Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida, USA.
Appl Environ Microbiol. 2022 Nov 22;88(22):e0093922. doi: 10.1128/aem.00939-22. Epub 2022 Nov 7.
Controlled gene expression is crucial for engineering bacteria for basic and applied research. Inducible systems enable tight regulation of expression, wherein a small-molecule inducer causes the transcription factor to activate or repress transcriptional initiation. The T7 expression system is one of the most widely used inducible systems, particularly for high overexpression of proteins. However, it is well known that the highly active T7 RNA polymerase (RNAP) has several drawbacks, including toxicity to the host and substantial leaky expression in the absence of an inducer. Much work has been done to address these issues; current solutions require special strains or additional plasmids, making the system more complicated and less accessible. Here, we challenge the assumption that the T7 expression system is the best choice for obtaining high protein titers. We hypothesized that expression from strong inducible promoters expressed from high-copy plasmids could compete with expression levels obtained from T7 RNAP but that such promoters would possess improved control of transcription. Employing inducible systems from a toolbox we developed previously, we demonstrate that our plasmids consistently give higher outputs and greater fold changes over basal expression than the T7 system across rich and minimal media. In addition, we show that they outperformed the T7 system when we used an engineered metabolic pathway to produce lycopene. Genetic systems for protein overexpression are required tools in microbiological and biochemical research. Ideally, these systems include standardized genetic parts with predictable behavior, enabling the construction of stable expression systems in the host organism. Modularity of a genetic system is advantageous, so that the expression system can be easily moved into a host that best suits the needs of a given experiment. The T7 expression system lacks both predictability and stability and requires special host strains to function. Despite these limitations, it remains one of the most popular systems for protein overproduction. This study directly compared the T7 system to four inducible systems from our broad-host-range plasmid toolbox and demonstrated these alternative expression systems have distinct advantages over the T7. The systems are entirely plasmid-based and not constrained to a specific bacterial host, expanding the options for high-level protein expression across strains.
基因表达的控制对于细菌的基础研究和应用研究至关重要。诱导型系统可实现表达的紧密调控，其中小分子诱导剂使转录因子激活或抑制转录起始。T7 表达系统是最广泛使用的诱导型系统之一，特别是用于蛋白质的高过表达。然而，众所周知，高活性的 T7 RNA 聚合酶（RNAP）有几个缺点，包括对宿主的毒性和在没有诱导剂的情况下大量漏表达。已经做了很多工作来解决这些问题；目前的解决方案需要特殊的菌株或额外的质粒，使系统更加复杂且不太容易获得。在这里，我们质疑 T7 表达系统是获得高蛋白滴度的最佳选择这一假设。我们假设，强诱导启动子的表达，来自高拷贝质粒，可以与 T7 RNAP 获得的表达水平竞争，但这种启动子将具有更好的转录控制。我们使用之前开发的工具包中的诱导系统，证明我们的质粒在丰富和基础培养基中都比 T7 系统具有更高的产量和更大的倍数变化，从而始终获得更高的输出和更高的基础表达。此外，当我们使用工程化代谢途径生产番茄红素时，它们的表现优于 T7 系统。
用于蛋白质过表达的遗传系统是微生物学和生物化学研究中必需的工具。理想情况下，这些系统包括具有可预测行为的标准化遗传元件，从而能够在宿主生物体内构建稳定的表达系统。遗传系统的模块化是有利的，因此表达系统可以很容易地转移到最适合特定实验需求的宿主中。T7 表达系统既缺乏可预测性又不稳定，并且需要特殊的宿主菌株才能发挥作用。尽管存在这些限制，但它仍然是最受欢迎的蛋白质过表达系统之一。本研究直接比较了 T7 系统和我们广泛宿主范围质粒工具包中的四个诱导系统，证明这些替代表达系统在 T7 系统上具有明显的优势。这些系统完全基于质粒，不受特定细菌宿主的限制，在不同菌株中扩大了高水平蛋白质表达的选择。
