# Copper-Induced In Vivo Gene Amplification in Budding Yeast

## TL;DR

This study introduces a method using copper to trigger gene amplification in yeast, enabling higher protein production for biotech applications.

## Contribution

A novel copper-induced gene amplification system in budding yeast for multicopy gene expression is developed.

## Key findings

- Deleting the CUP1 gene made yeast sensitive to copper, enabling its use as a selection pressure.
- Gene amplification at retrotransposon sites increased protein expression under copper stress.
- Tandem duplication at the CUP1 locus was achieved under high copper concentrations.

## Abstract

In the biotechnological industry, multicopy gene integration represents an effective strategy to maintain a high-level production of recombinant proteins and to assemble multigene biochemical pathways. In this study, we developed copper-induced in vivo gene amplification in budding yeast for multicopy gene expressions. To make copper as an effective selection pressure, we first constructed a copper-sensitive yeast strain by deleting the CUP1 gene encoding a small metallothionein-like protein for copper resistance. Subsequently, the reporter gene fused with a proline–glutamate–serine–threonine-destabilized CUP1 was integrated at the δ sites of retrotransposon (Ty) elements to counter the copper toxicity at 100 μM Cu2+. We further demonstrated the feasibility of modulating chromosomal rearrangements for increased protein expression under higher copper concentrations. In addition, we also demonstrated a simplified design of integrating the expression cassette at the CUP1 locus to achieve tandem duplication under high concentrations of copper. Taken together, we envision that this method of copper-induced in vivo gene amplification would serve as a robust and useful method for protein overproduction and metabolic engineering applications in budding yeast.