Department of Chemical and Biomolecular Engineering, National University of Singapore, Singapore, 117585, Singapore.
Department of Biomolecular Sciences, Kingston University, London, UK.
Appl Microbiol Biotechnol. 2018 Aug;102(16):7007-7015. doi: 10.1007/s00253-018-9141-z. Epub 2018 Jun 8.
Expression of recombinant proteins in Escherichia coli often requires use of inducible promoters to shorten the lag phase and improve protein productivity and final protein titer. Synthetic molecules that cannot be metabolized by E. coli, such as isopropyl thiogalactopyranoside (IPTG), have been frequently used to trigger the protein expression during early exponential growth phase. This practice has many drawbacks, including high cost and toxicity of IPTG, complex operating procedure, and non-uniform protein expression pattern (some cells in the population do not express recombinant proteins). A few auto-inducible protein expression systems have been developed recently to overcome some of these limitations, but they required use of an additional plasmid or presence of large (a few kilobases) DNA part to be functional, making plasmid construction to be difficult, especially when multiple genes need to be expressed. In this study, by using RNA sequencing, we identified a short, endogenous promoter (PthrC) that can be auto-induced during early exponential growth phase, and improved its performance by use of native and mutated regulatory elements. We found that the developed mutants of PthrC drove uniform protein expression-close to 100% of cells were fluorescent when green fluorescence protein was used as target protein-and cells carrying them could achieve much higher cell density than those with T7 promoter (PT7), a commonly used inducible promoter. In terms of promoter strength (product protein quantity per cell), the developed promoter mutants can cover a range of strength, from 30 to 150% of maximal strength of PT7. One strong mutant (PthrC3_8) was found to work well at a large range of temperature (22, 30, 37 °C) and in various media, and was also confirmed to cause less stress to host cell than PT7 when they were used to express a toxic protein. We foresee that PthrC3 and its mutants will be useful genetic parts for various applications including metabolic engineering and biocatalysis.
在大肠杆菌中表达重组蛋白通常需要使用诱导型启动子来缩短迟滞期，提高蛋白产率和最终蛋白滴度。不能被大肠杆菌代谢的合成分子，如异丙基-β-D-硫代半乳糖苷（IPTG），常被用来在早期指数生长期触发蛋白表达。这种做法有许多缺点，包括 IPTG 的成本高、毒性大、操作程序复杂以及蛋白表达模式不均匀（群体中的一些细胞不表达重组蛋白）。最近开发了一些自动诱导型蛋白表达系统来克服其中的一些限制，但它们需要使用额外的质粒或存在较大（几千个碱基）的 DNA 部分才能发挥作用，使得质粒构建变得困难，尤其是当需要表达多个基因时。在这项研究中，我们通过使用 RNA 测序，鉴定了一个短的、内源性的启动子（PthrC），它可以在早期指数生长期自动诱导，并且通过使用天然和突变的调控元件来提高其性能。我们发现，开发的 PthrC 突变体驱动均匀的蛋白表达——当绿色荧光蛋白作为靶蛋白时，几乎 100%的细胞都有荧光——并且携带它们的细胞可以达到比 T7 启动子（PT7）更高的细胞密度，PT7 是一种常用的诱导型启动子。在启动子强度（每个细胞的产物蛋白量）方面，开发的启动子突变体可以覆盖从 30%到 150%的 PT7 最大强度的范围。我们发现一个强突变体（PthrC3_8）在很宽的温度范围（22、30、37°C）和各种培养基中都能很好地工作，并且在表达毒性蛋白时比 PT7 对宿主细胞的应激更小。我们预计 PthrC3 及其突变体将成为各种应用（包括代谢工程和生物催化）的有用遗传元件。
