Zhang Chenglong, Chen Wang, Dong Tianyu, Wang Ying, Yao Mingdong, Xiao Wenhai, Li Bingzhi
Frontier Science Center for Synthetic Biology, Key Laboratory of Systems Bioengineering (Ministry of Education), School of Chemical Engineering and Technology, Tianjin University, Tianjin, China.
Georgia Tech Shenzhen Institute, Tianjin University, Shenzhen, China.
Front Bioeng Biotechnol. 2023 Feb 20;11:1141272. doi: 10.3389/fbioe.2023.1141272. eCollection 2023.
Taxadiene is an important precursor in taxol biosynthesis pathway, but its biosynthesis in eukaryotic cell factories is limited, which seriously hinders the biosynthesis of taxol. In this study, it is found that there was the catalysis compartmentalization between two key exogenous enzymes of geranylgeranyl pyrophosphate synthase and taxadiene synthase (TS) for taxadiene synthesis progress, due to their different subcellular localization. Firstly, the enzyme-catalysis compartmentalization was overcome by means of the intracellular relocation strategies of taxadiene synthase, including N-terminal truncation of taxadiene synthase and enzyme fusion of GGPPS-TS. With the help of two strategies for enzyme relocation, the taxadiene yield was increased by 21% and 54% respectively, among them the GGPPS-TS fusion enzyme is more effective. Further, the expression of GGPPS-TS fusion enzyme was improved the multi-copy plasmid, resulting that the taxadiene titer was increased by 38% to 21.8 mg/L at shake-flask level. Finally, the maximum taxadiene titer of 184.2 mg/L was achieved by optimization of the fed-batch fermentation conditions in 3 L bioreactor, which is the highest reported titer of taxadiene biosynthesis accomplished in eukaryotic microbes. This study provides a successful example for improving biosynthesis of complex natural products by solving the critical problem of multistep enzymes catalysis compartmentalization.
紫杉二烯是紫杉醇生物合成途径中的重要前体，但它在真核细胞工厂中的生物合成受到限制，这严重阻碍了紫杉醇的生物合成。在本研究中，发现由于香叶基香叶基焦磷酸合酶和紫杉二烯合酶（TS）这两种用于紫杉二烯合成的关键外源酶的亚细胞定位不同，它们在催化过程中存在区室化现象。首先，通过紫杉二烯合酶的细胞内重新定位策略克服了酶催化区室化，包括紫杉二烯合酶的N端截短和GGPPS-TS的酶融合。借助这两种酶重新定位策略，紫杉二烯产量分别提高了21%和54%，其中GGPPS-TS融合酶更有效。进一步地，通过多拷贝质粒提高了GGPPS-TS融合酶的表达，使得在摇瓶水平上紫杉二烯滴度提高了38%，达到21.8 mg/L。最后，通过优化3 L生物反应器中的补料分批发酵条件，实现了184.2 mg/L的最大紫杉二烯滴度，这是在真核微生物中报道的最高的紫杉二烯生物合成滴度。本研究通过解决多步酶催化区室化这一关键问题，为提高复杂天然产物的生物合成提供了一个成功范例。
