Enzyme-constrained genome-scale models (ecGEMs) have potential to predict phenotypes in a variety of conditions, such as growth rates or carbon sources. This study investigated if ecGEMs can guide metabolic engineering efforts to swap anaerobic redox-neutral ATP-providing pathways in yeast from alcoholic fermentation to equimolar co-production of 2,3-butanediol and glycerol. With proven pathways and low product toxicity, the ecGEM solution space aligned well with observed phenotypes. Since this catabolic pathway provides only one-third of the ATP of alcoholic fermentation (2/3 versus 2 ATP per glucose), the ecGEM predicted a growth decrease from 0.36 h in the reference to 0.175 h in the engineered strain. However, this <3-fold decrease would require the specific glucose consumption rate to increase. Surprisingly, after the pathway swap the engineered strain immediately grew at 0.15 h with a glucose consumption rate of 29 mmol (g CDW) h, which was indeed higher than reference (23 mmol (g CDW) h) and one of the highest reported for . The accompanying 2,3-butanediol- (15.8 mmol (g CDW) h) and glycerol (19.6 mmol (g CDW) h) production rates were close to predicted values. Proteomics confirmed that this increased consumption rate was facilitated by enzyme reallocation from especially ribosomes (from 25.5 to 18.5 %) towards glycolysis (from 28.7 to 43.5 %). Subsequently, 200 generations of sequential transfer did not improve growth of the engineered strain, showing the use of ecGEMs in predicting opportunity space for laboratory evolution. The observations in this study illustrate both the current potential, as well as future improvements, of ecGEMs as a tool for both metabolic engineering and laboratory evolution.

中文翻译：

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通过酿酒酵母厌氧联产 2,3-丁二醇和甘油的代谢工程评估酶约束基因组规模模型

酶约束基因组规模模型 (ecGEM) 有潜力预测各种条件下的表型，例如生长速率或碳源。本研究调查了 ecGEM 是否可以指导代谢工程工作，将酵母中的厌氧氧化还原中性 ATP 提供途径从酒精发酵转变为等摩尔共生产 2,3-丁二醇和甘油。凭借经过验证的途径和低产品毒性，ecGEM 解决方案空间与观察到的表型非常吻合。由于这种分解代谢途径仅提供酒精发酵 ATP 的三分之一（每葡萄糖 2/3 与 2 ATP），因此 ecGEM 预测生长会从参考文献中的 0.36 小时减少到工程菌株中的 0.175 小时。然而，这种<3倍的降低需要增加特定的葡萄糖消耗率。令人惊讶的是，在途径交换后，工程菌株立即在 0.15 小时内生长，葡萄糖消耗率为 29 mmol (g CDW) h，这确实高于参考值 (23 mmol (g CDW) h)，并且是报道的最高葡萄糖消耗率之一。 。伴随的 2,3-丁二醇 (15.8 mmol (g CDW) h) 和甘油 (19.6 mmol (g CDW) h) 生产率接近预测值。蛋白质组学证实，酶从核糖体（从 25.5% 到 18.5%）重新分配到糖酵解（从 28.7% 到 43.5%）促进了消耗率的增加。随后，200 代的连续转移并未改善工程菌株的生长，这表明 ecGEM 可用于预测实验室进化的机会空间。本研究中的观察结果说明了 ecGEM 作为代谢工程和实验室进化工具的当前潜力以及未来的改进。