In metabolic engineering, unbalanced microbial carbon distribution has long blocked the further improvement in yield and productivity of high-volume natural metabolites. Current studies mostly focus on regulating desired biosynthetic pathways, whereas few strategies are available to maximize L-threonine efficiently. Here, we present a strategy to guarantee the supply of reduced cofactors and actualize L-threonine maximization by regulating cellular carbon distribution in central metabolic pathways. A thermal switch system was designed and applied to divide the whole fermentation process into two stages: growth and production. This system could rebalance carbon substrates between pyruvate and oxaloacetate by controlling the heterogenous expression of pyruvate carboxylase and oxaloacetate decarboxylation that responds to temperature. The system was tested in an L-threonine producer Escherichia coli TWF001, and the resulting strain TWF106/pFT24rp overproduced L-threonine from glucose with 111.78% molar yield. The thermal switch system was then employed to switch off the L-alanine synthesis pathway, resulting in the highest L-threonine yield of 124.03%, which exceeds the best reported yield (87.88%) and the maximum available theoretical value of L-threonine production (122.47%). This inducer-free genetic circuit design can be also developed for other biosynthetic pathways to increase product conversion rates and shorten production cycles.

在代谢工程中，微生物碳分布不平衡长期以来阻碍了大量天然代谢物产量和生产力的进一步提高。目前的研究主要集中在调节所需的生物合成途径，而很少有策略可以有效地最大化 L-苏氨酸。在这里，我们提出了一种策略，通过调节中央代谢途径中的细胞碳分布来保证减少辅因子的供应并实现 L-苏氨酸最大化。设计并应用了热开关系统，将整个发酵过程分为生长和生产两个阶段。该系统可以通过控制丙酮酸羧化酶的异源表达和响应温度的草酰乙酸脱羧来重新平衡丙酮酸和草酰乙酸之间的碳底物。大肠杆菌TWF001 和由此产生的菌株 TWF106/pFT24rp 从葡萄糖中过量生产 L-苏氨酸，摩尔产率为 111.78%。然后使用热开关系统关闭 L-丙氨酸合成途径，导致最高的 L-苏氨酸产率达到 124.03%，超过了最佳报告产率 (87.88%) 和 L-苏氨酸生产的最大可用理论值(122.47%)。这种不含诱导剂的基因回路设计也可以用于其他生物合成途径，以提高产品转化率并缩短生产周期。