Research Article | Open Access

Volume 2024 |Article ID 0030 | https://doi.org/10.34133/bdr.0030

Copper-Induced In Vivo Gene Amplification in Budding Yeast

Junyi Wang,^1,4 Jingya Song,^1,4 Cong Fan,^1,4 Jiahao Duan,¹ Kaiyuan He,¹ Jifeng Yuan ^1,2,3

¹State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Faculty of Medicine and Life Sciences, Xiamen University, Fujian 361102, China
²Key Laboratory for Synthetic Biotechnology of Xiamen City, Xiamen University, Fujian 361005, China
³Shenzhen Research Institute of Xiamen University, Shenzhen 518057, China
⁴These authors contributed equally to this work

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.