Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.
Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.
Biotechnol J. 2023 Apr;18(4):e2200535. doi: 10.1002/biot.202200535. Epub 2023 Feb 13.
Engineered Saccharomyces cerevisiae expressing a lactic acid dehydrogenase can metabolize pyruvate into lactic acid. However, three pyruvate decarboxylase (PDC) isozymes drive most carbon flux toward ethanol rather than lactic acid. Deletion of endogenous PDCs will eliminate ethanol production, but the resulting strain suffers from C auxotrophy and struggles to complete a fermentation. Engineered yeast assimilating xylose or cellobiose produce lactic acid rather than ethanol as a major product without the deletion of any PDC genes. We report here that sugar flux, but not sensing, contributes to the partition of flux at the pyruvate branch point in S. cerevisiae expressing the Rhizopus oryzae lactic acid dehydrogenase (LdhA). While the membrane glucose sensors Snf3 and Rgt2 did not play any direct role in the option of predominant product, the sugar assimilation rate was strongly correlated to the partition of flux at pyruvate: fast sugar assimilation favors ethanol production while slow sugar assimilation favors lactic acid. Applying this knowledge, we created an engineered yeast capable of simultaneously converting glucose and xylose into lactic acid, increasing lactic acid production to approximately 17 g L from the 12 g L observed during sequential consumption of sugars. This work elucidates the carbon source-dependent effects on product selection in engineered yeast.
工程化酿酒酵母表达的乳酸脱氢酶可以将丙酮酸代谢为乳酸。然而，三种丙酮酸脱羧酶（PDC）同工酶将大部分碳通量导向乙醇而不是乳酸。敲除内源性 PDC 会消除乙醇的产生，但由此产生的菌株会出现 C 营养缺陷，并难以完成发酵。工程化酵母同化木糖或纤维二糖会产生乳酸而不是乙醇作为主要产物，而无需敲除任何 PDC 基因。我们在这里报告，糖通量而不是感应有助于在表达根霉乳酸脱氢酶（LdhA）的酿酒酵母中丙酮酸分支点的通量分配。虽然膜葡萄糖传感器 Snf3 和 Rgt2 没有在主要产物的选择中发挥任何直接作用，但糖同化率与丙酮酸处的通量分配密切相关：快速糖同化有利于乙醇的产生，而慢速糖同化有利于乳酸的产生。应用这一知识，我们创造了一种能够同时将葡萄糖和木糖转化为乳酸的工程化酵母，将乳酸的产量从 12 g/L 左右提高到 17 g/L，这是在顺序消耗糖时观察到的。这项工作阐明了碳源对工程化酵母产物选择的依赖性影响。
