Supplementary information to: Simpler is not always better: transplanting the Yarrowia lipolytica glycolytic pathway into Saccharomyces cerevisiae reveals essential synergetic regulatory mechanisms

This document contains supplementary Tables and Figures pertaining to the research article entitled: Simpler is not always better: transplanting the <i>Yarrowia lipolytica</i> glycolytic pathway into<i> Saccharomyces cerevisiae</i> reveals essential synergetic regulatory mechanisms.<br>Abstract The Embden-Meyerof-Parnas pathway of glycolysis is a widely distributed and intensively investigated metabolic route. While allosteric regulation is thought to be essential for glycolytic flux dynamics in many organisms including yeast, to date single enzyme complementation studies with non-allosteric glycolytic enzymes have failed to experimentally demonstrate this essentiality and quantify the overall contribution of allosteric regulation in tuning the glycolytic flux. This study brings new insight in the synergetic metabolic role of allosteric regulation by implementing pathway swapping, a strategy enabling to remodel, in two simple genetic interventions, the entire glycolytic pathway of <i>Saccharomyces cerevisiae.</i> <i>S. cerevisiae</i> equipped with the full set of non-allosteric glycolytic enzymes from the oleaginous yeast <i>Y. lipolytica</i> lost the ability to grow on media containing 2% glucose and displayed dynamic responses suggesting metabolic imbalance between upper and lower glycolysis. Single and combined gene complementation demonstrated that this phenotype was caused by the simultaneous deregulation of the three key kinases: hexokinase, phosphofructokinase and pyruvate kinase. ‘Deregulated glycolysis’ <i>S. cerevisiae</i> strains could naturally restore glycolytic stability and growth on glucose by evolving mutations in the <i>Y. lipolytica</i> glucokinase, causing a strong decrease in glucokinase activity and glycolytic flux. This solution could be recapitulated in non-evolved deregulated glycolysis <i>S. cerevisiae</i> strains by experimentally tuning glucose import. Supported by kinetic modelling, the present work demonstrates the major synergetic role played by allosteric regulations in preventing metabolic imbalance in glycolysis and highlights the power of synthetic biology in addressing long-standing questions in systems biology.<br>

本文件包含与下述研究论文配套的补充表格与附图：《化繁未必简：将产油酵母解脂耶氏酵母（Yarrowia lipolytica）糖酵解通路移植至酿酒酵母（Saccharomyces cerevisiae）揭示关键协同调控机制》。
摘要 糖酵解的Embden-Meyerhof-Parnas（EMP）通路是一类分布广泛且被深入研究的代谢途径。尽管包括酵母在内的诸多生物体中，变构调控（allosteric regulation）被认为对糖酵解流量动态至关重要，但迄今为止，使用非变构糖酵解酶开展的单酶互补研究，均未能通过实验证实这一必要性，也无法量化变构调控在调节糖酵解流量中的整体贡献。本研究通过实施通路置换策略——仅需两次简单的遗传操作即可重塑酿酒酵母的完整糖酵解通路——为阐明变构调控的协同代谢功能提供了全新见解。携带产油酵母解脂耶氏酵母全套非变构糖酵解酶的酿酒酵母，无法在含2%葡萄糖的培养基中生长，且其动态响应提示糖酵解上下区段存在代谢失衡。单基因与组合基因互补实验表明，该表型源于三种关键激酶：己糖激酶、磷酸果糖激酶与丙酮酸激酶的调控同时失常。携带"失调型糖酵解"特征的酿酒酵母菌株，可通过在解脂耶氏酵母葡萄糖激酶中积累突变，自然恢复糖酵解稳定性与葡萄糖生长能力，该突变可显著降低葡萄糖激酶活性与糖酵解流量。通过实验手段调控葡萄糖摄入，可在未经过定向进化的失调型糖酵解酿酒酵母菌株中重现这一恢复表型。本研究结合动力学建模，证实了变构调控在预防糖酵解代谢失衡中发挥的核心协同作用，并彰显了合成生物学在解答系统生物学领域长期遗留问题上的强大能力。