Mammalian enzymes and chaperones are often needed to express human proteins with complex folding and glycosylation requirements. In alternative hosts, however, exogenous secretory pathway proteins can be inactive or toxic when overexpressed or localized to the wrong cellular compartment. Engineered promoters have been traditionally used to rationally tune the expression of heterologous genes, but the toolbox of expression cassettes is limited for non-model organisms. Native promoters are an elegant alternative to engineered promoters, encompassing native induction and <i>trans</i> regulation when driving expression of a heterologous gene. Recently, simplified gene editing tools and the accessibility of genomic and transcriptomic sequencing have expanded the toolbox of expression cassettes to include the entire host genome. Here, we use native promoters in <i>Komagataella phaffii</i> to achieve both rational tuning and inducible expression of both endogenous and exogenous secretory pathway genes to achieve uniform engineered glycosylation and protein folding while avoiding toxicity. Native promoters and regulation provide a nearly unlimited toolbox of available gene expression cassettes in non-model organisms, exemplifying the advantages and flexibility of efficient, markerless gene editing tools.

Utilizing native regulation of heterologous secretory pathway genes in yeast engineered for expression of human proteins

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.