Zhang Yaojie, Zhou Lu, Tang Kexuan, Xu Meihui, Miao Zhiqi
School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China.
ACS Omega. 2020 Dec 16;5(51):33028-33038. doi: 10.1021/acsomega.0c04391. eCollection 2020 Dec 29.
The valuable terpenoids, such as artemisinin acid, have achieved bioproduction in the chassis of microbes recently. In this study, L, a promising plant synthetic biology chassis, was used to explore the possibility of patchoulol production by constructing a synthetic biology pathway composed of and . The experiment results show that the maximum yields based on the cytoplasm and plastid pathway were 621.56 and 1006.45 μg/g, respectively. However, there is no statistically significant difference in the yield of patchoulol between transformant plants with different subcellular compartment-targeting pathways. However, it was found that the highest yield of patchoulol was achieved in transformant plants with similar transcription levels of and . Also, the optimized transcription ratio between and is determined at 1.12 based on statistical analysis and model simulation. Therefore, two kinds of new optimized pathway vectors were constructed. One is based on the fusion protein method, and the other is based on protein expression individually, in which the same promoter and terminator were used to derive the expression of both and . The effect of pathway optimization was tested by transient and stable transformation. The production of patchoulol in transient transformation was the same for the two abovementioned kinds of matching pathway and higher than that for the original pathway. Also, in stable transformation, the yield of patchoulol reached up to 3250.30 μg/g, being three times the maximum content before optimization. It is suggested that is a powerful plant chassis for terpenoid synthetic biology and the matching between enzymes may be the key factor in determining the metabolic flux of the pathway in the study of synthetic biology.
诸如青蒿酸等有价值的萜类化合物近来已在微生物底盘中实现了生物合成。在本研究中，L作为一种有前景的植物合成生物学底盘，被用于通过构建由[具体基因1]和[具体基因2]组成的合成生物学途径来探索广藿香醇生产的可能性。实验结果表明，基于细胞质和质体途径的最大产量分别为621.56和1006.45μg/g。然而，具有不同亚细胞区室靶向途径的转化植株之间广藿香醇产量在统计学上并无显著差异。不过，发现[具体基因1]和[具体基因2]转录水平相似的转化植株中广藿香醇产量最高。此外，基于统计分析和模型模拟确定[具体基因1]和[具体基因2]之间的优化转录比为1.12。因此，构建了两种新的优化途径载体。一种基于融合蛋白方法，另一种基于蛋白单独表达，其中使用相同的启动子和终止子来驱动[具体基因1]和[具体基因2]的表达。通过瞬时和稳定转化测试了途径优化的效果。上述两种匹配途径在瞬时转化中广藿香醇的产量相同且高于原始途径。同样，在稳定转化中，广藿香醇产量高达3250.30μg/g，是优化前最大含量的三倍。结果表明，L是萜类合成生物学的强大植物底盘，在合成生物学研究中，酶之间的匹配可能是决定该途径代谢通量的关键因素。
