmitters to hormones. It therefore plays a big role in the pharmaceutical industry. Tyrosine is also used by the chemical industry to make non-biological products such as polymers, adhesives, and semiconductors. Yeasts are unicellular eukaryotic organisms capable of producing tyrosine through the shikimate pathway. We seek to evolve specific genes in the Shikimate pathway – ARO2, ARO3, ARO4 – to increase yeast tyrosine production and improve efficiency. These genes are evolved through a method called in vivo continuous evolution, or ICE, which is a novel way to generate mutations using a retrotransposon. ICE allows for mutations to target genes instead of random mutations throughout the genome. We used ICE to induce mutations to the shikimate genes and produced a library of mutant yeast cells. We screened the cells for high tyrosine production and found four possible mutants. Three of the four strains could not be sequenced due to failure to isolate the mutant gene through PCR. The mutant gene from the AROp-G strain was successfully isolated and amplified. Sequencing showed that AROp-G strain was a mutant with a truncated C-terminus at the ARO4 gene. We inserted this mutant gene into a plasmid which was then transformed into wild-type yeast. There was a significant increase in tyrosine production, confirming that increased tyrosine production was due to the mutant gene. We will continue to make additional mutant libraries and find more mutant sequences.