Gibisch Martin, Gorecki Pawel, Tauer Christopher, Egger Esther, Müller Matthias, Albrecht Bernd, Hahn Rainer, Striedner Gerald, Cserjan-Puschmann Monika
Christian Doppler Laboratory for Production of Next-Level Biopharmaceuticals in 𝐸. 𝐶𝑜𝑙𝑖, Institute of Bioprocess Science and Engineering, BOKU University, Muthgasse 18, 1190, Vienna, Austria.
Boehringer-Ingelheim RCV, GmbH & Co KG, Dr.-Boehringer-Gasse 5-11, Vienna, 1120, Austria.
Appl Microbiol Biotechnol. 2025 Jun 4;109(1):136. doi: 10.1007/s00253-025-13524-z.
Despite its many benefits, Escherichia coli only poorly secretes recombinant proteins and peptides into the medium. This complicates downstream processing and notably contributes to the production costs of biopharmaceuticals. The permeability of production strains can be increased by deletion of the lpp gene, coding for Braun's lipoprotein Lpp. Consequently, the outer membrane (OM) is destabilized, and periplasmic recombinant proteins/peptides can leak out of the cell into the cultivation medium. However, we observed poor process performance during C-limited fed-batch cultivations in bioreactors when production strains with lpp knockout were cultivated. In this study, we developed an inducible system for in-process Lpp downregulation (knockdown) in E. coli with the goal to facilitate the release of the periplasmic recombinant fusion peptide CASPON-SST into the cultivation medium. By plasmid-based overexpression of MicL sRNA, we were able to efficiently inhibit Lpp synthesis and increase the OM permeability of our production strains. With this approach, we were able to achieve the secretion of 80-100% of all peptide and increased production capacities. The system was further optimized by utilizing different promoter systems to induce peptide and MicL expression separately in order to coordinate them. We report here for the first time the extracellular production of a recombinant peptide by inducible downregulation of Lpp via MicL sRNA during C-limited fed-batch cultivations. By utilizing a flexible system for Lpp knockdown, potential drawbacks of lpp knockout can be counteracted, thus making our approach a valuable tool for the in-process adaptation of OM permeability in production hosts. KEY POINTS: • Lpp can be downregulated on mRNA level by MicL sRNA overexpressed during fed-batch. • Recombinant peptides can leak out of the periplasm when Lpp is downregulated. • Leakiness and outer membrane permeability must be distinguished.
尽管大肠杆菌有诸多益处，但它向培养基中分泌重组蛋白和肽的能力很差。这使得下游加工变得复杂，尤其增加了生物制药的生产成本。通过缺失编码布劳恩脂蛋白Lpp的lpp基因，可以提高生产菌株的通透性。因此，外膜会变得不稳定，周质中的重组蛋白/肽会从细胞中泄漏到培养基中。然而，我们观察到，在生物反应器中进行碳源限制补料分批培养时，培养具有lpp基因敲除的生产菌株时工艺性能不佳。在本研究中，我们开发了一种可诱导系统，用于在大肠杆菌中进行过程中Lpp的下调（敲低），目的是促进周质重组融合肽CASPON-SST释放到培养基中。通过基于质粒的MicL sRNA过表达，我们能够有效抑制Lpp的合成并提高生产菌株的外膜通透性。通过这种方法，我们能够实现所有肽80%-100%的分泌，并提高生产能力。通过利用不同的启动子系统分别诱导肽和MicL的表达以协调它们，该系统得到了进一步优化。我们首次报道了在碳源限制补料分批培养过程中，通过MicL sRNA诱导下调Lpp实现重组肽的胞外生产。通过利用一种灵活的Lpp敲低系统，可以抵消lpp基因敲除的潜在缺点，从而使我们的方法成为生产宿主中外膜通透性过程中适应性调整的有价值工具。要点：• 在补料分批培养期间过表达的MicL sRNA可在mRNA水平上下调Lpp。• 当Lpp下调时，重组肽可从周质中泄漏出来。• 必须区分渗漏性和外膜通透性。
