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异戊二烯焦磷酸 1

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Development of a dual temperature control system for isoprene biosynthesis in

《化学科学与工程前沿（英文）》 2022年第16卷第7期页码 1079-1089 doi: 10.1007/s11705-021-2088-0

摘要： Conflict between cell growth and product accumulation is frequently encountered in the biosynthesis of secondary metabolites. To address the growth-production conflict in yeast strains harboring the isoprene synthetic pathway in the mitochondria, the dynamic control of isoprene biosynthesis was explored. A dual temperature regulation system was developed through engineering and expression regulation of the transcriptional activator Gal4p. A cold-sensitive mutant, Gal4ep19, was created by directed evolution of Gal4p based on an internally developed growth-based high-throughput screening method and expressed under the heat-shock promoter P_SSA4 to control the expression of P_GAL-driven pathway genes in the mitochondria. Compared to the control strain with constitutively expressed wild-type Gal4p, the dual temperature regulation strategy led to 34.5% and 72% improvements in cell growth and isoprene production, respectively. This study reports the creation of the first cold-sensitive variants of Gal4p by directed evolution and provides a dual temperature control system for yeast engineering that may also be conducive to the biosynthesis of other high-value natural products.

关键词： transcriptional activator directed evolution dynamic control heat-shock isoprene

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重构异戊二烯焦磷酸代谢以促进萜类化合物的合成 Article

徐显皓, 吕雪芹, 崔世修, 刘延峰, 夏洪志, 李江华, 堵国成, 李兆丰, Rodrigo Ledesma-Amaro, 陈坚, 刘龙

《工程（英文）》 2023年第28卷第9期页码 166-178 doi: 10.1016/j.eng.2023.03.019

摘要：

萜类化合物是最大的一类天然产物。它们以异戊二烯焦磷酸(IPP)作为基本单元构建而成。使用细胞工厂合成萜类化合物已经引起了极大关注。而迄今为止，IPP的供应不足仍然是高效合成萜类化合物的主要挑战。在本项研究中，我们发现在枯草芽孢杆菌（Bacillus subtilis）中，中心代谢和IPP供应之间的代谢通量分配不平衡阻碍了IPP的积累。因此，我们构建了一系列响应丙酮酸和/或丙二酰辅酶A的双输入多输出（two-input-multi-output, TIMO）基因回路对IPP的代谢途径进行了改造，使IPP的积累增加了将近4倍。随后，设计了一种IPP代谢网络重构策略以提高三种萜类化合物的产量，包括维生素K2 (MK-7, 4.1倍)、番茄红素(9倍)和β-胡萝卜素（0.9倍）。在50-L的生物反应器中，MK-7的产量达到1549.6 mg∙L⁻¹，是迄今为止报道的最高产量。本文提出了一种基于TIMO基因回路的IPP代谢网络重构框架，可用于复杂代谢网络的协同精细调控，从而实现萜类化合物的高效合成。

关键词：萜类基因回路枯草芽孢杆菌异戊二烯焦磷酸