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<p>A high-throughput and sensitive screen for the improved expression of gene targets in <i>Saccharomyces cerevisiae</i> is described that is based upon the activity of the luciferase from <i>Gaussia princeps.</i> Using the Unspecific Peroxygenase (UPO) from <i>Agrocybe aegerita</i> (<i>Aae</i>UPO) as a model protein, improvements in expression, effected through error-prone PCR-based mutation within the signal peptide (SP) domain, can be detected using fusion of the target to <i>Gaussia</i> luciferase encoded downstream of the first folded domain of the <i>Aae</i>UPO protein and luminescent assay of expression supernatants. In this way, previously undiscovered mutations within the SP of <i>Aae</i>UPO that improve expression were revealed, and then applied to the expression of full-length <i>Aae</i>UPO in <i>S. cerevisiae</i>. The system was validated against control expression constructs that were well or poorly expressed and indicated a 13.9-fold improvement in expression for the best mutant over the wild-type SP sequence. It is envisaged that this protocol may be applied generally to the high-throughput detection of improved expression in <i>S. cerevisiae</i> for constructs that are engineered using directed evolution techniques.</p>

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Identification of improved signal peptides for heterologous expression in Saccharomyces using a screen that exploits Gaussia luciferase

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年7月4日

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  • Ginevra Camboni,
  • Jared Cartwright,
  • Gideon Grogan

摘要

A high-throughput and sensitive screen for the improved expression of gene targets in Saccharomyces cerevisiae is described that is based upon the activity of the luciferase from Gaussia princeps. Using the Unspecific Peroxygenase (UPO) from Agrocybe aegerita (AaeUPO) as a model protein, improvements in expression, effected through error-prone PCR-based mutation within the signal peptide (SP) domain, can be detected using fusion of the target to Gaussia luciferase encoded downstream of the first folded domain of the AaeUPO protein and luminescent assay of expression supernatants. In this way, previously undiscovered mutations within the SP of AaeUPO that improve expression were revealed, and then applied to the expression of full-length AaeUPO in S. cerevisiae. The system was validated against control expression constructs that were well or poorly expressed and indicated a 13.9-fold improvement in expression for the best mutant over the wild-type SP sequence. It is envisaged that this protocol may be applied generally to the high-throughput detection of improved expression in S. cerevisiae for constructs that are engineered using directed evolution techniques.