Institute of Molecular Biology, Slovak Academy of Sciences, Dubravska cesta 21, 845 51, Bratislava, Slovak Republic.
Institute of Chemistry, Slovak Academy of Sciences, 845 38, Bratislava, Slovak Republic.
Appl Microbiol Biotechnol. 2021 Mar;105(5):2123-2137. doi: 10.1007/s00253-021-11161-w. Epub 2021 Feb 10.
The bacteria of the genus Streptomyces are among the most important producers of biologically active secondary metabolites. Moreover, recent genomic sequence data have shown their enormous genetic potential for new natural products, although many new biosynthetic gene clusters (BGCs) are silent. Therefore, efficient and stable genome modification techniques are needed to activate their production or to manipulate their biosynthesis towards increased production or improved properties. We have recently developed an efficient markerless genome modification system for streptomycetes based on positive blue/white selection of double crossovers using the bpsA gene from indigoidine biosynthesis, which has been successfully applied for markerless deletions of genes and BGCs. In the present study, we optimized this system for markerless insertion of large BGCs. In a pilot test experiment, we successfully inserted a part of the landomycin BGC (lanFABCDL) under the control of the ermEp* promoter in place of the actinorhodin BGC (act) of Streptomyces lividans TK24 and RedStrep 1.3. The resulting strains correctly produced UWM6 and rabelomycin in twice the yield compared to S. lividans strains with the same construct inserted using the PhiBT1 phage-based integration vector system. Moreover, the system was more stable. Subsequently, using the same strategy, we effectively inserted the entire BGC for mithramycin (MTM) in place of the calcium-dependent antibiotic BGC (cda) of S. lividans RedStrep 1.3 without antibiotic-resistant markers. The resulting strain produced similar levels of MTM when compared to the previously described S. lividans RedStrep 1.3 strain with the VWB phage-based integration plasmid pMTMF. The system was also more stable. KEY POINTS: • Optimized genome editing system for markerless insertion of BGCs into Streptomyces genomes • Efficient heterologous production of MTM in the stable engineered S. lividans strain.
链霉菌属的细菌是产生生物活性次生代谢物的最重要的生产者之一。此外，最近的基因组序列数据表明，它们具有产生新天然产物的巨大遗传潜力，尽管许多新的生物合成基因簇（BGCs）是沉默的。因此，需要有效的和稳定的基因组修饰技术来激活它们的生产或操纵它们的生物合成以增加产量或改善性质。我们最近基于靛蓝生物合成的 bpsA 基因的双交叉阳性蓝/白选择，开发了一种用于链霉菌的高效无标记基因组修饰系统，该系统已成功应用于基因和 BGC 的无标记缺失。在本研究中，我们优化了该系统用于无标记插入大型 BGC。在一个试点实验中，我们成功地将 landomycin BGC 的一部分（lanFABCDL）在 ermEp*启动子的控制下插入到 Streptomyces lividans TK24 和 RedStrep 1.3 的放线紫红素 BGC（act）中，所得菌株与使用 PhiBT1 噬菌体整合载体系统插入相同构建体的 S. lividans 菌株相比，正确地产生了 UWM6 和雷博霉素，产量增加了两倍。此外，该系统更稳定。随后，我们使用相同的策略，有效地将 mithramycin（MTM）的整个 BGC 插入到 S. lividans RedStrep 1.3 的钙依赖性抗生素 BGC（cda）中，而没有抗生素抗性标记。与之前描述的 S. lividans RedStrep 1.3 菌株使用基于 VWB 噬菌体的整合质粒 pMTMF 相比，所得菌株产生的 MTM 水平相似。该系统也更稳定。关键点：• 优化了无标记插入 BGC 到链霉菌基因组中的基因组编辑系统• 在稳定的工程化 S. lividans 菌株中高效异源生产 MTM。
