Bassett Shane, Da Silva Nancy A
Department of Chemical & Biomolecular Engineering, University of California, Irvine, CA, 92697-2580, USA.
Metab Eng Commun. 2024 May 20;18:e00238. doi: 10.1016/j.mec.2024.e00238. eCollection 2024 Jun.
Many desired biobased chemicals exhibit a range of toxicity to microbial cell factories, making industry-level biomanufacturing more challenging. Separating microbial growth and production phases is known to be beneficial for improving production of toxic products. Here, we developed a novel synthetic carbon-responsive promoter for use in the rapidly growing, stress-tolerant yeast , by fusing carbon-source responsive elements of the native promoter to the strong or native promoter cores. Two hybrids, P and P , were validated via EGFP fluorescence and demonstrated exceptional strength, partial repression during growth, and late phase activation in glucose- and lactose-based medium, respectively. Expressing the 2-pyrone synthase (2-PS) for synthesis of the polyketide triacetic acid lactone (TAL) under the control of P increased TAL more than 50% relative to the native promoter, and additional promoter engineering further increased TAL titer to 1.39 g/L in tube culture. Expression of the 6-methylsalicylic acid synthase (6-MSAS) under the control of P resulted in a 6.6-fold increase in 6-MSA titer to 1.09 g/L and a simultaneous 1.5-fold increase in cell growth. Finally, we used P to express the IaaM and IaaH proteins and the sabinene synthase protein to improve production of the auxin hormone indole-3-acetic acid and the monoterpene sabinene, respectively, both extremely toxic to yeast. The development of carbon-responsive promoters adds to the synthetic biology toolbox and available metabolic engineering strategies for , allowing greater control over heterologous protein expression and improved production of toxic metabolites.
许多理想的生物基化学品对微生物细胞工厂具有一系列毒性，这使得工业规模的生物制造更具挑战性。众所周知，分离微生物生长阶段和生产阶段有利于提高有毒产品的产量。在此，我们通过将天然启动子的碳源响应元件与强启动子或天然启动子核心融合，开发了一种新型的合成碳响应启动子，用于快速生长、耐胁迫的酵母。通过绿色荧光蛋白（EGFP）荧光验证了两个杂交启动子P 和P ，它们分别表现出超强的强度、生长阶段的部分抑制以及在基于葡萄糖和乳糖的培养基中的后期激活。在P 的控制下表达用于合成聚酮三乙酸内酯（TAL）的2-吡喃合酶（2-PS），相对于天然启动子，TAL产量提高了50%以上，进一步的启动子工程在摇瓶培养中将TAL滴度提高到1.39 g/L。在P 的控制下表达6-甲基水杨酸合酶（6-MSAS），使6-MSA滴度提高了6.6倍，达到1.09 g/L，同时细胞生长提高了1.5倍。最后，我们使用P 分别表达IaaM和IaaH蛋白以及桧烯合酶蛋白，以提高生长素吲哚-3-乙酸和单萜桧烯的产量，这两种物质对酵母都具有极高的毒性。碳响应启动子的开发丰富了合成生物学工具库以及可用于酿酒酵母的代谢工程策略，从而能够更好地控制异源蛋白表达并提高有毒代谢产物的产量。
