Abstract

With the goal of expanding the diversity of tools available for controlling gene expression in cyanobacteria, the T7-RNA polymerase gene expression system from E. coli BL21(DE3) was adapted and systematically engineered for robust function Synechococcus sp. PCC 7002, a fast-growing saltwater strain. Expression of T7-RNA polymerase was controlled via LacI regulation, while functionality was optimized by both further tuning its expression level along with optimizing the translation initiation region of the expressed gene, in this case an enhanced YFP reporter. Under high CO₂ conditions, the resulting system displayed a 60-fold dynamic range in expression levels. Furthermore, when maximally induced, T7-RNA polymerase-dependent protein production constituted up to two-thirds of total cellular protein content in Synechococcus sp. PCC 7002. Ultimately, however, this came at the cost of 40% reductions in both biomass and pigmentation levels. Taken together, the developed T7-RNA polymerase gene expression system is effective for controlling and achieving high-level expression of heterologous genes in Synechococcus sp. PCC 7002, making it a valuable tool for cyanobacterial research.

Key Points

• Promoter driving T7-RNA polymerase was optimized.

• Up to 60-fold dynamic range in expression, depending on CO ₂ conditions.

• Two-thirds of total protein is T7-RNA polymerase dependent.

中文翻译：

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Synechococcus sp中T7-RNA聚合酶系统的优化。PCC 7002 反映了大肠杆菌 BL21(DE3) 的蛋白质过度生产表型

摘要

为了扩大可用于控制蓝藻中基因表达的工具的多样性，来自大肠杆菌BL21(DE3) 的 T7-RNA 聚合酶基因表达系统经过改造和系统工程，以实现强大的功能Synechococcus sp。PCC 7002，一种快速生长的盐水菌株。T7-RNA 聚合酶的表达通过 LacI 调节来控制，而功能通过进一步调整其表达水平以及优化表达基因的翻译起始区域来优化，在这种情况下是增强的 YFP 报告基因。在高 CO _2下条件下，所得系统的表达水平显示出 60 倍的动态范围。此外，当最大限度地诱导时，T7-RNA 聚合酶依赖性蛋白的产生占Synechococcus sp.中细胞总蛋白含量的三分之二。PCC 7002。然而，最终，这是以生物量和色素沉着水平降低 40% 为代价的。总之，开发的 T7-RNA 聚合酶基因表达系统可有效控制和实现聚球藻中异源基因的高水平表达。PCC 7002，使其成为蓝藻研究的宝贵工具。

关键点

• 优化了驱动T7-RNA 聚合酶的启动子。

• 表达的动态范围高达 60 倍，具体取决于 CO ₂条件。

• 三分之二的总蛋白质依赖于 T7-RNA 聚合酶。