Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts.
Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts.
Biotechnol Bioeng. 2020 Feb;117(2):543-555. doi: 10.1002/bit.27209. Epub 2019 Nov 12.
Komagataella phaffii, also known as Pichia pastoris, is a common host for the production of biologics and enzymes, due to fast growth, high productivity, and advancements in host engineering. Several K. phaffii variants are commonly used as interchangeable base strains, which confounds efforts to improve this host. In this study, genomic and transcriptomic analyses of Y-11430 (CBS7435), GS115, X-33, and eight other variants enabled a comparative assessment of the relative fitness of these hosts for recombinant protein expression. Cell wall integrity explained the majority of the variation among strains, impacting transformation efficiency, growth, methanol metabolism, and secretion of heterologous proteins. Y-11430 exhibited the highest activity of genes involved in methanol utilization, up to two-fold higher transcription of heterologous genes, and robust growth. With a more permeable cell wall, X-33 displayed a six-fold higher transformation efficiency and up to 1.2-fold higher titers than Y-11430. X-33 also shared nearly all mutations, and a defective variant of HIS4, with GS115, precluding robust growth. Transferring two beneficial mutations identified in X-33 into Y-11430 resulted in an optimized base strain that provided up to four-fold higher transformation efficiency and three-fold higher protein titers, while retaining robust growth. The approach employed here to assess unique banked variants in a species and then transfer key beneficial variants into a base strain should also facilitate rational assessment of a broad set of other recombinant hosts.
毕赤酵母（Komagataella phaffii），又称巴斯德毕赤酵母（Pichia pastoris），由于其生长迅速、生产能力高、宿主工程技术不断进步，是生物制品和酶类生产的常用宿主。几种毕赤酵母变体通常作为可互换的基础菌株使用，这使得提高该宿主的性能变得复杂。在这项研究中，对 Y-11430（CBS7435）、GS115、X-33 和其他八种变体进行了基因组和转录组分析，从而能够对这些宿主用于重组蛋白表达的相对适应性进行比较评估。细胞壁完整性解释了菌株间大部分变异，影响转化效率、生长、甲醇代谢和异源蛋白分泌。Y-11430 表现出参与甲醇利用的基因的最高活性，异源基因的转录高达两倍，生长健壮。X-33 的细胞壁更具渗透性，转化效率比 Y-11430 高六倍，产量高达 1.2 倍。X-33 还与 GS115 共享几乎所有的突变和一个缺陷型 HIS4 变体，这使得 GS115 无法健壮生长。将在 X-33 中鉴定的两个有益突变转移到 Y-11430 中，得到了一个优化的基础菌株，其转化效率提高了四倍，蛋白产量提高了三倍，同时保持了健壮的生长。本研究采用的方法评估了一个物种中独特的库存变体，然后将关键的有益变体转移到基础菌株中，这也应该有助于对广泛的其他重组宿主进行合理评估。
