Explogen LLC, Lviv, Ukraine.
Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava, Slovak Republic.
Methods Mol Biol. 2023;2555:213-260. doi: 10.1007/978-1-0716-2795-2_16.
The choice of an expression system for the metagenomic DNA of interest is of vital importance for the detection of any particular gene or gene cluster. Most of the screens to date have used the Gram-negative bacterium Escherichia coli as a host for metagenomic gene libraries. However, the use of E. coli introduces a potential host bias since only 40% of the enzymatic activities may be readily recovered by random cloning in E. coli. To recover some of the remaining 60%, alternative cloning hosts such as Streptomyces spp. have been used. Streptomycetes are high-GC Gram-positive bacteria belonging to the Actinomycetales and they have been studied extensively for more than 25 years as an alternative expression system. They are extremely well suited for the expression of DNA from other actinomycetes and genomes of high GC content. Furthermore, due to its high innate, extracellular secretion capacity, Streptomyces can be a better system than E. coli for the production of many extracellular proteins. In this article, an overview is given about the materials and methods for growth and successful expression and secretion of heterologous proteins from diverse origin using Streptomyces lividans as a host. More in detail, an overview is given about the protocols of transformation, type of plasmids used and of vectors useful for integration of DNA into the host chromosome, and accompanying cloning strategies. In addition, various control elements for gene expression including synthetic promoters are discussed, and methods to compare their strength are described. Stable and efficient marker-less integration of the gene of interest under the control of the promoter of choice into S. lividans chromosome via homologous recombination using pAMR23A-based system will be explained. Finally, a basic protocol for bench-top bioreactor experiments which can form the start in the production process optimization and up-scaling will be provided.
选择感兴趣的宏基因组 DNA 的表达系统对于检测任何特定的基因或基因簇都至关重要。迄今为止，大多数筛选都使用革兰氏阴性细菌大肠杆菌作为宏基因组基因文库的宿主。然而，使用大肠杆菌会引入潜在的宿主偏倚，因为只有 40%的酶活性可以通过随机克隆在大肠杆菌中轻易回收。为了回收其余的 60%，已经使用了替代克隆宿主，例如链霉菌属。链霉菌是属于放线菌目的高 GC 革兰氏阳性细菌，它们作为替代表达系统已经被广泛研究了超过 25 年。它们非常适合表达来自其他放线菌的 DNA 和高 GC 含量的基因组。此外，由于其固有的、细胞外分泌能力强，链霉菌可以成为比大肠杆菌更好的系统，用于生产许多细胞外蛋白。本文概述了使用链霉菌作为宿主生长和成功表达及分泌异源蛋白的材料和方法。更详细地说，概述了转化的方案、使用的质粒类型和用于将 DNA 整合到宿主染色体的载体，以及伴随的克隆策略。此外，还讨论了各种基因表达控制元件，包括合成启动子，并描述了比较它们强度的方法。通过使用基于 pAMR23A 的系统，在选择的启动子控制下，通过同源重组将目的基因稳定、高效地无标记整合到链霉菌染色体中，这将得到解释。最后，将提供一个基本的台式生物反应器实验方案，这可以作为生产过程优化和放大的起点。
