Vrancken Kristof, Van Mellaert Lieve, Anné Jozef
Department of Microbiology and Immunology, Rega Institute for Medical Research, K.U. Leuven, Leuven, Belgium.
Methods Mol Biol. 2010;668:97-107. doi: 10.1007/978-1-60761-823-2_6.
The choice of an expression system for the meta-genomic 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 the meta-genomic 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 (Gabor et al., Environ Microbiol 6:879-886, 2004). To recover some of the remaining 60%, alternative cloning hosts such as Streptomyces spp. have been used (Lorenz and Eck, Nat Rev Microbiol 3:510-516, 2005). Streptomycetes are high-GC Gram-positive bacteria that belong to the Actinomycetales, and they have been studied extensively in the last 10 years as an alternative expression system (reviewed in Vrancken and Anné, Future Microbiol 4:181-188, 2009). Streptomyces is extremely well suited for the expression of DNA from other actinomycetes and genomes of high GC content (Wang et al., Org Lett 2:2401-2404, 2000). Furthermore, due to its high innate secretion capacity, it can be a superior system than E. coli for the production of many extra-cellular proteins.
对于感兴趣的宏基因组DNA而言，选择一种表达系统对于检测任何特定基因或基因簇至关重要。迄今为止，大多数筛选都使用革兰氏阴性菌大肠杆菌作为宏基因组基因文库的宿主。然而，使用大肠杆菌存在潜在的宿主偏差，因为通过在大肠杆菌中随机克隆，只有40%的酶活性能够轻易恢复（加博尔等人，《环境微生物学》6:879 - 886，2004年）。为了恢复其余60%中的一部分，已使用诸如链霉菌属等替代克隆宿主（洛伦兹和埃克，《自然评论：微生物学》3:510 - 516，2005年）。链霉菌是属于放线菌目的高GC含量革兰氏阳性菌，在过去10年中，它们作为一种替代表达系统受到了广泛研究（弗兰肯和安妮在《未来微生物学》4:181 - 188，2009年中进行了综述）。链霉菌非常适合表达来自其他放线菌的DNA以及高GC含量的基因组（王等人，《有机快报》2:2401 - 2404，2000年）。此外，由于其较高的固有分泌能力，对于许多细胞外蛋白质的生产而言，它可能是比大肠杆菌更优越的系统。
