School of Food Science and Pharmaceutical Engineering, Nanjing Normal University, Qixia District, Nanjing, People's Republic of China.
College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, People's Republic of China.
Biotechnol Bioeng. 2022 Oct;119(10):2819-2830. doi: 10.1002/bit.28176. Epub 2022 Jul 21.
The sesquiterpene α-humulene is an important plant natural product, which has been used in the pharmaceutical industry due to its anti-inflammatory and anticancer activities. Although phytoextraction and chemical synthesis have previously been applied in α-humulene production, the low efficiency and high costs limit the development. In this study, Yarrowia lipolytica was engineered as the robust cell factory for sustainable α-humulene production. First, a chassis with high α-humulene output in the cytoplasm was constructed by integrating α-humulene synthases with high catalytic activity, optimizing the flux of mevalonate and acetyl-CoA pathways. Subsequently, the strategy of dual cytoplasmic-peroxisomal engineering was adopted in Y. lipolytica; the best strain GQ3006 generated by introducing 31 copies of 12 different genes could produce 2280.3± 38.2 mg/l (98.7 ± 4.2 mg/g dry cell weight) α-humulene, a 100-fold improvement relative to the baseline strain. To further improve the titer, a novel strategy for downregulation of squalene biosynthesis based on Cu -repressible promoters was firstly established, which significantly improved the α-humulene titer by 54.2% to 3516.6 ± 34.3 mg/l. Finally, the engineered strain could produce 21.7 g/l α-humulene in a 5-L bioreactor, 6.8-fold higher than the highest α-humulene titer reported before this study. Overall, system metabolic engineering strategies used in this study provide a valuable reference for the highly sustainable production of terpenoids in Y. lipolytica.
倍半萜 α- 葎草烯是一种重要的植物天然产物，由于其具有抗炎和抗癌活性，已在制药行业中得到应用。尽管植物提取法和化学合成法以前曾用于 α- 葎草烯的生产，但由于效率低、成本高，限制了其发展。在这项研究中，利用酿酒酵母（Yarrowia lipolytica）作为生产可持续 α- 葎草烯的强大细胞工厂。首先，通过整合具有高催化活性的 α- 葎草烯合酶，优化甲羟戊酸和乙酰辅酶 A 途径的通量，构建了在细胞质中具有高 α- 葎草烯产量的底盘。随后，在酿酒酵母中采用了双细胞质-过氧化物酶体工程策略；通过引入 31 个拷贝的 12 个不同基因，得到的最佳菌株 GQ3006 可以产生 2280.3±38.2mg/L（98.7±4.2mg/g 干细胞重量）的 α- 葎草烯，与基线菌株相比提高了 100 倍。为了进一步提高产量，首次建立了基于 Cu 可诱导启动子下调角鲨烯生物合成的新策略，使 α- 葎草烯的产量提高了 54.2%，达到 3516.6±34.3mg/L。最后，该工程菌株在 5L 生物反应器中可以生产 21.7g/L 的 α- 葎草烯，比本研究之前报道的最高 α- 葎草烯产量提高了 6.8 倍。总之，本研究中使用的系统代谢工程策略为酿酒酵母中萜类化合物的高度可持续生产提供了有价值的参考。
