Wong Ryan Wei Kwan, Foo Marissa, Lay Jasmine R S, Wai Tiffany L T, Moore Jackson, Dutreux Fabien, Molzahn Cristen, Nislow Corey, Measday Vivien, Schacherer Joseph, Mayor Thibault
Department of Biochemistry and Molecular Biology, Michael Smith Laboratories, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada.
Department of Food Science, Wine Research Centre, Faculty of Land and Food Systems, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada.
Microb Cell Fact. 2025 Mar 10;24(1):60. doi: 10.1186/s12934-025-02677-1.
The budding yeast Saccharomyces cerevisiae is a widely utilized host cell for recombinant protein production due to its well studied and annotated genome, its ability to secrete large and post-translationally modified proteins, fast growth and cost-effective culturing. However, recombinant protein yields from S. cerevisiae often fall behind that of other host systems. To address this, we developed a high-throughput screen of wild, industrial and laboratory S. cerevisiae isolates to identify strains with a natural propensity for greater recombinant protein production, specifically focussing on laccase multicopper oxidases from the fungi Trametes trogii and Myceliophthora thermophila. Using this method, we identified 20 non-laboratory strains with higher capacity to produce active laccase. Interestingly, lower levels of laccase mRNA were measured in most cases, indicating that the drivers of elevated protein production capacity lie beyond the regulation of recombinant gene expression. We characterized the identified strains using complementary genomic and proteomic approaches to reveal several potential pathways driving the improved expression phenotype. Gene ontology analysis suggests broad changes in cellular metabolism, specifically in genes/proteins involved in carbohydrate catabolism, thiamine biosynthesis, transmembrane transport and vacuolar degradation. Targeted deletions of the hexose transporter HXT11 and the Coat protein complex II interacting paralogs PRM8 and 9, involved in ER to Golgi transport, resulted in significantly improved laccase production from the S288C laboratory strain. Whereas the deletion of the Hsp110 SSE1 gene, guided by our proteomic analysis, also led to higher laccase activity, we did not observe major changes of the protein homeostasis network within the strains with higher laccase activity. This study opens new avenues to leverage the vast diversity of Saccharomyces cerevisiae for recombinant protein production, as well as offers new strategies and insights to enhance recombinant protein yields of current strains.
出芽酵母酿酒酵母是一种广泛用于重组蛋白生产的宿主细胞，因其基因组已得到充分研究和注释，能够分泌大量经翻译后修饰的蛋白，生长迅速且培养成本效益高。然而，酿酒酵母的重组蛋白产量往往落后于其他宿主系统。为解决这一问题，我们开发了一种高通量筛选野生、工业和实验室酿酒酵母分离株的方法，以鉴定具有更高重组蛋白生产天然倾向的菌株，特别关注来自真菌糙皮侧耳和嗜热毁丝霉的漆酶多铜氧化酶。通过这种方法，我们鉴定出20株具有更高活性漆酶生产能力的非实验室菌株。有趣的是，在大多数情况下，漆酶mRNA水平较低，这表明蛋白生产能力提高的驱动因素超出了重组基因表达的调控范围。我们使用互补的基因组学和蛋白质组学方法对鉴定出的菌株进行了表征，以揭示驱动表达表型改善的几种潜在途径。基因本体分析表明细胞代谢发生了广泛变化，特别是在参与碳水化合物分解代谢、硫胺素生物合成、跨膜运输和液泡降解的基因/蛋白质方面。对参与内质网到高尔基体运输的己糖转运蛋白HXT11以及与衣被蛋白复合物II相互作用的旁系同源物PRM8和9进行靶向缺失，导致S288C实验室菌株的漆酶产量显著提高。而在我们的蛋白质组学分析指导下删除Hsp110 SSE1基因，也导致漆酶活性更高，但我们没有观察到漆酶活性较高的菌株内蛋白质稳态网络的重大变化。这项研究为利用酿酒酵母的广泛多样性进行重组蛋白生产开辟了新途径，同时也为提高现有菌株的重组蛋白产量提供了新策略和见解。
