Institute of Molecular Biology, Slovak Academy of Sciences, Dubravska cesta 21, 845 51, Bratislava, Slovak Republic.
Appl Microbiol Biotechnol. 2022 Nov;106(21):7285-7299. doi: 10.1007/s00253-022-12187-4. Epub 2022 Sep 29.
The bacteria of the genus Streptomyces are important producers of a large number of biologically active natural products. Examination of their genomes has revealed great biosynthetic potential for the production of new products, but many of them are silent under laboratory conditions. One of the promising avenues for harnessing this biosynthetic potential is the refactoring and heterologous expression of relevant biosynthetic gene clusters (BGCs) in suitable optimized chassis strains. Although several Streptomyces strains have been used for this purpose, the efficacy is relatively low, and some BGCs have not been expressed. In this study, we optimized our long-term genetically studied Streptomyces lavendulae subsp. lavendulae CCM 3239 strain as a potential host for heterologous expression along with its stable large linear plasmid pSA3239 as a vector system. Two reporter genes, mCherry and gusA under the control of ermEp* promoter, were successfully integrated into pSA3239. The activity of GUS reporter was four-fold higher in pSA3239 than in a single site in S. lavendulae subsp. lavendulae CCM 3239 chromosome, consistent with a higher copy number of pSA3239 (4 copies per chromosome). In addition, the two Att/Int systems (based on PhiC31 and pSAM2) were able to integrate into the corresponding individual attB sites in the chromosome. The BGC for actinorhodin was successfully integrated into pSA3239. However, the resulting strain produced very low amounts of actinorhodin. Its level increased dramatically after integration of the actII-ORF4 gene for the positive regulator under the control of the kasOp* promoter into this strain using the PhiC31 phage integration system. KEY POINTS: • New Streptomyces chassis for heterologous expression of genes and BGCs • Optimized strategy for insertion of heterologous genes into linear plasmid pSA3239 • Efficient heterologous production of actinorhodin after induction of its regulator.
放线菌属的细菌是大量生物活性天然产物的重要生产者。对其基因组的研究表明，它们具有产生新产品的巨大生物合成潜力，但在实验室条件下，许多基因簇处于沉默状态。利用这种生物合成潜力的一种有前途的方法是在合适的优化底盘菌株中对相关生物合成基因簇（BGCs）进行重构和异源表达。虽然已经使用了几种链霉菌菌株来实现这一目标，但效果相对较低，并且一些 BGCs 尚未表达。在这项研究中，我们优化了我们长期研究的薰衣草链霉菌亚种。薰衣草 CCM 3239 菌株作为异源表达的潜在宿主，以及其稳定的大型线性质粒 pSA3239 作为载体系统。两个报告基因 mCherry 和 gusA 在 ermEp启动子的控制下成功整合到 pSA3239 中。与 S. lavendulae subsp. lavendulae CCM 3239 染色体中单一位点相比，pSA3239 中的 GUS 报告基因活性高四倍，这与 pSA3239 的更高拷贝数一致（每个染色体 4 个拷贝）。此外，两个 Att/Int 系统（基于 PhiC31 和 pSAM2）能够整合到染色体中相应的单个 attB 位点。放线紫红素生物合成基因簇成功整合到 pSA3239 中。然而，该菌株产生的放线紫红素非常少。在用 PhiC31 噬菌体整合系统将阳性调节基因 actII-ORF4 整合到该菌株中，受 kasOp启动子控制后，其水平显著增加。关键点：• 用于基因和 BGC 异源表达的新型链霉菌底盘• 将异源基因插入线性质粒 pSA3239 的优化策略• 在诱导其调节剂后，有效异源生产放线紫红素。
