Institute for Synthetic Microbiology, Heinrich Heine University Duesseldorf, 40225 Duesseldorf, Germany.
Department of Chemistry - Ångström, Uppsala University, 75120 Uppsala, Sweden.
ACS Synth Biol. 2020 Apr 17;9(4):843-855. doi: 10.1021/acssynbio.9b00505. Epub 2020 Mar 17.
Design and implementation of synthetic biological circuits highly depends on well-characterized, robust promoters with predictable input-output responses. While great progress has been made with heterotrophic model organisms such as , the available variety of tunable promoter parts for phototrophic cyanobacteria is still limited. Commonly used synthetic and semisynthetic promoters show weak dynamic ranges or no regulation at all in cyanobacterial models. Well-controlled alternatives such as native metal-responsive promoters, however, pose the problems of inducer toxicity and lacking orthogonality. Here, we present the comparative assessment of dose-response functions of four different inducible promoter systems in the model cyanobacterium sp. PCC 6803. Using the novel bimodular reporter plasmid pSHDY, dose-response dynamics of the re-established vanillate-inducible promoter P was compared to the previously described rhamnose-inducible P, the anhydrotetracycline-inducible P, and the Co-inducible P. We estimate individual advantages and disadvantages regarding dynamic range and strength of each promoter, also in comparison with well-established constitutive systems. We observed a delicate balance between transcription factor toxicity and sufficient expression to obtain a dose-dependent response to the inducer. In summary, we expand the current understanding and employability of inducible promoters in cyanobacteria, facilitating the scalability and robustness of synthetic regulatory network designs and of complex metabolic pathway engineering strategies.
合成生物学电路的设计和实现高度依赖于特征良好、稳健的启动子，这些启动子具有可预测的输入-输出响应。虽然在异养模式生物如 中取得了巨大进展，但用于光养蓝藻的可调启动子部件的种类仍然有限。常用的合成和半合成启动子在蓝藻模型中表现出较弱的动态范围或根本没有调节。然而，像天然金属响应启动子这样的良好控制的替代方案存在诱导剂毒性和缺乏正交性的问题。在这里，我们在模式蓝藻 sp. PCC 6803 中对四种不同诱导型启动子系统的剂量反应函数进行了比较评估。使用新型双模块报告质粒 pSHDY，我们比较了重新建立的香草酸盐诱导型启动子 P 的剂量反应动力学与先前描述的鼠李糖诱导型 P、安非他命四环素诱导型 P 和共诱导型 P。我们估计了每个启动子在动态范围和强度方面的个体优势和劣势，同时也与成熟的组成型系统进行了比较。我们观察到转录因子毒性和足够表达之间的微妙平衡，以获得对诱导剂的剂量依赖性响应。总之，我们扩展了目前对蓝藻中诱导型启动子的理解和可利用性，促进了合成调控网络设计和复杂代谢途径工程策略的可扩展性和稳健性。
