Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China.
Science Center for Future Foods, Ministry of Education, Jiangnan University, Wuxi 214122, China.
ACS Synth Biol. 2023 Apr 21;12(4):1146-1153. doi: 10.1021/acssynbio.2c00635. Epub 2023 Apr 4.
The metabolic burden caused by terpenoid accumulation limits the development of highly efficient microbial cell factories, which can be circumvented using exporter-mediated product secretion. Although our previous study showed that the pleiotropic drug resistance exporter (PDR11) mediates the export of rubusoside in , the underlying mechanism is still unclear. Herein, we used GROMACS software to simulate PDR11-mediated rubusoside recruitment and found six residues (D116, D167, Y168, P521, R663, and L1146) on PDR11 that are critical for this process. We also explored the exportation potential of PDR11 for 39 terpenoids by calculating their binding affinity using batch molecular docking. Then, we verified the accuracy of the predicted results by conducting experiments with squalene, lycopene, and β-carotene as examples. We found that PDR11 can efficiently secrete terpenoids with binding affinities lower than -9.0 kcal/mol. Combining the computer-based prediction and experimental verification, we proved that binding affinity is a reliable parameter to screen exporter substrates and might potentially enable rapid screening of exporters for natural products in microbial cell factories.
萜类化合物积累所导致的代谢负担限制了高效微生物细胞工厂的发展，而利用外排蛋白介导的产物分泌则可以规避这一问题。尽管我们之前的研究表明，多药耐药外排蛋白（PDR11）介导了毛蕊花糖苷在 中的输出，但其中的具体机制仍不清楚。在此，我们使用 GROMACS 软件模拟了 PDR11 介导的毛蕊花糖苷募集过程，并发现 PDR11 上有六个关键残基（D116、D167、Y168、P521、R663 和 L1146）对这一过程至关重要。我们还通过批量分子对接计算了 39 种萜类化合物的结合亲和力，从而探究了 PDR11 对它们的外排潜力。然后，我们以角鲨烯、番茄红素和β-胡萝卜素为例进行实验，验证了预测结果的准确性。结果发现，PDR11 能够有效地分泌结合亲和力低于-9.0 kcal/mol 的萜类化合物。通过计算机预测与实验验证相结合，我们证明了结合亲和力是筛选外排蛋白底物的可靠参数，这可能使我们能够快速筛选微生物细胞工厂中天然产物的外排蛋白。
