代谢工程改造酿酒酵母从头合成赤霉素A3

Metabolic engineering of Saccharomyces cerevisiae for de novo biosynthesis of gibberellin A3

摘要:

赤霉素A₃ (gibberellin A3, GA₃)作为一种高活性的植物生长调节剂，在农业生产中广泛应用。为构建高效合成GA₃的微生物细胞工厂，本研究以酿酒酵母C800为底盘菌株成功实现了GA₃的从头合成。首先，基于藤仓赤霉菌的赤霉素合成途径，整合GA₃合成的关键基因，同时结合甲羟戊酸途径关键基因(tHMG1、IDI1)过表达，使GA₃产量达到595 μg/L。之后，为增强P450酶的催化效率，优化了胞内还原型辅酶II (nicotinamide adenine dinucleotide phosphate, NADPH)和血红素供应，通过过表达ZWF1、POS5Δ17及HME3，使GA₃产量提高至1.73 mg/L。接着，通过整合拟南芥来源的膜结合支架蛋白AtMSBP1，将P450酶与细胞色素P450酶氧化还原伴侣(cytochrome P450 reductase, CPR)共定位于内质网膜区域，增强二者的空间邻近性与功能耦合，GA₃产量进一步提高至4.32 mg/L。最后，经5 L发酵罐高密度补料发酵，GA₃产量提升至14.17 mg/L。结果表明，本研究所构建的工程菌株具备良好放大潜力与代谢稳定性，为赤霉素的微生物制造提供了可行策略。

Abstract:

Gibberellin A₃ (GA₃), a highly active plant growth regulator, is widely used in agricultural production. To develop a microbial cell factory for efficient GA₃ synthesis, this study achieved the de novo biosynthesis of GA₃ with Saccharomyces cerevisiae as the chassis strain. First, on the basis of the gibberellin biosynthesis pathway of Gibberella fujikuroi, key genes for GA₃ synthesis were integrated, which, combined with the overexpression of mevalonate pathway genes (tHMG1 and IDI1), resulting in a GA₃ titer of 595 μg/L. Next, to enhance the catalytic efficiency of P450 enzymes, we optimized intracellular NADPH and heme supply by overexpressing ZWF1, POS5Δ17, and HME3, increasing the GA₃ titer to 1.73 mg/L. Subsequently, the integration of Arabidopsis thaliana-derived membrane-bound scaffold protein AtMSBP1 enabled the co-localization of P450 enzymes and CPR on the endoplasmic reticulum membrane, enhancing their spatial proximity and functional coupling, thereby improving the electron transfer efficiency and raising the GA₃ titer to 4.32 mg/L. Finally, high-cell-density fed-batch fermentation in a 5 L bioreactor further increased the GA₃ titer to 14.17 mg/L. These results demonstrate that the engineered strain exhibits promising scalability and metabolic stability, providing a feasible strategy for the microbial production of gibberellins.