Molecular Plant Biology, Department of Biochemistry, University of Turku, 20014, Turku, Finland.
Microb Cell Fact. 2018 Mar 2;17(1):34. doi: 10.1186/s12934-018-0882-2.
Photosynthetic cyanobacteria have been studied as potential host organisms for direct solar-driven production of different carbon-based chemicals from CO and water, as part of the development of sustainable future biotechnological applications. The engineering approaches, however, are still limited by the lack of comprehensive information on most optimal expression strategies and validated species-specific genetic elements which are essential for increasing the intricacy, predictability and efficiency of the systems. This study focused on the systematic evaluation of the key translational control elements, ribosome binding sites (RBS), in the cyanobacterial host Synechocystis sp. PCC 6803, with the objective of expanding the palette of tools for more rigorous engineering approaches.
An expression system was established for the comparison of 13 selected RBS sequences in Synechocystis, using several alternative reporter proteins (sYFP2, codon-optimized GFPmut3 and ethylene forming enzyme) as quantitative indicators of the relative translation efficiencies. The set-up was shown to yield highly reproducible expression patterns in independent analytical series with low variation between biological replicates, thus allowing statistical comparison of the activities of the different RBSs in vivo. While the RBSs covered a relatively broad overall expression level range, the downstream gene sequence was demonstrated in a rigorous manner to have a clear impact on the resulting translational profiles. This was expected to reflect interfering sequence-specific mRNA-level interaction between the RBS and the coding region, yet correlation between potential secondary structure formation and observed translation levels could not be resolved with existing in silico prediction tools.
The study expands our current understanding on the potential and limitations associated with the regulation of protein expression at translational level in engineered cyanobacteria. The acquired information can be used for selecting appropriate RBSs for optimizing over-expression constructs or multicistronic pathways in Synechocystis, while underlining the complications in predicting the activity due to gene-specific interactions which may reduce the translational efficiency for a given RBS-gene combination. Ultimately, the findings emphasize the need for additional characterized insulator sequence elements to decouple the interaction between the RBS and the coding region for future engineering approaches.
作为开发可持续未来生物技术应用的一部分，人们已经研究了光合蓝藻作为从 CO 和水中直接太阳能驱动生产不同碳基化学品的潜在宿主生物。然而，这些工程方法仍然受到缺乏全面的最佳表达策略信息和经过验证的物种特异性遗传元件的限制，这些元件对于提高系统的复杂性、可预测性和效率至关重要。本研究侧重于系统评估蓝藻宿主集胞藻 PCC 6803 中的关键翻译控制元件——核糖体结合位点 (RBS)，旨在扩展更严格的工程方法的工具库。
为了在集胞藻中比较 13 个选定的 RBS 序列，建立了一个表达系统，使用几种替代报告蛋白（sYFP2、密码子优化 GFPmut3 和乙烯形成酶）作为相对翻译效率的定量指标。该系统在独立的分析系列中表现出高度可重复的表达模式，生物重复之间的变化很小，因此允许在体内比较不同 RBS 的活性。虽然 RBS 覆盖了相对较宽的整体表达水平范围，但下游基因序列以严格的方式表明对最终翻译谱有明显影响。这预计反映了 RBS 和编码区之间的干扰序列特异性 mRNA 水平相互作用，但现有基于计算机的预测工具无法解决潜在二级结构形成与观察到的翻译水平之间的相关性。
该研究扩展了我们目前对工程蓝藻中翻译水平调节蛋白表达的潜力和限制的理解。所获得的信息可用于选择合适的 RBS 来优化集胞藻中的过表达构建体或多顺反子途径，同时强调由于基因特异性相互作用导致翻译效率降低的复杂性，这些相互作用可能会降低给定 RBS-基因组合的翻译效率。最终，这些发现强调了需要额外的特征化绝缘子序列元件来解耦 RBS 和编码区之间的相互作用，以用于未来的工程方法。
