Supplementary data to the publication: What’s wrong with SwYG? Reaching the limits of the Saccharomyces cerevisiae molecular and analytical toolbox

Supplementary Material pertaining to the manuscript:<br><b>What’s wrong with SwYG? Reaching the limits of <i>S. cerevisiae</i> molecular and analytical toolbox</b> Ewout Knibbe, Eline D. Postma, Francine J. Boonekamp, Sofiia Dashko, Jordi Geelhoed, Anne-Marijn Maat, Marijke A.H. Luttik, Marcel van den Broek, Pascale Daran-Lapujade<br><b>Abstract:</b>The construction of powerful cell factories requires extensive remodelling of microbial genomes, entailing many rounds of transformations to perform the large number of desired gene modifications. However, increasing the number of genetic interventions inevitably increases the occurrence of unwanted mutations and effects. Using glycolysis as paradigm, a previous study developed a <i>Saccharomyces cerevisiae</i> strain in which the glycolytic genes, relocated to a single locus, can be easily swapped by any new design, thereby enabling fast and easy remodelling of the entire pathway. After 27 genetic modifications performed in 43 transformation rounds, the Switchable Yeast Glycolysis (SwYG) strain grew ca. 20% slower than its ancestor with the same glycolytic genes with native glycolysis design. Exploring the cause of this slower growth rate, the present study reflects on the genetic and analytical challenges encountered by extensive strain construction programs and provides design guidelines for integration of large constructs in the yeast genome. This study also suggests a potential involvement of the yeast glycolytic enzyme phosphoglycerate kinase (Pgk1) in PI(3)P synthesis and autophagy, as found in mammalian cells.<br>Includes supplemental figures and tables with descriptions of their content. <br>

本补充材料对应学术论文：**《SwYG存在哪些问题？触及酿酒酵母（*Saccharomyces cerevisiae*）分子与分析工具库的极限》**
作者：Ewout Knibbe、Eline D. Postma、Francine J. Boonekamp、Sofiia Dashko、Jordi Geelhoed、Anne-Marijn Maat、Marijke A.H. Luttik、Marcel van den Broek、Pascale Daran-Lapujade

**摘要：**
构建高性能细胞工厂需对微生物基因组进行大规模重构，该过程需通过多轮转化操作完成大量目标基因修饰。然而，遗传干预次数的增加不可避免地会提升意外突变与不良效应的发生概率。
此前一项研究以糖酵解为范式，构建了一株酿酒酵母菌株：该菌株的糖酵解基因已被整合至单一基因座，可通过任意新设计轻松进行置换，从而实现整条代谢通路的快速简便重构。在经过43轮转化操作、完成27次基因修饰后，该可切换酵母糖酵解（Switchable Yeast Glycolysis, SwYG）菌株的生长速度较其亲本菌株（采用天然糖酵解设计、携带相同糖酵解基因）慢约20%。
本研究针对该生长速率减缓的原因展开探究，回顾了大规模菌株构建项目中面临的遗传与分析挑战，并为大型基因元件整合至酵母基因组提供了设计指南。此外，本研究还发现，酵母糖酵解酶磷酸甘油酸激酶（phosphoglycerate kinase, Pgk1）可能参与了磷脂酰肌醇3-磷酸（PI(3)P）合成与自噬过程，这一现象与哺乳动物细胞中的相关发现一致。

本补充材料包含附带内容说明的补充图表。