School of Food Science and Pharmaceutical Engineering, Nanjing Normal University, Nanjing, 210046, Jiangsu, China.
State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, 211816, Jiangsu, China.
Bioprocess Biosyst Eng. 2021 Jun;44(6):1033-1047. doi: 10.1007/s00449-020-02503-5. Epub 2021 Jan 24.
To construct a Saccharomyces cerevisiae strain for efficient lycopene production, we used a pathway engineering strategy based on expression modules comprising fusion proteins and a strong constitutive promoter. The two recombinant plasmids pEBI encoding the fusion genes with an inducible promoter, as well as pIETB with a constitutive promoter and terminator were introduced into S. cerevisiae YPH499 and BY4741 to obtain the four recombinant strains ypEBI, ypIETB, byEBI and byIETB. The lycopene production and the transcription levels of key genes were higher in the BY4741 chassis than in YPH499. Accordingly, the content of total and unsaturated fatty acids was also higher in BY4741, which also exhibited a decrease of glucose, increase of trehalose, increase of metabolite in citrate cycle, and low levels of amino acids. These changes rerouted metabolic fluxes toward lycopene synthesis, indicating that the BY4741 chassis was more suitable for lycopene synthesis. The lycopene content of bpIETB in SG-Leu medium supplemented with 100 mg/L of linolenic acid reached 10.12 mg/g dry cell weight (DCW), which was 85.7% higher than without the addition of unsaturated fatty acids. The constitutive promoter expression strategy employed in this study achieved efficient lycopene synthesis in S. cerevisiae, and the strain bpIETB was obtained a suitable chassis host for lycopene production, which provides a basis for further optimization of lycopene production in artificial synthetic cells and a reference for the multi-enzyme synthesis of other similar complex terpenoids.
为了构建高效生产番茄红素的酿酒酵母菌株，我们采用了基于包含融合蛋白和强组成型启动子的表达模块的途径工程策略。将编码带有诱导型启动子的融合基因的两个重组质粒 pEBI 以及带有组成型启动子和终止子的 pIETB 引入酿酒酵母 YPH499 和 BY4741 中，得到了四个重组菌株 ypEBI、ypIETB、byEBI 和 byIETB。与 YPH499 相比，BY4741 底盘中的番茄红素产量和关键基因的转录水平更高。因此，BY4741 底盘中的总脂肪酸和不饱和脂肪酸含量也更高，同时葡萄糖消耗减少，海藻糖增加，柠檬酸循环中的代谢物增加，氨基酸水平降低。这些变化改变了代谢通量，有利于番茄红素的合成，表明 BY4741 底盘更适合番茄红素的合成。在补充有 100mg/L 亚油酸的 SG-Leu 培养基中，bpIETB 的番茄红素含量达到 10.12mg/g 干细胞重量（DCW），比不添加不饱和脂肪酸时提高了 85.7%。本研究中采用的组成型启动子表达策略在酿酒酵母中实现了高效的番茄红素合成，并且获得了一个适合番茄红素生产的底盘宿主菌株 bpIETB，为在人工合成细胞中进一步优化番茄红素生产以及为其他类似复杂类异戊二烯的多酶合成提供了基础。
