Transcriptomic profiling of the yeast Komagataella phaffii in response to environmental alkalinization

Adaptation to alkalinization of the medium in fungi involves an extensive remodeling of gene expression. Komagataella phaffii is an ascomycetous yeast that has become an organism widely used for heterologous protein expression. We explore here the transcriptional impact of moderate alkalinization in this yeast. In spite of a minor effect on growth, shifting the cultures from pH 5.5 to 8.0 or 8.2 provokes significant changes in the mRNA levels of over 700 genes. Functional categories such as arginine and methionine biosynthesis, non-reductive iron uptake and phosphate metabolism are enriched in induced genes, whereas many genes encoding iron-sulfur proteins or members of the respirasome were repressed. We also show that alkalinization is accompanied by oxidative stress and we propose this circumstance as a common trigger of a subset of the observed changes. PHO89, encoding a Na+/Pi cotransporter, appears among the most potently induced genes by high pH. We demonstrate that this response is mainly based on two calcineurin-dependent response elements located in its promoter, thus indicating that alkalinization triggers a calcium-mediated signal in K. phaffii. Further characterization of alkaline pH responsive promoters may lead to developing novel pH-controlled systems for heterologous protein expression in this fungus. comparison of mRNA levels by RNA-Seq between Komagataella phaffii yeast cells grown at pH 5.5 (referene) and 8.0 (grown on glycerol or glucose) or 8.2 (grown on glycerol). Replicates are included.

真菌对培养基碱化的适应过程涉及基因表达的广泛重塑。科马加塔毕赤酵母（Komagataella phaffii）是一类子囊菌酵母，现已广泛应用于异源蛋白表达。本研究探讨了中等强度碱化对该酵母的转录影响。
尽管对菌体生长仅存在轻微影响，将培养体系从pH 5.5调整至pH 8.0或8.2，可导致超过700个基因的mRNA水平发生显著改变。
诱导上调基因富集于精氨酸与甲硫氨酸生物合成、非还原性铁摄取及磷酸代谢等功能类别；而大量编码铁硫蛋白或呼吸体组分的基因则被抑制。
本研究同时证实，碱化伴随氧化应激的产生，我们据此提出氧化应激是部分观测到的表达变化的共同触发因素。编码Na+/Pi共转运蛋白的PHO89基因，是高pH条件下诱导效应最强的基因之一。实验证明，该响应主要依赖于其启动子区域内的两个钙调磷酸酶依赖性应答元件，这表明碱化在科马加塔毕赤酵母中可触发钙介导的信号通路。
对碱性pH应答启动子的进一步表征，有望开发出适用于该真菌的新型pH调控表达系统，用于异源蛋白生产。
本数据集通过RNA测序（RNA-Seq）对比了在pH 5.5（参照组）、pH 8.0（以甘油或葡萄糖为碳源培养）及pH 8.2（以甘油为碳源培养）条件下培养的科马加塔毕赤酵母细胞的mRNA水平，且包含生物学重复。