Wang Rufeng, Cress Brady F, Yang Zheng, Hordines John C, Zhao Shujuan, Jung Gyoo Yeol, Wang Zhengtao, Koffas Mattheos A G
The MOE Key Laboratory for Standardization of Chinese Medicines and the SATCM Key Laboratory for New Resources and Quality Evaluation of Chinese Medicines, Institute of Chinese Materia Medica , Shanghai University of Traditional Chinese Medicine , Shanghai 201203 , China.
Center for Biotechnology and Interdisciplinary Studies, Department of Chemical and Biological Engineering , Rensselaer Polytechnic Institute , Troy , New York 12180 , United States.
ACS Synth Biol. 2019 Sep 20;8(9):2121-2130. doi: 10.1021/acssynbio.9b00212. Epub 2019 Aug 28.
A common challenge in the assembly and optimization of plant natural product biosynthetic pathways in recombinant hosts is the identification of gene orthologues that will result in best production titers. Here, we describe the modular assembly of a naringenin biosynthetic pathway in that was facilitated by optimized naringenin-inducible prokaryotic transcription activators used as biosensors. The biosensors were designed and developed in by a multiparametric engineering strategy, which further was applied for the , high-throughput screening of the established yeast library. The workflow for assembling naringenin biosynthetic pathways involved Golden gate-directed combinatorial assembly of genes and promoters, resulting in a strain library ideally covering 972 combinations in . For improving the performance of our screening biosensor, a series of fundamental components was optimized, affecting the efficiency of the biosensor such as nuclear localization signal (NLS), the detector module and the effector module. One biosensor (NLS_FdeR-N-_mcherry_MV2) showed better performance, defined as better dynamic range and sensitivity than others established in this study as well as other previously reported naringenin biosensors. Using this biosensor, we were able to identify a recombinant strain as the most efficient candidate for the production of naringenin from the established naringenin biosynthetic library. This approach can be exploited for the optimization of other metabolites derived from the flavonoid biosynthetic pathways and more importantly employed in the characterization of putative flavonoid biosynthetic genes.
在重组宿主中组装和优化植物天然产物生物合成途径时，一个常见的挑战是鉴定能产生最佳生产效价的基因直系同源物。在此，我们描述了在大肠杆菌中通过使用优化的柚皮素诱导型原核转录激活因子作为生物传感器来促进柚皮素生物合成途径的模块化组装。这些生物传感器是通过多参数工程策略在大肠杆菌中设计和开发的，该策略进一步应用于对已建立的酵母文库进行高通量筛选。组装柚皮素生物合成途径的工作流程涉及基因和启动子的金门定向组合组装，从而在大肠杆菌中产生一个理想情况下涵盖972种组合的菌株文库。为了提高我们筛选生物传感器的性能，对一系列基本组件进行了优化，这些组件会影响生物传感器的效率，如核定位信号（NLS）、检测模块和效应模块。一种生物传感器（NLS_FdeR-N-_mcherry_MV2）表现出更好的性能，其定义为与本研究中建立的其他生物传感器以及其他先前报道的柚皮素生物传感器相比，具有更好的动态范围和灵敏度。使用这种生物传感器，我们能够从已建立的柚皮素生物合成文库中鉴定出一种重组大肠杆菌菌株作为生产柚皮素的最有效候选菌株。这种方法可用于优化源自黄酮类生物合成途径的其他代谢物，更重要的是可用于鉴定假定的黄酮类生物合成基因。
